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WO2020032366A1 - Immunoreactant carrier - Google Patents

Immunoreactant carrier Download PDF

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Publication number
WO2020032366A1
WO2020032366A1 PCT/KR2019/006682 KR2019006682W WO2020032366A1 WO 2020032366 A1 WO2020032366 A1 WO 2020032366A1 KR 2019006682 W KR2019006682 W KR 2019006682W WO 2020032366 A1 WO2020032366 A1 WO 2020032366A1
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WO
WIPO (PCT)
Prior art keywords
silica particles
porous silica
antibody
hours
cytokine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/KR2019/006682
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French (fr)
Korean (ko)
Other versions
WO2020032366A9 (en
Inventor
원철희
민달희
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Lemonex Inc
Original Assignee
Lemonex Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to EP19846510.6A priority Critical patent/EP3834820A4/en
Priority to CA3108251A priority patent/CA3108251C/en
Priority to US17/265,692 priority patent/US12343406B2/en
Priority to CN201980052469.0A priority patent/CN112543631B/en
Priority to AU2019317939A priority patent/AU2019317939A1/en
Priority to SG11202101258VA priority patent/SG11202101258VA/en
Priority to JP2021506399A priority patent/JP7688394B2/en
Priority claimed from KR1020190065620A external-priority patent/KR102316722B1/en
Application filed by Lemonex Inc filed Critical Lemonex Inc
Publication of WO2020032366A1 publication Critical patent/WO2020032366A1/en
Anticipated expiration legal-status Critical
Publication of WO2020032366A9 publication Critical patent/WO2020032366A9/en
Priority to AU2023208105A priority patent/AU2023208105A1/en
Priority to AU2025226757A priority patent/AU2025226757A1/en
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • the present invention relates to an immunoreactive substance carrier having an excellent immunotherapeutic effect by delivering an immunoreactive substance stably to a target site.
  • Drug delivery system refers to a medical technology that can efficiently deliver the required amount of drugs, such as proteins, nucleic acids, or other small molecules by minimizing the side effects and maximizing the efficacy and effects of existing drugs.
  • This technology which saves the cost and time required for the development of new drugs, has recently become one of the cutting-edge technologies that create new added value in the pharmaceutical industry, in combination with nanotechnology.
  • the company has been focusing on the development of drug delivery systems along with the development of new drugs, especially companies and companies.
  • Efficient delivery systems are needed for the study of the function of bioactive substances in cells or for intracellular delivery.
  • a universal delivery system capable of delivering a wide range of bioactive substances, a system capable of accommodating and delivering a large amount of drugs, and a system for releasing drugs in a sustained manner is far from under development.
  • An object of the present invention is to stably deliver various immunoreactive substances including antibodies and cytokines to a target site, thereby providing an immunoreactive substance carrier having excellent immunotherapeutic effects.
  • the porous silica particles react with silica particles having pores less than 5 nm in diameter at 120 ° C. to 180 ° C. for 24 to 96 hours to expand the pores less than 5 nm in diameter; And calcining the pores of expanded silica particles at a temperature of 400 ° C. or higher for at least 3 hours.
  • the average diameter of the porous silica particles is 150 nm to 1000 nm, the BET surface area is 200m 2 / g to 700m 2 / g, the volume per g is 0.7ml to 2.2ml,
  • the porous silica particles have an immunoreactive substance carrier in which t is 24 or more such that the ratio of absorbance of Equation 1 is 1/2:
  • a 0 is the absorbance of the porous silica particles measured by placing 5 ml of the 1 mg / ml suspension of the porous silica particles into a cylindrical permeable membrane having pores having a diameter of 50 kDa,
  • the pH of the suspension is 7.4,
  • a t is the absorbance of the porous silica particles measured after t hours have elapsed since the measurement of A 0 ).
  • the antibody of 1 above wherein the antibody is an IgG; PD-1, CTLA-4, TIM-3, BTLA, VISTA, LAG-3, PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD27, CD137, HVEM, GITR, VEGFR, VEGF,
  • the antibody is an antibody that specifically binds to at least one protein selected from the group consisting of PD-1, PD-L1, and CTLA-4.
  • the cytokine is IL-7, IL-10, IL-12, IL-13, IL-15, IL-21, IL-23, IL-24, IL-27, G-CSF , GM-CSF, HGF, EGF, VEGF, LTF, TGF- ⁇ , and at least one carrier selected from the group consisting of IL-2.
  • cytokine is at least one selected from the group consisting of IL-2, IL-12, IL-15, IL-21, IL-24, and IL-13.
  • porous silica particles have hydrophilic or hydrophobic functional groups on the outer surface or inside the pores.
  • composition for immunotherapy comprising the carrier of any one of the above 1 to 10.
  • a pharmaceutical composition for preventing or treating cancer or immune disease comprising the carrier of any one of 1 to 10 above.
  • the immune-responsive substance carrier of the present invention stably delivers various immune-responsive substances, including antibodies and cytokines, to the target site, and has an excellent immunotherapeutic effect.
  • FIG. 1 is a micrograph of porous silica particles according to an embodiment of the present invention.
  • FIG. 2 is a micrograph of porous silica particles according to one embodiment of the present invention.
  • Figure 3 is a micrograph of the small pore particles during the manufacturing process of the porous silica particles according to an embodiment of the present invention.
  • Figure 4 is a micrograph of the small pore particles according to an embodiment of the present invention.
  • Figure 5 is a micrograph of the pore diameter of the porous silica particles according to an embodiment of the present invention.
  • DDV Delivery Vehicle
  • the number in parenthesis means the diameter of the particle
  • the number of subscripts means the pore diameter.
  • DDV (200) 10 means the particles of the embodiment having a particle diameter of 200 nm
  • the pore diameter is 10 nm.
  • Figure 6 is a micrograph to confirm the biodegradability of the porous silica particles according to an embodiment of the present invention.
  • FIG. 7 is a tube having a cylindrical permeable membrane according to one example.
  • Figure 11 is a result of reducing the absorbance for each pH of the environment over time of the porous silica particles according to an embodiment of the present invention.
  • 13 to 15 show cumulative amounts released after supporting IgG, PD-1, and PD-L1 antibodies on porous silica particles, respectively.
  • FIG. 16 shows TEM images of porous silica particles used to support IL-2 cytokines.
  • 17 to 21 show the cumulative amount released after supporting IL-10, IL-15, HGF, EGF, IL-2 on porous silica particles.
  • an immunoreactive substance transporter comprising; porous silica particles carrying an antibody or cytokine.
  • the cytokine may be specifically interleukins or interferons.
  • the cytokines are IL-7, IL-10, IL-12, IL-13, IL-15, IL-21, IL-23, IL-24, IL-27, G-CSF, GM-CSF, HGF, EGF, VEGF, LTF, TGF- ⁇ , IL-2, A2M, ABI3BP, acidic fibroblast growth factor, ACVR1B, ADAM17, ADAMTS6, ADMLX, aFGF, AGPAT1, AGPAT2, AIF1, AIMP1, AKR1C1, AKT1S1, allograft-inflammatory factor- 1, amac-1, AMH, ANGPTL2, ANGPTL3, ANKFN1, ANKRD1, ankyrin repeat and SOCS box-containing protein 3 isoform a variant, ankyrin repeat domain-containing SOCS box protein ASB11, ankyrin repeat domain-containing SOCS box protein Asb-13 , ankyrin repeat domain-containing SOCS box protein Asb-14, ankyrin repeat domain-containing
  • the antibody may be an antibody that specifically binds to an interleukins or interferons protein.
  • the antibody is an IgG; Or PD-1, CTLA-4, TIM-3, BTLA, VISTA, LAG-3, PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD27, CD137, HVEM, GITR, VEGFR, VEGF Antibodies that specifically bind to at least one protein selected from the group consisting of EGFR, EGF, IL-1, IL-6, IL-23, TGF-beta, CTGF, TSLP, TNF-alpha, Notch, and OX40 But, if it is recognized as a conventional antibody from a biological point of view is not necessarily limited thereto.
  • the porous silica particles are particles of silica (SiO 2 ) material and have a particle size of nano size.
  • Porous silica nanoparticles of the present invention is a porous particle, having nano-sized pores, and can carry an antibody or cytokine on its surface and / or inside the pores.
  • Porous silica particles of the present invention can carry an antibody or cytokine and release the antibody or cytokine while being biodegraded in the body when administered to the body. Porous silica particles of the present invention can be slowly degraded in the body to allow the sustained release of the supported antibody (cytokine) or cytokine (cytokine).
  • t which is the ratio of the absorbance of the following formula 1 to 1/2, is 24 or more:
  • a 0 is the absorbance of the porous silica particles measured by placing 5 ml of the 1 mg / ml suspension of the porous silica particles into a cylindrical permeable membrane having pores having a diameter of 50 kDa,
  • the pH of the suspension is 7.4,
  • a t is the absorbance of the porous silica particles measured after t hours have elapsed since the measurement of A 0 ).
  • Equation 1 means that the rate at which the porous silica particles are degraded in an environment similar to the body.
  • Absorbance A 0 , A t in Equation 1 may be measured by putting porous silica particles and a suspension in a cylindrical permeable membrane and putting the same suspension outside the permeable membrane, as illustrated in FIG. 34, for example.
  • the porous silica particles of the present invention are biodegradable, and can be slowly decomposed in suspension, 50 kDa in diameter corresponds to about 5 nm, and biodegradable porous silica particles can pass through a permeable membrane of 50 kDa in diameter, and a cylindrical permeable membrane is 60 rpm horizontal. Under stirring, the suspension can be mixed evenly and the degraded porous silica particles can come out of the permeable membrane.
  • the absorbance in Equation 1 may be measured, for example, under an environment in which the suspension outside the permeable membrane is replaced with a new suspension.
  • the suspension can be one that is constantly replaced, one that can be replaced every period, and the period can be periodic or irregular. For example, within the range of 1 hour to 1 week, 1 hour interval, 2 hours interval, 3 hours interval, 6 hours interval, 12 hours interval, 24 hours interval, 2 days interval, 3 days interval, 4 days interval, 7 It may be replaced at day intervals, but is not limited thereto.
  • the ratio of the absorbance to 1/2 means that the absorbance is half of the initial absorbance after t hours, which means that approximately half of the porous silica particles are decomposed.
  • the suspension may be a buffer solution, for example, at least one selected from the group consisting of phosphate buffered saline (PBS) and simulated body fluid (SBF), and more specifically, PBS.
  • PBS phosphate buffered saline
  • SBF simulated body fluid
  • T of the absorbance ratio of Equation 1 of the present invention is 1/2 or more, for example, t may be 24 to 120, for example, 24 to 96, 24 to 72, 30 within the above range To 70, 40 to 70, 50 to 65 and the like, but is not limited thereto.
  • t for example, the absorbance ratio of Equation 1 is 1/5 may be, for example, 70 to 140, for example, 80 to 140, 80 to 120, and 80 to 110 within the above range. , 70 to 140, 70 to 120, 70 to 110, and the like, but is not limited thereto.
  • t may be 130 to 220, for example, wherein the ratio of absorbance of Equation 1 is 1/20, for example, 130 to 200, 140 to 200, 140 to 180 within the above range. , 150 to 180, and the like, but is not limited thereto.
  • the porous silica particles of the present invention may have a measured absorbance of 0.01 or less, for example, 250 or more, for example, 300 or more, 350 or more, 400 or more, 500 or more, 1000 or more, and the upper limit thereof is 2000 days. May be, but is not limited thereto.
  • the ratio of the absorbance of Formula 1 and t have a high positive correlation.
  • the Pearson correlation coefficient may be 0.8 or more, for example, 0.9 or more and 0.95 or more. .
  • T in Equation 1 means how fast the porous silica particles decompose in an environment similar to the body, for example, the surface area, particle diameter, pore diameter, surface and / or inside the pores of the porous silica particles. It can be controlled by controlling the substituent, the degree of compactness of the surface, and the like.
  • the surface area of the particles can be increased to reduce t, or the surface area can be reduced to increase t.
  • the surface area can be adjusted by adjusting the diameter of the particles and the diameter of the pores.
  • substituents on the surface and / or within the pores, it is possible to increase t by reducing the direct exposure of porous silica particles to the environment (such as solvents).
  • the porous silica silica particles are directly exposed to the environment.
  • the surface may be made more densely at the time of preparation of the particles to increase t.
  • Porous silica particles of the present invention may be, for example, spherical particles, but is not limited thereto.
  • the porous silica particles of the present invention may have an average diameter of, for example, 150 nm to 1000 nm, for example, within the above range, for example, 150 nm to 800 nm, 150 nm to 500 nm, 150 nm to 400 nm, 150 nm to 300 nm, and 150 nm to 200 nm. May be, but is not limited thereto.
  • the porous silica particles of the present invention may have an average pore diameter of, for example, 1 nm to 100 nm, for example, within the above range, for example, 5 nm to 100 nm, 7 nm to 100 nm, 7 nm to 50 nm, 10 nm to 50 nm, 10 nm to 30 nm. , 7 nm to 30 nm, but is not limited thereto. Having such a large diameter can carry a large amount of antibody (cytokine) or cytokine (cytokine), it is also possible to carry a large antibody (cytokine) or cytokine (cytokine).
  • the porous silica particles of the present invention may have a BET surface area of, for example, 200 m 2 / g to 700 m 2 / g.
  • a BET surface area of, for example, 200 m 2 / g to 700 m 2 / g, 200 m 2 / g to 650 m 2 / g, 250 m 2 / g to 650 m 2 / g, 300 m 2 / g to 700 m 2 / g, 300 m 2 / g to 650m 2 / g, 300m 2 / g to 600m 2 / g, 300m 2 / g to 550m 2 / g, 300m 2 / g to 500m 2 / g, 300m 2 / g to 450m 2 / g, etc. It is not limited to this.
  • the porous silica nanoparticles of the present invention may have a volume per g, for example, 0.7 ml to 2.2 ml.
  • a volume per g for example, 0.7 ml to 2.2 ml.
  • within the above range may be 0.7ml to 2.0ml, 0.8ml to 2.2ml, 0,8ml to 2.0ml, 0.9ml to 2.0ml, 1.0ml to 2.0ml and the like, but is not limited thereto. If the volume per gram is too small, the rate of decomposition may be too high, and excessively large particles may be difficult to manufacture or may not have an intact shape.
  • the porous silica particles of the present invention may have hydrophilic substituents and / or hydrophobic substituents on the outer surface and / or inside the pores.
  • hydrophilic substituents may exist on both the surface and inside of the pores, or only hydrophobic substituents may exist, hydrophilic substituents may exist on the surface or inside of the pores, hydrophobic substituents may exist on the surface, hydrophilic substituents on the surface, and hydrophobic substituents inside the pores. It may be present and vice versa.
  • the release of an antibody or cytokine supported on the porous silica particles of the present invention is mainly carried out by decomposition of the nanoparticles, and the antibody or cytokine is controlled by the control of the substituent.
  • the interaction of the porous silica particles with respect to the release environment of is controlled to control the rate of degradation of the nanoparticles themselves, thereby controlling the release rate of the antibody or cytokine, and also the antibody or cytokine.
  • the cytokine may be diffused from the nanoparticles to be released, and the binding force of the antibody or cytokine to the nanoparticles is controlled by the control of the substituents, thereby controlling the antibody or cytokine. Release can be controlled.
  • hydrophobic substituents are present inside the pores to enhance binding to poorly soluble (hydrophobic) antibodies, cytokines, or substances, and the surface of the particles is characterized by hydrophilic substituents in terms of ease of use and formulation. May be present.
  • Hydrophilic substituents are, for example, hydroxyl groups, carboxy groups, amino groups, carbonyl groups, sulfhydryl groups, phosphate groups, thiol groups, ammonium groups, ester groups, imide groups, thiimide groups, keto groups, ether groups, indene groups, sulfonyl groups, polyethylene Glycol groups and the like
  • the hydrophobic substituent is, for example, substituted or unsubstituted C1 to C30 alkyl group, substituted or unsubstituted C3 to C30 cycloalkyl group, substituted or unsubstituted C6 to C30 aryl group, substituted Or an unsubstituted C2 to C30 heteroaryl group, a halogen group, a C1 to C30 ester group, a halogen-containing group, and the like.
  • the porous silica particles of the present invention may be one in which the outer surface and / or the inside of the pores are positively and / or negatively charged.
  • both the surface and the inside of the pore may be positively charged, or may be negatively charged, only the surface or the inside of the pore may be positively charged, or may be negatively charged, the surface may be positively charged, and the interior of the pore may be negatively charged. The opposite is also true.
  • the charging may be, for example, by the presence of a cationic substituent or an anionic substituent.
  • the cationic substituent may be, for example, an amino group, other nitrogen-containing groups, etc. as the basic group, and the anionic substituent may be, for example, a carboxy group (-COOH), a sulfonic acid group (-SO 3 H), a thiol group as an acidic group, and the like. (-SH) and the like, but is not limited thereto.
  • the interaction of the porous silica particles with respect to the release environment of the antibody or cytokine is controlled by the control of the substituent, thereby controlling the rate of decomposition of the nanoparticles themselves, thereby controlling the antibody or cytokine.
  • the release rate of the cytokine may be controlled, and the antibody or the cytokine may be released from the nanoparticles by being diffused, and by controlling the substituent, the antibody or the cytokine may be released.
  • the binding force to the nanoparticles of the can be controlled to control the release of antibody or cytokine (cytokine).
  • porous silica particles of the present invention may carry an antibody or cytokine in addition to the above, inside the surface and / or the pores, transfer the antibody or cytokine to target cells, and the like.
  • Substituents for supporting other materials or binding of other additional substituents may be present, and may further include antibodies, ligands, cell permeable peptides, or aptamers bound thereto.
  • Substituents, charges, binders and the like within the aforementioned surfaces and / or pores may be added, for example, by surface modification.
  • Surface modification can be carried out, for example, by reacting a compound having a substituent to be introduced with the particles, which may be, for example, an alkoxysilane having a C1 to C10 alkoxy group, but is not limited thereto.
  • the alkoxysilane has one or more alkoxy groups, and may have, for example, 1 to 3, and there may be a substituent to be introduced into a site where the alkoxy group is not bonded or a substituent substituted therewith.
  • the porous silica particles of the present invention may be manufactured through a small pore particle preparation and a pore expansion process, and may be manufactured through a calcination process, a surface modification process, and the like, as necessary. If both the calcination and the surface modification process has gone through may be surface modified after calcination.
  • the small pore particles may be, for example, particles having an average pore diameter of 1 nm to 5 nm.
  • the small pore particles can be obtained by adding a surfactant and a silica precursor in a solvent, stirring and homogenizing.
  • the solvent may be water and / or an organic solvent
  • the organic solvent may be, for example, ethers such as 1,4-dioxane (particularly cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Acetone, methyl isobutyl ketone, ⁇ -butyrolactone, 1,3-dimethyl-imidazolidinone, methyl ethyl ketone, cyclohexanone, cyclopentanone, 4-hydroxy-4-methyl-2-pentanone, etc.
  • ethers such as 1,4-dioxane (particularly cyclic ethers)
  • Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachlor
  • Ketones Carbon-based aromatics such as benzene, toluene, xylene and tetramethylbenzene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; Ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, diethylene glycol monoethyl ether, diethylene glycol monomethyl ether, diethylene glycol monobutyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether Glycol ethers (cellosolve) such as dipropylene glycol diethyl ether and triethylene glycol monoethyl ether; Dimethylacetamide (DMAc), N, N-diethylacetamide,
  • the ratio may be, for example, water and the organic solvent in a volume ratio of 1: 0.7 to 1.5, for example, 1: 1: 0.8 to 1.3, but is not limited thereto.
  • the surfactant may be, for example, cetyltrimethylammonium bromide (CTAB), hexadecyltrimethylammonium bromide (TMABr), hexadecyltrimethylpyridinium chloride (TMPrCl), tetramethylammonium chloride (TMACl), and the like, and specifically, CTAB may be used.
  • CTAB cetyltrimethylammonium bromide
  • TMABr hexadecyltrimethylammonium bromide
  • TMPrCl hexadecyltrimethylpyridinium chloride
  • TMACl tetramethylammonium chloride
  • the surfactant may be added, for example, in an amount of 1 g to 10 g, for example, 1 g to 8 g, 2 g to 8 g, 3 g to 8 g, etc., per liter of solvent, but is not limited thereto.
  • the silica precursor may be added after stirring with the addition of a surfactant to the solvent.
  • the silica precursor may be, for example, tetramethyl orthosilicate (TMOS), but is not limited thereto.
  • the stirring may be performed, for example, for 10 minutes to 30 minutes, but is not limited thereto.
  • the silica precursor may be added, for example, 0.5 ml to 5 ml per liter of solvent, for example, 0.5 ml to 4 ml, 0.5 ml to 3 ml, 0.5 ml to 2 ml, 1 ml to 2 ml, etc. within the above range, but is not limited thereto. It doesn't happen.
  • sodium hydroxide may further be used as a catalyst, which may be added with stirring after adding the surfactant to the solvent and before adding the silica precursor.
  • the sodium hydroxide may be, for example, 0.5 ml to 8 ml per liter of solvent, for example, 0.5 ml to 5 ml, 0.5 ml to 4 ml, 1 ml to 4 ml, 1 ml to 3 ml, 2 ml to 3 ml, etc., based on 1 M aqueous sodium hydroxide solution. However, it is not limited thereto.
  • the solution can be reacted with stirring.
  • the stirring may be performed for example, for 2 hours to 15 hours, for example, within the above range, for example, 3 hours to 15 hours, 4 hours to 15 hours, 4 hours to 13 hours, 5 hours to 12 hours, 6 hours to 12 hours , 6 hours to 10 hours, and the like, but is not limited thereto. If the stirring time (reaction time) is too short, nucleation may be insufficient.
  • the solution may be aged. Aging may be performed for example, from 8 hours to 24 hours, for example, within the range of 8 hours to 20 hours, 8 hours to 18 hours, 8 hours to 16 hours, 8 hours to 14 hours, 10 hours to 16 hours. , 10 hours to 14 hours, and the like, but is not limited thereto.
  • reaction product may be washed and dried to obtain porous silica particles, and if necessary, separation of unreacted material may be preceded before washing.
  • Separation of the unreacted material may be carried out by separating the supernatant, for example by centrifugation, centrifugation may be carried out, for example at 6,000 to 10,000 rpm, the time is for example 3 minutes to 60 minutes, For example, it may be performed within 3 minutes to 30 minutes, 3 minutes to 30 minutes, 5 minutes to 30 minutes, and the like, but is not limited thereto.
  • the washing may be performed with water and / or an organic solvent, and in particular, since a substance that can be dissolved in each solvent may be different, water and an organic solvent may be used once or several times, or once or even with water or an organic solvent alone. Can be washed several times.
  • the number of times may be, for example, two or more, ten or less, for example, three or more and ten or less, four or more and eight or less, four or more and six or less.
  • the organic solvent may be, for example, ethers such as 1,4-dioxane (particularly cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Acetone, methyl isobutyl ketone, ⁇ -butyrolactone, 1,3-dimethyl-imidazolidinone, methyl ethyl ketone, cyclohexanone, cyclopentanone, 4-hydroxy-4-methyl-2-pentanone, etc.
  • ethers such as 1,4-dioxane (particularly cyclic ethers)
  • Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichlor
  • Ketones Carbon-based aromatics such as benzene, toluene, xylene and tetramethylbenzene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; Ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, diethylene glycol monoethyl ether, diethylene glycol monomethyl ether, diethylene glycol monobutyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether Glycol ethers (cellosolve) such as dipropylene glycol diethyl ether and triethylene glycol monoethyl ether; Dimethylacetamide (DMAc), N, N-diethylacetamide,
  • the washing may be carried out under centrifugation, for example at 6,000 to 10,000 rpm, the time being for example 3 to 60 minutes, for example 3 to 30 minutes, 3 within the above range. It may be performed in minutes to 30 minutes, 5 minutes to 30 minutes and the like, but is not limited thereto.
  • the washing may be performed by filtering out particles with a filter without centrifugation.
  • the filter may have pores less than or equal to the diameter of the porous silica particles. Filtering the reaction liquid with such a filter leaves only particles on the filter, which can be washed by pouring water and / or an organic solvent on the filter.
  • water and an organic solvent may be used alternately once or several times, and may be washed once or several times even with water or an organic solvent alone.
  • the number of times may be, for example, two or more, ten or less, for example, three or more and ten or less, four or more and eight or less, four or more and six or less.
  • the drying may be performed at 20 ° C. to 100 ° C., but is not limited thereto, and may be performed in a vacuum state.
  • the pores of the obtained porous silica particles are expanded, and the pore expansion may be performed using a pore swelling agent.
  • the pore swelling agent may be trimethylbenzene, triethylbenzene, tripropylbenzene, tributylbenzene, tripentylbenzene, trihexylbenzene, toluene, benzene, and the like, and specifically, trimethylbenzene may be used. It is not limited.
  • the pore swelling agent may use, for example, N, N-dimethylhexadecylamine (N, N-dimethylhexadecylamine, DMHA), but is not limited thereto.
  • the pore expansion can be carried out, for example, by mixing porous silica particles in a solvent with a pore swelling agent and heating to react.
  • the solvent may be, for example, water and / or an organic solvent
  • the organic solvent may be, for example, ethers such as 1,4-dioxane (particularly cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Ketones such as acetone, methyl isobutyl ketone and cyclohexanone; Carbon-based aromatics such as benzene, toluene and xylene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; And the like
  • the porous silica particles are, for example, 10 g to 200 g per liter of solvent, for example, 10 g to 150 g, 10 g to 100 g, 30 g to 100 g, 40 g to 100 g, 50 g to 100 g, 50 g to 80 g, 60 g to 80 g, etc., within the above range. It may be added in a ratio of, but is not limited thereto.
  • the porous silica particles may be evenly dispersed in a solvent, for example, the porous silica particles may be added to the solvent and ultrasonically dispersed.
  • the second solvent may be added after the porous silica particles are dispersed in the first solvent.
  • the pore swelling agent is for example 10 to 200 parts by volume, 100 to 150 parts by volume, 10 to 100 parts by volume, 10 to 80 parts by volume, 30 to 80 parts by volume, 30 to 80 parts by volume based on 100 parts by volume of solvent. 70 parts by volume may be added, but is not limited thereto.
  • the reaction can be carried out, for example, at 120 ° C to 190 ° C.
  • 120 ° C to 190 ° C For example, within the range of 120 °C to 190 °C, 120 °C to 180 °C, 120 °C to 170 °C, 130 °C to 170 °C, 130 °C to 160 °C, 130 °C to 150 °C, 130 °C to 140 °C It may be performed, but is not limited thereto.
  • the reaction may be performed, for example, for 6 hours to 96 hours.
  • 6 hours to 96 hours within the range of 30 hours to 96 hours, 30 hours to 96 hours, 30 hours to 80 hours, 30 hours to 72 hours, 24 hours to 80 hours, 24 hours to 72 hours, 36 hours to 96 hours, 36 36 hours to 80 hours, 36 hours to 72 hours, 36 hours to 66 hours, 36 hours to 60 hours, 48 hours to 96 hours, 48 hours to 88 hours, 48 hours to 80 hours, 48 hours to 72 hours, 6 hours to 96 hours, 7 hours to 96 hours, 8 hours to 80 hours, 9 hours to 72 hours, 9 hours to 80 hours, 6 hours to 72 hours, 9 hours to 96 hours, 10 hours to 80 hours, 10 hours to 72 hours , 12 hours to 66 hours, 13 hours to 60 hours, 14 hours to 96 hours, 15 hours to 88 hours, 16 hours to 80 hours, 17 hours to 72 hours, and the like, but is not limited thereto.
  • the time and temperature can be adjusted within the ranges exemplified above so that the reaction can be carried out sufficiently without excess. For example, when the reaction temperature is lowered, the reaction time may be increased, or when the reaction temperature is lowered, the reaction time may be shortened. If the reaction is not sufficient, the expansion of the pores may not be sufficient, and if the reaction proceeds excessively, the particles may collapse due to the expansion of the pores.
  • the reaction can be carried out, for example, by gradually raising the temperature. Specifically, it may be carried out by gradually raising the temperature at a rate of 0.5 °C / min to 15 °C / min from the room temperature to the above temperature, for example, 1 °C / min to 15 °C / min, 3 °C / min within the above range To 15 ° C./minute, 3 ° C./minute to 12 ° C./minute, 3 ° C./minute to 10 ° C./minute, and the like, but are not limited thereto.
  • the reaction can be carried out under stirring. For example, it may be stirred at a speed of 100 rpm or more, and specifically, may be performed at a speed of 100 rpm to 1000 rpm, but is not limited thereto.
  • the reaction solution may be cooled slowly, for example, it may be cooled by gradually reducing the temperature. Specifically, it may be carried out by gradually decreasing the temperature at a rate of 0.5 °C / min to 20 °C / min from the temperature to room temperature, for example, 1 °C / min to 20 °C / min, 3 °C / min to within the above range 20 ° C./minute, 3 ° C./minute to 12 ° C./minute, 3 ° C./minute to 10 ° C./minute, and the like, but is not limited thereto.
  • reaction product After cooling, the reaction product may be washed and dried to obtain porous silica particles having expanded pores, and if necessary, separation of unreacted material may be preceded before washing.
  • Separation of the unreacted material may be carried out by separating the supernatant, for example by centrifugation, centrifugation may be carried out, for example at 6,000 to 10,000 rpm, the time is for example 3 minutes to 60 minutes, For example, it may be performed within 3 minutes to 30 minutes, 3 minutes to 30 minutes, 5 minutes to 30 minutes, and the like, but is not limited thereto.
  • the washing may be performed with water and / or an organic solvent, and in particular, since a substance that can be dissolved in each solvent may be different, water and an organic solvent may be used once or several times, or once or even with water or an organic solvent alone. Can be washed several times.
  • the number of times may be, for example, two or more times, ten times or less, for example, three times, four times, five times, six times, seven times, eight times, and the like.
  • the organic solvent may be, for example, ethers such as 1,4-dioxane (particularly cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Ketones such as acetone, methyl isobutyl ketone and cyclohexanone; Carbon-based aromatics such as benzene, toluene and xylene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; And the like, and specifically, alcohol, more specifically ethanol may be used, but is not limited
  • the washing may be carried out under centrifugation, for example at 6,000 to 10,000 rpm, the time being for example 3 to 60 minutes, for example 3 to 30 minutes, 3 within the above range. It may be performed in minutes to 30 minutes, 5 minutes to 30 minutes and the like, but is not limited thereto.
  • the washing may be performed by filtering out particles with a filter without centrifugation.
  • the filter may have pores less than or equal to the diameter of the porous silica particles. Filtering the reaction liquid with such a filter leaves only particles on the filter, which can be washed by pouring water and / or an organic solvent on the filter.
  • water and an organic solvent may be used alternately once or several times, and may be washed once or several times even with water or an organic solvent alone.
  • the number of times may be, for example, two or more, ten or less, for example, three or more and ten or less, four or more and eight or less, four or more and six or less.
  • the drying may be performed at 20 ° C. to 100 ° C., but is not limited thereto, and may be performed in a vacuum state.
  • the obtained particles may be calcined, which is a process of heating the particles to remove silanol groups on the surface and inside thereof to lower the reactivity of the particles, to have a more compact structure, and to remove organic substances filling the pores.
  • it may be heated to a temperature of 400 °C or more.
  • the upper limit thereof is not particularly limited, and may be, for example, 1000 ° C, 900 ° C, 800 ° C, 700 ° C, or the like. Heating can be carried out for example for 3 hours or more.
  • the upper limit is not particularly limited and may be, for example, 24 hours, 12 hours, 10 hours, 8 hours, 6 hours, or the like. More specifically, it may be performed for 3 hours to 8 hours at 400 ° C to 700 ° C, specifically 4 hours to 5 hours at 500 ° C to 600 ° C, but is not limited thereto.
  • the porous silica particles obtained can then be surface modified, and the surface modification can be carried out on the surface and / or inside the pores.
  • the particle surface and the inside of the pore may be surface-modified identically or may be surface-modified differently.
  • the surface modification can cause the particles to charge or to have hydrophilic and / or hydrophobic properties.
  • At least one selected from the group consisting of an amino group, an aminoalkyl group, an alkylamino group, a heterocyclic aromatic compound group containing a nitrogen atom, a cyan group and a guanidine group for effective loading of an antibody or cytokine By having a substituent of, the surface modification of the porous silica particles can be carried out.
  • Surface modification can be carried out, for example, by reacting a compound having substituents such as hydrophilic, hydrophobic, cationic, anionic and the like to be introduced with the particles, and the compound can be, for example, an alkoxysilane having a C1 to C10 alkoxy group. However, it is not limited thereto.
  • the alkoxysilane has one or more alkoxy groups, and may have, for example, 1 to 3, and there may be a substituent to be introduced into a site where the alkoxy group is not bonded or a substituent substituted therewith.
  • the alkoxysilane reacts with the porous silicon particles, a covalent bond is formed between the silicon atom and the oxygen atom so that the alkoxysilane may be bonded to the surface and / or the inside of the pores of the porous silicon particle, and the alkoxysilane has a substituent to be introduced.
  • the corresponding substituents may be introduced into the surface of the porous silicon particles and / or within the pores.
  • the reaction may be carried out by reacting porous silica particles dispersed in a solvent with an alkoxysilane.
  • the solvent may be water and / or an organic solvent
  • the organic solvent may be, for example, ethers such as 1,4-dioxane (particularly cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Acetone, methyl isobutyl ketone, ⁇ -butyrolactone, 1,3-dimethyl-imidazolidinone, methyl ethyl ketone, cyclohexanone, cyclopentanone, 4-hydroxy-4-methyl-2-pentanone, etc.
  • ethers such as 1,4-dioxane (particularly cyclic ethers)
  • Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachlor
  • Ketones Carbon-based aromatics such as benzene, toluene, xylene and tetramethylbenzene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; Ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, diethylene glycol monoethyl ether, diethylene glycol monomethyl ether, diethylene glycol monobutyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether Glycol ethers (cellosolve) such as dipropylene glycol diethyl ether and triethylene glycol monoethyl ether; Dimethylacetamide (DMAc), N, N-diethylacetamide,
  • the charge to the positive charge can be carried out by reacting with an alkoxysilane having a basic group such as a nitrogen-containing group such as an amino group, an aminoalkyl group, for example.
  • an alkoxysilane having a basic group such as a nitrogen-containing group such as an amino group, an aminoalkyl group, for example.
  • Charging to the negative charge may be carried out by reacting with an alkoxysilane having an acidic group such as, for example, a carboxyl group, a sulfonic acid group, a thiol group, and the like.
  • an alkoxysilane having an acidic group such as, for example, a carboxyl group, a sulfonic acid group, a thiol group, and the like.
  • 3-Mercaptopropyl) trimethoxysilane may be used, but is not limited thereto.
  • the hydrophilic property is a hydrophilic group such as hydroxy group, carboxy group, amino group, carbonyl group, sulfhydryl group, phosphate group, thiol group, ammonium group, ester group, imide group, thiimide group, keto group, ether group, indene group, sulfo It may be made to react with the alkoxysilane which has a silyl group, a polyethyleneglycol group, etc.
  • the hydrophobic nature may include hydrophobic substituents such as substituted or unsubstituted C1 to C30 alkyl groups, substituted or unsubstituted C3 to C30 cycloalkyl groups, substituted or unsubstituted C6 to C30 aryl groups, substituted or unsubstituted It can be made to react with the alkoxysilane which has a C2-C30 heteroaryl group, a halogen group, C1-C30 ester group, a halogen containing group, etc.
  • Trimethoxy (octadecyl) silane, Trimethoxy-n-octylsilane, Trimethoxy (propyl) silane, Isobutyl (trimethoxy) silane, Trimethoxy (7-octen-1-yl) silane, Trimethoxy (3,3,3-trifluoropropyl) Silane, Trimethoxy (2-phenylethyl) silane, Vinyltrimethoxysilane, Cyanomethyl, 3- (trimethoxysilyl) propyl] trithiocarbonate, (3-Bromopropyl) trimethoxysilane, etc. may be used, but is not limited thereto.
  • hydrophobic substituents are present in the pores to enhance binding to poorly soluble (hydrophobic) antibodies, cytokines or substances through the surface modification, and particles in terms of ease of use and formulation.
  • the surface of may also be treated such that a hydrophilic substituent is present, and a substituent may be present on the surface to bind other antibodies, cytokines, or substances.
  • the surface modification may be carried out in combination.
  • two or more surface modifications may be performed on the outer surface or inside the pores.
  • a compound including a carboxyl group may be bonded to silica particles into which amino groups are introduced by amide bonds to change the positively charged particles to have different surface properties, but is not limited thereto.
  • the reaction of the porous silica particles with the alkoxysilane can be carried out, for example, under heating, and the heating is for example from 80 ° C. to 180 ° C., for example from 80 ° C. to 160 ° C., from 80 ° C. to 150 ° C. within the above range. , 100 ° C. to 160 ° C., 100 ° C. to 150 ° C., 110 ° C. to 150 ° C., etc., but is not limited thereto.
  • the reaction of the porous silica particles with the alkoxysilane is, for example, 4 hours to 20 hours, for example, 4 hours to 18 hours, 4 hours to 16 hours, 6 hours to 18 hours, 6 hours to 16 hours within the above range. , 8 hours to 18 hours, 8 hours to 16 hours, 8 hours to 14 hours, 10 hours to 14 hours, etc., but is not limited thereto.
  • the reaction temperature, time, and the amount of the compound used for surface modification may be selected according to the degree to which the surface is to be modified, and the degree of hydrophilicity, hydrophobicity, and charge of an antibody, cytokine, or materials may be selected. Accordingly, by controlling the hydrophilicity, hydrophobicity, and charge of the porous silica particles by varying the reaction conditions, the release rate of the antibody, cytokine, or substances can be controlled. For example, if antibodies, cytokines or substances have a strong negative charge at neutral pH, the reaction temperature may be increased or the reaction time may be increased in order for the porous silica particles to have a strong positive charge. It is possible to increase the compound throughput, but is not limited thereto.
  • porous silica particles of the present invention may be produced through, for example, the preparation of small pores, pore expansion, surface modification, and internal pore modification.
  • the small pore particle production and pore expansion process may be based on the above-described process, and the washing and drying process may be performed after the small pore particle production and after the pore expansion process.
  • separation of the unreacted material may be preceded before washing, and separation of the unreacted material may be performed by separating the supernatant, for example, by centrifugation.
  • the centrifugation may be performed, for example, at 6,000 to 10,000 rpm, and the time may be, for example, 3 to 60 minutes, specifically, 3 to 30 minutes, 3 to 30 minutes, and 5 minutes within the above range. To 30 minutes, etc., but is not limited thereto.
  • the washing after the preparation of the particles of the small pores may be performed by a method / condition within the above-described range, but is not limited thereto.
  • the washing after the pore expansion may be performed under more relaxed conditions than the above example.
  • washing may be performed within three times, but is not limited thereto.
  • the surface modification and internal pore modification may be by the processes described above, respectively, the process may be performed in the order of surface modification and internal pore modification, and the washing process of the particles may be further performed between the two processes. Can be.
  • the reaction solution such as a surfactant used for particle production and pore expansion is filled in the pores so that the inside of the pores is not modified during surface modification. Only the surface can be modified. Then, washing the particles may remove the reaction solution in the pores.
  • Particle washing between the surface modification and the internal pore reforming process may be water and / or an organic solvent, and in particular, water and an organic solvent may be alternately used once or several times because different materials may be dissolved for each solvent.
  • Water or organic solvents alone may be washed once or several times. The number of times may be, for example, two or more, ten or less, specifically, three or more and ten or less, four or more and eight or less, four or more and six or less.
  • the washing may be carried out under centrifugation, for example at 6,000 to 10,000 rpm, the time being for example 3 to 60 minutes, specifically 3 to 30 minutes, 3 within the above range. It may be performed in minutes to 30 minutes, 5 minutes to 30 minutes and the like, but is not limited thereto.
  • the washing may be performed by filtering out particles with a filter without centrifugation.
  • the filter may have pores less than or equal to the diameter of the porous silica particles. Filtering the reaction liquid with such a filter leaves only particles on the filter, which can be washed by pouring water and / or an organic solvent on the filter.
  • water and an organic solvent may be used alternately once or several times, and may be washed once or several times even with water or an organic solvent alone.
  • the number of times may be, for example, two or more, ten or less, specifically, three or more and ten or less, four or more and eight or less, four or more and six or less.
  • the drying may be performed at 20 ° C. to 100 ° C., but is not limited thereto, and may be performed in a vacuum state.
  • the antibody or cytokine may be supported on the surface and / or inside the pores of the porous silica particles, and the support may be mixed with, for example, the porous silica particles and the antibody or cytokine in a solvent. Can be performed.
  • the solvent may be water and / or an organic solvent
  • the organic solvent may be, for example, ethers such as 1,4-dioxane (particularly cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Ketones such as acetone, methyl isobutyl ketone and cyclohexanone; Carbon-based aromatics such as benzene, toluene and xylene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; Etc. can be used.
  • PBS phosphate buffered saline solution
  • SBF Simulated Body Fluid
  • Borate-buffered saline Borate-buffered saline
  • Tris-buffered saline may be used as the solvent.
  • the ratio of the porous silica particles and the oligonucleotide of the present invention is not particularly limited, for example, the weight ratio is 1: 0.05 to 0.8, for example within the above range 1: 0.05 to 0.7, 1: 0.05 to 0.6, 1: 0.1 to 0.8, 1: 0.1 to 0.6, 1: 0.2 to 0.8, 1: 0.2 to 0.6, and the like.
  • Antibodies or cytokines supported on the porous silica particles may be gradually released over an extended time. Such slow release may be continuous or discontinuous, linear or nonlinear, and may vary due to the characteristics of the porous silica particles and / or their interaction with antibodies or cytokines.
  • Antibodies or cytokines supported on the porous silica particles are released as the porous silica particles are biodegraded, and the porous silica particles according to the present invention are gradually decomposed and supported on an antibody or cytokine. This can be released slowly. This may be controlled by, for example, adjusting the surface area, particle diameter, pore diameter, substituents on the surface and / or pores, degree of compactness of the porous silica particles, and the like, but are not limited thereto.
  • the antibody or cytokine supported on the porous silica particles may be released while being diffused from the porous silica particles, which may be released. It is affected by the relationship with the release environment of the antibody or cytokine (cytokine), it can be controlled by controlling the release of the antibody (cytokine) or cytokine (cytokine). For example, it can be controlled by strengthening or weakening the binding strength of the porous silica particles to the antibody or cytokine by surface modification.
  • the surface of the particles and / or the inside of the pores may have a hydrophobic substituent, so that the porous silica particles and the antibody
  • the cytokine (cytokine) or the binding force with the substance may be increased, whereby the antibody (cytokine) or the substance (cytokine) or the substance may be released in a sustained manner.
  • This may be, for example, the surface-modified porous silica particles with an alkoxysilane having a hydrophobic substituent.
  • “poorly soluble” means to be insoluble (practically insoluble) or only slightly soluble (with respect to water), which means “Pharmaceutical Science” 18 th Edition ( USP, Remington, Mack Publishing Company).
  • the poorly water-soluble material may be, for example, water solubility of less than 10 g / L, specifically less than 5 g / L, more specifically less than 1 g / L at 1 atmosphere and 25 ° C., but is not limited thereto.
  • the surface and / or the inside of the pores have a hydrophilic substituent which has a porous silica particle and an antibody, cytokine or The binding force with the substance may be increased, whereby an antibody, cytokine or substance may be released in a sustained manner.
  • This may be, for example, the surface of the porous silica particles modified with an alkoxysilane having a hydrophilic substituent.
  • the water-soluble substance may have a water solubility of 10 g / L or more at 1 atmosphere and 25 ° C., but is not limited thereto.
  • the surface of the particles and / or the interior of the pores are charged with opposite charges so that the porous silica particles, antibodies, and cytokines ), Or the binding force with the substance may be increased, whereby an antibody, cytokine or substance may be released in a sustained manner.
  • This may be, for example, the surface-modified porous silica particles with an alkoxysilane having an acidic group or a basic group.
  • porous silica particles may be surface-modified with an alkoxysilane having an acidic group such as a carboxyl group (-COOH) and a sulfonic acid group (-SO 3 H).
  • the surface of the particles and / or the inside of the pores may be positively charged, whereby the porous silica particles and the antibody (antibody) ), Cytokines (cytokine) or the binding force with the substance is increased, the antibody (cytokine) or the substance can be released in a sustained release.
  • the porous silica particles may be surface-modified with an alkoxysilane having a basic group such as an amino group or another nitrogen-containing group.
  • Antibodies, cytokines or substances may be released for a period of, for example, 7 days to 1 year or more, depending on the type of treatment required, the release environment, and the porous silica particles used.
  • porous silica particles of the present invention are 100% degradable as biodegradable, the antibody, cytokine or substance supported thereon may be released 100%.
  • the present invention provides a composition for immunotherapy comprising the carrier described above.
  • Immunotherapy refers to the planning of patient treatment by immunological methods, which include specific immunotherapy involving only the immune response to specific antigens and nonspecific immunotherapy that is effective for the entire immune system but not limited to specific antigens.
  • the method of administering an antibody as said specific therapy is the typical example of the method of administering cytokines as said non-specific method.
  • the immunotherapeutic composition of the present invention comprises porous silica particles carrying an immunoreactive substance containing the above-described antibody or cytokine, and delivers the supported immunoreactive substance to the body stably and sustainably, and immunoreactive substance.
  • porous silica particles carrying an immunoreactive substance containing the above-described antibody or cytokine and delivers the supported immunoreactive substance to the body stably and sustainably, and immunoreactive substance.
  • it has an effective superiority to reduce the inherent side effects, it can be used in immunotherapy to exert an excellent effect in the prevention or treatment of various cancers or immune diseases.
  • the cancer includes carcinoma including skin cancer including bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, gastric cancer, cervical cancer, thyroid cancer and squamous cell carcinoma; Lymphoid hematopoietic tumors including leukemia, acute lymphocytic leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma and Bucket lymphoma; Myeloid hematopoietic tumors, including acute and chronic myeloid leukemia and promyelocytic leukemia; Mesenchymal-derived tumors including fibrosarcoma and rhabdomyosarcoma; Other tumors including melanoma, normal carcinoma, teratocarcinoma, neuroblastoma and glioma; Tumors of the central and peripheral nervous system,
  • the immune disease refers to a disease in which components of the mammalian immune system cause, mediate or otherwise contribute to a mammalian pathology, and all diseases in which stimulation or interruption of an immune response has a compensatory effect on the progression of the disease. And may include all autoimmune diseases, infectious diseases, inflammatory diseases or transplant rejection diseases of cells, tissues or organs, and the like, specifically, Behcet's disease, multiple myositis / skin myositis, autologous diseases Immunocytopenia, Autoimmune myocarditis, Atopic dermatitis, Asthma, Primary cirrhosis, Dermatitis, Goodfiction syndrome, Autoimmune meningitis, Obesity, Sjogren's syndrome, Ankylosing myelitis, Systemic lupus erythematosus, Addison's disease, Alopecia areata, Autoimmunity Hepatitis, autoimmune mumps, Crohn's disease, insulin dependent diabetes mellitus, dystrophic
  • Autoimmune diseases as one type of the immune disease is not limited to the type, Crohn's disease, erythema, atopy, rheumatoid arthritis, Hashimoto's thyroiditis, pernicious anemia, Edison's disease, type 1 diabetes, Lupus, chronic fatigue syndrome, fiber Myalgia, hypothyroidism and hyperplasia, scleroderma, Behcet's disease, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, Meniere's syndrome, Guilian-Barre syndrome, Sjogren's syndrome, vitiligo Endometriosis, psoriasis, systemic scleroderma, asthma or ulcerative colitis.
  • the infectious disease may be an infectious disease caused by bacteria, parasites, fungi, viruses, viroids and prions.
  • the virus may be enterovirus, rotorvirus, adenovirus and hepatitis virus, and in addition to the virus, the retroviral family (e.g., HIV-I (also HTLV-III, LAV or HTLV-III / LAV, or HIV-III) Human immunodeficiency virus such as; and other isolates such as HIV-LP; Picornaviridae (eg, polio virus, hepatitis A virus, enterovirus, human Coxsackie virus, rhinovirus, echovirus); Calciviridae (eg, species responsible for gastroenteritis); Togaviridae (eg, equine encephalitis virus, rubella virus); Flaviviridae (eg, dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (eg, coronavirus); Rhabdoviridae (eg, vesicular stomatitis virus, rabies virus); Phyllovirus family (eg,
  • Pox virus and Iridoviridae (e.g. African swine fever virus); and unclassified Viruses (for example delta hepatitis (presumed to be a satellite deficient in hepatitis B), hepatitis C); Norwalk and related viruses, and astroviruses ( preparations of astro virus), but are not limited thereto.
  • Iridoviridae e.g. African swine fever virus
  • unclassified Viruses for example delta hepatitis (presumed to be a satellite deficient in hepatitis B), hepatitis C); Norwalk and related viruses, and astroviruses ( preparations of astro virus), but are not limited thereto.
  • the present invention also provides a pharmaceutical composition for the prophylaxis or treatment of cancer or immune disease, including the above-mentioned carrier.
  • composition of the present invention has a prophylactic or therapeutic effect of cancer or immune disease, which stably delivers the supported antibody or cytokine to the body and releases it to the target in a slow manner, thereby inhibiting the growth of cancer (or tumor). And the effect achieved by suppressing the transition.
  • composition of the present invention may further comprise a pharmaceutically acceptable carrier, and may be formulated with the carrier.
  • pharmaceutically acceptable carrier refers to a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound.
  • Acceptable pharmaceutical carriers in compositions formulated in liquid solutions are sterile and biocompatible, which include saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary.
  • Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • composition of the present invention is applicable to any formulation containing the carrier of the present invention as an active ingredient, and can be prepared in oral or parenteral formulations.
  • Pharmaceutical formulations of the present invention may be oral, rectal, nasal, topical (including the cheek and sublingual), subcutaneous, vaginal or parenteral (intramuscular, subcutaneous). And forms suitable for administration by inhalation or insufflation.
  • composition of the present invention is administered in a pharmaceutically effective amount. Effective dose levels depend on the type of disease, severity, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent medications, and other factors well known in the medical field. Can be determined.
  • the pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.
  • the dosage of the composition of the present invention varies widely depending on the weight, age, sex, health condition, diet, time of administration, administration method, excretion rate and severity of the disease, and the appropriate dosage is, for example, Depending on the amount of drug accumulated in the body and / or the specific efficacy of the carrier of the invention used. It can be calculated on the basis of EC50, which is generally determined to be effective in in vivo animal models and in vitro, for example from 0.01 ⁇ g to 1 g per kg of body weight, in unit periods of daily, weekly, monthly or yearly It may be administered once or several times per unit period, or may be continuously administered for a long time using an infusion pump. The number of repeated doses is determined in consideration of the time the drug stays in the body, the drug concentration in the body, and the like. Even after treatment according to the course of the disease treatment, the composition can be administered for relapse.
  • composition of the present invention may further contain a compound which maintains / increases the solubility and / or absorption of one or more of the active ingredients or the active ingredients exhibiting the same or similar function in the treatment of cancer. It may also optionally further comprise chemotherapeutic agents, anti-inflammatory agents, antiviral agents and / or immunomodulators and the like.
  • compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.
  • the formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.
  • porous silica particles of the present invention may be abbreviated as 'DegradaBALL or DDV'
  • the DegradaBALL loaded with IL-2 may be abbreviated as 'BALLkine-2'.
  • peri-tumoral injection may be abbreviated as pt, intra-peritoreal injection as ip, subcutaneous injection as sc, and intravenous injection as iv. .
  • Example 1 Porous Silica Particles (DDV or DegradaBALL)
  • reaction solution was then centrifuged at 8000 rpm for 10 minutes at 25 ° C. to remove the supernatant, centrifuged at 8000 rpm for 10 minutes at 25 ° C., and washed five times with alternating ethanol and distilled water.
  • the reaction was carried out starting at 25 ° C. and warming up at a rate of 10 ° C./min, then slowly cooling at a rate of 1-10 ° C./min in the autoclave.
  • the cooled reaction solution was centrifuged at 8000 rpm for 10 minutes at 25 ° C. to remove the supernatant, and centrifuged at 8000 rpm for 10 minutes at 25 ° C. and washed five times with ethanol and distilled water.
  • porous silica particles prepared in 2) were placed in a glass vial, heated at 550 ° C. for 5 hours, and cooled to room temperature after completion of the reaction to prepare particles.
  • Porous silica particles were prepared in the same manner as in 1-1- (1), except that the reaction conditions at the time of pore expansion were changed to 140 ° C. and 72 hours.
  • Porous silica particles were prepared in the same manner as in Example 1-1- (1), except that a 5-fold large container was used, and each material was used in 5-fold volume.
  • Porous silica particles were prepared in the same manner as in 1-1- (1), except that 920 ml of distilled water and 850 ml of methanol were used to prepare the small pore particles.
  • Porous silica particles were prepared in the same manner as in 1-1- (1), except that 800 ml of distilled water, 1010 ml of methanol, and 10.6 g of CTAB were used to prepare the small pore particles.
  • Porous silica particles were prepared in the same manner as in 1-1- (1), except that 620 ml of distilled water, 1380 ml of methanol, and 7.88 g of CTAB were used to prepare the small pore particles.
  • Porous silica particles were prepared in the same manner as in 1-1- (1), except that 2.5 mL of TMB was used for pore expansion.
  • Porous silica particles were prepared in the same manner as in 1-1- (1), except that 4.5 mL of TMB was used for pore expansion.
  • Porous silica particles were prepared in the same manner as in 1-1- (1), except that 11 mL of TMB was used for pore expansion.
  • Porous silica particles were prepared in the same manner as in 1-1- (1), except that 12.5 mL of TMB was used for pore expansion.
  • Example 1-1- (1) -2 In the same manner as in Example 1-1- (1) -2), the small pore particles were reacted with TMB, cooled, and centrifuged to remove the supernatant. Thereafter, centrifuged under the same conditions as in Example 1-1- (1) -2), washed three times with alternating ethanol and distilled water, and then dried under the same conditions as in Example 1-1- (1) -2). Powdery porous silica particles (pore diameter 10-15 nm, particle diameter 200 nm) were obtained.
  • N-Hydroxysuccinimide 200 mg was dispersed in 30 mL of PBS and allowed to react for 12 hours while stirring at room temperature. The product is then washed and dried.
  • reaction solution of the previous step remains inside the pore, so that the inside of the pore is not modified.
  • the cooled reaction solution was centrifuged at 8000 rpm for 10 minutes to remove the supernatant, centrifuged at 8000 rpm for 10 minutes at 25 ° C, and washed five times with alternating ethanol and distilled water.
  • Example 1-1- (4) The porous silica particles of Example 1-1- (4) were reacted with (3-Aminopropyl) triethoxysilane (APTES) to charge with a positive charge.
  • APTES (3-Aminopropyl) triethoxysilane
  • porous silica particles in a 100 mL round bottom flask was dispersed in 10 mL of toluene with a bath sonicator. Then 1 mL of APTES was added and stirred at 400 rpm and stirred at 130 ° C. for 12 hours.
  • Example 1-1- (1) The porous silica particles of Example 1-1- (1) were charged with positive charge by reacting with (3-Aminopropyl) triethoxysilane (APTES), except that 0.4 ml of APTES was added and the reaction time was 3 hours.
  • APTES (3-Aminopropyl) triethoxysilane
  • Example 1-1- (9) The porous silica particles of Example 1-1- (9) were charged with positive charge by reacting with (3-Aminopropyl) triethoxysilane (APTES), and the other method was the method of 9-2- (1) -1). Modified in the same manner as
  • Example 1-1- (10) were charged with positive charge by reacting with (3-Aminopropyl) triethoxysilane (APTES), and were modified in the same manner as in the method of 9-2- (1) -1). It was.
  • APTES (3-Aminopropyl) triethoxysilane
  • Example 1-1- (1) The porous silica particles of Example 1-1- (1) were reacted with Trimethoxy (propyl) silane to introduce a propyl group into the surface and the pores, and 0.35ml of Trimethoxy (propyl) silane was added instead of APTES, followed by reaction for 12 hours. Modification was carried out in the same manner as in Example 1-2- (1) except for the above.
  • Example 1-1- (1) The porous silica particles of Example 1-1- (1) were reacted with Trimethoxy-n-octylsilane to introduce propyl groups into the surface and the pores, and 0.5 ml of Trimethoxy-n-octylsilane was added instead of APTES, followed by 12 hours of reaction. Modification was carried out in the same manner as in Example 1-2- (1) except for the above.
  • the porous silica particles of Example 1-1- (1) were charged with negative charge by reacting with succinic anhydride,
  • DMSO Dimethyl sulfoxide
  • 80 mg of succinic anhydride was added instead of APTES, and reacted at room temperature for 24 hours, except that DMSO was used instead of distilled water.
  • the modification was carried out in the same manner as in the method of -2- (1) -1).
  • Example 1-2- (1) -1) It was modified in the same manner as in Example 1-2- (1) -1) except that 1.1 mL of MPTES was used instead of APTES.
  • Example 1-2- (3) -2 100 mg of the porous silica nanoparticles of Example 1-2- (3) -2) were dispersed in 1 mL of 1 M aqueous sulfuric acid solution and 20 mL of 30% hydrogen peroxide solution, and stirred at room temperature to induce an oxidation reaction. Oxidized to a group. After the same washing and drying in the same manner as in Example 1-2- (1) -1).
  • FIG. 1 is a photograph of porous silica particles of Example 1-1- (1)
  • FIG. 2 is a photograph of porous silica particles of Example 1-1- (2). You can see that it was created
  • Figure 3 is a photograph of the small pore particles of Example 1-1- (1)
  • Figure 4 is a comparison photograph of the small pore particles of Example 1-1- (1) and 1-1- (3), spherical It can be seen that the small pore particles of evenly generated.
  • the surface area and pore volume of the small pore particles of Example 1-1- (1) and the porous silica particles of Examples 1-1- (1), (7), (8) and (10) were calculated.
  • the surface area was calculated by Brunauer-Emmett-Teller (BET) method, and the pore size distribution was calculated by Barrett-Joyner-Halenda (BJH) method.
  • Example 1-1- (1) 2.1 1337 0.69
  • Example 1-1- (7) 4.3 630 0.72
  • Example 1-1- (8) 6.9 521 0.79
  • Example 1-1- (1) 10.4 486 0.82
  • Example 1-1- (10) 23 395 0.97
  • porous silica particles are biodegraded and nearly decomposed after 360 hours.
  • a 0 is the absorbance of the porous silica particles measured by placing 5 ml of the 1 mg / ml suspension of the porous silica particles into a cylindrical permeable membrane having pores having a diameter of 50 kDa,
  • a t is the absorbance of the porous silica particles measured after t hours have elapsed since the measurement of A 0 ).
  • porous silica particle powder was dissolved in 5 ml of SBF (pH 7.4). Thereafter, 5 ml of the porous silica particle solution was placed in a permeable membrane having pores having a diameter of 50 kDa shown in FIG. 7. 15 ml of SBF was added to the outer membrane, and the SBF of the outer membrane was replaced every 12 hours. Decomposition of the porous silica particles was performed at 37 ° C. with 60 rpm horizontal stirring.
  • porous silica particles of the example have a significantly larger t than the control.
  • t which is a ratio of absorbance 1/2
  • t which has a ratio of absorbance 1/2 of the positively charged particles, was 24 or more.
  • IgG is released in both SBF and PBS in a sustained manner, and it can be seen that IgG, PD-1, and PD-L1 are released to almost 100% over 250 hours or more.
  • Porous silica particles bearing FITC-bound cytokines were resuspended in SBF (pH 7.4) at 37 ° C. and then observed for a predetermined period (1, 2, 3, 4, 5, 6, 7 days). The solution was centrifuged at 8,000 rpm for 10 minutes and the release of cytokines was confirmed by fluorescence intensity spectra of FITC.
  • IL-10, IL-15, HGF, EGF, IL-2 are all released slowly, it can be seen that the release to almost 100% over 7 hours or more.
  • porous silica particles of the present invention 31.25, 62.5, 125, and 250 mg / kg of porous silica particles (DegradaBALL) were respectively administered to all mice (four per group), and 5 days after the administration. Mice were sacrificed to separate major organs (liver, spleen, lung, heart and kidney).
  • porous silica particles of the present invention 0.5, 5, 50 mg / kg of porous silica particles (DegradaBALL) were added up to 9 times every 3 or 4 days to all mice (8 per group). Subcutaneously, 48 hours after the last administration, all mice were sacrificed to isolate the major organs (liver, spleen, lung, heart and kidney).
  • the porous silica particles of the present invention are organs (liver, spleen, kidney, thymus, heart, Lung and lymph nodes) did not induce significant weight loss (data is expressed as mean ⁇ standard deviation; significant difference from normal group ( * p ⁇ 0.05)) and did not affect the various parameters obtained by whole blood analysis and serum biochemical analysis.
  • ALT Alanine transaminase
  • AST aspartate transaminase
  • ALP alkaline phosphatase
  • GGT gamma-glutamyltransferase
  • MUSST myeloid U937 skin sensitivity test
  • the fluorescence intensity of PD-L1 antibody and DDV can be measured. It can be seen that DDV and anti-PD-L1 antibody remain at the administration site for a long time, which is the anti-PD- loaded carrier of the present invention.
  • the L1 antibody can be delivered stably, and it can be long-lived in the tumor and at the site of administration, demonstrating that it can have long-term efficacy.
  • IL-2 and DDV after labeling Cy7 and Cy5 to IL-2 and DDV, and subcutaneously administering DDV (BALLkine-2) carrying IL-2 (SC injection), IL-2 and DDV at each hour. It can be confirmed that the result of measuring the fluorescence intensity of, DDV and BALLkine-2 can be confirmed that it remains at the administration site for a long time, which proves that the present invention can be delivered stably delivered with the IL-2.
  • FITC fluorescence was added to IL-2, TAMRA fluorescence was applied to DDV, and subcutaneously administered. After 24 hours, lectin labeled with Cy5 fluorescence was injected into the tail vein. After staining the blood vessels, the tumor was extracted to confirm the results of the fluorescence image. It can be seen that DDV and BALLkine-2 are specifically distributed in the tumor tissue, which indicates IL-2 carrying the carrier of the present invention. To demonstrate tumor-specific targeting and delivery.
  • FIG. 52 shows histological cross-sectional images of tumor tissue showing tumor cells (DAPI), vehicle (TAMRA), IL-2 (FITC) and dylight 649 conjugated lectin
  • FIG. 53 shows BALLkine-2 (pt) ) And IL-2 (ip or pt) intratumoral pharmacokinetics (graphs represent mean ⁇ standard deviation of 3 mice per measurement time), followed by subcutaneous injection to determine the amount of IL-2 in the tumor
  • DAPI tumor cells
  • TAMRA vehicle
  • IL-2 FITC
  • FIG. 53 shows BALLkine-2 (pt) ) And IL-2 (ip or pt) intratumoral pharmacokinetics (graphs represent mean ⁇ standard deviation of 3 mice per measurement time), followed by subcutaneous injection to determine the amount of IL-2 in the tumor
  • the porous silica particles of the present invention can keep IL-2 in the tumor and the administration site for a long time, and stably deliver IL-2.
  • Figures 54 and 55 show subcutaneous infusion of 1xPBS buffer (100 ⁇ L) and Cy5 conjugated DegradaBALL (2.5 mg / kg) and sacrifice of mice at 1, 3 and 5 days after administration to organs (skin, thymus, spleen and lymph nodes). After separation), the results of photographing the fluorescence image of the organ by the FOBI imaging system can be confirmed, which can be confirmed through the fluorescence image to be distributed to the immune organs on day 1, 3, 5, the porous silica of the present invention It is to prove that the particles can carry a variety of immunoreactive substances to the immune system stably.
  • mice normal and melanoma tumor mice were injected subcutaneously or around tumors, respectively.
  • FIG. 57 all mice were injected subcutaneously with DegradaBALL with TAMRA at doses of 2.5, 10, and 50 mg / kg, respectively, and skin tissues were extracted at 1, 3, 5, and 7 days later to fluoresce.
  • the porous silica particles of the present invention have a high in vivo stability, and thus can carry the sustained sustained release of the immunoreactive substance stably for a long time. Imply that there is.
  • Dosing Group Dosing Protocol Total capacity Buffer i.p 7 injections every 3 days (0,3,6,9,12,15,18 days)
  • aPD-1 antibody 70 mg / kg IL-2: 10 mg / kg aPD-1 antibody (i.p) + IL-2
  • aPD-1 antibody 7 intraperitoneal injections every 3 days (0,3,6,9,12,15,18 days) IL-2: 4 mass peripher
  • TIL assays were isolated from mice on the target day (1,3,5,7 days after BALLkine-2 administration) to activate immune cell populations ((b) CD8 + T cells, (c) CD4 + T cells, (d) activation).
  • NK cells, (e) Treg cells, (f) CD8 + / Treg cells) were analyzed by flow cytometry (dots represent individual tumor tissues, lines represent the average of tumor tissues of 8 mice), 3, 5 days Analyzes were performed by staining the immune tissues of CD8 + T cells at the time point. Referring to the representative image of the BALLkine-2 administration group (FIG. 70), the number of CD8 + T cells increased dramatically and it was confirmed that they were distributed among a wide range of tumor cells. Can be.
  • spleen and drainage lymph nodes (iliac and inguinal lymph nodes) were isolated on days 1 and 3 and 5 after BALLkine-2 was injected into the right flank of mice with B16F10 melanoma tumors.
  • CD8 + T cells and NK cells were isolated to evaluate immune cell populations (data is expressed as mean ⁇ standard deviation of three mice), indicating that the number of immune cells compared to control group was significantly increased in BALLkine-2 administration group.
  • kidney size of BALLkine-2 was significantly smaller than that of other groups, and H & E staining of the kidney tissue of FIG. It can be seen that the degree is significantly higher.
  • wet lungs were harvested and weighed, and then dried in a 60 ° C. oven for 3 days and then weighed (FIG. 81, data are mean ⁇ SD, 3 mice per group, # #p ⁇ 0.01: significantly different than control group (Buffer group), ** p ⁇ 0.01: significantly different group than IL-2 group), the weight ratio of wet and dry lungs compared to IL-2 group in BALLkine-2 group It was found to be significantly lower.
  • mice results of analysis of leukocytes, neutrophils and lymphocytes in the blood of mice (FIG. 82, data are mean ⁇ SD, 3 mice per group, #p ⁇ 0.05: significantly different from the control group (Buffer administration group), * p ⁇ 0.05: Significantly different from IL-2 administration group), the number of BALLkine-2 treatment group showed significantly higher level than the other groups, and thus the remarkable excellence in the immune response promoting effect was confirmed.
  • the experiment was carried out according to the administration schedule of FIG. 83 and the CLS analysis protocol. Specifically, three days after the administration of IL-2 (15 ⁇ g) and BALLkine-2 (15 ⁇ g) to C57BL / 6 mice, the analysis was performed on the fourth day. It was. Referring to a representative H & E staining image of mouse lung tissue administered buffer (iv or sc), DegradaBALL (iv or sc), IL-2 (iv or sc) or BALLkine-2 (iv or sc) (FIG.
  • the number of inflammatory cells in and around the blood vessels was reduced compared to the IL-2 (iv or sc) administration group (the rod indicates 200 ⁇ m).
  • wet lungs were separated and weighed to evaluate pulmonary edema, and lungs were dried and weighed for 3 days in an oven at 60 ° C. (FIG. 85), IL-2 (iv or sc) in the BALLkine-2 administration group. It was confirmed that the weight ratio (wet lung weight / dry weight) of the dry lung to the wet lung was significantly lower.
  • mice were injected 2 hours before sacrificing Evans blue dye through the tail vein, and after sacrifice, the liver (Fig.
  • Vascular Leakage Syndrome (VLS) analysis was performed in a tumor bearing mouse model according to the protocol of FIG. 89 to determine if vascular leakage syndrome could be reduced even in actual cancer immunotherapy.
  • VLS Vascular Leakage Syndrome
  • wet lungs were separated and weighed, and lungs were dried and weighed for 3 days in an oven at 60 ° C. (FIG. 90), compared to IL-2 (iv or sc) -administered groups in the BALLkine-2 administration group. The weight ratio was significantly lower and showed similar values to the normal group.
  • mice injected 2 hours before sacrificing Evans blue dye through the tail vein, and then separated the liver ( Figure 91) and lung ( Figure 92) and measured VLS through extravasation of Evans Blue It was confirmed that the BALLkine-2 administration group was significantly lower than the IL-2 (iv or sc) administration group.

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Abstract

An immunoreactant carrier of the present invention has an excellent immunotherapeutic effect by stably delivering various immunoreactants including antibodies and cytokines to a target site. Therefore, a composition comprising a carrier of the present invention can be used for immunotherapy, thereby having excellent effects in the prevention or treatment of cancer or various immune diseases.

Description

면역반응 물질 전달체Immune Response Mass Carrier

본 발명은 면역반응 물질을 안정적으로, 표적부위에 전달하여 우수한 면역치료 효과를 갖는 면역반응 물질 전달체에 관한 것이다.The present invention relates to an immunoreactive substance carrier having an excellent immunotherapeutic effect by delivering an immunoreactive substance stably to a target site.

약물전달 시스템은 기존 의약품의 부작용을 최소화하고 효능 및 효과를 극대화시켜 필요한 양의 약물, 예를 들어, 단백질, 핵산, 또는 기타 저분자 등을 효율적으로 전달할 수 있도록 하는 의약 기술을 의미한다. 신약개발에 필요한 비용과 시간을 절감해 주는 상기 기술은 최근 나노기술과 결합하면서 의약계에서 새로운 부가가치를 창출하는 첨단기술의 한 분야로 자리 잡고 있으며, 미국과 일본 등 기술선진국들은 지난 80년대 후반부터 제약회사 등 기업을 중심으로 신약개발과 함께 약물전달시스템의 개발에 전력을 쏟아 왔다.Drug delivery system refers to a medical technology that can efficiently deliver the required amount of drugs, such as proteins, nucleic acids, or other small molecules by minimizing the side effects and maximizing the efficacy and effects of existing drugs. This technology, which saves the cost and time required for the development of new drugs, has recently become one of the cutting-edge technologies that create new added value in the pharmaceutical industry, in combination with nanotechnology. The company has been focusing on the development of drug delivery systems along with the development of new drugs, especially companies and companies.

지금까지는, 바이러스 유전자, 재조합 단백질, 리포좀(liposome), 양이온성 고분자, 그리고 다양한 형태의 나노입자와 나노물질들이 동물 세포 내로의 약물전달을 위해 이용되었다. 그러나 많은 양이온성 리포좀들과 양이온성 고분자들은 임상에 적용하기에는 세포에 독성이 강하여 부적합한 것으로 밝혀졌다. 또한, 안정적인 핵산의 세포막 투과를 위하여 핵산의 주 사슬을 화학적으로 변형시키는 방법도 시도되었다. 그러나, 이러한 방법은 비용이 비싸고 오랜 시간이 걸리며 노동 집약적인 공정이 요구되므로 임상 적용에는 적합하지 않다. 의미 있는 시도로서, 양자점, 자성 입자, 또는 금 나노입자를 포함한 다양한 형태의 나노입자를 이용한 약물전달 시스템(drug delivery system, DDS)이 개발된 바 있다. 그러나, 이러한 입자들은 세포에 독성을 가지며 핵산 등의 생체 고분자의 도입에 용이하지 않은 구조를 가지며, 세포 내로의 도입 효율도 낮다는 단점이 있었다.To date, viral genes, recombinant proteins, liposomes, cationic polymers, and various types of nanoparticles and nanomaterials have been used for drug delivery into animal cells. However, many cationic liposomes and cationic polymers have been found to be unsuitable because of their high toxicity to cells for clinical applications. In addition, a method of chemically modifying the main chain of the nucleic acid has been attempted for stable cell membrane penetration of the nucleic acid. However, this method is not suitable for clinical applications because it is expensive, time consuming, and requires labor intensive processes. As a significant attempt, drug delivery systems (DDS) have been developed that utilize various types of nanoparticles, including quantum dots, magnetic particles, or gold nanoparticles. However, these particles have a disadvantage in that they are toxic to cells, have a structure that is not easy to introduce biopolymers such as nucleic acids, and have low efficiency of introduction into cells.

세포 내에서의 생리활성물질의 기능의 연구 또는 세포내 전달을 위해서는 효율적인 전달 시스템이 필요하다. 그러나, 광범위한 생리활성물질을 전달할 수 있는 범용적인 전달 시스템, 다량의 약물을 수용 및 전달할 수 있는 시스템, 약물을 서방적으로 방출하는 시스템에 대한 개발은 아직 미진한 상황이다.Efficient delivery systems are needed for the study of the function of bioactive substances in cells or for intracellular delivery. However, the development of a universal delivery system capable of delivering a wide range of bioactive substances, a system capable of accommodating and delivering a large amount of drugs, and a system for releasing drugs in a sustained manner is far from under development.

본 발명은 항체와 사이토카인을 포함하는 다양한 면역반응 물질을 안정적으로, 표적부위에 전달하여, 우수한 면역치료 효과를 갖는 면역반응 물질 전달체를 제공함에 그 목적이 있다.An object of the present invention is to stably deliver various immunoreactive substances including antibodies and cytokines to a target site, thereby providing an immunoreactive substance carrier having excellent immunotherapeutic effects.

1. 항체(antibody) 또는 사이토카인(cytokine)을 담지한 다공성 실리카 입자를 포함하고, 1. Contains porous silica particles carrying an antibody or cytokine,

상기 다공성 실리카 입자는 직경 5nm 미만의 기공을 갖는 실리카 입자를 120℃ 내지 180℃에서 24시간 내지 96시간 동안 팽창제와 반응시켜 상기 직경 5nm 미만의 기공을 팽창시키는 단계; 및 상기 기공이 팽창된 실리카 입자를 400℃ 이상의 온도에서 3시간 이상 하소하는 단계를 포함하여 제조되며,The porous silica particles react with silica particles having pores less than 5 nm in diameter at 120 ° C. to 180 ° C. for 24 to 96 hours to expand the pores less than 5 nm in diameter; And calcining the pores of expanded silica particles at a temperature of 400 ° C. or higher for at least 3 hours.

상기 다공성 실리카 입자의 평균 직경은 150 nm 내지 1000nm이고, 그 BET 표면적은 200m 2/g 내지 700m 2/g이고, 그 g당 부피는 0.7ml 내지 2.2ml이며,The average diameter of the porous silica particles is 150 nm to 1000 nm, the BET surface area is 200m 2 / g to 700m 2 / g, the volume per g is 0.7ml to 2.2ml,

상기 다공성 실리카 입자는 하기 수학식 1의 흡광도의 비가 1/2이 되는 t가 24 이상인 것인 면역반응 물질 전달체:The porous silica particles have an immunoreactive substance carrier in which t is 24 or more such that the ratio of absorbance of Equation 1 is 1/2:

[수학식 1] [Equation 1]

A t/A 0 A t / A 0

(식 중, A 0는 상기 다공성 실리카 입자 1mg/ml 현탁액 5ml를 직경 50kDa의 기공을 갖는 원통형 투과막에 넣고 측정된 다공성 실리카 입자의 흡광도이고,Wherein A 0 is the absorbance of the porous silica particles measured by placing 5 ml of the 1 mg / ml suspension of the porous silica particles into a cylindrical permeable membrane having pores having a diameter of 50 kDa,

상기 투과막 외부에는 상기 투과막과 접하며, 상기 현탁액과 동일한 용매 15ml가 위치하고, 상기 투과막 내외부는 37℃에서 60rpm 수평 교반되며,Outside of the permeable membrane is in contact with the permeable membrane, 15ml of the same solvent as the suspension is located, the inside and outside of the permeable membrane is stirred 60 rpm at 37 ℃ horizontal,

상기 현탁액의 pH는 7.4이고,The pH of the suspension is 7.4,

A t는 상기 A 0의 측정시로부터 t시간 경과 후에 측정된 다공성 실리카 입자의 흡광도임).A t is the absorbance of the porous silica particles measured after t hours have elapsed since the measurement of A 0 ).

2. 위 1에 있어서, 상기 항체는 인터류킨(interleukins) 또는 인터페론(interferons) 단백질에 특이적으로 결합하는 항체인 전달체.2. The carrier according to 1 above, wherein the antibody is an antibody that specifically binds to an interleukins or interferons protein.

3. 위 1에 있어서, 상기 항체는 IgG 이거나; PD-1, CTLA-4, TIM-3, BTLA, VISTA, LAG-3, PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD27, CD137, HVEM, GITR, VEGFR, VEGF, EGFR, EGF, IL-1, IL-6, IL-23, TGF-beta, CTGF, TSLP, TNF-alpha, Notch 및 OX40으로 이루어진 군에서 선택된 적어도 하나의 단백질에 특이적으로 결합하는 항체인 전달체.3. The antibody of 1 above, wherein the antibody is an IgG; PD-1, CTLA-4, TIM-3, BTLA, VISTA, LAG-3, PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD27, CD137, HVEM, GITR, VEGFR, VEGF, A carrier that is an antibody that specifically binds to at least one protein selected from the group consisting of EGFR, EGF, IL-1, IL-6, IL-23, TGF-beta, CTGF, TSLP, TNF-alpha, Notch and OX40.

4. 위 1에 있어서, 상기 항체는 PD-1, PD-L1 및 CTLA-4로 이루어진 군에서 선택된 적어도 하나의 단백질에 특이적으로 결합하는 항체인 전달체.4. The carrier according to 1 above, wherein the antibody is an antibody that specifically binds to at least one protein selected from the group consisting of PD-1, PD-L1, and CTLA-4.

5. 위 1에 있어서, 상기 사이토카인은 인터류킨(interleukins) 또는 인터페론(interferons)인 전달체.5. The carrier of 1 above, wherein the cytokine is interleukins or interferons.

6. 위 1에 있어서, 상기 사이토카인은 IL-7, IL-10, IL-12, IL-13, IL-15, IL-21, IL-23, IL-24, IL-27, G-CSF, GM-CSF, HGF, EGF, VEGF, LTF, TGF-β 및 IL-2로 이루어진 군에서 선택된 적어도 하나인 전달체.6. In above 1, the cytokine is IL-7, IL-10, IL-12, IL-13, IL-15, IL-21, IL-23, IL-24, IL-27, G-CSF , GM-CSF, HGF, EGF, VEGF, LTF, TGF-β, and at least one carrier selected from the group consisting of IL-2.

7. 위 1에 있어서, 상기 사이토카인은 IL-2, IL-12, IL-15, IL-21, IL-24 및 IL-13으로 이루어진 군에서 선택된 적어도 하나인 전달체.7. The carrier according to 1 above, wherein the cytokine is at least one selected from the group consisting of IL-2, IL-12, IL-15, IL-21, IL-24, and IL-13.

8. 위 1에 있어서, 상기 다공성 실리카 입자는 외부 표면 또는 기공 내부가 중성의 pH에서 전하를 띠는 것인 전달체.8. The carrier of 1 above, wherein the porous silica particles are charged at an external pH or inside the pore at a neutral pH.

9. 위 1에 있어서, 상기 다공성 실리카 입자는 외부 표면 또는 기공 내부에 친수성 또는 소수성 작용기를 갖는 것인 전달체.9. The carrier according to the above 1, wherein the porous silica particles have hydrophilic or hydrophobic functional groups on the outer surface or inside the pores.

10. 위 1에 있어서, 항체(antibody)를 담지한 다공성 실리카 입자; 및 사이토카인(cytokine)을 담지한 다공성 실리카 입자를 포함하는 전달체.10. In the above 1, porous silica particles carrying an antibody (antibody); And a porous silica particle carrying a cytokine.

11. 위 1 내지 10 중 어느 한 항의 전달체를 포함하는 면역요법(immunotherapy)용 조성물.11. The composition for immunotherapy (immunotherapy) comprising the carrier of any one of the above 1 to 10.

12. 위 1 내지 10 중 어느 한 항의 전달체를 포함하는 암 또는 면역질환의 예방 또는 치료용 약학적 조성물.12. A pharmaceutical composition for preventing or treating cancer or immune disease, comprising the carrier of any one of 1 to 10 above.

본 발명의 면역반응 물질 전달체는 항체와 사이토카인을 포함하는 다양한 면역반응 물질을 안정적으로, 표적부위에 전달하여, 우수한 면역치료 효과를 갖는다.The immune-responsive substance carrier of the present invention stably delivers various immune-responsive substances, including antibodies and cytokines, to the target site, and has an excellent immunotherapeutic effect.

도 1은 본 발명의 일 구현예에 따른 다공성 실리카 입자의 현미경 사진이다.1 is a micrograph of porous silica particles according to an embodiment of the present invention.

도 2는 본 발명의 일 구현예에 따른 다공성 실리카 입자의 현미경 사진이다.2 is a micrograph of porous silica particles according to one embodiment of the present invention.

도 3은 본 발명의 일 구현예에 따른 다공성 실리카 입자의 제조 공정 중의 소기공 입자의 현미경 사진이다.Figure 3 is a micrograph of the small pore particles during the manufacturing process of the porous silica particles according to an embodiment of the present invention.

도 4는 본 발명의 일 구현예에 따른 소기공 입자의 현미경 사진이다.Figure 4 is a micrograph of the small pore particles according to an embodiment of the present invention.

도 5는 본 발명의 일 구현예에 따른 다공성 실리카 입자의 기공 직경별 현미경 사진이다. DDV(Degradable Delivery Vehicle)는 실시예의 입자로서 괄호안의 숫자는 입자의 직경, 아래첨자의 숫자는 기공 직경을 의미한다. 예를 들어, DDV(200) 10은 입자 직경은 200 nm, 기공 직경은 10 nm인 실시예의 입자를 의미한다.Figure 5 is a micrograph of the pore diameter of the porous silica particles according to an embodiment of the present invention. DDV (Degradable Delivery Vehicle) is the particle of the embodiment, the number in parenthesis means the diameter of the particle, the number of subscripts means the pore diameter. For example, DDV (200) 10 means the particles of the embodiment having a particle diameter of 200 nm, the pore diameter is 10 nm.

도 6은 본 발명의 일 구현예에 따른 다공성 실리카 입자의 생분해성을 확인할 수 있는 현미경 사진이다.Figure 6 is a micrograph to confirm the biodegradability of the porous silica particles according to an embodiment of the present invention.

도 7은 일 예시에 따른 원통형 투과막을 구비한 튜브이다.7 is a tube having a cylindrical permeable membrane according to one example.

도 8은 본 발명의 일 구현예에 따른 다공성 실리카 입자의 시간 경과에 따른 흡광도 감소 결과이다.8 is a result of the decrease in absorbance over time of the porous silica particles according to an embodiment of the present invention.

도 9는 본 발명의 일 구현예에 따른 다공성 실리카 입자의 시간 경과에 따른 입경별 흡광도 감소 결과이다.9 is a result of decreasing absorbance for each particle diameter over time of the porous silica particles according to an embodiment of the present invention.

도 10은 본 발명의 일 구현예에 따른 다공성 실리카 입자의 시간 경과에 따른 기공 직경별 흡광도 감소 결과이다.10 is a result of decreasing absorbance for each pore diameter of porous silica particles according to an embodiment of the present invention over time.

도 11은 본 발명의 일 구현예에 따른 다공성 실리카 입자의 시간 경과에 따른 환경의 pH별 흡광도 감소 결과이다.Figure 11 is a result of reducing the absorbance for each pH of the environment over time of the porous silica particles according to an embodiment of the present invention.

도 12는 본 발명의 일 구현예에 따른 다공성 실리카 입자의 시간 경과에 따른 흡광도 감소 결과이다.12 is a result of decreasing absorbance over time of the porous silica particles according to an embodiment of the present invention.

도 13 내지 15는 각각 IgG, PD-1, PD-L1 항체를 다공성 실리카 입자에 담지한 후 방출한 누적량을 나타낸 것이다.13 to 15 show cumulative amounts released after supporting IgG, PD-1, and PD-L1 antibodies on porous silica particles, respectively.

도 16은 IL-2 사이토카인을 담지하는 데에 사용된 다공성 실리카 입자의 TEM 이미지를 나타낸 것이다.FIG. 16 shows TEM images of porous silica particles used to support IL-2 cytokines. FIG.

도 17 내지 21은 IL-10, IL-15, HGF, EGF, IL-2를 다공성 실리카 입자에 담지한 후 방출한 누적량을 나타낸 것이다.17 to 21 show the cumulative amount released after supporting IL-10, IL-15, HGF, EGF, IL-2 on porous silica particles.

도 22 내지 43은 본 발명 다공성 실리카 입자의 독성여부를 확인한 결과이다.22 to 43 are the results confirming the toxicity of the present invention porous silica particles.

도 44, 45는 다공성 실리카 입자에 담지된 면역반응 물질의 생물학적 활성 보존여부를 확인한 결과이다.44 and 45 are results confirming the biological activity of the immunoreactive substance supported on the porous silica particles.

도 46은 본 발명 전달체에 의한 과민반응 여부를 확인한 결과이다.46 is a result confirming the hypersensitivity reaction by the carrier of the present invention.

도 47 내지 55는 본 발명 전달체의 담지된 항체 또는 사이토카인의 안정적인 전달성 및 표적성을 확인한 것이다.47 to 55 confirm the stable delivery and target of the supported antibody or cytokine of the carrier of the present invention.

도 56 내지 82는 본 발명 전달체의 암 면역치료 효능을 확인한 결과이다.56 to 82 is a result confirming the cancer immunotherapy efficacy of the carrier of the present invention.

도 83 내지 92는 본 발명 전달체의 부작용(VLS 또는 CLS) 감소 효과를 확인한 결과이다.83 to 92 is a result of confirming the side effects (VLS or CLS) reduction effect of the present invention carrier.

이하, 본 발명을 구체적으로 설명한다.Hereinafter, the present invention will be described in detail.

항체(antibody) 또는 사이토카인(cytokine)을 담지한 다공성 실리카 입자;를 포함하는 면역반응 물질 전달체를 제공한다.It provides an immunoreactive substance transporter comprising; porous silica particles carrying an antibody or cytokine.

상기 사이토카인은 구체적으로 인터류킨(interleukins) 또는 인터페론(interferons)일 수 있다.The cytokine may be specifically interleukins or interferons.

상기 사이토카인은 IL-7, IL-10, IL-12, IL-13, IL-15, IL-21, IL-23, IL-24, IL-27, G-CSF, GM-CSF, HGF, EGF, VEGF, LTF, TGF-β, IL-2, A2M, ABI3BP, acidic fibroblast growth factor, ACVR1B, ADAM17, ADAMTS6, ADMLX, aFGF, AGPAT1, AGPAT2, AIF1, AIMP1, AKR1C1, AKT1S1, allograft-inflammatory factor-1, amac-1, AMH, ANGPTL2, ANGPTL3, ANKFN1, ANKRD1, ankyrin repeat and SOCS box-containing protein 3 isoform a variant, ankyrin repeat domain-containing SOCS box protein ASB11, ankyrin repeat domain-containing SOCS box protein Asb-13, ankyrin repeat domain-containing SOCS box protein Asb-14, ankyrin repeat domain-containing SOCS box protein Asb-15, ankyrin repeat-containing protein ASB-2, ankyrin repeat-containing SOCS box protein 7, ANOS1, Apo-2 ligand, APOC1, ARHGAP10, ASB1, ASB-1 protein, ASB10, ASB-10, ASB11, ASB12, ASB13, ASB14, ASB15, ASB16, ASB17, ASB18, ASB2, ASB-2, ASB3, ASB-3 protein, ASB4, ASB-4 protein, ASB5, ASB6, ASB7, ASB8, ASB9, ASTN2, ATP10A, ATP1A1, ATP8A1, AXL, B219/OB receptor isoform HuB219.1 precursor, B219/OB receptor isoform HuB219.2 precursor, B219/OB receptor isoform HuB219.3 precursor, BATF, BCL2A1, BCL3, BCO2, BIG-2, BMP6, BOC, BTNL2, byk, C10orf99, C11orf40, C17, C17orf99, C19orf66, C1GALT1C1, C1QTNF4, C22orf39, C2orf83, C3orf39, C6 beta-chemokine, C6orf120, C8orf44-SGK3, CALL, CAMKMT, CARD14, cardiotrophin-like cytokine CLC, CASP1, cathepsin X, CBLB, CBLN4, CCDC134, CCDC86, CCL1, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL3L3, CCL4, CCL4L, CCL4L1, CCL4L2, CCL5, CCL7, CCL8, CCR4, CCR8, CD200R1, CD274, CD28, CD300LB, CD34, CD36, CD3D, CD40LG, CD53, CD70, CD74, CD74-ROS1_C6,R32, CD80, CD83, CD84, CD86, CDO, CDON, CEBPD, CER1, CFC1, chemokine (C-C motif) ligand 13, chemokine (C-C motif) ligand 3, CHL1, CHUK, CIAPIN1, ciliary neurotrophic factor receptor, CIP29, CIS2, CIS3, CIS4, CISH, CISH6, CITED2, CKId, CKIe, CKLF, CKLF1, CKLFSF1, CLC, CLCF1, CLEC11A, CLEC1B, CLEC2D, CLF-1, CLNK, c-mer, c-mpl-K, c-mpl-P, CMYA5, CNAIP, CNTF, CNTFR, CNTN1, CNTN2, CNTN3, CNTN4, CNTN5, CNTN6, CNTNAP4, CNTRL, Col VII, COL12A1, COL14A1, COL20A1, COL28A1, COL7A1, collagen VII, colony stimulating factor 2 receptor alpha subunit splice variant, colony stimulating factor 3 receptor isoform c precursor variant, CPEB 4 variant, CPEB4, CR6, CREME9, CRF2/12, CRIP2, CRL1, CRL2, CRL3, CRLF1, CRLF2, CRLF3, CRNDE, CRP, CRTAM, CS box-containing WD protein, CSA2, CSBP1, CSBP2, CSF1, CSF1R, CSF2, CSF2RA, CSF2RB, CSF3, CSF3R, CSN1S1, CTF1, CTLA4, CTLA8, CTSG, CUA001, CX3CL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL16, CXCL3, CXCL5, CXCL6, CXCL8, CXCL9, CXCR2, CXCR3, CXCR5, CXXC5, cyclon, CYP4F3, CYTIP, CYTL1, cytokine CX2 precusor, cytokine receptor CRL2 precursor, cytokine receptor-like factor 2 variant, cytokine SDF-1-beta, cytokine type 1 receptor CRLP-1 precursor, cytokine-inducible inhibitor of signalling type 1b, cytokine-inducible SH2-containing protein, cytokine-like nuclear factor n-pac, cytokine-like nuclear factor n-pac-like protein, cytokine-like protein 2-21, cytokine-like protein EF-7, CYTOR4, cytotoxic lymphocyte maturation factor 40 kDa subunit, DAGLA, DCC, DCSTAMP, DDT, DEFB103B, delta 4-delta 7/11 truncated prolactin receptor, delta 4-SF1b truncated prolactin receptor, dendritic cell associated lectin 2, density enhanced phosphatase-1, DHX58, DIP2C, DNAH3, DNAH9, Down syndrome cell adhesion molecule isoform CHD2-42 precursor variant, DRT, DSCAM, DSCAM2, DSCAML1, DTK, DTL, DUB3, DVL2, EBI3, ECK, EDIL3, eEF1A2 binding protein, EFNA5, EGFLAM, EGR4, ELAC1, ELAVL1, ELAVL3, ELP2, endothelial-monocyte activating polypeptide II, EOMES, eotaxin precursor, EPGN, EPHA1, EPHA10, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHB1, EPHB2, EPHB2 variant protein, EPHB3, EPHB4, EPHB6, EphB6 variant protein, ephrin receptor EphA5 isoform a variant, ephrin receptor EphA7 variant, ephrin receptor EphB4 precursor variant, EPO, EPOR, ERK, ESOP1, FABP5, factor for adipocyte differentiation 104 variant, fad104, FAM19A4, FAM213A, FAM3A, FAM3B, FAM3C, FAM3D, FANK1, FBRS, FCAR, FCER1A, FCER1G, FCGR2C, FCN3, FES, FGF19, FGF9, FGF-9, fibronectin, Fibronectin 1 variant, FIL1-theta, FILIP1L, FLJ00133, FLJ00148, FLJ00154, FLJ00236, FLJ00376, FLT3, FLT3LG, FN1, FNDC1, FNDC3A, FNDC3B, FNDC4, FNDC5, FNDC7, FNDC8, FOSB, FOSL1, FOXP3, FRMD1, FSD1, FSD1L, FSD2, FSTL3, FUCA2, G0S3, G18, G-26, GAB2, GAB3, gamma.1, GBP4, G-CSFR-1, G-CSFR-2, GDF15, GDF2, GHR, GJA1, GLEPP1, GLYR1, GM-CSF receptor beta chain, gp130, gp130-like monocyte receptor, gp250 precursor, GPI, GPR15, GPR75-ASB3, GPSM1, GRAIL, granulocyte colony-stimulating factor receptor, granulocyte macrophage-colony stimulating factor, granulocyte-macrophage colony stimulating factor 2, granulocyte-macrophage colony-stimulating factor precusor, GRAP2, GRO-beta, GRO-gamma, growth-inhibiting protein 45, GZMK, HAVCR2, HAX1, HBD, HCF-2, HCFC1, HCFC2, HCV NS5A-binding protein NS5ABP37, HDAC11, hDDM36, HEK, HEK11, HEK5, HEK7, HEK8, hematopoietic stem/progenitor cell induced protein 1, hematopoietic stem/progenitor cell induced protein 2, hematopoietic stem/progenitor cell induced protein 3, HES4, HGF, HGS, HIBDL, hIL-2Rg, HILPDA, HIN-1 putative cytokine, HMGB1, hmrp-2a, hmrp-2b, hNB-2, hNB-2s, hNB-3, host cell factor C2 variant, HOXB5, hp40, HSD13, hSHIP, HSP90B1, HSSOCS-2, HTK, HTPZP2, hTroy, Humig, HXB, hypothetical protein, ICOSLG, IFI16, IFI30, IFNA1, IFNA10, IFNA17, IFNA2, IFNA4, IFNA8, IFNAR1, IFNAR2, IFNB1, IFNG, IFNGR1, IFNGR2, IFNK, IFNL1, IFNL2, IFNL3, IFNLR1, IFNW1, IGANRP, IGDCC3, IGDCC4, IGF1R, IGFN1, IGSF22, IGSF9, IGSF9B, IK, IKBKB, IKK alpha, Interleukins, IL10, IL10RB, IL11, IL11RA, Il-12 receptor beta2, IL12 receptor component, IL12A, IL12B, IL12RB1, IL12RB2, IL13, IL-13Ra, IL13RA1, IL13RA2, IL15, IL15RA, IL16, IL-17, IL-17 receptor, IL17A, IL17B, IL17BR, IL17C, IL17D, IL-17D, IL17D precursor, IL17E, IL17F, IL17RA, IL17RB, IL17RC, IL-17RC, IL-17RD, IL-17RE, IL18, IL-18 receptor beta splice variant, IL18BP, IL18R1, IL18RAP, IL19, IL1A, IL1B, IL1BCE, IL1F10, IL1F7, IL1L1, IL1R1, IL1R2, IL1RAP, IL1RAPL1, IL1RAPL2, IL1RL1, IL1RL2, IL1RN, IL2, IL15, IL20, IL20RA, IL20RB, IL21, IL-21, IL21R, IL22, IL22BP, IL22R, IL22RA1, IL22RA2, IL23A, IL23R, IL-23R, IL-24 splice variant delE3, IL-24 splice variant delE5, IL25, IL26, IL27, IL27RA, IL28A, IL28B, IL28C, IL28RA, IL29, IL30, IL2RA, IL2RB, IL2RG, IL3, IL31, IL31RA, IL32, IL33, IL34, IL36A, IL36B, IL36G, IL36RN, IL37, IL3RA, IL4, IL4I1, IL4R, IL5, IL5RA, IL6, IL6R, IL6ST, IL7, IL7R, IL9, IL9R, il-xr, ING3, INPP5D, INSR, INSRR, insulin receptor variant, insulin responsive sequence DNA binding protein-1, integrin beta 4 isoform 3 precursor variant, interferon alpha 2b, interferon gamma, interferon-lambda1, interferon-lambda2, interferon-lambda3, interleukin 10 receptor, beta, interleukin 11 receptor, alpha, interleukin 12, P40, interleukin 13, interleukin 13 receptor, alpha 1 precursor variant, interleukin 17A, interleukin 17B, interleukin 17C, interleukin 18 precursor, interleukin 23 p19 subunit, interleukin 23 receptor isoform 1, interleukin 32 variant, interleukin 34 isoform 1 precursor, interleukin 34 isoform 2 precursor, interleukin 4 receptor alpha chain isoform a precursor variant, interleukin 6 receptor isoform 1 precursor variant, interleukin-1 homolog 1, interleukin-1 homolog 2, interleukin-1 homolog 4, interleukin-11 receptor alpha chain, interleukin-13 receptor, interleukin-17 beta, interleukin-1-related protein long isoform, interleukin-1-related protein long isoform a, interleukin-1-related protein short isoform, interleukin-2, interleukin-32 small, interleukin-32 theta, interleukin-33, intermediate prolactin receptor isoform, IRAK1, IRAK3, IREB2 variant, IRF9, IRS2, ISG15, ITGA1, ITGAL, ITGB2, ITGB4, ITPK1, JAB, JAK2, JAK3, JAKMIP1, JOSD2, KAL, KAL1, KALIG-1, KALRN, KAT6B, KCNA3, KCNJ12, KIAA0273, KIAA0282, KIAA0283, KIAA0318, KIAA0343, KIAA0671, KIAA0756, KIAA0970, KIAA1030, KIAA1132, KIAA1146, KIAA1297, KIAA1355, KIAA1397, KIAA1459, KIAA1496, KIAA1497, KIAA1510, KIAA1514, KIAA1568, KIAA1628, KIAA1666, KIAA1866, KLF8, KLRF1, L1CAM, LAR, LBP, LD78 alpha beta, LEPR, leptin receptor, LGI3, LICR2, LIF, LIFR, LILRA2, LIMK2, LOC57019, LOC728835, long myosin light chain kinase, LRG1, LRRC20, LRRC70, LRRFIP2, LRRN1, LRRN4, LTA, LTB, LTBP1, LTBP2, lymphokine, lymphotoxin beta isoform variant, lysophosphatidic acid acyltransferase-alpha, lysophosphatidic acid acyltransferase-beta, macrophage colony-stimulating factor 1, MAF, magic roundabout, MAPK14, MCP3, MCP-3, MCP4, MCP-4, MDK, MEP1B, MERTK, MEX3C, MGDF, MIC-1, microtubule-associated protein GLFND, MIP-1 delta, MIP1a, MIP2a, mitochondria associated granulocyte macrophage CSF signaling molecule Magmas, ML1, MLCK, MMP9, MNAT1, MON1B, MOV10, MPIF-1, MPL, MRPL17, MSLN, MTHFD2L, MTUS1, MUSK, MYBPC1, MYBPC2, MYBPC3, MYBPH, MYBPHL, MYDGF, myeloproliferative leukemia virus oncogene, MYLK, MYOM1, MYOM2, MYOM3, NAA35, NAMPT, NC28, NCAM, N-CAM, NCAM1, NCAM2, NCAM21, Nck, Ash and phospholipase C gamma-binding protein NAP4, NCR1, NCR3, NCR3LG1, NCSTN, NDFIP1, NDUFA2, NDUFB6, NEO1, neogenin, neogenin homolog 1 variant, neshbp, NET PTK, Neural cell adhesion molecule 1, 120 kDa isoform precursor variant, neural cell adhesion molecule CD56, Neural cell adhesion molecule variant, neurotrophin-1/B-cell stimulating factor-3, NFAM1, NFASC, NFAT1, NFATC1, NFATC4, NHLH1, NILR, NIPSNAP1, NIPSNAP2, NKSF1, NKSF2, NLRC5, NLRP11, NLRP12, NLRP13, NLRP2, NLRP4, NLRP6, NLRP7, NLRR-1, NOX5, N-PAC, NR2C2, NR4A1, NRCAM, OBSCN, OC-116KDa, OCLN, OPTC, OR2H1, OSM, OSMR, OSMRB, osteoprotegerin ligand, OTUB1, OTUD6B, OTUD7B, P2RY8/CRLF2 fusion, p48, PANX2, PARC, PDCD1LG2, PGLYRP1, PHF11, PHYHIP, PHYHIPL, PI9, PIAS1, PIBF1, PIK3R3, PLA2G2D, PLAC8, pLD78 peptide, PLK3, PLTP, PLXNC1, POMGNT2, PP14212, PPBP, PPIA, PPM1F, PPM1H, PPP2R5A, PPP3CC, PPP5C, PPY, PREX1, prk, PRLR, PRO0915, prolactin receptor, prolactin receptor delta 7/11, prolactin receptor isoform delta S1 precursor, proliferation associated cytokine-inducible protein CIP29, protein tyrosine phosphatase delta, protein tyrosine phosphatase sigma, protein tyrosine phosphatase, receptor type, D isoform 4 precursor variant, protein tyrosine phosphatase, receptor type, F isoform 2 precursor variant, protein tyrosine phosphatase, receptor type, G precursor variant, protein tyrosine phosphatase, receptor type, K precursor variant, protein tyrosine phosphatase, receptor type, sigma isoform 3 precursor variant, protein-tyrosine phosphatase, PRR16, PRSS27, PRTG, PSMA4, PTCHD1, PTGDR, PTGES, PTN, PTPRB, PTPRC, PTPRD, PTPRE, PTPRF, PTPRG, PTPRH, PTPRJ, PTPRK, PTPRM, PTPRO, PTPRQ, PTPRS, PTPRT, PTPRU, PTPRZ, PTPRZ1, PTPsigma, PTX4, PUS7L, putative cytokine receptor CRL4 precusor, PYPAF6, PYRIN-containing Apaf1-like protein 2, PYRIN-containing Apaf1-like protein 3, PYRIN-containing APAF1-like protein 4, PYRIN-containing APAF1-like protein 5, PYRIN-containing APAF1-like protein 7, PYY, RAB40A, RAB40AL, RAB40B, RAB40B, member RAS oncogene family variant, RAB40C, RAB6B, RANTES precursor, Rar protein, Rar-2 protein, RBM15, RBM5 variant, RCAN3, receptor protein tyrosine phosphatase hPTP-J precursor, Receptor protein tyrosine phosphatase hPTP-J precursor variant, receptor tyrosine kinase, receptor-type protein tyrosine phosphatase O isoform a precursor variant, RGS2, RIMBP2, RIMBP3, RIMBP3B, RIMBP3C, RIPK4, RMC1, RNASE10, RNASE7, RNASE9, RNASET2, RNF113A, RNF128, RNF41, ROBO1, ROBO2, ROBO2 isoform a, ROBO2 isoform b, ROBO3, ROBO4, RORC, ROS1, RPL27, RPL36, RPS21, R-PTP-kappa, RPTP-rho, rse, RSL24D1, S100A12, S100A8, S100A9, S1PR4, SAC3D1, SAP/SH2D1A, SAP-1, SARNP, SASH1, SCAMP5, SCGB3A1, SCGB3A2, SCM-1, SCYA13, SCYA16, SCYA26, SCYE1, SDC4, SDC4-ROS1_S2,R32, SDC4-ROS1_S4,R32, SDK1, SDK2, secreted osteoclastogenic factor of activated T cells, SELE, SEMA7A, SENP3, SERPINB9, serum/glucocorticoid regulated kinase-like isoform 1 variant, SETD1B, SGK3, SGK-like protein SGKL, SH2 domain containing SOCS box protein SOCS4, SH2B1, SH2B3, shen-dan, SIGIRR, SIGLEC14, Similar to RIKEN cDNA 3930401K13 gene, SKIP1, SKIP3, sky, SLAMF9, SLC2A5, SLC2A9, SLC34A2/ROS fusion, SLC34A2-ROS1, SLC39A13, SLC39A3, SLC4A3, SLC9A8, SLC9B2, SLURP1, small cytokine B subfamily member 11 SCYB11 precursor, SMIM27, SMPD2, SNED1, SNX10, SOCS box containing protein RAR2A, SOCS box protein ASB-5, SOCS1, SOCS-1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, SOCS7, SORL1, SPATA2, SPOCK3, SPP1, SPPL2B, SPRY domain-containing SOCS box protein SSB-1, SPRY domain-containing SOCS box protein SSB-1 variant, SPRY domain-containing SOCS box protein SSB-2, SPRY domain-containing SOCS box protein SSB-3, SPRY domain-containing SOCS box protein SSB-4, SPSB1, SPSB2, SPSB3, SPSB4, STAM, STAM2, STAMBP, STAT induced STAT inhibitor-1, STAT induced STAT inhibitor-2, STAT induced STAT inhibitor-3, STAT1, STAT5A, STAT5B, STAT-induced STAT inhibitor-2, stem cell factor, STIMATE, STK35, STK40, striated muscle preferentially expressed protein, suppressor of cytokine signaling 3, surface glycoprotein, Ig superfamily member variant, SUSD2, SWIP1, SYNJ2BP, synleurin, TACC2, TAIF, TARM1, TBX21, TBX5, TCEB1, TCIRG1, TEC, TEK, TEK tyrosine kinase variant, tenascin XB isoform 1 variant, tenascin-C isoform 14/AD1/16, testicular tissue protein Li 126, testicular tissue protein Li 64, testis tissue sperm-binding protein Li 57p, TEX12, TFPI, THEMIS2, THNSL2, THPO, thymic stromal lymphopoietin protein receptor TSLPR, thymic stromal lymphopoietin protein TSLP, TIE, tie receptor tyrosine kinase, TIE1, Tie-2, TIGIT, TIMP1, tip3, TIRAP, Titin, TLR1, TLR3, TLR6, TM7SF3, TMEM102, TMEM110-MUSTN1, TMSB4X, TMX2, TNC, TNC variant protein, TNF, TNFAIP3, TNFAIP8L2, TNFRSF11B, TNFRSF12A, TNFRSF13C, TNFRSF21, TNFRSF25, TNFRSF4, TNFRSF8, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF13B, TNFSF15, TNFSF18, TNFSF4, TNFSF8, TNFSF9, TNL, TNN, TNR, TNXB, TOPORS, Trad, TRAF6, TRIM21, TRIM3, TRIM42, TRIM46, TRIM67, TRIM8, TRIM9, TRPM4, TSLP, TTN, TULP4, tumor necrosis factor receptor, TUSP, TUT4, TUT7, TWIST2, TYK2, type VII collagen, type XII collagen, type-I T cell cytokine receptor, TYRO3, tyrosyl-tRNA synthetase, tyrosyl-tRNA synthetase variant, U2AF1L4, UBA6, UBAP1, ubiquitin ligase E3 alpha-I, ubiquitin ligase E3 alpha-II, UBR2, UFO, undulin 1, undulin 2, UNQ1942, UNQ2421, UNQ288, UNQ296, UNQ3121, UNQ421, UNQ5793, UNQ604, UNQ6309, UNQ6368, UNQ6389, UNQ6504, UNQ693, UQCRH, USH2A, USMG5, USP15, USP17L10, USP17L11, USP17L12, USP17L13, USP17L15, USP17L17, USP17L18, USP17L19, USP17L2, USP17L20, USP17L21, USP17L22, USP17L24, USP17L25, USP17L26, USP17L27, USP17L28, USP17L29, USP17L3, USP17L30, USP17L4, USP17L5, USP17L7, USP17L8, USP18, USP36, UTY, V alpha 1, V alpha 13, VCAM1, VESPR, VNN2, VSIG2, VWA1, WARS, WD SOCS-box protein 1 isoform 1 variant, WDR26, WSB1, WSB-1, WSB-1 isoform, WSB-1 protein, WSB2, WSB-2, WSX1, WWOX, XA, XB, XBP1, XCL1, XCL2, XKR4, YARS, ZBED3, ZBP1, ZC3H12D, ZC3H15, ZCYTO10, zcytor5, ZCYTOR7, ZFP36, ZNF366, ZNF580 및 ZNF827으로 이루어진 군에서 선택된 적어도 하나일 수 있으나, 반드시 이에 제한되지 않는다.The cytokines are IL-7, IL-10, IL-12, IL-13, IL-15, IL-21, IL-23, IL-24, IL-27, G-CSF, GM-CSF, HGF, EGF, VEGF, LTF, TGF-β, IL-2, A2M, ABI3BP, acidic fibroblast growth factor, ACVR1B, ADAM17, ADAMTS6, ADMLX, aFGF, AGPAT1, AGPAT2, AIF1, AIMP1, AKR1C1, AKT1S1, allograft-inflammatory factor- 1, amac-1, AMH, ANGPTL2, ANGPTL3, ANKFN1, ANKRD1, ankyrin repeat and SOCS box-containing protein 3 isoform a variant, ankyrin repeat domain-containing SOCS box protein ASB11, ankyrin repeat domain-containing SOCS box protein Asb-13 , ankyrin repeat domain-containing SOCS box protein Asb-14, ankyrin repeat domain-containing SOCS box protein Asb-15, ankyrin repeat-containing protein ASB-2, ankyrin repeat-containing SOCS box protein 7, ANOS1, Apo-2 ligand, APOC1, ARHGAP10, ASB1, ASB-1 protein, ASB10, ASB-10, ASB11, ASB12, ASB13, ASB14, ASB15, ASB16, ASB17, ASB18, ASB2, ASB-2, ASB3, ASB-3 protein, ASB4, ASB- 4 protein, ASB5, ASB6, ASB7, ASB8, ASB9, ASTN2, ATP10A, ATP1A1, ATP8A1, AXL, B219 / OB receptor isoform HuB219.1 precursor, B219 / OB receptor isoform HuB219.2 precursor, B219 / OB receptor isoform HuB219.3 precursor, BATF, BCL2A1, BCL3, BCO2, BIG-2, BMP6, BOC, BTNL2, byk, C10orf99, C11orf40, C17, C17orf99, C19orf66, C1GALT1C1, C1QTNF4, C22orf39, C2orf83, C3orf39, C6 beta-chemokine, C6orf120, C8orf44-SGK3, CALL, CAMKMT, CARD14, cardiotrophin-like cytoPKNBL cat, CBP, CCLBL, CNPCLC, CLC, CNP, CCL, CNP, CCL, CNP, CCL, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, C, P, C, C use, C, C, C, C, C, C use, C, C, C, C, C, C use, and CCDC86, CCL1, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL3L4, CCL3L4, CCL4L1, CCL4L2, CCL5, CCL7, CCL8, CCR4, CCR8, CD200R1, CD274, CD28, CD300LB, CD34, CD36, CD3D, CD40LG, CD53, CD70, CD74, CD74-ROS1_C6, R32, CD80, CD83, CD84, CD86, CDO, CDON, CEBPD, CER1, CFC1, chemokine (CC motif) ligand 13, chemokine (CC motif) ligand 3, CHL1, CHUK, CIAPIN1, ciliary neurotrophic factor receptor, CIP29, CIS2, CIS3, CIS4, CISH, CISH6, CITED2 , CKId, CKIe, CKLF, CKLF1, CKLFSF1, CLC, CLCF1, CLEC11A, CLEC1B, CLEC2D, CLF-1, CLNK, c-mer, c-mpl-K, c-mpl-P, CMYA5, CNAIP, CNTF, CNTFR, CNTN1, CNTN2, CNTN3, CNTN4, CNTN5, CNTN6, CNTNAP4, CNTRL, Col VII, COL12A1, COL14A1, COL20A1, COL28A1, COL7A1, collagen VII, colony stimulating factor 2 receptor alpha subunit splice variant, colony stimulating factor 3 receptor isoform c precursor variant, CPEB 4 variant, CPEB4, CR6, CREME9, CRF2 / 12, CRIP2, CRL1, CRL2, CRL3, CRLF1, CRLF2, CRLF3, CRNDE, CRP, CRTAM, CS box-containing WD protein, CSA2, CSBP1, CSBP2, CSF1, CSF1R, CSF2, CSF2RA, CSF2RB, CSF3, CSF3R, CSN1S1 , CTLA4, CTLA8, CTSG, CUA001, CX3CL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL16, CXCL3, CXCL5, CXCL6, CXCL8, CXCL9, CXCR2, CXCR3, CXCR5, CXXC5, CXXC5, cyclone CX2 precusor, cytokine receptor CRL2 precursor, cytokine receptor-like factor 2 variant, cytokine SDF-1-beta, cytokine type 1 receptor CRLP-1 precursor, cytokine-inducible inhibitor of signaling type 1b, cytokine-inducible SH2-containing protein, cytok ine-like nuclear factor n-pac, cytokine-like nuclear factor n-pac-like protein, cytokine-like protein 2-21, cytokine-like protein EF-7, CYTOR4, cytotoxic lymphocyte maturation factor 40 kDa subunit, DAGLA, DCC , DCSTAMP, DDT, DEFB103B, delta 4-delta 7/11 truncated prolactin receptor, delta 4-SF1b truncated prolactin receptor, dendritic cell associated lectin 2, density enhanced phosphatase-1, DHX58, DIP2C, DNAH3, DNAH9, Down syndrome cell adhesion molecule isoform CHD2-42 precursor variant, DRT, DSCAM, DSCAM2, DSCAML1, DTK, DTL, DUB3, DVL2, EBI3, ECK, EDIL3, eEF1A2 binding protein, EFNA5, EGFLAM, EGR4, ELAC1, ELAVL1, ELAVL3, ELP2, endothelial- monocyte activating polypeptide II, EOMES, eotaxin precursor, EPGN, EPHA1, EPHA10, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHB1, EPHB2, EPHB2 variant protein, EPHB3, EPHB4, EPHB6, EphB6 variant protein, ephrin6 EphA5 isoform a variant, ephrin receptor EphA7 variant, ephrin receptor EphB4 precursor variant, EPO, EPOR, ERK, ESOP1, F ABP5, factor for adipocyte differentiation 104 variant, fad104, FAM19A4, FAM213A, FAM3A, FAM3B, FAM3C, FAM3D, FANK1, FBRS, FCAR, FCER1A, FCER1G, FCGR2C, FCN3, FES, FGF19, FGF9, FGF-9, fibronectin 1 variant, FIL1-theta, FILIP1L, FLJ00133, FLJ00148, FLJ00154, FLJ00236, FLJ00376, FLT3, FLT3LG, FN1, FNDC1, FNDC3A, FNDC3B, FNDC4, FNDC5, FNDC7, FNDC8, FOSB, FOS3, FOSB1, XP , FSD2, FSTL3, FUCA2, G0S3, G18, G-26, GAB2, GAB3, gamma.1, GBP4, G-CSFR-1, G-CSFR-2, GDF15, GDF2, GHR, GJA1, GLEPP1, GLYR1, GM -CSF receptor beta chain, gp130, gp130-like monocyte receptor, gp250 precursor, GPI, GPR15, GPR75-ASB3, GPSM1, GRAIL, granulocyte colony-stimulating factor receptor, granulocyte macrophage-colony stimulating factor, granulocyte-macrophage colony stimulating factor 2 , granulocyte-macrophage colony-stimulating factor precusor, GRAP2, GRO-beta, GRO-gamma, growth-inhibiting protein 45, GZMK, HAVCR2, HAX1, HBD, HCF-2, HCFC1, HCFC2, HCV NS5A-binding protein NS5ABP37, HDAC11 , hDDM36, HEK, HEK11, HEK5, HEK7, HEK8, hematopoietic stem / progenitor cell induced protein 1, hematopoietic stem / progenitor cell induced protein 2, hematopoietic stem / progenitor cell induced protein 3, HES4, HGF, HGS, HIBDL, hIL-2Rg , HILPDA, HIN-1 putative cytokine, HMGB1, hmrp-2a, hmrp-2b, hNB-2, hNB-2s, hNB-3, host cell factor C2 variant, HOXB5, hp40, HSD13, hSHIP, HSP90B1, HSSOCS-2 , HTK, HTPZP2, hTroy, Humig, HXB, hypothetical protein, ICOSLG, IFI16, IFI30, IFNA1, IFNA10, IFNA17, IFNA2, IFNA4, IFNA8, IFNAR1, IFNAR2, IFNB1, IFNG, IFNGR1, IFNGR2, IFNK, IFNL1 IFNL3, IFNLR1, IFNW1, IGANRP, IGDCC3, IGDCC4, IGF1R, IGFN1, IGSF22, IGSF9, IGSF9B, IK, IKBKB, IKK alpha, Interleukins, IL10, IL10RB, IL11, IL11RA, Il-12 receptor component beta2, IL12 , IL12B, IL12RB1, IL12RB2, IL13, IL-13Ra, IL13RA1, IL13RA2, IL15, IL15RA, IL16, IL-17, IL-17 receptor, IL17A, IL17B, IL17BR, IL17C, IL17D, IL-17D, IL17D precursor, IL17E , IL17F, IL17RA, IL17RB, IL17RC, IL-17RC, IL-17RD, IL-17RE, IL18, IL-18 receptor beta splice variant, IL18BP, IL18R1, IL18RAP, IL19, IL1A, IL1B, IL1BCE, IL1F10, IL1F7, IL1L1, IL1R1, IL1R2, IL1RAP, IL1RAPL1, IL1RAPL2, IL1RL1, IL1RL2, IL1RN2, IL1RN2 IL20, IL20RA, IL20RB, IL21, IL-21, IL21R, IL22, IL22BP, IL22R, IL22RA1, IL22RA2, IL23A, IL23R, IL-23R, IL-24 splice variant delE3, IL-24 splice variant delE5, IL25, IL26, IL27, IL27RA, IL28A, IL28B, IL28C, IL28RA, IL29, IL30, IL2RA, IL2RB, IL2RG, IL3, IL31, IL31RA, IL32, IL33, IL34, IL36A, IL36B, IL36G, IL36RN, IL37, IL3RA, IL4, IL4I1, IL4R, IL5, IL5RA, IL6, IL6R, IL6ST, IL7, IL7R, IL9, IL9R, il-xr, ING3, INPP5D, INSR, INSRR, insulin receptor variant, insulin responsive sequence DNA binding protein-1, integrin beta 4 isoform 3 precursor variant, interferon alpha 2b, interferon gamma, interferon-lambda1, interferon-lambda2, interferon-lambda3, interleukin 10 receptor, beta, interleukin 11 receptor, alpha, interleukin 12, P40, interleukin 13, interleukin 13 receptor, alpha 1 precursor variant , interl eukin 17A, interleukin 17B, interleukin 17C, interleukin 18 precursor, interleukin 23 p19 subunit, interleukin 23 receptor isoform 1, interleukin 32 variant, interleukin 34 isoform 1 precursor, interleukin 34 isoform 2 precursor, interleukin 4 receptor alpha chain isoform a precursor variant, interleukin 6 receptor isoform 1 precursor variant, interleukin-1 homolog 1, interleukin-1 homolog 2, interleukin-1 homolog 4, interleukin-11 receptor alpha chain, interleukin-13 receptor, interleukin-17 beta, interleukin-1-related protein long isoform, interleukin-1-related protein long isoform a, interleukin-1-related protein short isoform, interleukin-2, interleukin-32 small, interleukin-32 theta, interleukin-33, intermediate prolactin receptor isoform, IRAK1, IRAK3, IREB2 variant , IRF9, IRS2, ISG15, ITGA1, ITGAL, ITGB2, ITGB4, ITPK1, JAB, JAK2, JAK3, JAKMIP1, JOSD2, KAL, KAL1, KALIG-1, KALRN, KAT6B, KCNA3, KCNJ12, KIAA0273, KIA0282, KIAA0282 , KIAA0343, KIAA0671, KIAA0756, KIAA0970, KIAA1030, KIAA1132, KIAA1146, KIAA1297, KIAA1355, KIAA1397, KIAA1459, KIAA1496, KIAA1497, KIAA1510, KIAA1514, KIAA1568, KIAA1628, KIAA1666, KIAA1866, KLF8, KLRA, LLRi , LICR2, LIF, LIFR, LILRA2, LIMK2, LOC57019, LOC728835, long myosin light chain kinase, LRG1, LRRC20, LRRC70, LRRFIP2, LRRN1, LRRN4, LTA, LTB, LTBP1, LTBP2, lymphokine, lymphotoxin beta isoform variant acyltransferase-alpha, lysophosphatidic acid acyltransferase-beta, macrophage colony-stimulating factor 1, MAF, magic roundabout, MAPK14, MCP3, MCP-3, MCP4, MCP-4, MDK, MEP1B, MERTK, MEX3C, MGDF, MIC-1, microtubule-associated protein GLFND, MIP-1 delta, MIP1a, MIP2a, mitochondria associated granulocyte macrophage CSF signaling molecule Magmas, ML1, MLCK, MMP9, MNAT1, MON1B, MOV10, MPIF-1, MPL, MRPL17, MSLN, MTHFD2L, MTUS1, MUSK, MYBPC1, MYBPC2, MYBPC3, MYBPH, MYBPHL, MYDGF, myeloproliferative leukemia virus oncogene, MYLK, MYOM1, MYOM2, MYOM3, N AA35, NAMPT, NC28, NCAM, N-CAM, NCAM1, NCAM2, NCAM21, Nck, Ash and phospholipase C gamma-binding protein NAP4, NCR1, NCR3, NCR3LG1, NCSTN, NDFIP1, NDUFA2, NDUFB6, NEO1, neogenin homo 1 variant, neshbp, NET PTK, Neural cell adhesion molecule 1, 120 kDa isoform precursor variant, neural cell adhesion molecule CD56, Neural cell adhesion molecule variant, neurotrophin-1 / B-cell stimulating factor-3, NFAM1, NFASC, NFAT1, NFATC1, NFATC4, NHLH1, NILR, NIPSNAP1, NIPSNAP2, NKSF1, NKSF2, NLRC5, NLRP11, NLRP12, NLRP13, NLRP2, NLRP4, NLRP6, NLRP7, NLRR-1, NOX5, N-PAC, NR2CNR OC-116KDa, OCLN, OPTC, OR2H1, OSM, OSMR, OSMRB, osteoprotegerin ligand, OTUB1, OTUD6B, OTUD7B, P2RY8 / CRLF2 fusion, p48, PANX2, PARC, PDCD1LG2, PGLYRP1, PHF11, PHIPLIP, PHIPLIP PIBF1, PIK3R3, PLA2G2D, PLAC8, pLD78 peptide, PLK3, PLTP, PLXNC1, POMGNT2, PP14212, PPBP, PPIA, PPM1F, PPM1H, PPP2R5A, PPP3CC, PPP5C, PPY, PREX1, prk, PRLR, PRO09 receptor protin15 delt a 7/11, prolactin receptor isoform delta S1 precursor, proliferation associated cytokine-inducible protein CIP29, protein tyrosine phosphatase delta, protein tyrosine phosphatase sigma, protein tyrosine phosphatase, receptor type, D isoform 4 precursor variant, protein tyrosine phosphatase, receptor type, F isoform 2 precursor variant, protein tyrosine phosphatase, receptor type, G precursor variant, protein tyrosine phosphatase, receptor type, K precursor variant, protein tyrosine phosphatase, receptor type, sigma isoform 3 precursor variant, protein-tyrosine phosphatase, PRR16, PRSS27, PRTG, PSMA4, PTCHD1, PTGDR, PTGES, PTN, PTPRB, PTPRC, PTPRD, PTPRE, PTPRF, PTPRG, PTPRH, PTPRJ, PTPRK, PTPRM, PTPRO, PTPRQ, PTPRS, PTPRT, PTPRU, PTPRZ, PTPRZ1, PTPsigma, PTPsigma PUS7L, putative cytokine receptor CRL4 precusor, PYPAF6, PYRIN-containing Apaf1-like protein 2, PYRIN-containing Apaf1-like protein 3, PYRIN-containing APAF1-like protein 4, PYRIN-containing APAF1-like protein 5, PYRIN-containing APAF1-like protein 7, PYY, RAB40A, RAB40AL, RAB40B, RAB40B, member RAS oncogene family variant, RAB40C, RAB6B, RANTES precursor, Rar protein, Rar-2 protein, RBM15, RBM5 variant, RCAN3, receptor protein tyrosine phosphatase hPTP- J precursor, Receptor protein tyrosine phosphatase hPTP-J precursor variant, receptor tyrosine kinase, receptor-type protein tyrosine phosphatase O isoform a precursor variant, RGS2, RIMBP2, RIMBP3, RIMBP3B, RIMBP3C, RIPK4, RMC1, RNASE10, RNASE7, RNASE9, RNASE9, RNASE9, RNASE9 , RNF113A, RNF128, RNF41, ROBO1, ROBO2, ROBO2 isoform a, ROBO2 isoform b, ROBO3, ROBO4, RORC, ROS1, RPL27, RPL36, RPS21, R-PTP-kappa, RPTP-rho, rse, RSL24D1, SA8A12, S100A12 , S100A9, S1PR4, SAC3D1, SAP / SH2D1A, SAP-1, SARNP, SASH1, SCAMP5, SCGB3A1, SCGB3A2, SCM-1, SCYA13, SCYA16, SCYA26, SCYE1, SDC4, SDC4-ROS1_S2, R32, SDC4-ROS1_S4, R32 , SDK1, SDK2, secreted osteoclastogenic factor of activated T cells, SELE, SEMA7A, SENP3, SERPINB9, serum / glucocorticoid regulated kinase-like isoform 1 variant, SETD1B, SGK3 , SGK-like protein SGKL, SH2 domain containing SOCS box protein SOCS4, SH2B1, SH2B3, shen-dan, SIGIRR, SIGLEC14, Similar to RIKEN cDNA 3930401K13 gene, SKIP1, SKIP3, sky, SLAMF9, SLC2A5, SLC2A9, SLC34A2 / ROS , SLC34A2-ROS1, SLC39A13, SLC39A3, SLC4A3, SLC9A8, SLC9B2, SLURP1, small cytokine B subfamily member 11 SCYB11 precursor, SMIM27, SMPD2, SNED1, SNX10, SOCS box containing protein RAR2A, SOCS box protein ASB-5, SOCS1 -1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, SOCS7, SORL1, SPATA2, SPOCK3, SPP1, SPPL2B, SPRY domain-containing SOCS box protein SSB-1, SPRY domain-containing SOCS box protein SSB-1 variant, SPRY domain -containing SOCS box protein SSB-2, SPRY domain-containing SOCS box protein SSB-3, SPRY domain-containing SOCS box protein SSB-4, SPSB1, SPSB2, SPSB3, SPSB4, STAM, STAM2, STAMBP, STAT induced STAT inhibitor- 1, STAT induced STAT inhibitor-2, STAT induced STAT inhibitor-3, STAT1, STAT5A, STAT5B, STAT-induced STAT inhibitor-2, stem cell factor, STIMATE, STK35, STK40, str iated muscle preferentially expressed protein, suppressor of cytokine signaling 3, surface glycoprotein, Ig superfamily member variant, SUSD2, SWIP1, SYNJ2BP, synleurin, TACC2, TAIF, TARM1, TBX21, TBX5, TCEB1, TCIRG1, TEC, TEK, TEK tyrosine kinase variant , tenascin XB isoform 1 variant, tenascin-C isoform 14 / AD1 / 16, testicular tissue protein Li 126, testicular tissue protein Li 64, testis tissue sperm-binding protein Li 57p, TEX12, TFPI, THEMIS2, THNSL2, THPO, thymic stromal lymphopoietin protein receptor TSLPR, thymic stromal lymphopoietin protein TSLP, TIE, tie receptor tyrosine kinase, TIE1, Tie-2, TIGIT, TIMP1, tip3, TIRAP, Titin, TLR1, TLR3, TLR6, TM7SF3, TMEM102, TMEM110-MUSTN1, TMSB4 TMX2, TNC, TNC variant protein, TNF, TNFAIP3, TNFAIP8L2, TNFRSF11B, TNFRSF12A, TNFRSF13C, TNFRSF21, TNFRSF25, TNFRSF4, TNFRSF8, TNFSF10, TNFSF11, TNFSF13, TNFSF13, TNFSF13, TNFSF13 TNN, TNR, TNXB, TOPORS, Trad, TRAF6, TRIM21, TRIM3, TRIM42, TRIM46, TRIM67, T RIM8, TRIM9, TRPM4, TSLP, TTN, TULP4, tumor necrosis factor receptor, TUSP, TUT4, TUT7, TWIST2, TYK2, type VII collagen, type XII collagen, type-I T cell cytokine receptor, TYRO3, tyrosyl-tRNA synthetase, tyrosyl-tRNA synthetase variant, U2AF1L4, UBA6, UBAP1, ubiquitin ligase E3 alpha-I, ubiquitin ligase E3 alpha-II, UBR2, UFO, undulin 1, undulin 2, UNQ1942, UNQ2421, UNQ288, UNQ296, UNQ3121, UN933 UNQ604, UNQ6309, UNQ6368, UNQ6389, UNQ6504, UNQ693, UQCRH, USH2A, USMG5, USP15, USP17L10, USP17L11, USP17L12, USP17L13, USP17L15, USP17L17, USP17L18, USP17L20, USP17L19, USP17L20 USP17L27, USP17L28, USP17L29, USP17L3, USP17L30, USP17L4, USP17L5, USP17L7, USP17L8, USP18, USP36, UTY, V alpha 1, V alpha 13, VCAM1, VESPR, VNN2, VSIG2, VWA1, WARS protein, WD isoform 1 variant, WDR26, WSB1, WSB-1, WSB-1 isoform, WSB-1 protein, WSB2, WSB-2, WSX1, WWOX, XA, XB, XBP1, XCL1, XCL2, XKR4, YARS, ZBED3, ZBP1, ZC3H12D, ZC3H15, ZCYTO10, At least one selected from the group consisting of zcytor5, ZCYTOR7, ZFP36, ZNF366, ZNF580, and ZNF827, but is not necessarily limited thereto.

상기 항체는 구체적으로, 인터류킨(interleukins) 또는 인터페론(interferons) 단백질에 특이적으로 결합하는 항체일 수 있다.Specifically, the antibody may be an antibody that specifically binds to an interleukins or interferons protein.

상기 항체는 IgG; 또는 PD-1, CTLA-4, TIM-3, BTLA, VISTA, LAG-3, PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD27, CD137, HVEM, GITR, VEGFR, VEGF, EGFR, EGF, IL-1, IL-6, IL-23, TGF-beta, CTGF, TSLP, TNF-alpha, Notch 및 OX40으로 이루어진 군에서 선택된 하나의 적어도 하나의 단백질에 특이적으로 결합하는 항체일 수 있으나, 생물학적 관점에서 통상의 항체로 인식되는 것이라면 반드시 이에 제한되지 않는다.The antibody is an IgG; Or PD-1, CTLA-4, TIM-3, BTLA, VISTA, LAG-3, PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD27, CD137, HVEM, GITR, VEGFR, VEGF Antibodies that specifically bind to at least one protein selected from the group consisting of EGFR, EGF, IL-1, IL-6, IL-23, TGF-beta, CTGF, TSLP, TNF-alpha, Notch, and OX40 But, if it is recognized as a conventional antibody from a biological point of view is not necessarily limited thereto.

상기 다공성 실리카 입자는 실리카(SiO 2) 소재의 입자이며, 나노 사이즈의 입경을 갖는다.The porous silica particles are particles of silica (SiO 2 ) material and have a particle size of nano size.

본 발명의 다공성 실리카 나노입자는 다공성 입자로서, 나노사이즈의 기공을 갖고, 그 표면 및/또는 기공 내부에 항체(antibody) 또는 사이토카인(cytokine)을 담지할 수 있다.Porous silica nanoparticles of the present invention is a porous particle, having nano-sized pores, and can carry an antibody or cytokine on its surface and / or inside the pores.

본 발명의 다공성 실리카 입자는 생분해성 입자로서, 항체(antibody) 또는 사이토카인(cytokine)을 담지하여 체내에 투여되었을 때 체내에서 생분해되면서 항체(antibody) 또는 사이토카인(cytokine)을 방출할 수 있는데, 본 발명의 다공성 실리카 입자는 체내에서 서서히 분해되어 담지된 항체(antibody) 또는 사이토카인(cytokine)이 서방적으로 방출되도록 할 수 있다. 예를 들면, 하기 수학식 1의 흡광도의 비가 1/2이 되는 t가 24 이상이다:Porous silica particles of the present invention, as a biodegradable particle, can carry an antibody or cytokine and release the antibody or cytokine while being biodegraded in the body when administered to the body. Porous silica particles of the present invention can be slowly degraded in the body to allow the sustained release of the supported antibody (cytokine) or cytokine (cytokine). For example, t, which is the ratio of the absorbance of the following formula 1 to 1/2, is 24 or more:

[수학식 1][Equation 1]

A t/A 0 A t / A 0

(식 중, A 0는 상기 다공성 실리카 입자 1mg/ml 현탁액 5ml를 직경 50kDa의 기공을 갖는 원통형 투과막에 넣고 측정된 다공성 실리카 입자의 흡광도이고,Wherein A 0 is the absorbance of the porous silica particles measured by placing 5 ml of the 1 mg / ml suspension of the porous silica particles into a cylindrical permeable membrane having pores having a diameter of 50 kDa,

상기 투과막 외부에는 상기 투과막과 접하며, 상기 현탁액과 동일한 용매 15ml가 위치하고, 상기 투과막 내외부는 37℃에서 60rpm 수평 교반되며,Outside of the permeable membrane is in contact with the permeable membrane, 15ml of the same solvent as the suspension is located, the inside and outside of the permeable membrane is stirred 60 rpm at 37 ℃ horizontal,

상기 현탁액의 pH는 7.4이고,The pH of the suspension is 7.4,

A t는 상기 A 0의 측정시로부터 t시간 경과 후에 측정된 다공성 실리카 입자의 흡광도임).A t is the absorbance of the porous silica particles measured after t hours have elapsed since the measurement of A 0 ).

상기 수학식 1은 다공성 실리카 입자가 체내와 유사한 환경에서 어느 정도의 속도로 분해되는지를 의미하는 것이다.Equation 1 means that the rate at which the porous silica particles are degraded in an environment similar to the body.

상기 수학식 1에서의 흡광도 A 0, A t는 예를 들면 도 34에 예시된 바와 같이, 원통형 투과막에 다공성 실리카 입자 및 현탁액을 넣고, 투과막 외부에도 동일한 현탁액을 넣고 측정된 것일 수 있다.Absorbance A 0 , A t in Equation 1 may be measured by putting porous silica particles and a suspension in a cylindrical permeable membrane and putting the same suspension outside the permeable membrane, as illustrated in FIG. 34, for example.

본 발명의 다공성 실리카 입자는 생분해성으로서, 현탁액 내에서 서서히 분해될 수 있고, 직경 50kDa는 약 5nm에 해당하는 것으로서 생분해된 다공성 실리카 입자는 직경 50kDa의 투과막을 통과할 수 있고, 원통형 투과막은 60rpm 수평 교반 하에 있으므로 현탁액이 고루 섞일 수 있으며 분해된 다공성 실리카 입자는 투과막 외부로 나올 수 있다.The porous silica particles of the present invention are biodegradable, and can be slowly decomposed in suspension, 50 kDa in diameter corresponds to about 5 nm, and biodegradable porous silica particles can pass through a permeable membrane of 50 kDa in diameter, and a cylindrical permeable membrane is 60 rpm horizontal. Under stirring, the suspension can be mixed evenly and the degraded porous silica particles can come out of the permeable membrane.

상기 수학식 1에서의 흡광도는 예를 들어 투과막 외부의 현탁액이 새로운 현탁액으로 교체되는 환경 하에 측정된 것일 수 있다. 현탁액은 지속적으로 교체되는 것일 수 있고, 일정 기간마다 교체되는 것일 수 있으며, 상기 일정 기간은 정기 또는 비정기적인 기간일 수 있다. 예를 들어 1시간 내지 1주일의 범위 내에서, 1시간 간격, 2시간 간격, 3시간 간격, 6시간 간격, 12시간 간격, 24시간 간격, 2일 간격, 3일 간격, 4일 간격, 7일 간격 등으로 교체될 수 있으나 이에 제한되는 것은 아니다The absorbance in Equation 1 may be measured, for example, under an environment in which the suspension outside the permeable membrane is replaced with a new suspension. The suspension can be one that is constantly replaced, one that can be replaced every period, and the period can be periodic or irregular. For example, within the range of 1 hour to 1 week, 1 hour interval, 2 hours interval, 3 hours interval, 6 hours interval, 12 hours interval, 24 hours interval, 2 days interval, 3 days interval, 4 days interval, 7 It may be replaced at day intervals, but is not limited thereto.

상기 흡광도의 비가 1/2가 된다는 것은 t시간 후에 흡광도가 초기 흡광도의 절반이 된다는 것인 바, 이는 다공성 실리카 입자의 대략 절반이 분해되었다는 의미이다.The ratio of the absorbance to 1/2 means that the absorbance is half of the initial absorbance after t hours, which means that approximately half of the porous silica particles are decomposed.

상기 현탁액은 완충용액일 수 있고, 구체적인 예를 들면, PBS(phosphate buffered saline) 및 SBF(simulated body fluid)로 이루어진 군에서 선택된 1종 이상일 수 있으며, 보다 구체적으로는 PBS일 수 있다.The suspension may be a buffer solution, for example, at least one selected from the group consisting of phosphate buffered saline (PBS) and simulated body fluid (SBF), and more specifically, PBS.

본 발명의 상기 수학식 1의 흡광도의 비가 1/2이 되는 t가 24 이상으로, 예를 들면 t는 24 내지 120일 수 있고, 예를 들어 상기 범위 내에서 24 내지 96, 24 내지 72, 30 내지 70, 40 내지 70, 50 내지 65 등일 수 있으나, 이에 제한되는 것은 아니다.T of the absorbance ratio of Equation 1 of the present invention is 1/2 or more, for example, t may be 24 to 120, for example, 24 to 96, 24 to 72, 30 within the above range To 70, 40 to 70, 50 to 65 and the like, but is not limited thereto.

본 발명의 다공성 실리카 입자는 상기 수학식 1의 흡광도의 비가 1/5가 되는 t가 예를 들면 70 내지 140일 수 있고, 예를 들어 상기 범위 내에서 80 내지 140, 80 내지 120, 80 내지 110, 70 내지 140, 70 내지 120, 70 내지 110 등일 수 있으나, 이에 제한되는 것은 아니다.In the porous silica particles of the present invention, t, for example, the absorbance ratio of Equation 1 is 1/5 may be, for example, 70 to 140, for example, 80 to 140, 80 to 120, and 80 to 110 within the above range. , 70 to 140, 70 to 120, 70 to 110, and the like, but is not limited thereto.

본 발명의 다공성 실리카 입자는 상기 수학식 1의 흡광도의 비가 1/20가 되는 t가 예를 들면 130 내지 220일 수 있고, 예를 들어 상기 범위 내에서 130 내지 200, 140 내지 200, 140 내지 180, 150 내지 180 등일 수 있으나, 이에 제한되는 것은 아니다.In the porous silica particles of the present invention, t may be 130 to 220, for example, wherein the ratio of absorbance of Equation 1 is 1/20, for example, 130 to 200, 140 to 200, 140 to 180 within the above range. , 150 to 180, and the like, but is not limited thereto.

본 발명의 다공성 실리카 입자는 측정되는 흡광도가 0.01 이하가 되는 t가 예를 들면 250 이상, 예를 들어, 300 이상, 350 이상, 400 이상, 500 이상, 1000 이상 등일 수 있으며, 그 상한은 2000일 수 있으나, 이에 제한되는 것은 아니다.The porous silica particles of the present invention may have a measured absorbance of 0.01 or less, for example, 250 or more, for example, 300 or more, 350 or more, 400 or more, 500 or more, 1000 or more, and the upper limit thereof is 2000 days. May be, but is not limited thereto.

본 발명의 다공성 실리카 입자에서 상기 수학식 1의 흡광도의 비와 t는 높은 양의 상관 관계를 갖는 것으로서, 예를 들면 피어슨 상관 계수가 0.8 이상일 수 있고, 예를 들어, 0.9 이상, 0.95 이상일 수 있다.In the porous silica particles of the present invention, the ratio of the absorbance of Formula 1 and t have a high positive correlation. For example, the Pearson correlation coefficient may be 0.8 or more, for example, 0.9 or more and 0.95 or more. .

상기 수학식 1의 t는 다공성 실리카 입자가 체내와 유사한 환경에서 어느 정도의 속도로 분해되는지를 의미하는 것으로서, 이는 예를 들면 다공성 실리카 입자의 표면적, 입경, 기공 직경, 표면 및/또는 기공 내부의 치환기, 표면의 치밀함 정도 등을 조절함으로써 조절될 수 있다.T in Equation 1 means how fast the porous silica particles decompose in an environment similar to the body, for example, the surface area, particle diameter, pore diameter, surface and / or inside the pores of the porous silica particles. It can be controlled by controlling the substituent, the degree of compactness of the surface, and the like.

예를 들면, 입자의 표면적을 증가시켜 t를 감소시키거나, 표면적을 감소시켜 t를 증가시킬 수 있다. 표면적은 입자의 직경, 기공의 직경을 조절함으로써 조절될 수 있다. 또한, 표면 및/또는 기공 내부에 치환기를 위치시켜 다공성 실리카 입자가 환경(용매 등)에 직접 노출되는 것을 줄여 t를 증가시킬 수 있다. 또한, 다공성 실리카 입자에 항체(antibody) 또는 사이토카인(cytokine)을 담지시키고 항체(antibody) 또는 사이토카인(cytokine)과 다공성 실리카 입자 간의 친화도를 증가시켜, 다공성 실리카 실리카 입자가 환경에 직접 노출되는 것을 줄여 t를 증가시킬 수 있다. 또한, 입자의 제조시에 표면을 보다 치밀하게 제조하여 t를 증가시킬 수도 있다. 상기에는 수학식 1의 t를 조절할 수 있는 다양한 예시를 서술하였으나, 이에 제한되는 것은 아니다.For example, the surface area of the particles can be increased to reduce t, or the surface area can be reduced to increase t. The surface area can be adjusted by adjusting the diameter of the particles and the diameter of the pores. In addition, by placing substituents on the surface and / or within the pores, it is possible to increase t by reducing the direct exposure of porous silica particles to the environment (such as solvents). In addition, by supporting an antibody or cytokine on the porous silica particles and increasing the affinity between the antibody or cytokine and the porous silica particles, the porous silica silica particles are directly exposed to the environment. To increase t. In addition, the surface may be made more densely at the time of preparation of the particles to increase t. In the above, various examples of adjusting t in Equation 1 have been described, but are not limited thereto.

본 발명의 다공성 실리카 입자는 예를 들면 구형 입자일 수 있으나, 이에 제한되는 것은 아니다.Porous silica particles of the present invention may be, for example, spherical particles, but is not limited thereto.

본 발명의 다공성 실리카 입자는 평균 직경이 예를 들면 150nm 내지 1000nm일 수 있고, 예를 들어 상기 범위 내에서 예를 들면 150nm 내지 800nm, 150nm 내지 500nm, 150nm 내지 400nm, 150nm 내지 300nm, 150nm 내지 200nm일 수 있으나, 이에 제한되는 것은 아니다.The porous silica particles of the present invention may have an average diameter of, for example, 150 nm to 1000 nm, for example, within the above range, for example, 150 nm to 800 nm, 150 nm to 500 nm, 150 nm to 400 nm, 150 nm to 300 nm, and 150 nm to 200 nm. May be, but is not limited thereto.

본 발명의 다공성 실리카 입자는 평균 기공 직경이 예를 들면 1nm 내지 100nm일 수 있고, 예를 들어 상기 범위 내에서 예를 들면 5nm 내지 100nm, 7nm 내지 100nm, 7nm 내지 50nm, 10nm 내지 50nm, 10nm 내지 30nm, 7nm 내지 30nm일 수 있으나, 이에 제한되는 것은 아니다. 상기와 같은 큰 직경을 가져 다량의 항체(antibody) 또는 사이토카인(cytokine)을 담지할 수 있고, 크기가 큰 항체(antibody) 또는 사이토카인(cytokine)의 담지도 가능하다.The porous silica particles of the present invention may have an average pore diameter of, for example, 1 nm to 100 nm, for example, within the above range, for example, 5 nm to 100 nm, 7 nm to 100 nm, 7 nm to 50 nm, 10 nm to 50 nm, 10 nm to 30 nm. , 7 nm to 30 nm, but is not limited thereto. Having such a large diameter can carry a large amount of antibody (cytokine) or cytokine (cytokine), it is also possible to carry a large antibody (cytokine) or cytokine (cytokine).

본 발명의 다공성 실리카 입자는 BET 표면적이 예를 들면 200m 2/g 내지 700m 2/g일 수 있다. 예를 들어 상기 범위 내에서 200m 2/g 내지 700m 2/g, 200m 2/g 내지 650m 2/g, 250m 2/g 내지 650m 2/g, 300m 2/g 내지 700m 2/g, 300m 2/g 내지 650m 2/g, 300m 2/g 내지 600m 2/g, 300m 2/g 내지 550m 2/g, 300m 2/g 내지 500m 2/g, 300m 2/g 내지 450m 2/g 등일 수 있으나, 이에 제한되는 것은 아니다.The porous silica particles of the present invention may have a BET surface area of, for example, 200 m 2 / g to 700 m 2 / g. For example, within the above range 200 m 2 / g to 700 m 2 / g, 200 m 2 / g to 650 m 2 / g, 250 m 2 / g to 650 m 2 / g, 300 m 2 / g to 700 m 2 / g, 300 m 2 / g to 650m 2 / g, 300m 2 / g to 600m 2 / g, 300m 2 / g to 550m 2 / g, 300m 2 / g to 500m 2 / g, 300m 2 / g to 450m 2 / g, etc. It is not limited to this.

본 발명의 다공성 실리카 나노입자는 g당 부피가 예를 들면 0.7ml 내지 2.2ml일 수 있다. 예를 들어 상기 범위 내에서 0.7ml 내지 2.0ml, 0.8ml 내지 2.2ml, 0,8 ml 내지 2.0ml, 0.9 ml 내지 2.0ml, 1.0 ml 내지 2.0ml 등일 수 있으나, 이에 제한되는 것은 아니다. g당 부피가 과도하게 작아지면 분해 속도가 너무 빨라질 수 있고, 과도하게 큰 입자는 제조가 어렵거나, 온전한 형상을 가질 수 없을 수 있다.The porous silica nanoparticles of the present invention may have a volume per g, for example, 0.7 ml to 2.2 ml. For example, within the above range may be 0.7ml to 2.0ml, 0.8ml to 2.2ml, 0,8ml to 2.0ml, 0.9ml to 2.0ml, 1.0ml to 2.0ml and the like, but is not limited thereto. If the volume per gram is too small, the rate of decomposition may be too high, and excessively large particles may be difficult to manufacture or may not have an intact shape.

본 발명의 다공성 실리카 입자는 외부 표면 및/또는 기공 내부에 친수성 치환기 및/또는 소수성 치환기가 존재할 수 있다. 예를 들면 표면 및 기공 내부 모두 친수성 치환기만 존재하거나, 소수성 치환기만 존재할 수도 있고, 표면 또는 기공 내부에만 친수성 치환기가 존재하거나, 소수성 치환기가 존재할 수도 있고, 표면에는 친수성 치환기, 기공 내부에는 소수성 치환기가 존재할 수도 있고, 그 반대의 경우도 가능하다.The porous silica particles of the present invention may have hydrophilic substituents and / or hydrophobic substituents on the outer surface and / or inside the pores. For example, only hydrophilic substituents may exist on both the surface and inside of the pores, or only hydrophobic substituents may exist, hydrophilic substituents may exist on the surface or inside of the pores, hydrophobic substituents may exist on the surface, hydrophilic substituents on the surface, and hydrophobic substituents inside the pores. It may be present and vice versa.

본 발명의 다공성 실리카 입자에 담지된 항체(antibody) 또는 사이토카인(cytokine)의 방출은 주로 나노입자의 분해에 의해 수행되는 것인 바, 상기 치환기의 조절로 항체(antibody) 또는 사이토카인(cytokine)의 방출 환경에 대한 다공성 실리카 입자의 상호 작용이 조절되어 나노입자 자체의 분해 속도가 조절되어 항체(antibody) 또는 사이토카인(cytokine)의 방출 속도가 조절될 수 있고, 또한, 항체(antibody) 또는 사이토카인(cytokine)은 나노입자로부터 확산되어 방출될 수도 있는데, 상기 치환기의 조절로 항체(antibody) 또는 사이토카인(cytokine)의 나노입자에 대한 결합력이 조절되어 항체(antibody) 또는 사이토카인(cytokine)의 방출이 조절될 수 있다.The release of an antibody or cytokine supported on the porous silica particles of the present invention is mainly carried out by decomposition of the nanoparticles, and the antibody or cytokine is controlled by the control of the substituent. The interaction of the porous silica particles with respect to the release environment of is controlled to control the rate of degradation of the nanoparticles themselves, thereby controlling the release rate of the antibody or cytokine, and also the antibody or cytokine. The cytokine may be diffused from the nanoparticles to be released, and the binding force of the antibody or cytokine to the nanoparticles is controlled by the control of the substituents, thereby controlling the antibody or cytokine. Release can be controlled.

또한, 난용성(소수성) 항체(antibody), 사이토카인(cytokine) 또는 물질과의 결합력 증진을 위해 기공 내부에는 소수성 치환기가 존재하고, 사용, 제형화의 용이성 등의 측면에서 입자의 표면은 친수성 치환기가 존재하도록 하는 등의 처리도 가능하다.In addition, hydrophobic substituents are present inside the pores to enhance binding to poorly soluble (hydrophobic) antibodies, cytokines, or substances, and the surface of the particles is characterized by hydrophilic substituents in terms of ease of use and formulation. May be present.

친수성 치환기는 예를 들면 히드록시기, 카르복시기, 아미노기, 카르보닐기, 설프히드릴기, 포스페이트기, 티올기, 암모늄기, 에스터기, 이미드기, 티오이미드기, 케토기, 에터기, 인덴기, 설포닐기, 폴리에틸렌글리콜기 등을 들 수 있고, 소수성 치환기는 예를 들면 치환 또는 비치환된 C1 내지 C30의 알킬기, 치환 또는 비치환된 C3 내지 C30의 사이클로알킬기, 치환 또는 비치환된 C6 내지 C30의 아릴기, 치환 또는 비치환된 C2 내지 C30의 헤테로아릴기, 할로겐기, C1 내지 C30의 에스테르기, 및 할로겐 함유기 등을 들 수 있다.Hydrophilic substituents are, for example, hydroxyl groups, carboxy groups, amino groups, carbonyl groups, sulfhydryl groups, phosphate groups, thiol groups, ammonium groups, ester groups, imide groups, thiimide groups, keto groups, ether groups, indene groups, sulfonyl groups, polyethylene Glycol groups and the like, and the hydrophobic substituent is, for example, substituted or unsubstituted C1 to C30 alkyl group, substituted or unsubstituted C3 to C30 cycloalkyl group, substituted or unsubstituted C6 to C30 aryl group, substituted Or an unsubstituted C2 to C30 heteroaryl group, a halogen group, a C1 to C30 ester group, a halogen-containing group, and the like.

또한, 본 발명의 다공성 실리카 입자는 외부 표면 및/또는 기공 내부가 양전하 및/또는 음전하로 대전된 것일 수 있다. 예를 들면 표면 및 기공 내부 모두 양전하로 대전되거나, 음전하로 대전될 수 있고, 표면 또는 기공 내부만 양전하로 대전되거나, 음전하로 대전될 수 있고, 표면은 양전하, 기공 내부는 음전하로 대전될 수 있고, 그 반대의 경우도 가능하다.In addition, the porous silica particles of the present invention may be one in which the outer surface and / or the inside of the pores are positively and / or negatively charged. For example, both the surface and the inside of the pore may be positively charged, or may be negatively charged, only the surface or the inside of the pore may be positively charged, or may be negatively charged, the surface may be positively charged, and the interior of the pore may be negatively charged. The opposite is also true.

상기 대전은 예를 들면 양이온성 치환기 또는 음이온성 치환기가 존재함으로써 된 것일 수 있다.The charging may be, for example, by the presence of a cationic substituent or an anionic substituent.

상기 양이온성 치환기는 예를 들면 염기성기로서 아미노기, 그 외 질소함유기 등일 수 있고, 상기 음이온성 치환기는 예를 들면 산성기로서 카르복시기(-COOH), 술폰산기(-SO 3H), 티올기(-SH) 등일 수 있으나, 이에 제한되는 것은 아니다.The cationic substituent may be, for example, an amino group, other nitrogen-containing groups, etc. as the basic group, and the anionic substituent may be, for example, a carboxy group (-COOH), a sulfonic acid group (-SO 3 H), a thiol group as an acidic group, and the like. (-SH) and the like, but is not limited thereto.

마찬가지로 상기 대전에 의해 상기 치환기의 조절로 항체(antibody) 또는 사이토카인(cytokine)의 방출 환경에 대한 다공성 실리카 입자의 상호 작용이 조절되어 나노입자 자체의 분해 속도가 조절되어 항체(antibody) 또는 사이토카인(cytokine)의 방출 속도가 조절될 수 있고, 또한, 항체(antibody) 또는 사이토카인(cytokine)은 나노입자로부터 확산되어 방출될 수도 있는데, 상기 치환기의 조절로 항체(antibody) 또는 사이토카인(cytokine)의 나노입자에 대한 결합력이 조절되어 항체(antibody) 또는 사이토카인(cytokine)의 방출이 조절될 수 있다.Likewise, by the charging, the interaction of the porous silica particles with respect to the release environment of the antibody or cytokine is controlled by the control of the substituent, thereby controlling the rate of decomposition of the nanoparticles themselves, thereby controlling the antibody or cytokine. The release rate of the cytokine may be controlled, and the antibody or the cytokine may be released from the nanoparticles by being diffused, and by controlling the substituent, the antibody or the cytokine may be released. The binding force to the nanoparticles of the can be controlled to control the release of antibody or cytokine (cytokine).

또한, 본 발명의 다공성 실리카 입자는 그 표면 및/또는 기공 내부에 상기 외에 항체(antibody) 또는 사이토카인(cytokine)의 담지, 항체(antibody) 또는 사이토카인(cytokine)의 표적 세포로의 이동, 그외 기타 목적을 위한 물질의 담지 또는 그외 추가 치환기의 결합 등을 위한 치환기가 존재할 수 있으며, 이에 결합된 항체, 리간드, 세포투과성 펩타이드 또는 엡타머 등을 더 포함할 수 있다.In addition, the porous silica particles of the present invention may carry an antibody or cytokine in addition to the above, inside the surface and / or the pores, transfer the antibody or cytokine to target cells, and the like. Substituents for supporting other materials or binding of other additional substituents may be present, and may further include antibodies, ligands, cell permeable peptides, or aptamers bound thereto.

전술한 표면 및/또는 기공 내부의 치환기, 전하, 결합물질 등은 예를 들면 표면 개질에 의해 부가될 수 있다.Substituents, charges, binders and the like within the aforementioned surfaces and / or pores may be added, for example, by surface modification.

표면 개질은 예를 들면 도입하고자 하는 치환기를 갖는 화합물을 입자와 반응시켜 수행할 수 있고, 상기 화합물은 예를 들면 C1 내지 C10 알콕시기를 갖는 알콕시실란일 수 있으나, 이에 제한되는 것은 아니다. 상기 알콕시실란은 상기 알콕시기를 1개 이상 갖는 것으로서, 예를 들면 1 내지 3개 가질 수 있고, 알콕시기가 결합되지 않은 부위에 도입하고자 하는 치환기가 있거나 이로 치환된 치환기가 있을 수 있다.Surface modification can be carried out, for example, by reacting a compound having a substituent to be introduced with the particles, which may be, for example, an alkoxysilane having a C1 to C10 alkoxy group, but is not limited thereto. The alkoxysilane has one or more alkoxy groups, and may have, for example, 1 to 3, and there may be a substituent to be introduced into a site where the alkoxy group is not bonded or a substituent substituted therewith.

본 발명의 다공성 실리카 입자는 예를 들면 소기공의 입자 제조 및 기공 확장 공정을 거쳐 제조된 것일 수 있고, 필요에 따라 하소(calcination) 공정, 표면 개질 공정 등을 더 거쳐 제조된 것일 수 있다. 하소 및 표면 개질 공정을 모두 거친 경우는 하소 이후에 표면 개질 된 것일 수 있다.For example, the porous silica particles of the present invention may be manufactured through a small pore particle preparation and a pore expansion process, and may be manufactured through a calcination process, a surface modification process, and the like, as necessary. If both the calcination and the surface modification process has gone through may be surface modified after calcination.

상기 소기공의 입자는 예를 들면 평균 기공 직경이 1nm 내지 5nm인 입자일 수 있다.The small pore particles may be, for example, particles having an average pore diameter of 1 nm to 5 nm.

상기 소기공의 입자는 용매에 계면활성제와 실리카 전구물질을 넣고 교반 및 균질화시켜 얻어질 수 있다.The small pore particles can be obtained by adding a surfactant and a silica precursor in a solvent, stirring and homogenizing.

상기 용매는 물 및/또는 유기용매일 수 있고, 유기용매는 예를 들면 1,4-디옥산 등의 에테르류(특히 고리형상 에테르류); 클로로포름, 염화메틸렌, 4염화탄소, 1,2-디클로로에탄, 디클로로에틸렌, 트리클로로에틸렌, 퍼클로로에틸렌, 디클로로프로판, 염화아밀, 1,2-디브로모에탄 등의 할로겐화 탄화수소류; 아세톤, 메틸이소부틸케톤, γ-부티로락톤, 1,3-디메틸-이미다졸리디논, 메틸에틸케톤, 시클로헥사논, 시클로펜타논, 4-하이드록시-4-메틸-2-펜타논 등의 케톤류; 벤젠, 톨루엔, 크실렌, 테트라메틸벤젠 등의 탄소계 방향족류 ; N,N-디메틸포름아미드, N,N-디부틸포름아미드, N,N-디메틸아세트아미드, N-메틸피롤리돈 등의 알킬아미드류; 메탄올, 에탄올, 프로판올, 부탄올 등의 알코올류; 에틸렌글리콜모노에틸에테르, 에틸렌글리콜모노메틸에테르, 에틸렌글리콜모노부틸에테르, 디에틸렌글리콜모노에틸에테르, 디에틸렌글리콜모노메틸에테르, 디에틸렌글리콜모노부틸에테르, 프로필렌글리콜모노메틸에테르, 프로필렌글리콜모노에틸에테르, 디프로필렌글리콜디에틸에테르, 트리에틸렌글리콜모노에틸에테르 등의 글리콜에테르류(셀로솔브); 그외 디메틸아세트아미드(DMAc), N,N-디에틸아세트아미드, 디메틸포름아미드(DMF), 디에틸포름아미드(DEF), N,N-디메틸아세트아미드(DMAc), N-메틸피롤리돈(NMP), N-에틸피롤리돈(NEP), 1,3-디메틸-2-이미다졸리디논, N,N-디메틸메톡시아세트아미드, 디메틸술폭사이드, 피리딘, 디메틸술폰, 헥사메틸포스포아미드, 테트라메틸우레아, N-메틸카르로락탐, 테트라히드로퓨란, m-디옥산, P-디옥산, 1,2-디메톡시에탄 등을 사용할 수 있고, 구체적으로는 알코올, 보다 구체적으로 메탄올을 사용할 수 있으나, 이에 제한되는 것은 아니다.The solvent may be water and / or an organic solvent, and the organic solvent may be, for example, ethers such as 1,4-dioxane (particularly cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Acetone, methyl isobutyl ketone, γ-butyrolactone, 1,3-dimethyl-imidazolidinone, methyl ethyl ketone, cyclohexanone, cyclopentanone, 4-hydroxy-4-methyl-2-pentanone, etc. Ketones; Carbon-based aromatics such as benzene, toluene, xylene and tetramethylbenzene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; Ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, diethylene glycol monoethyl ether, diethylene glycol monomethyl ether, diethylene glycol monobutyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether Glycol ethers (cellosolve) such as dipropylene glycol diethyl ether and triethylene glycol monoethyl ether; Dimethylacetamide (DMAc), N, N-diethylacetamide, dimethylformamide (DMF), diethylformamide (DEF), N, N-dimethylacetamide (DMAc), N-methylpyrrolidone ( NMP), N-ethylpyrrolidone (NEP), 1,3-dimethyl-2-imidazolidinone, N, N-dimethylmethoxyacetamide, dimethyl sulfoxide, pyridine, dimethyl sulfone, hexamethylphosphoamide , Tetramethylurea, N-methylcarrolactam, tetrahydrofuran, m-dioxane, P-dioxane, 1,2-dimethoxyethane and the like can be used. Specifically, alcohol, more specifically methanol can be used. May be, but is not limited thereto.

상기 물과 유기 용매의 혼합 용매 사용시 그 비율은 예를 들면 물과 유기용매를 1: 0.7 내지 1.5의 부피비, 예를 들어, 1: 0.8 내지 1.3의 부피비로 사용할 수 있으나, 이에 제한되는 것은 아니다.When using a mixed solvent of the water and the organic solvent, the ratio may be, for example, water and the organic solvent in a volume ratio of 1: 0.7 to 1.5, for example, 1: 1: 0.8 to 1.3, but is not limited thereto.

상기 계면활성제는 예를 들면 CTAB(cetyltrimethylammonium bromide), TMABr(hexadecyltrimethylammonium bromide), TMPrCl(hexadecyltrimethylpyridinium chloride), TMACl(tetramethylammonium chloride) 등일 수 있고, 구체적으로는 CTAB를 사용할 수 있다.The surfactant may be, for example, cetyltrimethylammonium bromide (CTAB), hexadecyltrimethylammonium bromide (TMABr), hexadecyltrimethylpyridinium chloride (TMPrCl), tetramethylammonium chloride (TMACl), and the like, and specifically, CTAB may be used.

상기 계면활성제는 예를 들면 용매 1리터당 1g 내지 10g, 예를 들어 상기 범위 내에서 1g 내지 8g, 2g 내지 8g, 3g 내지 8g 등의 양으로 첨가될 수 있으나, 이에 제한되는 것은 아니다.The surfactant may be added, for example, in an amount of 1 g to 10 g, for example, 1 g to 8 g, 2 g to 8 g, 3 g to 8 g, etc., per liter of solvent, but is not limited thereto.

상기 실리카 전구 물질은 용매에 계면활성제를 첨가하여 교반한 후에 첨가될 수 있다. 실리카 전구물질은 예를 들면 TMOS(Tetramethyl orthosilicate)일 수 있으나, 이에 제한되는 것은 아니다.The silica precursor may be added after stirring with the addition of a surfactant to the solvent. The silica precursor may be, for example, tetramethyl orthosilicate (TMOS), but is not limited thereto.

상기 교반은 예를 들면 10분 내지 30분간 수행될 수 있으나, 이에 제한되는 것은 아니다.The stirring may be performed, for example, for 10 minutes to 30 minutes, but is not limited thereto.

상기 실리카 전구물질은 예를 들면 용매 1리터당 0.5ml 내지 5ml, 예를 들어 상기 범위 내에서 0.5ml 내지 4ml, 0.5ml 내지 3ml, 0.5ml 내지 2ml, 1ml 내지 2ml 등으로 첨가될 수 있으나, 이에 제한되는 것은 아니다.The silica precursor may be added, for example, 0.5 ml to 5 ml per liter of solvent, for example, 0.5 ml to 4 ml, 0.5 ml to 3 ml, 0.5 ml to 2 ml, 1 ml to 2 ml, etc. within the above range, but is not limited thereto. It doesn't happen.

필요에 따라 촉매로서 수산화나트륨을 더 사용할 수 있으며, 이는 용매에 계면활성제를 첨가한 후 실리카 전구물질의 첨가 전에 교반하면서 첨가될 수 있다.If necessary, sodium hydroxide may further be used as a catalyst, which may be added with stirring after adding the surfactant to the solvent and before adding the silica precursor.

상기 수산화나트륨은 예를 들면 1M 수산화나트륨 수용액 기준으로 용매 1리터당 0.5ml 내지 8ml, 예를 들어 상기 범위 내에서 0.5ml 내지 5ml, 0.5ml 내지 4ml, 1ml 내지 4ml, 1ml 내지 3ml 2ml 내지 3ml 등일 수 있으나, 이에 제한되는 것은 아니다.The sodium hydroxide may be, for example, 0.5 ml to 8 ml per liter of solvent, for example, 0.5 ml to 5 ml, 0.5 ml to 4 ml, 1 ml to 4 ml, 1 ml to 3 ml, 2 ml to 3 ml, etc., based on 1 M aqueous sodium hydroxide solution. However, it is not limited thereto.

상기 실리카 전구 물질의 첨가 후에 용액을 교반하며 반응시킬 수 있다. 교반은 예를 들면 2시간 내지 15시간 할 수 있고, 예를 들어 상기 범위 내에서 3시간 내지 15시간, 4시간 내지 15시간, 4시간 내지 13시간, 5시간 내지 12시간, 6 시간 내지 12시간, 6시간 내지 10시간 등일 수 있으나, 이에 제한되는 것은 아니다. 교반 시간(반응 시간)이 너무 짧은 경우에는 결정핵 생성(nucleation)이 부족할 수 있다.After addition of the silica precursor, the solution can be reacted with stirring. The stirring may be performed for example, for 2 hours to 15 hours, for example, within the above range, for example, 3 hours to 15 hours, 4 hours to 15 hours, 4 hours to 13 hours, 5 hours to 12 hours, 6 hours to 12 hours , 6 hours to 10 hours, and the like, but is not limited thereto. If the stirring time (reaction time) is too short, nucleation may be insufficient.

상기 교반 이후에는 용액을 숙성(aging)시킬 수 있다. 숙성은 예를 들면 8시간 내지 24시간 할 수 있고, 예를 들어 상기 범위 내에서 8시간 내지 20시간, 8시간 내지 18시간, 8시간 내지 16시간, 8시간 내지 14시간, 10시간 내지 16시간, 10시간 내지 14시간 등일 수 있으나, 이에 제한되는 것은 아니다.After the agitation, the solution may be aged. Aging may be performed for example, from 8 hours to 24 hours, for example, within the range of 8 hours to 20 hours, 8 hours to 18 hours, 8 hours to 16 hours, 8 hours to 14 hours, 10 hours to 16 hours. , 10 hours to 14 hours, and the like, but is not limited thereto.

이후, 반응산물을 세척 및 건조시켜 다공성 실리카 입자를 얻을 수 있고, 필요에 따라 세척 전에 미반응 물질의 분리가 선행될 수 있다.Thereafter, the reaction product may be washed and dried to obtain porous silica particles, and if necessary, separation of unreacted material may be preceded before washing.

상기 미반응 물질의 분리는 예를 들면 원심분리로 상등액을 분리하여 수행될 수 있고, 원심분리는 예를 들면 6,000 내지 10,000rpm으로 수행될 수 있으며, 그 시간은 예를 들면 3분 내지 60분, 예를 들어 상기 범위 내에서 3분 내지 30분, 3분 내지 30분, 5분 내지 30분 등으로 수행될 수 있으나, 이에 제한되는 것은 아니다.Separation of the unreacted material may be carried out by separating the supernatant, for example by centrifugation, centrifugation may be carried out, for example at 6,000 to 10,000 rpm, the time is for example 3 minutes to 60 minutes, For example, it may be performed within 3 minutes to 30 minutes, 3 minutes to 30 minutes, 5 minutes to 30 minutes, and the like, but is not limited thereto.

상기 세척은 물 및/또는 유기용매로 할 수 있고, 구체적으로는 용매별로 녹일 수 있는 물질이 상이하므로 물과 유기용매를 1회 또는 수회 번갈아 사용할 수 있으며, 물 또는 유기용매 단독으로도 1회 또는 수회 세척할 수 있다. 상기 수회는 예를 들면 2회 이상, 10회 이하, 예를 들어, 3회 이상 10회 이하, 4회 이상 8회 이하, 4회 이상 6회 이하 등일 수 있다.The washing may be performed with water and / or an organic solvent, and in particular, since a substance that can be dissolved in each solvent may be different, water and an organic solvent may be used once or several times, or once or even with water or an organic solvent alone. Can be washed several times. The number of times may be, for example, two or more, ten or less, for example, three or more and ten or less, four or more and eight or less, four or more and six or less.

상기 유기용매는 예를 들면 1,4-디옥산 등의 에테르류(특히 고리형상 에테르류); 클로로포름, 염화메틸렌, 4염화탄소, 1,2-디클로로에탄, 디클로로에틸렌, 트리클로로에틸렌, 퍼클로로에틸렌, 디클로로프로판, 염화아밀, 1,2-디브로모에탄 등의 할로겐화 탄화수소류; 아세톤, 메틸이소부틸케톤, γ-부티로락톤, 1,3-디메틸-이미다졸리디논, 메틸에틸케톤, 시클로헥사논, 시클로펜타논, 4-하이드록시-4-메틸-2-펜타논 등의 케톤류; 벤젠, 톨루엔, 크실렌, 테트라메틸벤젠 등의 탄소계 방향족류 ; N,N-디메틸포름아미드, N,N-디부틸포름아미드, N,N-디메틸아세트아미드, N-메틸피롤리돈 등의 알킬아미드류; 메탄올, 에탄올, 프로판올, 부탄올 등의 알코올류; 에틸렌글리콜모노에틸에테르, 에틸렌글리콜모노메틸에테르, 에틸렌글리콜모노부틸에테르, 디에틸렌글리콜모노에틸에테르, 디에틸렌글리콜모노메틸에테르, 디에틸렌글리콜모노부틸에테르, 프로필렌글리콜모노메틸에테르, 프로필렌글리콜모노에틸에테르, 디프로필렌글리콜디에틸에테르, 트리에틸렌글리콜모노에틸에테르 등의 글리콜에테르류(셀로솔브); 그외 디메틸아세트아미드(DMAc), N,N-디에틸아세트아미드, 디메틸포름아미드(DMF), 디에틸포름아미드(DEF), N,N-디메틸아세트아미드(DMAc), N-메틸피롤리돈(NMP), N-에틸피롤리돈(NEP), 1,3-디메틸-2-이미다졸리디논, N,N-디메틸메톡시아세트아미드, 디메틸술폭사이드, 피리딘, 디메틸술폰, 헥사메틸포스포아미드, 테트라메틸우레아, N-메틸카르로락탐, 테트라히드로퓨란, m-디옥산, P-디옥산, 1,2-디메톡시에탄 등을 사용할 수 있고, 구체적으로는 알코올, 보다 구체적으로 에탄올을 사용할 수 있으나, 이에 제한되는 것은 아니다.The organic solvent may be, for example, ethers such as 1,4-dioxane (particularly cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Acetone, methyl isobutyl ketone, γ-butyrolactone, 1,3-dimethyl-imidazolidinone, methyl ethyl ketone, cyclohexanone, cyclopentanone, 4-hydroxy-4-methyl-2-pentanone, etc. Ketones; Carbon-based aromatics such as benzene, toluene, xylene and tetramethylbenzene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; Ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, diethylene glycol monoethyl ether, diethylene glycol monomethyl ether, diethylene glycol monobutyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether Glycol ethers (cellosolve) such as dipropylene glycol diethyl ether and triethylene glycol monoethyl ether; Dimethylacetamide (DMAc), N, N-diethylacetamide, dimethylformamide (DMF), diethylformamide (DEF), N, N-dimethylacetamide (DMAc), N-methylpyrrolidone ( NMP), N-ethylpyrrolidone (NEP), 1,3-dimethyl-2-imidazolidinone, N, N-dimethylmethoxyacetamide, dimethyl sulfoxide, pyridine, dimethyl sulfone, hexamethylphosphoamide , Tetramethylurea, N-methylcarrolactam, tetrahydrofuran, m-dioxane, P-dioxane, 1,2-dimethoxyethane and the like can be used, specifically, alcohol, more specifically ethanol can be used. May be, but is not limited thereto.

상기 세척은 원심분리 하에 수행될 수 있으며, 예를 들면 6,000 내지 10,000rpm으로 수행될 수 있으며, 그 시간은 예를 들면 3분 내지 60분, 예를 들어 상기 범위 내에서 3분 내지 30분, 3분 내지 30분, 5분 내지 30분 등으로 수행될 수 있으나, 이에 제한되는 것은 아니다.The washing may be carried out under centrifugation, for example at 6,000 to 10,000 rpm, the time being for example 3 to 60 minutes, for example 3 to 30 minutes, 3 within the above range. It may be performed in minutes to 30 minutes, 5 minutes to 30 minutes and the like, but is not limited thereto.

상기 세척은 원심분리를 하지 않고, 필터로 입자를 걸러내어 수행될 수도 있다. 필터는 다공성 실리카 입자의 직경 이하의 기공을 가지는 것일 수 있다. 반응액을 그러한 필터로 걸러내면 입자만이 필터 위에 남고, 그 필터 위에 물 및/또는 유기용매를 부어 세척할 수 있다.The washing may be performed by filtering out particles with a filter without centrifugation. The filter may have pores less than or equal to the diameter of the porous silica particles. Filtering the reaction liquid with such a filter leaves only particles on the filter, which can be washed by pouring water and / or an organic solvent on the filter.

상기 세척 시에 물과 유기용매를 1회 또는 수회 번갈아 사용할 수 있으며, 물 또는 유기용매 단독으로도 1회 또는 수회 세척할 수 있다. 상기 수회는 예를 들면 2회 이상, 10회 이하, 예를 들어, 3회 이상 10회 이하, 4회 이상 8회 이하, 4회 이상 6회 이하 등일 수 있다.In the washing, water and an organic solvent may be used alternately once or several times, and may be washed once or several times even with water or an organic solvent alone. The number of times may be, for example, two or more, ten or less, for example, three or more and ten or less, four or more and eight or less, four or more and six or less.

상기 건조는 예를 들면 20℃ 내지 100℃로 수행될 수 있으나, 이에 제한되는 것은 아니고, 진공 상태에서 수행될 수도 있다.For example, the drying may be performed at 20 ° C. to 100 ° C., but is not limited thereto, and may be performed in a vacuum state.

이후, 상기 얻어진 다공성 실리카 입자의 기공을 확장하고, 기공 확장은 기공 팽창제를 사용하여 수행될 수 있다.Thereafter, the pores of the obtained porous silica particles are expanded, and the pore expansion may be performed using a pore swelling agent.

상기 기공 팽창제는 예를 들면 트리메틸벤젠, 트리에틸벤젠, 트리프로필벤젠, 트리부틸벤젠, 트리펜틸벤젠, 트리헥실벤젠, 톨루엔, 벤젠 등을 사용할 수 있고, 구체적으로, 트리메틸벤젠을 사용할 수 있으나, 이에 제한되는 것은 아니다.For example, the pore swelling agent may be trimethylbenzene, triethylbenzene, tripropylbenzene, tributylbenzene, tripentylbenzene, trihexylbenzene, toluene, benzene, and the like, and specifically, trimethylbenzene may be used. It is not limited.

또한, 상기 기공 팽창제는 예를 들면 N,N-디메틸헥사데실아민(N,N-dimethylhexadecylamine,DMHA)를 사용할 수 있으나, 이에 제한되는 것은 아니다.In addition, the pore swelling agent may use, for example, N, N-dimethylhexadecylamine (N, N-dimethylhexadecylamine, DMHA), but is not limited thereto.

상기 기공 확장은 예를 들면 용매 중의 다공성 실리카 입자를 기공 팽창제와 혼합하고, 가열하여 반응시켜 수행될 수 있다.The pore expansion can be carried out, for example, by mixing porous silica particles in a solvent with a pore swelling agent and heating to react.

상기 용매는 예를 들면 물 및/또는 유기용매일 수 있고, 유기용매는 예를 들면 1,4-디옥산 등의 에테르류(특히 고리형상 에테르류); 클로로포름, 염화메틸렌, 4염화탄소, 1,2-디클로로에탄, 디클로로에틸렌, 트리클로로에틸렌, 퍼클로로에틸렌, 디클로로프로판, 염화아밀, 1,2-디브로모에탄 등의 할로겐화 탄화수소류; 아세톤, 메틸이소부틸케톤, 시클로헥산온 등의 케톤류; 벤젠, 톨루엔, 크실렌 등의 탄소계 방향족류 ; N,N-디메틸포름아미드, N,N-디부틸포름아미드, N,N-디메틸아세트아미드, N-메틸피롤리돈 등의 알킬아미드류; 메탄올, 에탄올, 프로판올, 부탄올 등의 알코올류; 등을 사용할 수 있고, 구체적으로는 알코올, 보다 구체적으로 에탄올을 사용할 수 있으나, 이에 제한되는 것은 아니다.The solvent may be, for example, water and / or an organic solvent, and the organic solvent may be, for example, ethers such as 1,4-dioxane (particularly cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Ketones such as acetone, methyl isobutyl ketone and cyclohexanone; Carbon-based aromatics such as benzene, toluene and xylene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; And the like, and specifically, alcohol, more specifically ethanol may be used, but is not limited thereto.

상기 다공성 실리카 입자는 예를 들면 용매 1리터당 10g 내지 200g, 예를 들어 상기 범위 내에서 10g 내지 150g, 10g 내지 100g, 30g 내지 100g, 40g 내지 100g, 50g 내지 100g, 50g 내지 80g, 60g 내지 80g 등의 비율로 첨가될 수 있으나, 이에 제한되는 것은 아니다.The porous silica particles are, for example, 10 g to 200 g per liter of solvent, for example, 10 g to 150 g, 10 g to 100 g, 30 g to 100 g, 40 g to 100 g, 50 g to 100 g, 50 g to 80 g, 60 g to 80 g, etc., within the above range. It may be added in a ratio of, but is not limited thereto.

상기 다공성 실리카 입자는 용매 중에 고르게 분산되어 있는 것일 수 있고, 예를 들면 용매에 다공성 실리카 입자를 첨가하고 초음파 분산시킨 것일 수 있다. 혼합용매를 사용하는 경우에는 제1 용매에 다공성 실리카 입자를 분산시킨 후에 제2 용매를 첨가한 것일 수 있다.The porous silica particles may be evenly dispersed in a solvent, for example, the porous silica particles may be added to the solvent and ultrasonically dispersed. In the case of using a mixed solvent, the second solvent may be added after the porous silica particles are dispersed in the first solvent.

상기 기공 팽창제는 예를 들면 용매 100부피부에 대하여 10 내지 200부피부, 상기 범위 내에서, 10 내지 150부피부, 10 내지 100부피부, 10 내지 80부피부, 30 내지 80부피부, 30 내지 70부피부 등의 비율로 첨가될 수 있으나, 이에 제한되는 것은 아니다.The pore swelling agent is for example 10 to 200 parts by volume, 100 to 150 parts by volume, 10 to 100 parts by volume, 10 to 80 parts by volume, 30 to 80 parts by volume, 30 to 80 parts by volume based on 100 parts by volume of solvent. 70 parts by volume may be added, but is not limited thereto.

상기 반응은 예를 들면 120℃ 내지 190℃로 수행될 수 있다. 예를 들어 상기 범위 내에서 120℃ 내지 190℃, 120℃ 내지 180℃, 120℃ 내지 170℃, 130℃ 내지 170℃, 130℃ 내지 160℃, 130℃ 내지 150℃, 130℃ 내지 140℃ 등으로 수행될 수 있으나, 이에 제한되는 것은 아니다.The reaction can be carried out, for example, at 120 ° C to 190 ° C. For example, within the range of 120 ℃ to 190 ℃, 120 ℃ to 180 ℃, 120 ℃ to 170 ℃, 130 ℃ to 170 ℃, 130 ℃ to 160 ℃, 130 ℃ to 150 ℃, 130 ℃ to 140 ℃ It may be performed, but is not limited thereto.

상기 반응은 예를 들면 6시간 내지 96시간 수행 수행될 수 있다. 예를 들어 상기 범위 내에서 30시간 내지 96시간, 30시간 내지 96시간, 30시간 내지 80시간, 30시간 내지 72시간, 24시간 내지 80시간, 24시간 내지 72시간, 36시간 내지 96시간, 36시간 내지 80시간, 36시간 내지 72시간, 36시간 내지 66시간, 36시간 내지 60시간, 48시간 내지 96시간, 48시간 내지 88시간, 48시간 내지 80시간, 48시간 내지 72시간, 6시간 내지 96시간, 7시간 내지 96시간, 8시간 내지 80시간, 9시간 내지 72시간, 9시간 내지 80시간, 6시간 내지 72시간, 9시간 내지 96시간, 10시간 내지 80시간, 10시간 내지 72시간, 12시간 내지 66시간, 13시간 내지 60시간, 14시간 내지 96시간, 15시간 내지 88시간, 16시간 내지 80시간, 17시간 내지 72시간 등일 수 있으나, 이에 제한되는 것은 아니다.The reaction may be performed, for example, for 6 hours to 96 hours. For example, within the range of 30 hours to 96 hours, 30 hours to 96 hours, 30 hours to 80 hours, 30 hours to 72 hours, 24 hours to 80 hours, 24 hours to 72 hours, 36 hours to 96 hours, 36 36 hours to 80 hours, 36 hours to 72 hours, 36 hours to 66 hours, 36 hours to 60 hours, 48 hours to 96 hours, 48 hours to 88 hours, 48 hours to 80 hours, 48 hours to 72 hours, 6 hours to 96 hours, 7 hours to 96 hours, 8 hours to 80 hours, 9 hours to 72 hours, 9 hours to 80 hours, 6 hours to 72 hours, 9 hours to 96 hours, 10 hours to 80 hours, 10 hours to 72 hours , 12 hours to 66 hours, 13 hours to 60 hours, 14 hours to 96 hours, 15 hours to 88 hours, 16 hours to 80 hours, 17 hours to 72 hours, and the like, but is not limited thereto.

상기 예시한 범위 내에서 시간 및 온도를 조절하여 반응이 과다하지 않으면서 충분히 수행될 수 있도록 할 수 있다. 예를 들면 반응 온도가 낮아지면 반응 시간을 늘리거나, 반응 온도가 낮아지면 반응 시간을 짧게하는 등에 의할 수 있다. 반응이 충분하지 않으면 기공의 확장이 충분하지 못할 수 있고, 반응이 과다하게 진행되면 기공의 과다 확장에 의해 입자가 붕괴될 수 있다.The time and temperature can be adjusted within the ranges exemplified above so that the reaction can be carried out sufficiently without excess. For example, when the reaction temperature is lowered, the reaction time may be increased, or when the reaction temperature is lowered, the reaction time may be shortened. If the reaction is not sufficient, the expansion of the pores may not be sufficient, and if the reaction proceeds excessively, the particles may collapse due to the expansion of the pores.

상기 반응은 예를 들면 단계적으로 승온시켜 수행될 수 있다. 구체적으로, 상온에서 상기 온도까지 0.5℃/분 내지 15℃/분의 속도로 단계적으로 승온시켜 수행될 수 있으며, 예를 들어 상기 범위 내에서 1℃/분 내지 15℃/분, 3℃/분 내지 15℃/분, 3℃/분 내지 12℃/분, 3℃/분 내지 10℃/분 등일 수 있으나, 이에 제한되는 것은 아니다.The reaction can be carried out, for example, by gradually raising the temperature. Specifically, it may be carried out by gradually raising the temperature at a rate of 0.5 ℃ / min to 15 ℃ / min from the room temperature to the above temperature, for example, 1 ℃ / min to 15 ℃ / min, 3 ℃ / min within the above range To 15 ° C./minute, 3 ° C./minute to 12 ° C./minute, 3 ° C./minute to 10 ° C./minute, and the like, but are not limited thereto.

상기 반응은 교반 하에 수행될 수 있다. 예를 들면 100rpm 이상의 속도로 교반될 수 있고, 구체적으로 100rpm 내지 1000rpm의 속도로 수행도리 수 있으나, 이에 제한되는 것은 아니다.The reaction can be carried out under stirring. For example, it may be stirred at a speed of 100 rpm or more, and specifically, may be performed at a speed of 100 rpm to 1000 rpm, but is not limited thereto.

상기 반응 이후에는 반응액을 서서히 냉각시킬 수 있으며, 예를 들면 단계적으로 감온하여 냉각시킬 수 있다. 구체적으로 상기 온도에서 상온까지 0.5℃/분 내지 20℃/분의 속도로 단계적으로 감온시켜 수행될 수 있으며, 예를 들어 상기 범위 내에서 1℃/분 내지 20℃/분, 3℃/분 내지 20℃/분, 3℃/분 내지 12℃/분, 3℃/분 내지 10℃/분 등일 수 있으나, 이에 제한되는 것은 아니다.After the reaction, the reaction solution may be cooled slowly, for example, it may be cooled by gradually reducing the temperature. Specifically, it may be carried out by gradually decreasing the temperature at a rate of 0.5 ℃ / min to 20 ℃ / min from the temperature to room temperature, for example, 1 ℃ / min to 20 ℃ / min, 3 ℃ / min to within the above range 20 ° C./minute, 3 ° C./minute to 12 ° C./minute, 3 ° C./minute to 10 ° C./minute, and the like, but is not limited thereto.

상기 냉각 이후에 반응 산물을 세척 및 건조시켜 기공이 확장된 다공성 실리카 입자를 얻을 수 있고, 필요에 따라 세척 전에 미반응 물질의 분리가 선행될 수 있다.After cooling, the reaction product may be washed and dried to obtain porous silica particles having expanded pores, and if necessary, separation of unreacted material may be preceded before washing.

상기 미반응 물질의 분리는 예를 들면 원심분리로 상등액을 분리하여 수행될 수 있고, 원심분리는 예를 들면 6,000 내지 10,000rpm으로 수행될 수 있으며, 그 시간은 예를 들면 3분 내지 60분, 예를 들어 상기 범위 내에서 3분 내지 30분, 3분 내지 30분, 5분 내지 30분 등으로 수행될 수 있으나, 이에 제한되는 것은 아니다.Separation of the unreacted material may be carried out by separating the supernatant, for example by centrifugation, centrifugation may be carried out, for example at 6,000 to 10,000 rpm, the time is for example 3 minutes to 60 minutes, For example, it may be performed within 3 minutes to 30 minutes, 3 minutes to 30 minutes, 5 minutes to 30 minutes, and the like, but is not limited thereto.

상기 세척은 물 및/또는 유기용매로 할 수 있고, 구체적으로는 용매별로 녹일 수 있는 물질이 상이하므로 물과 유기용매를 1회 또는 수회 번갈아 사용할 수 있으며, 물 또는 유기용매 단독으로도 1회 또는 수회 세척할 수 있다. 상기 수회는 예를 들면 2회 이상, 10회 이하, 예를 들어, 3회, 4회, 5회, 6회, 7회, 8회 등일 수 있다.The washing may be performed with water and / or an organic solvent, and in particular, since a substance that can be dissolved in each solvent may be different, water and an organic solvent may be used once or several times, or once or even with water or an organic solvent alone. Can be washed several times. The number of times may be, for example, two or more times, ten times or less, for example, three times, four times, five times, six times, seven times, eight times, and the like.

상기 유기용매는 예를 들면 1,4-디옥산 등의 에테르류(특히 고리형상 에테르류); 클로로포름, 염화메틸렌, 4염화탄소, 1,2-디클로로에탄, 디클로로에틸렌, 트리클로로에틸렌, 퍼클로로에틸렌, 디클로로프로판, 염화아밀, 1,2-디브로모에탄 등의 할로겐화 탄화수소류; 아세톤, 메틸이소부틸케톤, 시클로헥산온 등의 케톤류; 벤젠, 톨루엔, 크실렌 등의 탄소계 방향족류; N,N-디메틸포름아미드, N,N-디부틸포름아미드, N,N-디메틸아세트아미드, N-메틸피롤리돈 등의 알킬아미드류; 메탄올, 에탄올, 프로판올, 부탄올 등의 알코올류; 등을 사용할 수 있고, 구체적으로는 알코올, 보다 구체적으로 에탄올을 사용할 수 있으나, 이에 제한되는 것은 아니다.The organic solvent may be, for example, ethers such as 1,4-dioxane (particularly cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Ketones such as acetone, methyl isobutyl ketone and cyclohexanone; Carbon-based aromatics such as benzene, toluene and xylene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; And the like, and specifically, alcohol, more specifically ethanol may be used, but is not limited thereto.

상기 세척은 원심분리 하에 수행될 수 있으며, 예를 들면 6,000 내지 10,000rpm으로 수행될 수 있으며, 그 시간은 예를 들면 3분 내지 60분, 예를 들어 상기 범위 내에서 3분 내지 30분, 3분 내지 30분, 5분 내지 30분 등으로 수행될 수 있으나, 이에 제한되는 것은 아니다.The washing may be carried out under centrifugation, for example at 6,000 to 10,000 rpm, the time being for example 3 to 60 minutes, for example 3 to 30 minutes, 3 within the above range. It may be performed in minutes to 30 minutes, 5 minutes to 30 minutes and the like, but is not limited thereto.

상기 세척은 원심분리를 하지 않고, 필터로 입자를 걸러내어 수행될 수도 있다. 필터는 다공성 실리카 입자의 직경 이하의 기공을 가지는 것일 수 있다. 반응액을 그러한 필터로 걸러내면 입자만이 필터 위에 남고, 그 필터 위에 물 및/또는 유기용매를 부어 세척할 수 있다.The washing may be performed by filtering out particles with a filter without centrifugation. The filter may have pores less than or equal to the diameter of the porous silica particles. Filtering the reaction liquid with such a filter leaves only particles on the filter, which can be washed by pouring water and / or an organic solvent on the filter.

상기 세척 시에 물과 유기용매를 1회 또는 수회 번갈아 사용할 수 있으며, 물 또는 유기용매 단독으로도 1회 또는 수회 세척할 수 있다. 상기 수회는 예를 들면 2회 이상, 10회 이하, 예를 들어, 3회 이상 10회 이하, 4회 이상 8회 이하, 4회 이상 6회 이하 등일 수 있다.In the washing, water and an organic solvent may be used alternately once or several times, and may be washed once or several times even with water or an organic solvent alone. The number of times may be, for example, two or more, ten or less, for example, three or more and ten or less, four or more and eight or less, four or more and six or less.

상기 건조는 예를 들면 20℃ 내지 100℃로 수행될 수 있으나, 이에 제한되는 것은 아니고, 진공 상태에서 수행될 수도 있다.For example, the drying may be performed at 20 ° C. to 100 ° C., but is not limited thereto, and may be performed in a vacuum state.

이후, 얻어진 입자는 하소될 수 있는데, 하소는 입자를 가열하여 그 표면 및 내부의 실라놀기를 제거하여 입자의 반응성을 낮추고, 좀 더 치밀한 구조를 갖게 하고, 기공을 채우는 유기물들을 제거하는 공정으로, 예를 들면 400℃ 이상의 온도로 가열될 수 있다. 그 상한은 특별히 제한되지 않으며, 예를 들면 1000℃, 900℃, 800℃, 700℃ 등일 수 있다. 가열은 예를 들면 3시간 이상 수행될 수 있다. 그 상한은 특별히 한정되지 않으며, 예를 들면 24시간, 12시간, 10시간, 8시간, 6시간 등일 수 있다. 보다 구체적으로는 400℃ 내지 700℃에서 3시간 내지 8시간, 구체적으로 500℃ 내지 600℃에서 4시간 내지 5시간 수행될 수 있으나, 이에 제한되는 것은 아니다.Thereafter, the obtained particles may be calcined, which is a process of heating the particles to remove silanol groups on the surface and inside thereof to lower the reactivity of the particles, to have a more compact structure, and to remove organic substances filling the pores. For example, it may be heated to a temperature of 400 ℃ or more. The upper limit thereof is not particularly limited, and may be, for example, 1000 ° C, 900 ° C, 800 ° C, 700 ° C, or the like. Heating can be carried out for example for 3 hours or more. The upper limit is not particularly limited and may be, for example, 24 hours, 12 hours, 10 hours, 8 hours, 6 hours, or the like. More specifically, it may be performed for 3 hours to 8 hours at 400 ° C to 700 ° C, specifically 4 hours to 5 hours at 500 ° C to 600 ° C, but is not limited thereto.

기공을 채우는 유기물을 제거함으로써, 잔존 유기물에 의해 나타나는 세포 독성, 거품 발생 등의 문제를 방지할 수 있다.By removing the organic matter filling the pores, it is possible to prevent problems such as cytotoxicity and foaming caused by the remaining organic matter.

이후, 얻어진 다공성 실리카 입자는 표면개질 될 수 있고, 표면 개질은 표면 및/또는 기공 내부에 수행될 수 있다. 입자 표면과 기공 내부는 동일하게 표면개질될 수도 있고, 서로 다르게 표면개질될 수도 있다.The porous silica particles obtained can then be surface modified, and the surface modification can be carried out on the surface and / or inside the pores. The particle surface and the inside of the pore may be surface-modified identically or may be surface-modified differently.

상기 표면 개질을 통해 입자가 대전되도록 하거나, 친수성 및/또는 소수성 성질을 갖도록 할 수 있다. The surface modification can cause the particles to charge or to have hydrophilic and / or hydrophobic properties.

보다 구체적으로, 항체(antibody) 또는 사이토카인(cytokine)의 효과적인 담지를 위하여, 아미노기, 아미노알킬기, 알킬아미노기, 질소원자를 포함하는 헤테로고리 방향족화합물기, 시안기 및 구아니딘기로 이루어진 군에서 선택된 적어도 하나의 치환기를 갖도록 하여, 상기 다공성 실리카 입자의 표면개질을 수행할 수 있다.More specifically, at least one selected from the group consisting of an amino group, an aminoalkyl group, an alkylamino group, a heterocyclic aromatic compound group containing a nitrogen atom, a cyan group and a guanidine group for effective loading of an antibody or cytokine By having a substituent of, the surface modification of the porous silica particles can be carried out.

표면 개질은 예를 들면 도입하고자 하는 친수성, 소수성, 양이온성, 음이온성 등의 치환기를 갖는 화합물을 입자와 반응시켜 수행할 수 있고, 상기 화합물은 예를 들면 C1 내지 C10 알콕시기를 갖는 알콕시실란일 수 있으나, 이에 제한되는 것은 아니다.Surface modification can be carried out, for example, by reacting a compound having substituents such as hydrophilic, hydrophobic, cationic, anionic and the like to be introduced with the particles, and the compound can be, for example, an alkoxysilane having a C1 to C10 alkoxy group. However, it is not limited thereto.

상기 알콕시실란은 상기 알콕시기를 1개 이상 갖는 것으로서, 예를 들면 1 내지 3개 가질 수 있고, 알콕시기가 결합되지 않은 부위에 도입하고자 하는 치환기가 있거나 이로 치환된 치환기가 있을 수 있다. The alkoxysilane has one or more alkoxy groups, and may have, for example, 1 to 3, and there may be a substituent to be introduced into a site where the alkoxy group is not bonded or a substituent substituted therewith.

상기 알콕시실란을 다공성 실리콘 입자와 반응시키면 실리콘 원자와 산소 원자간 공유 결합이 형성되어 알콕시실란이 다공성 실리콘 입자의 표면 및/또는 기공 내부와 결합될 수 있고, 상기 알콕시실란은 도입하고자 하는 치환기를 가지고 있는 바, 해당 치환기가 다공성 실리콘 입자의 표면 및/또는 기공 내부에 도입될 수 있다.When the alkoxysilane reacts with the porous silicon particles, a covalent bond is formed between the silicon atom and the oxygen atom so that the alkoxysilane may be bonded to the surface and / or the inside of the pores of the porous silicon particle, and the alkoxysilane has a substituent to be introduced. As such, the corresponding substituents may be introduced into the surface of the porous silicon particles and / or within the pores.

상기 반응은 용매에 분산시킨 다공성 실리카 입자를 알콕시실란과 반응시켜 수행할 수 있다.The reaction may be carried out by reacting porous silica particles dispersed in a solvent with an alkoxysilane.

상기 용매는 물 및/또는 유기용매일 수 있고, 유기용매는 예를 들면 1,4-디옥산 등의 에테르류(특히 고리형상 에테르류); 클로로포름, 염화메틸렌, 4염화탄소, 1,2-디클로로에탄, 디클로로에틸렌, 트리클로로에틸렌, 퍼클로로에틸렌, 디클로로프로판, 염화아밀, 1,2-디브로모에탄 등의 할로겐화 탄화수소류; 아세톤, 메틸이소부틸케톤, γ-부티로락톤, 1,3-디메틸-이미다졸리디논, 메틸에틸케톤, 시클로헥사논, 시클로펜타논, 4-하이드록시-4-메틸-2-펜타논 등의 케톤류; 벤젠, 톨루엔, 크실렌, 테트라메틸벤젠 등의 탄소계 방향족류 ; N,N-디메틸포름아미드, N,N-디부틸포름아미드, N,N-디메틸아세트아미드, N-메틸피롤리돈 등의 알킬아미드류; 메탄올, 에탄올, 프로판올, 부탄올 등의 알코올류; 에틸렌글리콜모노에틸에테르, 에틸렌글리콜모노메틸에테르, 에틸렌글리콜모노부틸에테르, 디에틸렌글리콜모노에틸에테르, 디에틸렌글리콜모노메틸에테르, 디에틸렌글리콜모노부틸에테르, 프로필렌글리콜모노메틸에테르, 프로필렌글리콜모노에틸에테르, 디프로필렌글리콜디에틸에테르, 트리에틸렌글리콜모노에틸에테르 등의 글리콜에테르류(셀로솔브); 그외 디메틸아세트아미드(DMAc), N,N-디에틸아세트아미드, 디메틸포름아미드(DMF), 디에틸포름아미드(DEF), N,N-디메틸아세트아미드(DMAc), N-메틸피롤리돈(NMP), N-에틸피롤리돈(NEP), 1,3-디메틸-2-이미다졸리디논, N,N-디메틸메톡시아세트아미드, 디메틸술폭사이드, 피리딘, 디메틸술폰, 헥사메틸포스포아미드, 테트라메틸우레아, N-메틸카르로락탐, 테트라히드로퓨란, m-디옥산, P-디옥산, 1,2-디메톡시에탄 등을 사용할 수 있고, 구체적으로는 톨루엔을 사용할 수 있으나, 이에 제한되는 것은 아니다.The solvent may be water and / or an organic solvent, and the organic solvent may be, for example, ethers such as 1,4-dioxane (particularly cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Acetone, methyl isobutyl ketone, γ-butyrolactone, 1,3-dimethyl-imidazolidinone, methyl ethyl ketone, cyclohexanone, cyclopentanone, 4-hydroxy-4-methyl-2-pentanone, etc. Ketones; Carbon-based aromatics such as benzene, toluene, xylene and tetramethylbenzene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; Ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, diethylene glycol monoethyl ether, diethylene glycol monomethyl ether, diethylene glycol monobutyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether Glycol ethers (cellosolve) such as dipropylene glycol diethyl ether and triethylene glycol monoethyl ether; Dimethylacetamide (DMAc), N, N-diethylacetamide, dimethylformamide (DMF), diethylformamide (DEF), N, N-dimethylacetamide (DMAc), N-methylpyrrolidone ( NMP), N-ethylpyrrolidone (NEP), 1,3-dimethyl-2-imidazolidinone, N, N-dimethylmethoxyacetamide, dimethyl sulfoxide, pyridine, dimethyl sulfone, hexamethylphosphoamide , Tetramethylurea, N-methyl carrolactam, tetrahydrofuran, m-dioxane, P-dioxane, 1,2-dimethoxyethane, etc. may be used. Specifically, toluene may be used, but is not limited thereto. It doesn't happen.

상기 양전하로의 대전은 예를 들면 아미노기, 아미노알킬기 등 질소함유기 등의 염기성기를 갖는 알콕시실란과 반응시켜 수행할 수 있다. 구체적으로는 N-[3-(Trimethoxysilyl)propyl]ethylenediamine, N1-(3-Trimethoxysilylpropyl)diethylenetriamine, (3-Aminopropyl)trimethoxysilane, N-[3-(Trimethoxysilyl)propyl]aniline, Trimethoxy[3-(methylamino)propyl]silane, 3-(2-Aminoethylamino)propyldimethoxymethylsilane 등을 사용할 수 있으나, 이에 제한되는 것은 아니다.The charge to the positive charge can be carried out by reacting with an alkoxysilane having a basic group such as a nitrogen-containing group such as an amino group, an aminoalkyl group, for example. Specifically, N- [3- (Trimethoxysilyl) propyl] ethylenediamine, N1- (3-Trimethoxysilylpropyl) diethylenetriamine, (3-Aminopropyl) trimethoxysilane, N- [3- (Trimethoxysilyl) propyl] aniline, Trimethoxy [3- (methylamino) propyl] silane, 3- (2-Aminoethylamino) propyldimethoxymethylsilane, etc. may be used, but is not limited thereto.

상기 음전하로의 대전은 예를 들면 카르복시기, 술폰산기, 티올기 등의 산성기를 갖는 알콕시실란과 반응시켜 수행할 수 있다. 구체적으로는 (3-Mercaptopropyl) trimethoxysilane 등을 사용할 수 있으나, 이에 제한되는 것은 아니다.Charging to the negative charge may be carried out by reacting with an alkoxysilane having an acidic group such as, for example, a carboxyl group, a sulfonic acid group, a thiol group, and the like. Specifically, (3-Mercaptopropyl) trimethoxysilane may be used, but is not limited thereto.

상기 친수성 성질은 친수성기, 예를 들면 히드록시기, 카르복시기, 아미노기, 카르보닐기, 설프히드릴기, 포스페이트기, 티올기, 암모늄기, 에스터기, 이미드기, 티오이미드기, 케토기, 에터기, 인덴기, 설포닐기, 폴리에틸렌글리콜기 등을 갖는 알콕시실란과 반응시켜 갖도록 할 수 있다. 구체적으로는, N-[3-(Trimethoxysilyl)propyl]ethylenediamine, N1-(3-Trimethoxysilylpropyl)diethylenetriamine, (3-Aminopropyl)trimethoxysilane, (3-Mercaptopropyl) trimethoxysilane, Trimethoxy[3-(methylamino)propyl]silane, 3-(2-Aminoethylamino)propyldimethoxymethylsilane 등을 사용할 수 있으나, 이에 제한되는 것은 아니다.The hydrophilic property is a hydrophilic group such as hydroxy group, carboxy group, amino group, carbonyl group, sulfhydryl group, phosphate group, thiol group, ammonium group, ester group, imide group, thiimide group, keto group, ether group, indene group, sulfo It may be made to react with the alkoxysilane which has a silyl group, a polyethyleneglycol group, etc. Specifically, N- [3- (Trimethoxysilyl) propyl] ethylenediamine, N1- (3-Trimethoxysilylpropyl) diethylenetriamine, (3-Aminopropyl) trimethoxysilane, (3-Mercaptopropyl) trimethoxysilane, Trimethoxy [3- (methylamino) propyl] silane, 3- (2-Aminoethylamino) propyldimethoxymethylsilane may be used, but is not limited thereto.

상기 소수성 성질은 소수성 치환기, 예를 들면 치환 또는 비치환된 C1 내지 C30의 알킬기, 치환 또는 비치환된 C3 내지 C30의 사이클로알킬기, 치환 또는 비치환된 C6 내지 C30의 아릴기, 치환 또는 비치환된 C2 내지 C30의 헤테로아릴기, 할로겐기, C1 내지 C30의 에스테르기, 할로겐 함유기 등을 갖는 알콕시실란과 반응시켜 갖도록 할 수 있다. 구체적으로는, Trimethoxy(octadecyl)silane, Trimethoxy-n-octylsilane, Trimethoxy(propyl)silane, Isobutyl(trimethoxy)silane, Trimethoxy(7-octen-1-yl)silane, Trimethoxy(3,3,3-trifluoropropyl)silane, Trimethoxy(2-phenylethyl)silane, Vinyltrimethoxysilane, Cyanomethyl, 3-(trimethoxysilyl)propyl] trithiocarbonate, (3-Bromopropyl)trimethoxysilane 등을 사용할 수 있으나, 이에 제한되는 것은 아니다.The hydrophobic nature may include hydrophobic substituents such as substituted or unsubstituted C1 to C30 alkyl groups, substituted or unsubstituted C3 to C30 cycloalkyl groups, substituted or unsubstituted C6 to C30 aryl groups, substituted or unsubstituted It can be made to react with the alkoxysilane which has a C2-C30 heteroaryl group, a halogen group, C1-C30 ester group, a halogen containing group, etc. Specifically, Trimethoxy (octadecyl) silane, Trimethoxy-n-octylsilane, Trimethoxy (propyl) silane, Isobutyl (trimethoxy) silane, Trimethoxy (7-octen-1-yl) silane, Trimethoxy (3,3,3-trifluoropropyl) Silane, Trimethoxy (2-phenylethyl) silane, Vinyltrimethoxysilane, Cyanomethyl, 3- (trimethoxysilyl) propyl] trithiocarbonate, (3-Bromopropyl) trimethoxysilane, etc. may be used, but is not limited thereto.

그 외에 상기 표면 개질을 통해 난용성(소수성) 항체(antibody), 사이토카인(cytokine) 또는 물질과의 결합력 증진을 위해 기공 내부에는 소수성 치환기가 존재하고, 사용, 제형화의 용이성 등의 측면에서 입자의 표면은 친수성 치환기가 존재하도록 하는 등의 처리도 가능하고, 표면에 다른 항체(antibody), 사이토카인(cytokine) 또는 물질을 결합시키기 위한 치환기가 존재할 수도 있다.In addition, hydrophobic substituents are present in the pores to enhance binding to poorly soluble (hydrophobic) antibodies, cytokines or substances through the surface modification, and particles in terms of ease of use and formulation. The surface of may also be treated such that a hydrophilic substituent is present, and a substituent may be present on the surface to bind other antibodies, cytokines, or substances.

또한, 상기 표면 개질은 복합적으로 수행될 수도 있다. 예를 들어, 외부 표면 또는 기공 내부에 2회 이상의 표면 개질이 수행될 수도 있다. 구체적인 예를 들자면, 아미노기가 도입된 실리카 입자에 카르복실기를 포함하는 화합물을 아미드 결합으로 결합시켜 양전하로 대전된 입자를 다른 표면특성을 가지게 변화시킬 수 있으나, 이에 제한되는 것은 아니다.In addition, the surface modification may be carried out in combination. For example, two or more surface modifications may be performed on the outer surface or inside the pores. As a specific example, a compound including a carboxyl group may be bonded to silica particles into which amino groups are introduced by amide bonds to change the positively charged particles to have different surface properties, but is not limited thereto.

상기 다공성 실리카 입자의 알콕시실란과의 반응은 예를 들면 가열 하에 수행될 수 있고, 가열은 예를 들면 80℃ 내지 180℃, 예를 들어 상기 범위 내에서 80℃ 내지 160℃, 80℃ 내지 150℃, 100℃ 내지 160℃, 100℃ 내지 150℃, 110℃ 내지 150℃ 등으로 수행될 수 있으나, 이에 제한되는 것은 아니다.The reaction of the porous silica particles with the alkoxysilane can be carried out, for example, under heating, and the heating is for example from 80 ° C. to 180 ° C., for example from 80 ° C. to 160 ° C., from 80 ° C. to 150 ° C. within the above range. , 100 ° C. to 160 ° C., 100 ° C. to 150 ° C., 110 ° C. to 150 ° C., etc., but is not limited thereto.

상기 다공성 실리카 입자의 알콕시실란과의 반응은 예를 들면 4시간 내지 20시간, 예를 들어 상기 범위 내에서 4시간 내지 18시간, 4시간 내지 16시간, 6시간 내지 18시간, 6시간 내지 16시간, 8시간 내지 18시간, 8시간 내지 16시간, 8시간 내지 14시간, 10시간 내지 14시간 등으로 수행될 수 있으나, 이에 제한되는 것은 아니다.The reaction of the porous silica particles with the alkoxysilane is, for example, 4 hours to 20 hours, for example, 4 hours to 18 hours, 4 hours to 16 hours, 6 hours to 18 hours, 6 hours to 16 hours within the above range. , 8 hours to 18 hours, 8 hours to 16 hours, 8 hours to 14 hours, 10 hours to 14 hours, etc., but is not limited thereto.

상기 반응 온도, 시간, 그리고 표면개질에 사용되는 화합물의 양 등은 표면개질하고자 하는 정도에 따라 선택될 수 있는 것으로서, 항체(antibody), 사이토카인(cytokine) 또는 물질들의 친수성, 소수성, 전하 정도에 따라 반응 조건을 달리하여 다공성 실리카 입자의 친수성, 소수성, 전하 정도를 조절함으로써, 항체(antibody), 사이토카인(cytokine) 또는 물질들의 방출 속도를 조절할 수 있다. 예를 들면, 항체(antibody), 사이토카인(cytokine) 또는 물질들이 중성의 pH에서 강한 음전하를 띠는 경우에는 다공성 실리카 입자가 강한 양전하를 띠도록 하기 위해, 반응 온도를 높이거나 반응 시간을 길게 할 수 있으며, 화합물 처리량을 늘릴 수 있으나, 이에 제한되는 것은 아니다.The reaction temperature, time, and the amount of the compound used for surface modification may be selected according to the degree to which the surface is to be modified, and the degree of hydrophilicity, hydrophobicity, and charge of an antibody, cytokine, or materials may be selected. Accordingly, by controlling the hydrophilicity, hydrophobicity, and charge of the porous silica particles by varying the reaction conditions, the release rate of the antibody, cytokine, or substances can be controlled. For example, if antibodies, cytokines or substances have a strong negative charge at neutral pH, the reaction temperature may be increased or the reaction time may be increased in order for the porous silica particles to have a strong positive charge. It is possible to increase the compound throughput, but is not limited thereto.

또한, 본 발명의 다공성 실리카 입자는 예를 들면 소기공의 입자 제조, 기공 확장, 표면 개질, 기공 내부 개질 공정을 거쳐 제조된 것일 수도 있다.In addition, the porous silica particles of the present invention may be produced through, for example, the preparation of small pores, pore expansion, surface modification, and internal pore modification.

상기 소기공의 입자 제조 및 기공 확장 공정은 전술한 바의 공정에 의할 수 있으며, 소기공의 입자 제조 이후, 그리고 기공 확장 공정 이후에 세척 및 건조 공정을 수행할 수 있다.The small pore particle production and pore expansion process may be based on the above-described process, and the washing and drying process may be performed after the small pore particle production and after the pore expansion process.

필요에 따라 세척 전에 미반응 물질의 분리가 선행될 수 있고, 미반응 물질의 분리는 예를 들면 원심분리로 상등액을 분리하여 수행될 수 있다.If necessary, separation of the unreacted material may be preceded before washing, and separation of the unreacted material may be performed by separating the supernatant, for example, by centrifugation.

상기 원심분리는 예를 들면 6,000 내지 10,000rpm으로 수행될 수 있으며, 그 시간은 예를 들면 3분 내지 60분, 구체적으로, 상기 범위 내에서 3분 내지 30분, 3분 내지 30분, 5분 내지 30분 등으로 수행될 수 있으나, 이에 제한되는 것은 아니다.The centrifugation may be performed, for example, at 6,000 to 10,000 rpm, and the time may be, for example, 3 to 60 minutes, specifically, 3 to 30 minutes, 3 to 30 minutes, and 5 minutes within the above range. To 30 minutes, etc., but is not limited thereto.

상기 소기공의 입자 제조 이후의 세척은 앞서 예시한 범위 내의 방법/조건으로 수행될 수 있으나, 이에 제한되는 것은 아니다.The washing after the preparation of the particles of the small pores may be performed by a method / condition within the above-described range, but is not limited thereto.

상기 기공 확장 이후의 세척은 앞서 예시보다는 보다 완화된 조건으로 수행할 수 있다. 예를 들면, 세척을 3회 이내 수행할 수 있으나, 이에 제한되는 것은 아니다.The washing after the pore expansion may be performed under more relaxed conditions than the above example. For example, washing may be performed within three times, but is not limited thereto.

상기 표면 개질과 기공 내부 개질은 각각 전술한 바의 공정에 의할 수 있으며, 표면 개질과 기공 내부 개질의 순서로 공정이 수행될 수 있고, 상기 두 공정 사이에 입자의 세척 공정이 추가로 수행될 수 있다.The surface modification and internal pore modification may be by the processes described above, respectively, the process may be performed in the order of surface modification and internal pore modification, and the washing process of the particles may be further performed between the two processes. Can be.

상기 소기공의 입자 제조 및 기공 확장 이후에 세척을 보다 완화된 조건으로 수행하는 경우 기공 내부에 입자 제조, 기공 확장에 사용된 계면활성제 등의 반응액이 채워져 있어 표면 개질시에 기공 내부는 개질되지 않고 표면만 개질될 수 있다. 그러고 나서 입자를 세척하면 기공 내부의 반응액이 제거될 수 있다.When the washing is performed in a more relaxed condition after the particle production and pore expansion of the small pores, the reaction solution such as a surfactant used for particle production and pore expansion is filled in the pores so that the inside of the pores is not modified during surface modification. Only the surface can be modified. Then, washing the particles may remove the reaction solution in the pores.

상기 표면 개질과 기공 내부 개질 공정 사이의 입자 세척은 물 및/또는 유기용매로 할 수 있고, 구체적으로는 용매별로 녹일 수 있는 물질이 상이하므로 물과 유기용매를 1회 또는 수회 번갈아 사용할 수 있으며, 물 또는 유기용매 단독으로도 1회 또는 수회 세척할 수 있다. 상기 수회는 예를 들면 2회 이상, 10회 이하, 구체적으로, 3회 이상 10회 이하, 4회 이상 8회 이하, 4회 이상 6회 이하 등일 수 있다.Particle washing between the surface modification and the internal pore reforming process may be water and / or an organic solvent, and in particular, water and an organic solvent may be alternately used once or several times because different materials may be dissolved for each solvent. Water or organic solvents alone may be washed once or several times. The number of times may be, for example, two or more, ten or less, specifically, three or more and ten or less, four or more and eight or less, four or more and six or less.

상기 세척은 원심분리 하에 수행될 수 있으며, 예를 들면 6,000 내지 10,000rpm으로 수행될 수 있으며, 그 시간은 예를 들면 3분 내지 60분, 구체적으로, 상기 범위 내에서 3분 내지 30분, 3분 내지 30분, 5분 내지 30분 등으로 수행될 수 있으나, 이에 제한되는 것은 아니다.The washing may be carried out under centrifugation, for example at 6,000 to 10,000 rpm, the time being for example 3 to 60 minutes, specifically 3 to 30 minutes, 3 within the above range. It may be performed in minutes to 30 minutes, 5 minutes to 30 minutes and the like, but is not limited thereto.

상기 세척은 원심분리를 하지 않고, 필터로 입자를 걸러내어 수행될 수도 있다. 필터는 다공성 실리카 입자의 직경 이하의 기공을 가지는 것일 수 있다. 반응액을 그러한 필터로 걸러내면 입자만이 필터 위에 남고, 그 필터 위에 물 및/또는 유기용매를 부어 세척할 수 있다.The washing may be performed by filtering out particles with a filter without centrifugation. The filter may have pores less than or equal to the diameter of the porous silica particles. Filtering the reaction liquid with such a filter leaves only particles on the filter, which can be washed by pouring water and / or an organic solvent on the filter.

상기 세척 시에 물과 유기용매를 1회 또는 수회 번갈아 사용할 수 있으며, 물 또는 유기용매 단독으로도 1회 또는 수회 세척할 수 있다. 상기 수회는 예를 들면 2회 이상, 10회 이하, 구체적으로, 3회 이상 10회 이하, 4회 이상 8회 이하, 4회 이상 6회 이하 등일 수 있다.In the washing, water and an organic solvent may be used alternately once or several times, and may be washed once or several times even with water or an organic solvent alone. The number of times may be, for example, two or more, ten or less, specifically, three or more and ten or less, four or more and eight or less, four or more and six or less.

상기 건조는 예를 들면 20℃ 내지 100℃로 수행될 수 있으나, 이에 제한되는 것은 아니고, 진공 상태에서 수행될 수도 있다.For example, the drying may be performed at 20 ° C. to 100 ° C., but is not limited thereto, and may be performed in a vacuum state.

항체(antibody) 또는 사이토카인(cytokine)은 다공성 실리카 입자의 표면 및/또는 기공 내부에 담지될 수 있고, 담지는 예를 들면 용매 중의 다공성 실리카 입자와 항체(antibody) 또는 사이토카인(cytokine)을 혼합하여 수행될 수 있다.The antibody or cytokine may be supported on the surface and / or inside the pores of the porous silica particles, and the support may be mixed with, for example, the porous silica particles and the antibody or cytokine in a solvent. Can be performed.

상기 용매는 물 및/또는 유기용매일 수 있으며, 유기용매는 예를 들면 1,4-디옥산 등의 에테르류(특히 고리형상 에테르류); 클로로포름, 염화메틸렌, 4염화탄소, 1,2-디클로로에탄, 디클로로에틸렌, 트리클로로에틸렌, 퍼클로로에틸렌, 디클로로프로판, 염화아밀, 1,2-디브로모에탄 등의 할로겐화 탄화수소류; 아세톤, 메틸이소부틸케톤, 시클로헥산온 등의 케톤류; 벤젠, 톨루엔, 크실렌 등의 탄소계 방향족류; N,N-디메틸포름아미드, N,N-디부틸포름아미드, N,N-디메틸아세트아미드, N-메틸피롤리돈 등의 알킬아미드류; 메탄올, 에탄올, 프로판올, 부탄올 등의 알코올류; 등을 사용할 수 있다.The solvent may be water and / or an organic solvent, and the organic solvent may be, for example, ethers such as 1,4-dioxane (particularly cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Ketones such as acetone, methyl isobutyl ketone and cyclohexanone; Carbon-based aromatics such as benzene, toluene and xylene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; Etc. can be used.

또한, 상기 용매로 PBS(phosphate buffered saline solution), SBF(Simulated Body Fluid), Borate-buffered saline, Tris-buffered saline 등을 사용할 수도 있다. In addition, PBS (phosphate buffered saline solution), SBF (Simulated Body Fluid), Borate-buffered saline, Tris-buffered saline may be used as the solvent.

상기 다공성 실리카 입자와 본 발명의 올리고뉴클레오티드의 비율은 특별히 한정되지 않으며, 예를 들면 중량비가 1: 0.05 내지 0.8, 예를 들어 상기 범위 내에서 1: 0.05 내지 0.7, 1:0.05 내지 0.6, 1: 0.1 내지 0.8, 1: 0.1 내지 0.6, 1: 0.2 내지 0.8, 1: 0.2 내지 0.6 등일 수 있다.The ratio of the porous silica particles and the oligonucleotide of the present invention is not particularly limited, for example, the weight ratio is 1: 0.05 to 0.8, for example within the above range 1: 0.05 to 0.7, 1: 0.05 to 0.6, 1: 0.1 to 0.8, 1: 0.1 to 0.6, 1: 0.2 to 0.8, 1: 0.2 to 0.6, and the like.

상기 다공성 실리카 입자에 담지된 항체(antibody) 또는 사이토카인(cytokine)은 연장된 시간에 걸쳐 점진적으로 방출될 수 있다. 이와 같이 느린 방출은 연속성 또는 비연속성, 선형 또는 비선형일 수 있으며, 다공성 실리카 입자의 특징 및/또는 그와 항체(antibody) 또는 사이토카인(cytokine)과의 상호작용에 기인하여 달라질 수 있다.Antibodies or cytokines supported on the porous silica particles may be gradually released over an extended time. Such slow release may be continuous or discontinuous, linear or nonlinear, and may vary due to the characteristics of the porous silica particles and / or their interaction with antibodies or cytokines.

상기 다공성 실리카 입자에 담지된 항체(antibody) 또는 사이토카인(cytokine)은 다공성 실리카 입자가 생분해되면서 방출되는데, 본 발명에 따른 다공성 실리카 입자는 서서히 분해되어 담지된 항체(antibody) 또는 사이토카인(cytokine)이 서방적으로 방출되도록 할 수 있다. 이는 예를 들면 다공성 실리카 입자의 표면적, 입경, 기공 직경, 표면 및/또는 기공 내부의 치환기, 표면의 치밀함 정도 등을 조절함으로써 조절될 수 있으나, 이에 제한되는 것은 아니다.Antibodies or cytokines supported on the porous silica particles are released as the porous silica particles are biodegraded, and the porous silica particles according to the present invention are gradually decomposed and supported on an antibody or cytokine. This can be released slowly. This may be controlled by, for example, adjusting the surface area, particle diameter, pore diameter, substituents on the surface and / or pores, degree of compactness of the porous silica particles, and the like, but are not limited thereto.

또한, 상기 다공성 실리카 입자에 담지된 항체(antibody) 또는 사이토카인(cytokine)은 다공성 실리카 입자로부터 이탈되어 확산되면서도 방출될 수 있고, 이는 다공성 실리카 입자와 항체(antibody) 또는 사이토카인(cytokine), 항체(antibody) 또는 사이토카인(cytokine)의 방출 환경과의 관계에 영향을 받는 것인 바, 이를 조절하여 항체(antibody) 또는 사이토카인(cytokine)의 방출을 조절할 수 있다. 예를 들면 표면개질에 의해 다공성 실리카 입자의 항체(antibody) 또는 사이토카인(cytokine)과의 결합력을 강화 또는 약화시킴으로써 조절할 수 있다.In addition, the antibody or cytokine supported on the porous silica particles may be released while being diffused from the porous silica particles, which may be released. It is affected by the relationship with the release environment of the antibody or cytokine (cytokine), it can be controlled by controlling the release of the antibody (cytokine) or cytokine (cytokine). For example, it can be controlled by strengthening or weakening the binding strength of the porous silica particles to the antibody or cytokine by surface modification.

보다 구체적인 예를 들자면, 담지된 항체(antibody), 사이토카인(cytokine) 또는 물질이 난용성(소수성)인 경우에는 입자의 표면 및/또는 기공 내부가 소수성 치환기를 가져 다공성 실리카 입자와 항체(antibody), 사이토카인(cytokine) 또는 물질과의 결합력이 증가된 것일 수 있고, 이에 의해 항체(antibody), 사이토카인(cytokine) 또는 물질이 서방적으로 방출될 수 있다. 이는 예를 들면 다공성 실리카 입자가 소수성 치환기를 갖는 알콕시실란으로 표면개질된 것일 수 있다.More specifically, for example, when a supported antibody, cytokine, or substance is poorly soluble (hydrophobic), the surface of the particles and / or the inside of the pores may have a hydrophobic substituent, so that the porous silica particles and the antibody In addition, the cytokine (cytokine) or the binding force with the substance may be increased, whereby the antibody (cytokine) or the substance (cytokine) or the substance may be released in a sustained manner. This may be, for example, the surface-modified porous silica particles with an alkoxysilane having a hydrophobic substituent.

본 명세서에서 "난용성"은 (물에 대해) 불용성(insoluble), 실질적으로 불용성(practically insoluble) 또는 극히 약간의 가용성(only slightly soluble)인 것을 포함하는 의미로서 이는 "Pharmaceutical Science" 18 th Edition(U.S.P., Remington, Mack Publishing Company 발행)에 정의되어 있는 용어이다.As used herein, "poorly soluble" means to be insoluble (practically insoluble) or only slightly soluble (with respect to water), which means "Pharmaceutical Science" 18 th Edition ( USP, Remington, Mack Publishing Company).

상기 난용성 물질은 예를 들면 1기압, 25℃에서 수용해도가 10g/L 미만, 구체적으로 5g/L 미만, 보다 구체적으로 1g/L 미만일 수 있으나, 이에 제한되는 것은 아니다.The poorly water-soluble material may be, for example, water solubility of less than 10 g / L, specifically less than 5 g / L, more specifically less than 1 g / L at 1 atmosphere and 25 ° C., but is not limited thereto.

담지된 항체(antibody), 사이토카인(cytokine) 또는 물질이 수용성(친수성)인 경우에는 입자의 표면 및/또는 기공 내부가 친수성 치환기를 가져 다공성 실리카 입자와 항체(antibody), 사이토카인(cytokine) 또는 물질과의 결합력이 증가된 것일 수 있고, 이에 의해 항체(antibody), 사이토카인(cytokine) 또는 물질이 서방적으로 방출될 수 있다. 이는 예를 들면 다공성 실리카 입자가 친수성 치환기를 갖는 알콕시실란으로 표면개질된 것일 수 있다.If the supported antibody, cytokine or substance is water soluble (hydrophilic), the surface and / or the inside of the pores have a hydrophilic substituent which has a porous silica particle and an antibody, cytokine or The binding force with the substance may be increased, whereby an antibody, cytokine or substance may be released in a sustained manner. This may be, for example, the surface of the porous silica particles modified with an alkoxysilane having a hydrophilic substituent.

수용성 물질은 예를 들면 1기압, 25℃에서 수용해도가 10g/L 이상일 수 있으나, 이에 제한되는 것은 아니다.For example, the water-soluble substance may have a water solubility of 10 g / L or more at 1 atmosphere and 25 ° C., but is not limited thereto.

담지된 항체(antibody), 사이토카인(cytokine) 또는 물질이 전하를 띠는 경우에는 입자의 표면 및/또는 기공 내부가 그와 반대 전하로 대전되어 다공성 실리카 입자와 항체(antibody), 사이토카인(cytokine) 또는 물질과의 결합력이 증가된 것일 수 있고, 이에 의해 항체(antibody), 사이토카인(cytokine) 또는 물질이 서방적으로 방출될 수 있다. 이는 예를 들면 다공성 실리카 입자가 산성기 또는 염기성기를 갖는 알콕시실란으로 표면개질된 것일 수 있다.When a supported antibody, cytokine, or substance is charged, the surface of the particles and / or the interior of the pores are charged with opposite charges so that the porous silica particles, antibodies, and cytokines ), Or the binding force with the substance may be increased, whereby an antibody, cytokine or substance may be released in a sustained manner. This may be, for example, the surface-modified porous silica particles with an alkoxysilane having an acidic group or a basic group.

구체적으로, 항체(antibody), 사이토카인(cytokine) 또는 물질이 중성의 pH에서 양전하를 띠는 것이라면 입자의 표면 및/또는 기공 내부가 중성의 pH에서 음전하로 대전되는 것일 수 있고, 이에 의해 다공성 실리카 입자와 항체(antibody), 사이토카인(cytokine) 또는 물질과의 결합력이 증가되어 항체(antibody), 사이토카인(cytokine) 또는 물질이 서방적으로 방출될 수 있다. 이는 예를 들면 다공성 실리카 입자가 카르복시기(-COOH), 술폰산기(-SO 3H) 등의 산성기를 갖는 알콕시실란으로 표면개질된 것일 수 있다.Specifically, if an antibody, cytokine, or substance is positively charged at neutral pH, the surface of the particles and / or the interior of the pores may be negatively charged at neutral pH, whereby porous silica The binding force between the particle and the antibody, cytokine, or substance may be increased, and thus, the antibody, cytokine, or substance may be released in a sustained manner. For example, the porous silica particles may be surface-modified with an alkoxysilane having an acidic group such as a carboxyl group (-COOH) and a sulfonic acid group (-SO 3 H).

또한, 항체(antibody), 사이토카인(cytokine) 또는 물질이 중성의 pH에서 음전하를 띠는 것이라면 입자의 표면 및/또는 기공 내부가 양전하로 대전되는 것일 수 있고, 이에 의해 다공성 실리카 입자와 항체(antibody), 사이토카인(cytokine) 또는 물질과의 결합력이 증가되어 항체(antibody), 사이토카인(cytokine) 또는 물질이 서방적으로 방출될 수 있다. 이는 예를 들면 다공성 실리카 입자가 아미노기, 그 외 질소함유기 등의 염기성기를 갖는 알콕시실란으로 표면개질된 것일 수 있다.In addition, if an antibody, cytokine or substance is negatively charged at neutral pH, the surface of the particles and / or the inside of the pores may be positively charged, whereby the porous silica particles and the antibody (antibody) ), Cytokines (cytokine) or the binding force with the substance is increased, the antibody (cytokine) or the substance can be released in a sustained release. For example, the porous silica particles may be surface-modified with an alkoxysilane having a basic group such as an amino group or another nitrogen-containing group.

항체(antibody), 사이토카인(cytokine) 또는 물질은 필요한 치료 유형, 방출 환경, 사용되는 다공성 실리카 입자에 의존하여 예를 들면 7일 내지 1년 또는 그 이상의 기간 동안 방출될 수 있다. Antibodies, cytokines or substances may be released for a period of, for example, 7 days to 1 year or more, depending on the type of treatment required, the release environment, and the porous silica particles used.

또한, 본 발명의 다공성 실리카 입자는 생분해성으로서 100% 분해될 수 있으므로, 이에 담지된 항체(antibody), 사이토카인(cytokine) 또는 물질은 100% 방출될 수 있다.In addition, since the porous silica particles of the present invention are 100% degradable as biodegradable, the antibody, cytokine or substance supported thereon may be released 100%.

본 발명은 상술한 전달체를 포함하는 면역요법용 조성물을 제공한다.The present invention provides a composition for immunotherapy comprising the carrier described above.

면역요법(immunotherapy)이란 면역학적 방법으로 환자치료를 기획하는 것을 말하는 것으로서, 특정항원에 대한　면역 응답에만 관여하는 특이적 면역요법과 특정항원에 한정하지 않고　면역계 전체에 효과를 미치는 비특이적 면역요법이 있다. 상기 특이적 요법으로서 항체를 투여하는 방법이, 상기 비특이적 방법으로서 사이토카인을 투여하는 방법이 대표적인 예라고 할 수 있다.Immunotherapy refers to the planning of patient treatment by immunological methods, which include specific immunotherapy involving only the immune response to specific antigens and nonspecific immunotherapy that is effective for the entire immune system but not limited to specific antigens. . The method of administering an antibody as said specific therapy is the typical example of the method of administering cytokines as said non-specific method.

본 발명의 면역요법용 조성물은 상술한 항체 또는 사이토카인을 포함하는 면역반응 물질을 담지한 다공성 실리카 입자를 포함하고 있어, 담지한 면역반응 물질을 체내에 안정적이고 서방적으로 전달하며, 면역반응 물질 본래의 부작용을 감소시키는 효과적 우수성을 가짐에 따라, 면역요법에 사용되어 각종 암 또는 면역질환 등의 예방 또는 치료에 탁월한 효과를 발휘할 수 있다.The immunotherapeutic composition of the present invention comprises porous silica particles carrying an immunoreactive substance containing the above-described antibody or cytokine, and delivers the supported immunoreactive substance to the body stably and sustainably, and immunoreactive substance. As it has an effective superiority to reduce the inherent side effects, it can be used in immunotherapy to exert an excellent effect in the prevention or treatment of various cancers or immune diseases.

상기 암은 방광암, 유방암, 결장암, 신장암, 간암, 폐암, 난소암, 전립선암, 췌장암, 위암, 경부암, 갑상선암 및 편평세포암종을 포함하는 피부암을 포함하는 암종; 백혈병, 급성 림프성 백혈병, 급성 림프구성 백혈병, B-세포 림프종, T-세포 림프종, 호지킨스 림프종, 비-호지킨스 림프종, 모발상 세포 림프종 및 버켓 림프종을 포함하는 림프계 조혈모 종양; 급성 및 만성 골수성 백혈병 및 전골수구 백혈병을 포함하는 골수형의 조혈 종양; 섬유 육종 및 횡문 근육종을 포함하는 간엽 유래 종양; 흑색종, 정상피종, 기형암종, 신경모세포종 및 신경교종을 포함하는 다른 종양; 성상세포종, 신경아세포종, 신경교종 및 신경초종을 포함하는 중추 및 말초 신경 시스템의 종양; 섬유육종, 횡문근육종 및 골육종을 포함하는 중간엽 유래 종양; 및 흑색종, 색소성 건피증, 각화극세포종, 정상피종, 갑상선 여포상암 및 기형 암종을 포함하는 기타 종양으로 이루어진 군으로부터 선택되는 어느 하나일 수 있다.The cancer includes carcinoma including skin cancer including bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, gastric cancer, cervical cancer, thyroid cancer and squamous cell carcinoma; Lymphoid hematopoietic tumors including leukemia, acute lymphocytic leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma and Bucket lymphoma; Myeloid hematopoietic tumors, including acute and chronic myeloid leukemia and promyelocytic leukemia; Mesenchymal-derived tumors including fibrosarcoma and rhabdomyosarcoma; Other tumors including melanoma, normal carcinoma, teratocarcinoma, neuroblastoma and glioma; Tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma and schwannoma; Mesenchymal-derived tumors including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; And other tumors, including melanoma, pigmentary dry skin disease, keratinocytes, normal adenomas, thyroid follicular cancers, and malformed carcinomas.

상기 면역질환은 포유류 면역계의 구성성분들이 포유류의 병리상태를 야기하거나, 매개하거나 또는 기타 공헌하는 질환을 의미하는 것으로서, 면역 반응의 자극 또는 중단이 그 질병의 진행에 보상적인 효과를 갖는 질환을 모두 포함할 수 있으며, 자가면역질환, 감염질환, 염증성질환 또는 세포, 조직 또는 기관의 이식거부(transplantation rejection)질환 등을 모두 포함할 수 있으며, 구체적으로는, 베체트병, 다발성 근육염/피부 근육염, 자가면역 혈구감소증, 자가면역 심근염, 아토피피부염, 천식, 일차성간경변, 피부근염, 굿파이처 증후군, 자가면역 뇌수막염, 비만, 쇼그렌 증후군, 강직성 척수염, 전신 홍반성 루프스, 애디슨병, 원형 탈모증, 자가면역성 간염, 자가면역성 이하선염, 크론병, 인슐린 의존성 당뇨병, 이영양성 수포성 표피박리증, 부고환염, 사구체 신염, 그레이브스병, 길랑바레 증후군, 하시모토병, 용혈성 빈혈, 다발성 경화증, 중증 근무력증, 심상천포창, 건선, 류마티스열, 류마티스 관절염, 유육종증, 피부 경화증, 척추관절증, 갑상선염, 혈관염, 백반증, 점액수종, 악성빈혈, 궤양성 대장염 등을 포함할 수 있다.The immune disease refers to a disease in which components of the mammalian immune system cause, mediate or otherwise contribute to a mammalian pathology, and all diseases in which stimulation or interruption of an immune response has a compensatory effect on the progression of the disease. And may include all autoimmune diseases, infectious diseases, inflammatory diseases or transplant rejection diseases of cells, tissues or organs, and the like, specifically, Behcet's disease, multiple myositis / skin myositis, autologous diseases Immunocytopenia, Autoimmune myocarditis, Atopic dermatitis, Asthma, Primary cirrhosis, Dermatitis, Goodfiction syndrome, Autoimmune meningitis, Obesity, Sjogren's syndrome, Ankylosing myelitis, Systemic lupus erythematosus, Addison's disease, Alopecia areata, Autoimmunity Hepatitis, autoimmune mumps, Crohn's disease, insulin dependent diabetes mellitus, dystrophic bullous epidermal detachment, epididymitis, Glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, multiple sclerosis, myasthenia gravis, vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, Pernicious anemia, ulcerative colitis, and the like.

상기 면역질환의 일 종류로서 자가면역질환은 그 종류에 제한이 없으나, 크론병, 홍반병, 아토피, 류마티스 관절염, 하시모토 갑상선염, 악성빈혈, 에디슨병, 제1형 당뇨, 루프스, 만성피로증후군, 섬유근육통, 갑상선기능저하증과 항진증, 경피증, 베체트병, 염증성 장질환, 다발성 경화증, 중증 근무력증, 메니에르 증후군(Meniere's syndrome), 길리안-바레 증후근(Guilian-Barre syndrome), 쇼그렌 증후군(Sjogren's syndrome), 백반증, 자궁내막증, 건선, 전신성 경피증, 천식 또는 궤양성 대장염일 수 있다.Autoimmune diseases as one type of the immune disease is not limited to the type, Crohn's disease, erythema, atopy, rheumatoid arthritis, Hashimoto's thyroiditis, pernicious anemia, Edison's disease, type 1 diabetes, Lupus, chronic fatigue syndrome, fiber Myalgia, hypothyroidism and hyperplasia, scleroderma, Behcet's disease, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, Meniere's syndrome, Guilian-Barre syndrome, Sjogren's syndrome, vitiligo Endometriosis, psoriasis, systemic scleroderma, asthma or ulcerative colitis.

상기 면역질환의 일 종류로서 감염 질환은 박테리아, 기생충, 진균류, 바이러스, 비로이드 및 프리온에 의해 초래되는 감염 질환일 수 있다.As one type of immune disease, the infectious disease may be an infectious disease caused by bacteria, parasites, fungi, viruses, viroids and prions.

상기 바이러스는 장내바이러스, 로터바이러스, 아데노바이러스 및 간염 바이러스일 수 있고, 상기 바이러스 외에도 레트로바이러스과(예를 들어, HIV-I(또한 HTLV-III, LAV 또는 HTLV-III/LAV, 또는 HIV-III으로서도 언급됨)와 같은 인간 면역 결핍 바이러스; 및 HIV-LP와 같은 다른 분리물); 피코나바이러스과(Picornaviridae)(예를 들어, 폴리오 바이러스(polio virus), A형 간염 바이러스, 엔테로바이러스, 인간 콕사키바이러스(human Coxsackie virus), 리노바이러스(rhinovirus), 에코바이러스(echovirus)); 칼시바이러스과(Calciviridae)(예를 들어, 위장염의 원인이 되는 종); 토가바이러스과(Togaviridae)(예를 들어, 말 뇌염 바이러스(equine encephalitis virus), 풍진 바이러스(rubella virus)); 플라비비아러스과(Flaviviridae)(예를 들어, 뎅기 바이러스(dengue virus), 뇌염 바이러스(encephalitis virus), 황열 바이러스(yellow fever virus)); 코로나바이러스과(Coronaviridae)(예를 들어, 코로나바이러스(coronavirus)); 랍도바이러스과(Rhabdoviridae)(예를 들어, 수포성구내염바이러스(vesicular stomatitis virus), 광견병 바이러스(rabies virus)); 필로바이러스과(예를 들어, 에볼라 바이러스(ebola virus)); 파라믹소바이러스과(Paramyxoviridae)(예를 들어, 파라인플루엔자 바이러스(parainfluenza virus), 볼거리 바이러스(mumps virus), 홍역 바이러스(measles virus), 호흡기세포융합 바이러스(respiratory syncytial virus)); 오소믹소바이러스과(Orthomyxoviridae)(예를 들어, 인플루엔자 바이러스(influenza virus)); 부니아바이러스(Bunyaviridae)(예를 들어, 한탄 바이러스(Hantaan virus), 부니아 바이러스(bunya virus), 플레보바이러스(phlebovirus) 및 나이로 바이러스(Nairo virus)); 아레나바이러스과(출혈열 바이러스(hemorrhagic fever virus)); 레오바이러스과(Reoviridae)(예를 들어, 레오바이러스(reovirus), 오르비바이러스(orbivirus) 및 로타바이러스(rotavirus)); 보르나바이러스과(Bornaviridae); 헤파드나바이러스과(Hepadnaviridae)(B형 간염 바이러스(Hepatitis B virus)); 파보바이러스과(Parvoviridae)(파보바이러스(Parvoviridae)); 파포바바이러스과(Papovaviridae)(유두종 바이러스(papilloma virus), 폴리오마 바이러스(polyoma virus)); 아데노바이러스과(Adenoviridae)(대부분의 아데노바이러스(Adenovirus)); 헤르페스바이러스과(Herpesviridae)(단순 포진 바이러스(herpes simplex virus, HSV) 1 및 2, 수두 대상 포진 바이러스(varicella zoster virus), 사이토메갈로바이러스(cytomegalovirus, CMV), 헤르페스 바이러스(herpes virus); 폭스바이러스과(Poxviridae)(바리올라 바이러스(variola virus), 우두 바이러스(vaccinia virus). 폭스 바이러스(pox virus)); 및 이리도바이러스과(Iridoviridae)(예를 들어, 아프리카 돼지 콜레라 바이러스(African swine fever virus)); 및 미분류 바이러스들(예를 들어 델타 간염(delta hepatitis)(B형 간염의 결핍된 부수체(satellite)인 것으로 생각됨), C형 간염(Hepatitis C); 노워크(Norwalk) 및 관련 바이러스, 및 아스트로 바이러스(astro virus)의 제제)를 포함하지만 이에 제한되지 않는다.The virus may be enterovirus, rotorvirus, adenovirus and hepatitis virus, and in addition to the virus, the retroviral family (e.g., HIV-I (also HTLV-III, LAV or HTLV-III / LAV, or HIV-III) Human immunodeficiency virus such as; and other isolates such as HIV-LP; Picornaviridae (eg, polio virus, hepatitis A virus, enterovirus, human Coxsackie virus, rhinovirus, echovirus); Calciviridae (eg, species responsible for gastroenteritis); Togaviridae (eg, equine encephalitis virus, rubella virus); Flaviviridae (eg, dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (eg, coronavirus); Rhabdoviridae (eg, vesicular stomatitis virus, rabies virus); Phyllovirus family (eg, ebola virus); Pararamyxoviridae (eg, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (eg, influenza virus); Bunyaviridae (eg, Hantaan virus, bunya virus, phlebovirus and Nairo virus); Arenaviraceae (hemorrhagic fever virus); Reoviridae (eg, reovirus, orbivirus and rotavirus); Bornaviridae; Hepadnaviridae (Hepatitis B virus); Parvooviridae (Parvoviridae); Papovaviridae (papilloma virus, polyoma virus); Adenoviridae (most Adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxviridae) (Variola virus, vaccinia virus. Pox virus); and Iridoviridae (e.g. African swine fever virus); and unclassified Viruses (for example delta hepatitis (presumed to be a satellite deficient in hepatitis B), hepatitis C); Norwalk and related viruses, and astroviruses ( preparations of astro virus), but are not limited thereto.

본 발명은 또한, 상술한 전달체를 포함하는 암 또는 면역질환의 예방 또는 치료용 약학적 조성물을 제공한다.The present invention also provides a pharmaceutical composition for the prophylaxis or treatment of cancer or immune disease, including the above-mentioned carrier.

본 발명의 조성물은 암 또는 면역질환의 예방 또는 치료 효과를 갖는 것으로서, 이는 담지된 항체 또는 사이토카인을 체내에 안정적으로 전달하고, 타겟에 서방적으로 방출하여, 암(또는 종양)의 성장을 억제하고, 전이를 억제함으로써 달성되는 효과일 수 있다.The composition of the present invention has a prophylactic or therapeutic effect of cancer or immune disease, which stably delivers the supported antibody or cytokine to the body and releases it to the target in a slow manner, thereby inhibiting the growth of cancer (or tumor). And the effect achieved by suppressing the transition.

본 발명의 조성물은 약학적으로 허용가능한 담체를 추가로 포함할 수 있으며, 담체와 함께 제제화될 수 있다. 본 발명에서 용어, "약학적으로 허용가능한 담체"란 생물체를 자극하지 않고 투여 화합물의 생물학적 활성 및 특성을 저해하지 않는 담체 또는 희석제를 말한다. 액상 용액으로 제제화되는 조성물에 있어서 허용되는 약제학적 담체로는, 멸균 및 생체에 적합한 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사용액, 덱스트로오스 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다.The composition of the present invention may further comprise a pharmaceutically acceptable carrier, and may be formulated with the carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound. Acceptable pharmaceutical carriers in compositions formulated in liquid solutions are sterile and biocompatible, which include saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

본 발명의 조성물은 본 발명의 전달체를 유효성분으로 포함하는 어떠한 제형으로도 적용가능하며, 경구용 또는 비경구용 제형으로 제조할 수 있다. 본 발명의 약학적 제형은 구강(oral), 직장(rectal), 비강(nasal), 국소(topical; 볼 및 혀 밑을 포함), 피하, 질(vaginal) 또는 비경구(parenteral; 근육내, 피하 및 정맥내를 포함) 투여에 적당한 것 또는 흡입(inhalation) 또는 주입(insufflation)에 의한 투여에 적당한 형태를 포함한다.The composition of the present invention is applicable to any formulation containing the carrier of the present invention as an active ingredient, and can be prepared in oral or parenteral formulations. Pharmaceutical formulations of the present invention may be oral, rectal, nasal, topical (including the cheek and sublingual), subcutaneous, vaginal or parenteral (intramuscular, subcutaneous). And forms suitable for administration by inhalation or insufflation.

본 발명의 조성물은 약학적으로 유효한 양으로 투여한다. 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The composition of the present invention is administered in a pharmaceutically effective amount. Effective dose levels depend on the type of disease, severity, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent medications, and other factors well known in the medical field. Can be determined. The pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.

본 발명의 조성물의 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설률 및 질환의 중증도 등에 따라 그 범위가 매우 다양하며, 적정한 투여량은 예를 들면 환자의 체내에 축적된 약물의 양 및/또는 사용되는 본 발명의 전달체의 구체적 효능정도에 따라 달라질 수 있다. 일반적으로 인비보 동물모델 및 인비트로에서 효과적인 것으로 측정된 EC50을 기초로 계산될 수 있으며, 예를 들면 체중 1kg당 0.01 μg 내지 1 g 일 수 있으며, 일별, 주별, 월별 또는 연별의 단위 기간으로, 단위 기간 당 일회 내지 수회 나누어 투여될 수 있으며, 또는 인퓨전 펌프를 이용하여 장기간 연속적으로 투여될 수 있다. 반복투여 횟수는 약물이 체내 머무는 시간, 체내 약물 농도 등을 고려하여 결정된다. 질환 치료 경과에 따라 치료가 된 후라도, 재발을 위해 조성물이 투여될 수 있다.The dosage of the composition of the present invention varies widely depending on the weight, age, sex, health condition, diet, time of administration, administration method, excretion rate and severity of the disease, and the appropriate dosage is, for example, Depending on the amount of drug accumulated in the body and / or the specific efficacy of the carrier of the invention used. It can be calculated on the basis of EC50, which is generally determined to be effective in in vivo animal models and in vitro, for example from 0.01 μg to 1 g per kg of body weight, in unit periods of daily, weekly, monthly or yearly It may be administered once or several times per unit period, or may be continuously administered for a long time using an infusion pump. The number of repeated doses is determined in consideration of the time the drug stays in the body, the drug concentration in the body, and the like. Even after treatment according to the course of the disease treatment, the composition can be administered for relapse.

본 발명의 조성물은 암의 치료와 관련하여 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 또는 유효성분의 용해성 및/또는 흡수성을 유지/증가시키는 화합물을 추가로 함유할 수 있다. 또한 선택적으로, 화학치료제, 항염증제, 항바이러스제 및/또는 면역조절제 등을 추가로 포함할 수 있다.The composition of the present invention may further contain a compound which maintains / increases the solubility and / or absorption of one or more of the active ingredients or the active ingredients exhibiting the same or similar function in the treatment of cancer. It may also optionally further comprise chemotherapeutic agents, anti-inflammatory agents, antiviral agents and / or immunomodulators and the like.

또한, 본 발명의 조성물은 포유동물에 투여된 후 활성 성분의 신속, 지속 또는 지연된 방출을 제공할 수 있도록 당업계에 공지된 방법을 사용하여 제형화될 수 있다. 제형은 분말, 과립, 정제, 에멀젼, 시럽, 에어로졸, 연질 또는 경질 젤라틴 캅셀, 멸균 주사용액, 멸균 분말의 형태일 수 있다.In addition, the compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.

상기 예방 또는 치료의 대상으로서 암과 면역질환에 관련된 구체적 설명은 상술한 바와 같다.Specific description related to cancer and immune diseases as the object of the prophylaxis or treatment is as described above.

이하, 본 발명을 구체적으로 설명하기 위해 실시예를 들어 상세하게 설명하기로 한다. Hereinafter, the present invention will be described in detail with reference to Examples.

이하, 본 발명의 다공성 실리카 입자는 'DegradaBALL 또는 DDV'로, IL-2가 담지된 DegradaBALL은 'BALLkine-2'로 각각 약칭될 수 있다.Hereinafter, the porous silica particles of the present invention may be abbreviated as 'DegradaBALL or DDV', and the DegradaBALL loaded with IL-2 may be abbreviated as 'BALLkine-2'.

이하, 종괴주위주사(peri-tumoral injection)는 p.t로, 복강주사(intra-peritoreal injection)는 i.p로, 피하주사(subcutaneous injection)는 s.c로, 정맥주사(intravenous injection)는 i.v로 약칭될 수 있다.Hereinafter, peri-tumoral injection may be abbreviated as pt, intra-peritoreal injection as ip, subcutaneous injection as sc, and intravenous injection as iv. .

실시예 1. 다공성 실리카 입자(DDV 또는 DegradaBALL)Example 1. Porous Silica Particles (DDV or DegradaBALL)

1. 다공성 실리카 입자의 제조1. Preparation of Porous Silica Particles

(1) 다공성 실리카 입자의 제조(1) Preparation of Porous Silica Particles

1) 소기공 입자의 제조1) Preparation of Small Pore Particles

2 L 둥근바닥플라스크에 증류수 (DW) 960 mL 과 MeOH 810 mL을 넣었다. 상기 플라스크에 CTAB 7.88 g을 넣은 후 교반하면서 1M NaOH 4.52 mL를 빠르게 넣었다. 10분 동안 교반시켜 균일한 혼합액을 넣은 후 TMOS 2.6 mL를 넣었다. 6시간 동안 교반하여 균일하게 혼합한 후, 24시간 동안 숙성시켰다.Into a 2 L round bottom flask was placed 960 mL of distilled water (DW) and 810 mL of MeOH. 7.88 g of CTAB was added to the flask, followed by rapid addition of 4.52 mL of 1 M NaOH while stirring. After stirring for 10 minutes to add a uniform mixture, 2.6 mL of TMOS was added. After stirring for 6 hours to mix uniformly, it was aged for 24 hours.

이후 상기 반응액을 25℃에서 10분간 8000rpm에서 원심분리하여 상등액을 제거하고, 25℃에서 10분간 8000rpm에서 원심분리하며 에탄올 및 증류수로 번갈아가며 5회 세척하였다.The reaction solution was then centrifuged at 8000 rpm for 10 minutes at 25 ° C. to remove the supernatant, centrifuged at 8000 rpm for 10 minutes at 25 ° C., and washed five times with alternating ethanol and distilled water.

이후 70℃ 오븐에서 건조시켜 1.5g의 분말형의 소기공 다공성 실리카 입자(기공 평균 직경 2nm, 입경 200nm)를 얻었다.Thereafter, the resultant was dried in an oven at 70 ° C. to obtain 1.5 g of powdery microporous silica particles (pore average diameter of 2 nm and particle size of 200 nm).

2) 기공 확장2) pore expansion

1.5g의 소기공 다공성 실리카 입자 분말을 에탄올 10ml에 첨가하여 초음파 분산시키고, 물 10ml, TMB (trimethyl benzene) 10ml를 첨가하여 초음파 분산시켰다.1.5 g of small pore porous silica particle powder was added to 10 ml of ethanol for ultrasonic dispersion, and 10 ml of water and 10 ml of TMB (trimethyl benzene) were added for ultrasonic dispersion.

이후 상기 분산액을 오토클레이브에 넣고 160℃, 48시간 반응시켰다.Thereafter, the dispersion was placed in an autoclave and reacted at 160 ° C. for 48 hours.

반응은 25℃에서 시작하여 10℃/분의 속도로 승온시켜 수행하였고, 이후 오토클레이브 내에서 1~10℃/분의 속도로 서서히 냉각시켰다.The reaction was carried out starting at 25 ° C. and warming up at a rate of 10 ° C./min, then slowly cooling at a rate of 1-10 ° C./min in the autoclave.

냉각된 반응액을 25℃에서 10분간 8000rpm에서 원심분리하여 상등액을 제거하고, 25℃에서 10분간 8000rpm에서 원심분리하며 에탄올 및 증류수로 번갈아가며 5회 세척하였다.The cooled reaction solution was centrifuged at 8000 rpm for 10 minutes at 25 ° C. to remove the supernatant, and centrifuged at 8000 rpm for 10 minutes at 25 ° C. and washed five times with ethanol and distilled water.

이후 70℃ 오븐에서 건조시켜 분말형의 다공성 실리카 입자(기공 직경 10~15nm, 입경 200nm)를 얻었다.Then, dried in an oven at 70 ℃ to obtain a powdery porous silica particles (pore diameter 10 ~ 15nm, particle diameter 200nm).

3) 하소3) calcination

상기 2)에서 제조된 다공성 실리카 입자를 유리 vial에 담아 550℃에서 5시간 동안 가열하고, 반응 종료 후 상온으로 서서히 식혀 입자를 제조하였다.The porous silica particles prepared in 2) were placed in a glass vial, heated at 550 ° C. for 5 hours, and cooled to room temperature after completion of the reaction to prepare particles.

(2) 다공성 실리카 입자의 제조(2) Preparation of Porous Silica Particles

기공 확장시의 반응 조건을 140℃, 72시간으로 변경한 것을 제외하고는 상기 1-1-(1)과 동일한 방법으로 다공성 실리카 입자를 제조하였다.Porous silica particles were prepared in the same manner as in 1-1- (1), except that the reaction conditions at the time of pore expansion were changed to 140 ° C. and 72 hours.

(3) 다공성 실리카 입자의 제조 (10L 스케일)(3) Preparation of Porous Silica Particles (10L Scale)

5배 큰 용기를 사용하고, 각 물질을 모두 5배 용량으로 사용한 것을 제외하고는 실시예 1-1-(1)과 동일한 방법으로 다공성 실리카 입자를 제조하였다.Porous silica particles were prepared in the same manner as in Example 1-1- (1), except that a 5-fold large container was used, and each material was used in 5-fold volume.

(4) 다공성 실리카 입자의 제조 (입경 300nm)(4) Preparation of Porous Silica Particles (Particle Diameter 300nm)

소기공 입자의 제조시에 증류수 920ml, 메탄올 850ml를 사용한 것을 제외하고는 1-1-(1)과 동일한 방법으로 다공성 실리카 입자를 제조하였다.Porous silica particles were prepared in the same manner as in 1-1- (1), except that 920 ml of distilled water and 850 ml of methanol were used to prepare the small pore particles.

(5) 다공성 실리카 입자의 제조 (입경 500nm)(5) Preparation of Porous Silica Particles (Particle Size 500nm)

소기공 입자의 제조시에 증류수 800ml, 메탄올 1010 ml, CTAB 10.6g을 사용한 것을 제외하고는 1-1-(1)과 동일한 방법으로 다공성 실리카 입자를 제조하였다.Porous silica particles were prepared in the same manner as in 1-1- (1), except that 800 ml of distilled water, 1010 ml of methanol, and 10.6 g of CTAB were used to prepare the small pore particles.

(6) 다공성 실리카 입자의 제조 (입경 1000nm)(6) Preparation of Porous Silica Particles (Particle Diameter 1000nm)

소기공 입자의 제조시에 증류수 620ml, 메탄올 1380ml, CTAB 7.88g을 사용한 것을 제외하고는 1-1-(1)과 동일한 방법으로 다공성 실리카 입자를 제조하였다.Porous silica particles were prepared in the same manner as in 1-1- (1), except that 620 ml of distilled water, 1380 ml of methanol, and 7.88 g of CTAB were used to prepare the small pore particles.

(7) 다공성 실리카 입자의 제조 (기공 직경 4nm)(7) Preparation of Porous Silica Particles (Pore Diameter 4nm)

기공 확장시에 TMB를 2.5mL를 사용한 것을 제외하고는 1-1-(1)과 동일한 방법으로 다공성 실리카 입자를 제조하였다.Porous silica particles were prepared in the same manner as in 1-1- (1), except that 2.5 mL of TMB was used for pore expansion.

(8) 다공성 실리카 입자의 제조 (기공 직경 7nm)(8) Preparation of Porous Silica Particles (Pore Diameter 7nm)

기공 확장시에 TMB를 4.5mL를 사용한 것을 제외하고는 1-1-(1)과 동일한 방법으로 다공성 실리카 입자를 제조하였다.Porous silica particles were prepared in the same manner as in 1-1- (1), except that 4.5 mL of TMB was used for pore expansion.

(9) 다공성 실리카 입자의 제조 (기공 직경 17nm)(9) Preparation of Porous Silica Particles (Pore Diameter 17nm)

기공 확장시에 TMB를 11mL를 사용한 것을 제외하고는 1-1-(1)과 동일한 방법으로 다공성 실리카 입자를 제조하였다.Porous silica particles were prepared in the same manner as in 1-1- (1), except that 11 mL of TMB was used for pore expansion.

(10) 다공성 실리카 입자의 제조 (기공 직경 23nm)(10) Preparation of Porous Silica Particles (Pore Diameter 23nm)

기공 확장시에 TMB를 12.5mL를 사용한 것을 제외하고는 1-1-(1)과 동일한 방법으로 다공성 실리카 입자를 제조하였다.Porous silica particles were prepared in the same manner as in 1-1- (1), except that 12.5 mL of TMB was used for pore expansion.

(11) 다공성 실리카 입자의 제조 (이중개질)(11) Preparation of Porous Silica Particles (Dual Modification)

1) 소기공 입자의 제조1) Preparation of Small Pore Particles

실시예 1-1-(1)-1)과 동일한 방법으로 소기공 입자를 제조하였다.Small pore particles were prepared in the same manner as in Example 1-1- (1) -1).

2) 기공 확장2) pore expansion

실시예 1-1-(1)-2)와 동일한 방법으로 소기공 입자를 TMB와 반응시키고 냉각시키고 원심분리하여 상등액을 제거하였다. 이후 실시예 1-1-(1)-2)와 동일 조건으로 원심분리하며 에탄올 및 증류수로 번갈아가며 3회 세척하고, 이후 실시예 1-1-(1)-2)와 동일 조건으로 건조하여 분말형의 다공성 실리카 입자(기공 직경 10~15nm, 입경 200nm)를 얻었다.In the same manner as in Example 1-1- (1) -2), the small pore particles were reacted with TMB, cooled, and centrifuged to remove the supernatant. Thereafter, centrifuged under the same conditions as in Example 1-1- (1) -2), washed three times with alternating ethanol and distilled water, and then dried under the same conditions as in Example 1-1- (1) -2). Powdery porous silica particles (pore diameter 10-15 nm, particle diameter 200 nm) were obtained.

3) 표면 개질3) surface modification

기공이 확장된 다공성 실리카 입자 0.8g 내지 1g을 50mL의 톨루엔에 분산시킨 후, (3-aminopropyl)triethoxysilane를 5mL 넣어주어 120℃로 환류한 채로 12시간 가열하였다. 해당 과정은 상기 서술된 세척과정 및 건조 과정을 거친 뒤 1mL의 트레에틸렌글리콜 (PEG3, 2-[2-(2-methoxyethoxy)ethoxy]acetic acid)와 100mg의 EDC(1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) 및 200mg의 N-Hydroxysuccinimide (NHS)를 30mL의 PBS에 분산시켜서 상온에서 교반한 채로 12시간 동안 반응을 보낸다. 이후 생성물은 상기의 세척 및 건조과정을 거친다.After dispersing 0.8 g to 1 g of porous silica particles having expanded pores in 50 mL of toluene, 5 mL of (3-aminopropyl) triethoxysilane was added thereto, followed by heating at reflux at 120 ° C for 12 hours. The procedure is followed by the washing and drying procedures described above, followed by 1 mL of threethylene glycol (PEG3, 2- [2- (2-methoxyethoxy) ethoxy] acetic acid) and 100 mg of EDC (1-Ethyl-3- (3). -dimethylaminopropyl) carbodiimide) and 200 mg of N-Hydroxysuccinimide (NHS) were dispersed in 30 mL of PBS and allowed to react for 12 hours while stirring at room temperature. The product is then washed and dried.

기공 내부에 이전 단계의 반응액이 남아 있어, 기공 내부는 개질 되지 않는다.The reaction solution of the previous step remains inside the pore, so that the inside of the pore is not modified.

4) 기공 내부 세척4) pore inside washing

표면개질된 입자 분말 800mg을 2M HCl/에탄올 40ml에 녹이고, 12시간 강하게 교반 하에 환류시켰다.800 mg of surface modified particle powder was dissolved in 40 ml of 2M HCl / ethanol and refluxed under vigorous stirring for 12 hours.

이후 냉각된 반응액을 10분간 8000rpm에서 원심분리하여 상등액을 제거하고, 25℃에서 10분간 8000rpm에서 원심분리하며 에탄올 및 증류수로 번갈아가며 5회 세척하였다.Thereafter, the cooled reaction solution was centrifuged at 8000 rpm for 10 minutes to remove the supernatant, centrifuged at 8000 rpm for 10 minutes at 25 ° C, and washed five times with alternating ethanol and distilled water.

이후 70℃ 오븐에서 건조시켜 분말형의 다공성 실리카 입자를 얻었다.After drying in an oven at 70 ℃ to obtain a powdery porous silica particles.

5) 기공 내부 개질5) Pore internal reforming

① 후술하는 실시예 1-2-(2)-1)의 방법과 동일한 방법으로 기공 내부에 프로필기를 도입하였다.(1) A propyl group was introduced into the pores in the same manner as in Example 1-2- (2) -1) described later.

② 후술하는 실시예 1-2-(2)-2)의 방법과 동일한 방법으로 기공 내부에 옥틸기를 도입하였다.(2) An octyl group was introduced into the pores in the same manner as in Example 1-2- (2) -2) described later.

2. 다공성 실리카 입자의 표면 개질2. Surface Modification of Porous Silica Particles

(1) 양전하로의 대전(1) Approach with positive charge

1) 입경 300nm의 입자1) 300nm particle size

실시예 1-1-(4)의 다공성 실리카 입자를 (3-Aminopropyl)triethoxysilane (APTES)와 반응시켜 양전하로 대전시켰다.The porous silica particles of Example 1-1- (4) were reacted with (3-Aminopropyl) triethoxysilane (APTES) to charge with a positive charge.

구체적으로, 100 mL 둥근바닥플라스크에 100 mg의 다공성 실리카 입자를 10 mL의 톨루엔에 bath sonicator로 분산시켰다. 이후 1 mL의 APTES를 첨가하고 400 rpm으로 교반하며 130℃에서 교반하며 12시간 동안 반응시켰다.Specifically, 100 mg of porous silica particles in a 100 mL round bottom flask was dispersed in 10 mL of toluene with a bath sonicator. Then 1 mL of APTES was added and stirred at 400 rpm and stirred at 130 ° C. for 12 hours.

반응 후에 상온까지 서서히 식히고, 10분간 8000rpm에서 원심분리하여 상등액을 제거하고, 25℃에서 10분간 8000rpm에서 원심분리하며 에탄올 및 증류수로 번갈아가며 5회 세척하였다.After the reaction was slowly cooled to room temperature, the supernatant was removed by centrifugation at 8000rpm for 10 minutes, centrifuged at 8000rpm for 10 minutes at 25 ℃ and washed five times alternately with ethanol and distilled water.

이후 70℃ 오븐에서 건조시켜 표면 및 기공 내부에 아미노기를 갖는 분말형의 다공성 실리카 입자를 얻었다.After drying in an oven at 70 ℃ to obtain a porous porous silica particles having an amino group on the surface and inside the pores.

2) 입경 200nm의 입자2) particle of 200nm particle size

① 실시예 1-1-(1)의 다공성 실리카 입자를 (3-Aminopropyl)triethoxysilane (APTES)와 반응시켜 양전하로 대전시켰으며, APTES를 0.4ml 첨가하고, 반응 시간을 3시간으로 한 것을 제외하고는 상기 9-2-(1)-1)의 방법과 동일하게 개질하였다.① The porous silica particles of Example 1-1- (1) were charged with positive charge by reacting with (3-Aminopropyl) triethoxysilane (APTES), except that 0.4 ml of APTES was added and the reaction time was 3 hours. Was modified in the same manner as in the method of 9-2- (1) -1).

② 실시예 1-1-(9)의 다공성 실리카 입자를 (3-Aminopropyl)triethoxysilane (APTES)와 반응시켜 양전하로 대전시켰으며, 그 외 방법은 상기 9-2-(1)-1)의 방법과 동일하게 개질하였다.② The porous silica particles of Example 1-1- (9) were charged with positive charge by reacting with (3-Aminopropyl) triethoxysilane (APTES), and the other method was the method of 9-2- (1) -1). Modified in the same manner as

③ 실시예 1-1-(10)의 다공성 실리카 입자를 (3-Aminopropyl)triethoxysilane (APTES)와 반응시켜 양전하로 대전시켰으며, 상기 9-2-(1)-1)의 방법과 동일하게 개질하였다.③ The porous silica particles of Example 1-1- (10) were charged with positive charge by reacting with (3-Aminopropyl) triethoxysilane (APTES), and were modified in the same manner as in the method of 9-2- (1) -1). It was.

(2) 소수성기의 도입(2) Introduction of hydrophobic groups

1) 프로필기1) Profile

상기 실시예 1-1-(1)의 다공성 실리카 입자를 Trimethoxy(propyl)silane와 반응시켜 표면 및 기공 내부에 프로필기를 도입하였으며, APTES 대신에 Trimethoxy(propyl)silane를 0.35ml 첨가하고, 12시간 반응시킨 것을 제외하고는 상기 실시예 1-2-(1)과 동일한 방법으로 개질을 수행하였다.The porous silica particles of Example 1-1- (1) were reacted with Trimethoxy (propyl) silane to introduce a propyl group into the surface and the pores, and 0.35ml of Trimethoxy (propyl) silane was added instead of APTES, followed by reaction for 12 hours. Modification was carried out in the same manner as in Example 1-2- (1) except for the above.

2) 옥틸기2) octyl group

상기 실시예 1-1-(1)의 다공성 실리카 입자를 Trimethoxy-n-octylsilane와 반응시켜 표면 및 기공 내부에 프로필기를 도입하였으며, APTES 대신에 Trimethoxy-n-octylsilane를 0.5ml 첨가하고, 12시간 반응시킨 것을 제외하고는 상기 실시예 1-2-(1)과 동일한 방법으로 개질을 수행하였다.The porous silica particles of Example 1-1- (1) were reacted with Trimethoxy-n-octylsilane to introduce propyl groups into the surface and the pores, and 0.5 ml of Trimethoxy-n-octylsilane was added instead of APTES, followed by 12 hours of reaction. Modification was carried out in the same manner as in Example 1-2- (1) except for the above.

(3) 음전하로의 대전(3) Approach by negative charge

1) 카르복실기1) carboxyl group

상기 실시예 1-1-(1)의 다공성 실리카 입자를 succinic anhydride와 반응시켜 음전하로 대전시켰으며, The porous silica particles of Example 1-1- (1) were charged with negative charge by reacting with succinic anhydride,

톨루엔 대신에 DMSO(dimethyl sulfoxide)를 사용하고, APTES 대신에 80 mg의 succinic anhydride를 첨가하여 24시간 동안 상온에서 교반하며 반응시키고, 세척 시에 증류수 대신에 DMSO를 사용한 것을 제외하고는 상기 실시예 1-2-(1)-1)의 방법과 동일하게 개질하였다.Dimethyl sulfoxide (DMSO) was used instead of toluene, 80 mg of succinic anhydride was added instead of APTES, and reacted at room temperature for 24 hours, except that DMSO was used instead of distilled water. The modification was carried out in the same manner as in the method of -2- (1) -1).

2) 티올기2) thiol group

APTES 대신에 MPTES 1.1 mL를 사용한 것을 제외하고는 상기 실시예 1-2-(1)-1)의 방법과 동일하게 개질하였다.It was modified in the same manner as in Example 1-2- (1) -1) except that 1.1 mL of MPTES was used instead of APTES.

3) 술폰산기3) sulfonic acid group

상기 실시예 1-2-(3)-2)의 다공성 실리카 나노입자 100 mg를 1 M 황산수용액을 1 mL와 30% 과산화수소수 20 mL에 분산하여 상온에서 교반하여 산화반응을 유도하여 티올기를 술폰산기로 산화시켰다. 이후 상기 실시예 1-2-(1)-1)의 방법과 동일하게 세척 및 건조시켰다.100 mg of the porous silica nanoparticles of Example 1-2- (3) -2) were dispersed in 1 mL of 1 M aqueous sulfuric acid solution and 20 mL of 30% hydrogen peroxide solution, and stirred at room temperature to induce an oxidation reaction. Oxidized to a group. After the same washing and drying in the same manner as in Example 1-2- (1) -1).

3. 입자의 형성 및 기공의 확장 확인3. Confirm the formation of particles and the expansion of pores

실험방법 실시예 1-1-(1) 내지 (3)의 입자의 소기공 입자, 제조된 다공성 실리카 입자를 현미경으로 관찰하여, 소기공 입자가 균일하게 생성되었는지, 기공이 충분히 확장되어 다공성 실리카 입자가 균일하게 형성되었는지를 확인하였다(도 1 내지 4).Experimental Method Observing the small pore particles and the prepared porous silica particles of the particles of Examples 1-1- (1) to (3) under a microscope, whether the small pore particles were uniformly produced or the pores were sufficiently expanded to give the porous silica particles. It was confirmed whether was formed uniformly (Figs. 1 to 4).

도 1은 실시예 1-1-(1)의 다공성 실리카 입자의 사진, 도 2는 실시예 1-1-(2)의 다공성 실리카 입자의 사진으로 기공이 충분히 확장된 구형의 다공성 실리카 입자가 고르게 생성된 것을 확인할 수 있고, 1 is a photograph of porous silica particles of Example 1-1- (1), and FIG. 2 is a photograph of porous silica particles of Example 1-1- (2). You can see that it was created,

도 3은 실시예 1-1-(1)의 소기공 입자의 사진이고, 도 4는 실시예 1-1-(1)과 1-1-(3)의 소기공 입자의 비교 사진으로, 구형의 소기공 입자가 고르게 생성된 것을 확인할 수 있다.Figure 3 is a photograph of the small pore particles of Example 1-1- (1), Figure 4 is a comparison photograph of the small pore particles of Example 1-1- (1) and 1-1- (3), spherical It can be seen that the small pore particles of evenly generated.

4. BET 표면적 및 기공의 부피 계산4. Calculation of BET Surface Area and Pore Volume

실시예 1-1-(1)의 소기공 입자, 실시예 1-1-(1),(7),(8),(10)의 다공성 실리카 입자의 표면적과 기공 부피를 계산하였다. 표면적은 Brunauer-Emmett-Teller(BET) 방법에 의해 계산되었으며, 기공 크기의 분포는 Barrett-Joyner-Halenda(BJH) 방법에 의하여 계산되었다.The surface area and pore volume of the small pore particles of Example 1-1- (1) and the porous silica particles of Examples 1-1- (1), (7), (8) and (10) were calculated. The surface area was calculated by Brunauer-Emmett-Teller (BET) method, and the pore size distribution was calculated by Barrett-Joyner-Halenda (BJH) method.

상기 각 입자들의 현미경 사진은 도 5에 나타내었고, 계산 결과는 하기 표 1에 나타내었다.The micrographs of the particles are shown in FIG. 5, and the calculation results are shown in Table 1 below.

구분division 기공 직경(nm)Pore diameter (nm) BET 표면적(m 2/g)BET surface area (m 2 / g) 기공 부피(mL/g)Pore Volume (mL / g) 실시예 1-1-(1)의 소기공 입자Small Pore Particles of Example 1-1- (1) 2.12.1 13371337 0.690.69 실시예 1-1-(7)Example 1-1- (7) 4.34.3 630630 0.720.72 실시예 1-1-(8)Example 1-1- (8) 6.96.9 521521 0.790.79 실시예 1-1-(1)Example 1-1- (1) 10.410.4 486486 0.820.82 실시예 1-1-(10)Example 1-1- (10) 2323 395395 0.970.97

5. 생분해성 확인5. Check biodegradability

실시예 1-1-(1)의 다공성 실리카 입자의 생분해성 확인을 위해 37℃, SBF(pH 7.4)에서의 생분해 정도를 0시간, 120시간, 360시간에 현미경으로 관찰하였고, 이는 도 6에 나타내었다.In order to confirm biodegradability of the porous silica particles of Example 1-1- (1), the degree of biodegradation at 37 ° C. and SBF (pH 7.4) was observed under a microscope at 0 hours, 120 hours, and 360 hours. Indicated.

이를 참조하면 다공성 실리카 입자가 생분해되어 360시간 경과 후에는 거의 다 분해된 것을 확인할 수 있다.Referring to this, it can be seen that porous silica particles are biodegraded and nearly decomposed after 360 hours.

6. 흡광도비 측정6. Absorbance Ratio Measurement

시간별 하기 수학식 1에 따른 흡광도비를 측정하였다.The absorbance ratio according to Equation 1 according to time was measured.

[수학식 1][Equation 1]

A t/A 0 A t / A 0

(식 중, A 0는 상기 다공성 실리카 입자 1mg/ml 현탁액 5ml를 직경 50 kDa의 기공을 갖는 원통형 투과막에 넣고 측정된 다공성 실리카 입자의 흡광도이고,Wherein A 0 is the absorbance of the porous silica particles measured by placing 5 ml of the 1 mg / ml suspension of the porous silica particles into a cylindrical permeable membrane having pores having a diameter of 50 kDa,

상기 투과막 외부에는 상기 투과막과 접하며, 상기 현탁액과 동일한 용매 15ml가 위치하고, 상기 투과막 내외부는 37℃에서 60rpm 수평 교반되며,Outside of the permeable membrane is in contact with the permeable membrane, 15ml of the same solvent as the suspension is located, the inside and outside of the permeable membrane is stirred 60 rpm at 37 ℃ horizontal,

A t는 상기 A 0의 측정시로부터 t시간 경과 후에 측정된 다공성 실리카 입자의 흡광도임).A t is the absorbance of the porous silica particles measured after t hours have elapsed since the measurement of A 0 ).

구체적으로, 다공성 실리카 입자 분말 5mg을 SBF (pH 7.4) 5ml에 녹였다. 이후 5ml의 다공성 실리카 입자 용액을 도 7에 도시된 직경 50 kDa의 기공을 갖는 투과막에 넣었다. 외부막에 15ml의 SBF를 첨가하고, 외부막의 SBF는 12시간마다 교체하였다. 다공성 실리카 입자의 분해는 37℃에서 60rpm 수평 교반하며 수행되었다.Specifically, 5 mg of porous silica particle powder was dissolved in 5 ml of SBF (pH 7.4). Thereafter, 5 ml of the porous silica particle solution was placed in a permeable membrane having pores having a diameter of 50 kDa shown in FIG. 7. 15 ml of SBF was added to the outer membrane, and the SBF of the outer membrane was replaced every 12 hours. Decomposition of the porous silica particles was performed at 37 ° C. with 60 rpm horizontal stirring.

이후 UV-vis spectroscopy에 의해 흡광도를 측정하였고, λ = 640 nm에서 분석되었다.The absorbance was then measured by UV-vis spectroscopy and analyzed at λ = 640 nm.

(1) 흡광도 비 측정(1) absorbance ratio measurement

실시예 1-1-(1)의 다공성 실리카 입자의 흡광도비를 상기 방법에 따라 측정하였고, 그 결과는 도 8에 나타내었다.The absorbance ratio of the porous silica particles of Example 1-1- (1) was measured according to the above method, and the results are shown in FIG. 8.

이를 참조하면 흡광도비가 1/2가 되는 t가 약 58시간으로 굉장히 천천히 분해되는 것을 확인할 수 있다.Referring to this, it can be seen that t, which has an absorbance ratio of 1/2, decomposes very slowly in about 58 hours.

(2) 입경별(2) by particle size

실시예 1-1-(1),(5),(6)의 다공성 실리카 입자의 흡광도를 상기 수학식 1에 따라 측정하였고, 그 결과는 도 9에 나타내었다(현탁액과 용매로는 SBF를 사용).The absorbance of the porous silica particles of Examples 1-1- (1), (5), and (6) was measured according to Equation 1, and the results are shown in FIG. 9 (SBF was used as the suspension and the solvent. ).

이를 참조하면, 입경의 증가에 따라 t가 감소함을 알 수 있다.Referring to this, it can be seen that t decreases with increasing particle size.

(3) 기공 평균 직경별(3) by pore average diameter

실시예 1-1-(1),(9)의 다공성 실리카 입자, 그리고 컨트롤로서 실시예 1-1-(1)의 소기공 다공성 실리카 입자의 흡광도를 상기 수학식 1에 따라 측정하였고, 그 결과는 도 10에 나타내었다(현탁액과 용매로는 SBF를 사용).The absorbances of the porous silica particles of Examples 1-1- (1) and (9), and the microporous porous silica particles of Example 1-1- (1) as controls, were measured according to Equation 1 above, and as a result, Is shown in Figure 10 (suspension and solvent used SBF).

이를 참조하면, 실시예의 다공성 실리카 입자는 컨트롤에 비해 t가 상당히 큰 것을 확인할 수 있다.Referring to this, it can be seen that the porous silica particles of the example have a significantly larger t than the control.

(4) pH별(4) pH

실시예 1-1-(4)의 다공성 실리카 입자의 pH별 흡광도를 측정하였다. 흡광도는 SBF에서, 그리고 pH 2, 5, 및 7.4의 Tris에서 측정하였고, 그 결과는 도 11에 나타내었다.The absorbance for each pH of the porous silica particles of Example 1-1- (4) was measured. Absorbance was measured in SBF and in Tris at pH 2, 5, and 7.4 and the results are shown in FIG. 11.

이를 참조하면, pH 별 t의 차이는 있으나, 모두 흡광도의 비가 1/2이 되는 t가 24 이상이었다.Referring to this, although there is a difference in t for each pH, t, which is a ratio of absorbance 1/2, was 24 or more.

(5) 대전(5) Daejeon

실시예 1-2-(1)-1)의 다공성 실리카 입자의 흡광도를 측정하였고, 그 결과는 도 12에 나타내었다(현탁액과 용매로는 Tris(pH 7.4)를 사용).The absorbance of the porous silica particles of Example 1-2- (1) -1) was measured, and the results are shown in FIG. 12 (Tris (pH 7.4) was used as a suspension and a solvent).

이를 참조하면, 양전하로 대전된 입자도 흡광도의 비가 1/2이 되는 t가 24 이상이었다.Referring to this, t, which has a ratio of absorbance 1/2 of the positively charged particles, was 24 or more.

7. 항체 또는 사이토카인의 담지7. Loading of Antibodies or Cytokines

(1) 항체의 담지(1) Support of Antibody

1 x PBS 200 ㎕ 내에 실시예 1-2-(1)-2)-②의 다공성 실리카 입자 분말 100 ㎍과 anti-twist IgG(Santacruz, sc-81417) 10 ㎍을 혼합한 후, 실온에서 1시간 인큐베이션하여 IgG를 담지하였다. 상기와 동일한 조건 하에 타 항체들도 담지하였다.100 μg of the porous silica particle powder of Example 1-2- (1) -2) -② and 10 μg of anti-twist IgG (Santacruz, sc-81417) were mixed in 200 μl of 1 × PBS, and then 1 hour at room temperature. Incubate to support IgG. Other antibodies were also supported under the same conditions as above.

(2) 사이토카인의 담지(2) Support of cytokines

PBS 또는 증류수 100 μL 내에 실시예 1-2-(1)-2)-②의 다공성 실리카 입자 분말 25 ㎍과 IL-2 10 μg을 혼합한 후, 1시간 인큐베이션 하여 IL-2를 담지하였다. 상기와 동일한 조건 하에 타 사이토카인들도 담지하였다.After mixing 25 µg of the porous silica particle powder of Example 1-2- (1) -2) -② and 10 µg of IL-2 in 100 µL of PBS or distilled water, incubated for 1 hour to support IL-2. Under the same conditions as above, other cytokines were also supported.

8. 항체 또는 사이토카인의 방출8. Release of Antibodies or Cytokines

(1) 항체의 방출 (1) release of antibody

Fluorescein 형광이 표지된 IgG를 로딩한 다공성 실리카 입자 100 ㎍을 SBF(pH 7.4) 또는 PBS(pH 7.4) 200 ㎕에 재부유시켰다.100 μg of porous silica particles loaded with Fluorescein fluorescence labeled IgG were resuspended in 200 μl SBF (pH 7.4) or PBS (pH 7.4).

IgG, PD-1, PD-L1의 방출은 37℃에서 60rpm 수평 교반하며 수행되었고, 6 시간, 12 시간, 24 시간, 48 시간, 96 시간, 144 시간, 240 시간 지점에서 200 ㎕의 방출 용매를 형광 측정을 위해 회수하고 등량의 SBF 또는 PBS를 첨가하였다. Fluorescein 형광이 표지된 IgG의 형광 강도는 517 nm 파장(λ ex=492 nm)에서 측정하여 BSA의 방출 정도를 측정하였다.Release of IgG, PD-1, PD-L1 was carried out at 37 ° C. with 60 rpm horizontal stirring and 200 μl of the release solvent at 6, 12, 24, 48, 96, 144, 240 hours. Recovered for fluorescence measurements and an equivalent amount of SBF or PBS was added. Fluorescence intensity of Fluorescein fluorescence labeled IgG was measured at 517 nm wavelength (λ ex = 492 nm) to determine the degree of emission of BSA.

도 13 내지 15를 참조하면, IgG는 SBF와 PBS에서 모두 서방적으로 방출되며, IgG, PD-1, PD-L1 모두 250시간 이상에 걸쳐 거의 100%까지 방출되는 것을 확인할 수 있다.Referring to FIGS. 13 to 15, IgG is released in both SBF and PBS in a sustained manner, and it can be seen that IgG, PD-1, and PD-L1 are released to almost 100% over 250 hours or more.

(2) 사이토카인의 방출(2) release of cytokines

FITC가 결합된 사이토카인을 담지한 다공성 실리카 입자를 37℃에서 SBF(pH 7.4)에 재부유시킨 후, 기설정된 기간(1, 2, 3, 4, 5, 6, 7일) 동안 관찰하였다. 용액은 10분 동안 8,000 rpm 원심분리하였고, 사이토카인의 방출량은 FITC의 형광 세기 스펙트럼을 통해 확인하였다.Porous silica particles bearing FITC-bound cytokines were resuspended in SBF (pH 7.4) at 37 ° C. and then observed for a predetermined period (1, 2, 3, 4, 5, 6, 7 days). The solution was centrifuged at 8,000 rpm for 10 minutes and the release of cytokines was confirmed by fluorescence intensity spectra of FITC.

도 17 내지 21을 참조하면, IL-10, IL-15, HGF, EGF, IL-2는 모두 서방적으로 방출되며, 7시간 이상에 걸쳐 거의 100%까지 방출되는 것을 확인할 수 있다.Referring to Figure 17 to 21, IL-10, IL-15, HGF, EGF, IL-2 are all released slowly, it can be seen that the release to almost 100% over 7 hours or more.

실시예 2. 다공성 실리카 입자의 독성여부 확인Example 2. Checking the Toxicity of Porous Silica Particles

1. 실험방법1. Experiment Method

본 발명 다공성 실리카 입자의 단회 투여 독성을 확인하기 위해, 모든 마우스(군 당 4마리)에 각각 31.25, 62.5, 125, 250 mg/kg의 다공성 실리카 입자(DegradaBALL)를 투여하고, 투여 후 5일째에 마우스를 희생하여 주요 장기(간, 비장, 폐, 심장 및 신장)를 분리하였다.In order to confirm the single dose toxicity of the porous silica particles of the present invention, 31.25, 62.5, 125, and 250 mg / kg of porous silica particles (DegradaBALL) were respectively administered to all mice (four per group), and 5 days after the administration. Mice were sacrificed to separate major organs (liver, spleen, lung, heart and kidney).

본 발명 다공성 실리카 입자의 반복 투여 독성을 확인하기 위해, 모든 마우스(군 당 8마리)에 0.5, 5, 50 mg/kg의 다공성 실리카 입자(DegradaBALL)를 매 3일 또는 4일 간격으로 최대 9회 피하로 투여하였고, 마지막 투여 48시간 후 모든 마우스를 희생하여 주요 장기(간, 비장, 폐, 심장 및 신장)를 분리하였다.In order to confirm the repeated dose toxicity of the porous silica particles of the present invention, 0.5, 5, 50 mg / kg of porous silica particles (DegradaBALL) were added up to 9 times every 3 or 4 days to all mice (8 per group). Subcutaneously, 48 hours after the last administration, all mice were sacrificed to isolate the major organs (liver, spleen, lung, heart and kidney).

2. 실험결과2. Experimental Results

도 22 내지 43을 참조하면, 모든 군에서 명백한 병리학적 또는 임상적 독성 징후는 관찰되지 않음을 확인할 수 있는데, 보다 구체적으로, 본 발명 다공성 실리카 입자는 기관(간, 비장, 신장, 흉선, 심장, 폐 및 림프절)의 유의한 체중 감소를 유발하지 않았고(데이터는 평균±표준 편차로 나타냄. 정상군과 유의한 차이 값( * p<0.05)), 전혈 분석 및 혈청 생화학 분석으로 얻은 다양한 매개 변수에 있어서, 적혈구 (RBC), 백혈구 (WBC) 및 혈액 세포 부피 (즉, 평균 미립자 부피 (MCV)), 평균 혈소판 부피 (MPV), 평균 미립자 헤모글로빈 농도 (MCHC), 헤모글로빈, 헤마토크리트 및 적혈구 분포 폭(RDW) 수준은 정상 범위를 나타내었으며, ALT(Alanine transaminase), AST(aspartate transaminase), ALP(alkaline phosphatase) 및 GGT(gamma-glutamyltransferase) 수치에 있어서도 간 손상의 징후를 보이지 않았다. 신장 기능(혈액 우레아 질소(BUN), 크레아티닌)의 지표 및 전체 단백질과 빌리루빈도 역시 모든 군에서 정상범위였으며, 모든 군의 심장, 간, 폐, 신장 및 비장의 H&E 염색 (x200)의 조직학적 관찰에 있어서도 출혈은 관찰되지 않아 군 간 차이가 없었다(막대는 100 μm를 나타냄). 22 to 43, it can be seen that no clear pathological or clinical signs of toxicity were observed in all groups. More specifically, the porous silica particles of the present invention are organs (liver, spleen, kidney, thymus, heart, Lung and lymph nodes) did not induce significant weight loss (data is expressed as mean ± standard deviation; significant difference from normal group ( * p <0.05)) and did not affect the various parameters obtained by whole blood analysis and serum biochemical analysis. Red blood cell (RBC), white blood cell (WBC) and blood cell volume (ie, average particulate volume (MCV)), average platelet volume (MPV), average particulate hemoglobin concentration (MCHC), hemoglobin, hematocrit and erythrocyte distribution width (RDW). ) Levels were normal and there was no sign of hepatic damage in ALT (Alanine transaminase), AST (aspartate transaminase), ALP (alkaline phosphatase) and GGT (gamma-glutamyltransferase) levels. Indicators of renal function (blood urea nitrogen (BUN), creatinine) and total protein and bilirubin were also normal in all groups and histological observations of H & E staining (x200) in heart, liver, lung, kidney and spleen in all groups. No bleeding was observed, and there was no difference between the groups (the rod shows 100 μm).

실시예 3. 담지된 면역반응 물질의 생물학적 활성 보존 확인Example 3 Confirmation of Preservation of Biological Activity of Supported Immune Responsive Substances

1. 실험방법1. Experiment Method

다공성 실리카 입자에 담지된 항체 또는 사이토카인의 생물학적 활성이, 담지되지 아니한 경우와 마찬가지로 생물학적 활성이 보존되는지 확인하기 위하여, IL-2를 담지한 다공성 실리카 입자(BALLkine-2)를 처리한 마우스 T세포의 세포 증식(cell proliferation) 정도를 CCK assay로 확인하였고, IL-2를 담지한 다공성 실리카 입자(BALLkine-2)를 처리한 IL-HEK 세포에서의 STAT5 활성 정도를 HEK-Blue TM IL-2 assay (InvivoGen, CA, USA)을 이용하여 제조사의 프로토콜에 따라 분석하였다.Mouse T cells treated with IL-2-supported porous silica particles (BALLkine-2) to confirm that the biological activity of the antibody or cytokine supported on the porous silica particles is preserved as in the case of unsupported Cell proliferation was confirmed by CCK assay, and HEK-Blue TM IL-2 assay was used to measure the STAT5 activity in IL-HEK cells treated with IL-2 loaded porous silica particles (BALLkine-2). (InvivoGen, CA, USA) was used to analyze according to the manufacturer's protocol.

2. 실험결과2. Experimental Results

도 44, 45를 참조하면, IL-2가 담지되지 않은 경우 대비, IL-2가 다공성 실리카 입자에 담지된 경우, 마우스의 T세포의 증식과 STAT5 활성 정도가 처리 농도에 무관하게 유의미한 차이를 나타내지 않았다. 이는, 항체나 사이토카인을 본 발명 다공성 실리카 입자에 담지하여 처리하여도 생물학적 활성이 잘 보존됨을 보여주는 결과이다.44 and 45, compared with the case where IL-2 is not supported, when IL-2 is supported on the porous silica particles, the proliferation of T cells and STAT5 activity of mice do not show a significant difference regardless of the treatment concentration. Did. This result shows that biological activity is well preserved even when the antibody or cytokine is supported and treated on the porous silica particles of the present invention.

실시예 4. 본 발명 전달체에 의한 과민반응 여부 확인Example 4. Confirmation of hypersensitivity by the present invention carrier

1. 실험방법1. Experiment Method

DegradaBALL, IL-2 및 IL-2를 담지한 DegradaBALL(BALLkine-2)의 잠재 면역원성, 특히 지연형 과민반응(type 4 hypersensitivity)을 평가하기 위해 시험관 내 MUSST(골수성 U937 피부 민감성 시험) 분석을 수행하였다. Stimulation index가 3 이하이면 반응이 없다는 것을 의미한다(NC: 음성대조군(negative control), PC: 양성대조군(positive control), 자극 지수 = 시험 샘플의 양성 세포의 평균% / NC의 양성 세포의 평균%, NC 군과 유의한 차이( ### p<0.05)). NC와 PC 군에서 3회, 다른 군에서 2회 반복시험하였다.In vitro MUSST (myeloid U937 skin sensitivity test) assays are performed to assess the potential immunogenicity of DegradaBALL (BALLkine-2) carrying DegradaBALL, IL-2 and IL-2, in particular type 4 hypersensitivity It was. A stimulation index of 3 or less means no response (NC: negative control, PC: positive control, stimulation index = average% of positive cells in test sample / average% of positive cells in NC) , Significant difference from NC group ( ### p <0.05)). The test was repeated three times in the NC and PC groups and twice in the other group.

2. 실험결과2. Experimental Results

도 46을 참조하면, 모든 군의 모든 자극 지수(이론적 혈장 농도의 10배에서 1/25배까지)는 3.0보다 낮아 양성 반응이 없음으로 나타났는데, 이는 본 발명 전달체에 의한 과민반응, 특히 지연형 과민반응이 없다는 것을 입증하는 결과이다.Referring to FIG. 46, all stimulation indices (from 10 times to 1/25 times the theoretical plasma concentration) of all groups were lower than 3.0, indicating no positive reaction, which is a hypersensitivity reaction by the carrier of the present invention, especially delayed type. This proves that there is no hypersensitivity reaction.

실시예 5. 본 발명 전달체의 안정적인 전달성 및 표적성 확인Example 5 Confirmation of Stable Delivery and Targeting of the Carrier of the Invention

1. 항체의 전달1. Delivery of Antibodies

도 47, 48을 참조하면, anti-PD-L1 항체와 DDV에 각각 FITC, Cy5를 표지하고, anti-PD-L1 항체를 담지한 DDV를 종양내투여(intratumoral injection)한 후, 각 시간 별로 anti-PD-L1 항체와 DDV의 형광세기를 측정한 결과를 확인할 수 있는데, DDV와 anti-PD-L1 항체가 오랫동안 투여부위에 남아있는 것을 확인할 수 있고, 이는 본 발명 전달체가 담지된 anti-PD-L1 항체를 안정적으로 전달할 수 있고, 종양 내 및 투여부위에 오래 머물게 하여, 오랫동안 약효를 가질수 있음을 입증하는 것이다.47 and 48, after labeling FITC and Cy5 on anti-PD-L1 antibody and DDV, and intratumoral injection of DDV carrying anti-PD-L1 antibody, The fluorescence intensity of PD-L1 antibody and DDV can be measured. It can be seen that DDV and anti-PD-L1 antibody remain at the administration site for a long time, which is the anti-PD- loaded carrier of the present invention. The L1 antibody can be delivered stably, and it can be long-lived in the tumor and at the site of administration, demonstrating that it can have long-term efficacy.

2. 사이토카인의 전달2. Delivery of cytokines

도 49를 참조하면, IL-2와 DDV에 각각 Cy7, Cy5를 표지하고, IL-2를 담지한 DDV(BALLkine-2)를 피하투여(SC injection)한 후, 각 시간 별로 IL-2와 DDV의 형광세기를 측정한 결과를 확인할 수 있는데, DDV와 BALLkine-2가 오랫동안 투여부위에 남아있는 것을 확인할 수 있고, 이는 본 발명 전달체가 담지된 IL-2를 안정적으로 전달할 수 있음을 입증하는 것이다.Referring to FIG. 49, after labeling Cy7 and Cy5 to IL-2 and DDV, and subcutaneously administering DDV (BALLkine-2) carrying IL-2 (SC injection), IL-2 and DDV at each hour. It can be confirmed that the result of measuring the fluorescence intensity of, DDV and BALLkine-2 can be confirmed that it remains at the administration site for a long time, which proves that the present invention can be delivered stably delivered with the IL-2.

도 50, 51을 참조하면, IL-2에 FITC형광을 달고, DDV에 TAMRA형광을 달아 피하로 투여하고, 24시간 후에 꼬리정맥으로 Cy5형광으로 라벨링(labeling) 되어있는 렉틴(lectin)을 주사하여 혈관을 염색한 뒤, 종양을 적출하여 형광이미지를 확인한 결과를 확인할 수 있는데, DDV와 BALLkine-2가 종양조직에 특이적으로 분포함을 확인할 수 있고, 이는 본 발명 전달체가 담지된 IL-2를 종양특이적으로 표적하여 전달할 수 있음을 입증하는 것이다.Referring to FIGS. 50 and 51, FITC fluorescence was added to IL-2, TAMRA fluorescence was applied to DDV, and subcutaneously administered. After 24 hours, lectin labeled with Cy5 fluorescence was injected into the tail vein. After staining the blood vessels, the tumor was extracted to confirm the results of the fluorescence image. It can be seen that DDV and BALLkine-2 are specifically distributed in the tumor tissue, which indicates IL-2 carrying the carrier of the present invention. To demonstrate tumor-specific targeting and delivery.

도 52는 종양 세포(DAPI), vehicle(TAMRA), IL-2(FITC) 및 혈관염색(dylight 649 접합 lectin)을 나타내는 종양 조직의 조직학적 단면 이미지를 나타내는 것이고, 도 53은 BALLkine-2(p.t) 및 IL-2(i.p 또는 p.t)의 종양 내 약물동태(그래프는 측정시간당 3마리 마우스의 평균±표준편차를 나타냄)를 나타낸 것으로서, 피하주사후, 종양 내에서의 IL-2의 양을 ELISA로 측정한 결과를 나타낸 것인데, 이들을 참조하면, 본 발명 다공성 실리카 입자가 IL-2를 종양 및 투여부위에 오랫동안 머무르게 하고, IL-2를 안정적으로 전달할 수 있음을 확인할 수 있다.FIG. 52 shows histological cross-sectional images of tumor tissue showing tumor cells (DAPI), vehicle (TAMRA), IL-2 (FITC) and dylight 649 conjugated lectin, FIG. 53 shows BALLkine-2 (pt) ) And IL-2 (ip or pt) intratumoral pharmacokinetics (graphs represent mean ± standard deviation of 3 mice per measurement time), followed by subcutaneous injection to determine the amount of IL-2 in the tumor As shown in the results, it can be seen that, referring to these, the porous silica particles of the present invention can keep IL-2 in the tumor and the administration site for a long time, and stably deliver IL-2.

3. 면역반응 물질의 면역기관으로의 전달능 확인3. Confirmation of delivery ability of immune reactant to immune organ

도 54, 55는 마우스에 1xPBS buffer (100 μL) 및 Cy5 접합 DegradaBALL (2.5 mg/kg)를 피하 주입하고, 투여 후 1, 3, 5일에 마우스를 희생하여 장기(피부, 흉선, 비장 및 림프절)를 분리한 후, FOBI 이미징 시스템으로 장기의 형광 이미지를 촬영한 결과를 확인할 수 있는데, 1, 3, 5일에 면역기관으로 분포되는 것을 형광이미지를 통해 확인할 수 있는 바, 본 발명의 다공성 실리카 입자가 담지된 다양한 면역반응 물질을 면역기관으로 안정적으로 전달할 수 있음을 입증하는 것이다.Figures 54 and 55 show subcutaneous infusion of 1xPBS buffer (100 μL) and Cy5 conjugated DegradaBALL (2.5 mg / kg) and sacrifice of mice at 1, 3 and 5 days after administration to organs (skin, thymus, spleen and lymph nodes). After separation), the results of photographing the fluorescence image of the organ by the FOBI imaging system can be confirmed, which can be confirmed through the fluorescence image to be distributed to the immune organs on day 1, 3, 5, the porous silica of the present invention It is to prove that the particles can carry a variety of immunoreactive substances to the immune system stably.

실시예 6. 본 발명 전달체의 암 면역치료 효능 확인Example 6 Confirmation of Cancer Immunotherapy Efficacy of the Carrier of the Present Invention

1. 흑색종(melanoma) 모델Melanoma model

도 56을 참조하면, 정상 마우스와 흑색종 종양 마우스에 DegradaBALL을 각각 피하 또는 종양주변으로 주사하였다. 도 57을 참조하면, 모든 마우스에 TAMRA가 달린 DegradaBALL을 각각 2.5, 10, 50 mg/kg의 용량으로 피하로 주입하고, 1, 3, 5, 7일 뒤 마우스를 희생하여 피부조직을 적출하여 형광 이미지를 확보한 결과를 확인할 수 있는데, 주입 후 7일째가 되어도 형광이 잔존하는 것으로 보아, 본 발명 다공성 실리카 입자의 생체 내 안정성이 매우 높아 담지된 면역반응 물질을 안정적으로 오랜기간 서방적으로 전달할 수 있음을 암시케 한다.Referring to FIG. 56, normal and melanoma tumor mice were injected subcutaneously or around tumors, respectively. Referring to FIG. 57, all mice were injected subcutaneously with DegradaBALL with TAMRA at doses of 2.5, 10, and 50 mg / kg, respectively, and skin tissues were extracted at 1, 3, 5, and 7 days later to fluoresce. As a result of obtaining the image, it can be confirmed that the fluorescence remains even on the 7th day after the injection, and thus, the porous silica particles of the present invention have a high in vivo stability, and thus can carry the sustained sustained release of the immunoreactive substance stably for a long time. Imply that there is.

본 발명 전달체의 흑색종 치료효과 우수성을 입증하기 위해, 도 58 및 하기 표 2의 프로토콜에 따라 흑색종 마우스 모델을 이용하여 실험을 진행하였다. 각 그룹별로 종양의 성장속도를 측정하고, 마우스 개체별 종양의 성장속도를 측정한 결과를 도 59에 나타내었는데, aPD-1 항체와 IL-2의 병용투여는 aPD-1 항체 단독투여군 대비 그 성장속도가 낮았는데, IL-2를 다공성 실리카 입자에 담지하여 병용투여한 경우(BALLkine-2) 그 성장속도가 현저하게 낮음을 확인할 수 있다. 또한, 각 군별 생존율 역시 IL-2를 다공성 실리카 입자에 담지하여 병용투여한 경우(BALLkine-2), 그러하지 않은 경우 대비 현저히 높음을 도 60에서 확인할 수 있다.In order to demonstrate the superior melanoma treatment effect of the present invention, an experiment was carried out using a melanoma mouse model according to the protocol of FIG. 58 and Table 2 below. The growth rate of the tumor was measured for each group, and the result of measuring the growth rate of the tumor for each mouse individual was shown in FIG. 59. The combination of aPD-1 antibody and IL-2 was compared with that of the aPD-1 antibody alone group. Although the rate was low, when IL-2 was supported on the porous silica particles and co-administered (BALLkine-2), the growth rate was remarkably low. In addition, it can be seen in FIG. 60 that the survival rate of each group is also significantly higher than that in the case of co-administering IL-2 on porous silica particles (BALLkine-2).

투여 군(Group)Dosing Group 투여 프로토콜Dosing Protocol 총 용량Total capacity Buffer(i.p)Buffer (i.p) 3일 간격으로 7회 복강주사(0,3,6,9,12,15,18일)7 injections every 3 days (0,3,6,9,12,15,18 days) DegradaBALL(s.c)DegradaBALL (s.c) 6일 간격으로 4회 종괴주위주사(0,6,12,18일)4 mass periphery injections every 6 days (0,6,12,18 days) aPD-1 항체(i.p)aPD-1 antibody (i.p) 3일 간격으로 7회 복강주사(0,3,6,9,12,15,18일)7 injections every 3 days (0,3,6,9,12,15,18 days) 70 mg/kg70 mg / kg aPD-1 항체(i.p) + IL-2(i.p)aPD-1 antibody (i.p) + IL-2 (i.p) aPD-1 항체: 3일 간격으로 7회 복강주사(0,3,6,9,12,15,18일)IL-2: 매 5일간 2사이클 복강주사(0,1,2,3,4,10,11,12,13,14일)aPD-1 antibody: 7 intraperitoneal injections every 3 days (0,3,6,9,12,15,18 days) IL-2: 2 cycles intraperitoneally every 5 days (0,1,2,3,4 , 10,11,12,13,14 days) aPD-1 항체: 70 mg/kgIL-2: 10 mg/kg aPD-1 antibody: 70 mg / kg IL-2: 10 mg / kg aPD-1 항체(i.p) + IL-2(s.c)aPD-1 antibody (i.p) + IL-2 (s.c) aPD-1 항체: 3일 간격으로 7회 복강주사(0,3,6,9,12,15,18일)IL-2: 6일 간격으로 4회 종괴주위주사(0,6,12,18일)aPD-1 antibody: 7 intraperitoneal injections every 3 days (0,3,6,9,12,15,18 days) IL-2: 4 mass periphery injections every 6 days (0,6,12,18) Work) aPD-1 항체: 70 mg/kgIL-2: 4 mg/kgaPD-1 antibody: 70 mg / kg IL-2: 4 mg / kg aPD-1 항체(i.p) + BALLkine-2(s.c)(1 또는 0.25 mg/kg)aPD-1 antibody (i.p) + BALLkine-2 (s.c) (1 or 0.25 mg / kg) aPD-1 항체: 3일 간격으로 7회 복강주사(0,3,6,9,12,15,18일)BALLkine-2: 6일 간격으로 4회 종괴주위주사(0,6,12,18일)aPD-1 antibody: 7 intraperitoneal injections every 3 days (0,3,6,9,12,15,18 days) BALLkine-2: 4 mass periphery injections every 6 days (0,6,12,18) Work) aPD-1 항체: 70 mg/kgBALLkine-2: 4 mg/kg 또는 1 mg/kgaPD-1 antibody: 70 mg / kg BALLkine-2: 4 mg / kg or 1 mg / kg

또한, 본 발명 전달체의 전이성 흑색종(metastatic melanoma) 치료효과 우수성을 입증하기 위해, 도 61의 프로토콜에 따라 전이성 흑색종의 폐전이 동물모델에서 실험을 진행하였는데, 상기 도 58 내지 60에서의 결과와 유사하게, aPD-1 항체와 IL-2의 병용투여는 aPD-1 항체 단독투여군 대비 그 성장속도가 낮았는데, IL-2를 다공성 실리카 입자에 담지하여 병용투여한 경우(BALLkine-2) 그 성장속도가 현저하게 낮음을 확인하였다. 구체적으로, 도 61에서 IL-2를 다공성 실리카 입자에 담지하여 aPD-1 항체와 병용투여한 경우(BALLkine-2), 그러하지 않은 경우 대비 검은색의 암 조직 부위가 현저히 적은 면적으로 퍼져있어, 폐 전이가 낮고, 폐종양 결절의 형성과 폐의 부피 증대를 현저히 억제함을 확인할 수 있고, 도 62에서 폐 전이가 진행된 각 폐의 무게 역시 IL-2를 다공성 실리카 입자에 담지하여 aPD-1 항체와 병용투여한 경우(BALLkine-2), 그러하지 않은 경우 대비 현저히 낮음을 확인할 수 있으며(데이터는 3마리의 마우스의 평균±표준편차로 나타냄), 도 63에서 생존율 역시 IL-2를 다공성 실리카 입자에 담지하여 aPD-1 항체와 병용투여한 경우(BALLkine-2), 그러하지 않은 경우 대비 현저히 높음을 확인할 수 있다(군 당 10마리의 마우스).본 발명 전달체의 흑색종 치료에 있어서 면역반응 기전을 탐구하기 위해, 도 64의 프로토콜에 따라 종양침윤림프구(TILs) 분석을 진행하였다. TIL 분석은 목표일(BALLkine-2 투여 후 1,3,5,7일)에 마우스로부터 분리하여, 면역 세포 집단((b) CD8 + T 세포, (c) CD4 + T 세포, (d) 활성화 NK 세포, (e) Treg 세포, (f) CD8 +/Treg 세포)을 유세포분석법으로 분석하였고(점은 개별 종양 조직을, 선은 8마리 마우스의 종양 조직의 평균을 나타냄), 3, 5일 시점의 CD8 + T 세포의 면역 조직을 염색하여 분석하였는데, BALLkine-2 투여군의 대표이미지(도 70)를 참조하면, CD8 + T세포의 수가 극적으로 증가해 광범위한 종양세포 사이에 분포하고 있음을 확인할 수 있다. 또한, BALLkine-2를 B16F10 흑색종 종양을 가진 마우스의 오른쪽 옆구리에 주입(종괴주위주사; peritumoral injection) 후 1,3,5일에 비장과 배수 림프절(장골과 사타구니 림프절)을 분리하고, 림프구(CD8 + T 세포과 NK 세포)를 분리해 면역 세포 집단을 평가하였는데(데이터는 3마리 마우스의 평균±표준편차로 나타냄), 대조군 대비 면역세포의 수가 BALLkine-2 투여군에서 현저히 증가함을 확인할 수 있다.In addition, in order to demonstrate the superior therapeutic effect of metastatic melanoma (metastatic melanoma) of the carrier of the present invention, the experiment was conducted in the lung metastasized animal model of metastatic melanoma according to the protocol of FIG. Similarly, the co-administration of aPD-1 antibody and IL-2 was slower than the aPD-1 antibody alone administration group, but the growth rate was increased when IL-2 was supported on porous silica particles (BALLkine-2). It was confirmed that the speed was remarkably low. In detail, in FIG. 61, IL-2 is supported on porous silica particles and coadministered with aPD-1 antibody (BALLkine-2). The metastasis was low, and the formation of lung tumor nodules and lung volume was significantly suppressed. In FIG. 62, the weight of each lung in which lung metastasis was progressed was also supported by IL-2 on porous silica particles. In the case of co-administration (BALLkine-2), it can be seen that it is significantly lower than the other cases (data is expressed as the mean ± standard deviation of the three mice), the survival rate in Figure 63 also supported IL-2 in porous silica particles In the case of co-administration with aPD-1 antibody (BALLkine-2), it was found to be significantly higher than that without (10 mice per group). , It was performed for tumor infiltrating lymphocytes (TILs) analysis according to the protocol of Figure 64 to obtain. TIL assays were isolated from mice on the target day (1,3,5,7 days after BALLkine-2 administration) to activate immune cell populations ((b) CD8 + T cells, (c) CD4 + T cells, (d) activation). NK cells, (e) Treg cells, (f) CD8 + / Treg cells) were analyzed by flow cytometry (dots represent individual tumor tissues, lines represent the average of tumor tissues of 8 mice), 3, 5 days Analyzes were performed by staining the immune tissues of CD8 + T cells at the time point. Referring to the representative image of the BALLkine-2 administration group (FIG. 70), the number of CD8 + T cells increased dramatically and it was confirmed that they were distributed among a wide range of tumor cells. Can be. In addition, the spleen and drainage lymph nodes (iliac and inguinal lymph nodes) were isolated on days 1 and 3 and 5 after BALLkine-2 was injected into the right flank of mice with B16F10 melanoma tumors. CD8 + T cells and NK cells) were isolated to evaluate immune cell populations (data is expressed as mean ± standard deviation of three mice), indicating that the number of immune cells compared to control group was significantly increased in BALLkine-2 administration group.

2. 신장암(renal cell carcinoma) 모델2. Renal cell carcinoma model

본 발명 전달체의 신장암 치료 우수성을 확인하기 위해, 도 75의 프로토콜에 따라 신장 세포 암종(RCC) 이종 이식 마우스 모델에서, 다공성 실리카 입자에 담지하지 않은 IL-2 대비, 다공성 실리카 입자에 담지한 IL-2(BALLkine-2)의 종양 성장 억제 정도를 확인하고자 하였다. 도 76에서 확인할 수 있듯, BALLkine-2는 IL-2 대비 유의한 종양 성장 억제 우수성을 보여, 신장암 치료 우수성을 입증하였다(데이터는 평균±SEM, 군당 7마리의 마우스, ***p< 0.001: 다른 군과 유의하게 다름).In order to confirm the excellence of renal cancer treatment of the carrier of the present invention, in the renal cell carcinoma (RCC) xenograft mouse model according to the protocol of FIG. 75, IL supported on porous silica particles compared to IL-2 not supported on porous silica particles To determine the extent of tumor growth inhibition of -2 (BALLkine-2). As can be seen in Figure 76, BALLkine-2 showed significant tumor growth inhibition superiority to IL-2, demonstrating excellence in renal cancer treatment (data is mean ± SEM, 7 mice per group, *** p <0.001 : Significantly different from other groups).

또한, RCC의 전이성(metastatic) 및 정위(orthotopic) 마우스 모델에서, 다공성 실리카 입자에 담지하지 않은 IL-2 대비, 다공성 실리카 입자에 담지한 IL-2(BALLkine-2)의 종양 성장 억제 정도를 확인하고자 도 77의 프로토콜에 따라 실험을 진행하였다. 투여 후 10일째에 마우스를 희생시키고, 신장을 적출하여 그 무게를 재었는데(신장무게/체중(%)을 계산함, 데이터는 평균±SD, 그룹당 3마리의 마우스, #p< 0.05, ###p< 0.001: 대조군(Buffer 투여군) 대비 유의하게 다름, *p<0.05: IL-2 투여군 대비 유의하게 다름), IL-2 투여군 대비, BALLkine-2 투여군 에서의 신장 무게가 유의하게 낮음을 도 78에서 확인할 수 있다. 도 79의 적출한 신장의 실측 이미지에서도 BALLkine-2의 신장 크기가 타 군 대비 현저히 작음을 확인할 수 있고, 도 80의 신장 조직의 H&E 염색 사진에서도 타 군 대비, BALLkine-2 처리군에서 조직염색의 정도가 현저히 높음을 확인할 수 있다. 또한, 폐부종을 평가하기 위해, 젖은 폐를 수확하고 무게를 측정한 후, 60℃ 오븐에서 3일 동안 건조시킨 후 무게를 측정한 결과(도 81, 데이터는 평균±SD, 그룹당 3마리 마우스, ##p<0.01: 대조군(Buffer 투여군) 대비 유의하게 다름, **p<0.01: IL-2 투여군 대비 유의하게 다름), BALLkine-2 처리군에서 젖은 폐와 건조한 폐의 무게비가 IL-2 투여군 대비 유의하게 낮음을 확인하였다. In addition, in the metastatic and orthotopic mouse models of RCC, the extent of tumor growth inhibition of IL-2 (BALLkine-2) supported on porous silica particles was compared to IL-2 not supported on porous silica particles. The experiment was conducted according to the protocol of FIG. 77. Mice were sacrificed at 10 days post-dosing, kidneys were removed and weighed (calculated weight / weight in%); data were mean ± SD, 3 mice per group, #p <0.05, ## #p <0.001: significantly different than control group (Buffer group), * p <0.05: significantly different group than IL-2 group), kidney weight of BALLkine-2 group compared to IL-2 group 78. In the measured image of the extracted kidney of FIG. 79, it can be seen that the kidney size of BALLkine-2 was significantly smaller than that of other groups, and H & E staining of the kidney tissue of FIG. It can be seen that the degree is significantly higher. In addition, to evaluate pulmonary edema, wet lungs were harvested and weighed, and then dried in a 60 ° C. oven for 3 days and then weighed (FIG. 81, data are mean ± SD, 3 mice per group, # #p <0.01: significantly different than control group (Buffer group), ** p <0.01: significantly different group than IL-2 group), the weight ratio of wet and dry lungs compared to IL-2 group in BALLkine-2 group It was found to be significantly lower.

또한, 마우스의 혈액 내 백혈구, 호중구 및 림프구를 분석한 결과(도 82, 데이터는 평균±SD, 그룹당 3마리 마우스, #p<0.05: 대조군(Buffer 투여군) 대비 유의하게 다름, *p<0.05: IL-2 투여군 대비 유의하게 다름), 그 수에 있어서, BALLkine-2 처리군에서 타 군 대비 유의하게 높은 수준을 보임에 따라, 면역반응 촉진 효과에 있어서 현저한 우수성을 확인하였다. In addition, the results of analysis of leukocytes, neutrophils and lymphocytes in the blood of mice (FIG. 82, data are mean ± SD, 3 mice per group, #p <0.05: significantly different from the control group (Buffer administration group), * p <0.05: Significantly different from IL-2 administration group), the number of BALLkine-2 treatment group showed significantly higher level than the other groups, and thus the remarkable excellence in the immune response promoting effect was confirmed.

3. 기타 암 모델3. Other cancer models

흑색종과 신장암을 포함한 다른 암종에 대한 본 발명 조성물의 효과를 하기 표 3에 나타내었다.The effects of the present compositions on other carcinomas, including melanoma and kidney cancer, are shown in Table 3 below.

암종carcinoma 세포주Cell line 측정변수Measurement variable 대조군(vehicle)Control 실험군 1Experimental group 1 실험군 2Experiment group 2 실험군 3Experiment group 3 흑색종Melanoma B16F10B16F10 상대적 종양크기(12일)Relative tumor size (12 days) 14.114.1 IL-2/aPD-1항체IL-2 / aPD-1 Antibody IL-2+DegradaBALL/aPD-1항체IL-2 + DegradaBALL / aPD-1 Antibody 9.19.1 4.34.3 상대적 종양크기(10일)Relative tumor size (10 days) 12.512.5 IL-15IL-15 IL-15+DegradaBALLIL-15 + DegradaBALL 8.28.2 3.93.9 신장암Kidney cancer RencaRenca 체중량 대비 신장무게(w/w%; 10일)Kidney Weight to Weight (w / w%; 10 days) 1.051.05 IL-2IL-2 IL-2+DegradaBALLIL-2 + DegradaBALL 0.860.86 0.780.78 간암Liver cancer BNL-h1BNL-h1 간 전이의 수Number of liver metastases 8585 IL-12IL-12 IL-12+DegradaBALLIL-12 + DegradaBALL 99 22 대장암Colorectal cancer CT26(s.c)CT26 (s.c) 종양크기(mm 3; 19일)Tumor size (mm 3 ; 19 days) 452452 aPD-L1항체aPD-L1 Antibody aPD-L1항체+DegradaBALLaPD-L1 Antibody + DegradaBALL 220220 187187 CT26(i.v)CT26 (i.v) 생체발광, 광자량/sBioluminescence, photon / s 3838 IL-2IL-2 IL-2+DegradaBALLIL-2 + DegradaBALL 1111 1.41.4 폐암Lung cancer LLCLLC 생체발광, 광자량/sBioluminescence, photon / s 3.93.9 IL-12IL-12 IL-12+DegradaBALLIL-12 + DegradaBALL 1.11.1 0.40.4 PTX/CDDPPTX / CDDP IL-2/PTX/CDDPIL-2 / PTX / CDDP IL-12+DegradaBALL/PTX/CDDPIL-12 + DegradaBALL / PTX / CDDP 3.83.8 0.20.2 0.010.01 유방암Breast cancer 4T14T1 종양크기(mm 3; 20일)Tumor size (mm 3 ; 20 days) 411411 IL-12IL-12 IL-12+DegradaBALLIL-12 + DegradaBALL 32.432.4 8.38.3 EMT-6EMT-6 종양크기(mm 3; 28일)Tumor size (mm 3 ; 28 days) 953953 IL-21IL-21 IL-21+DegradaBALLIL-21 + DegradaBALL 521521 120120 aCTLA-4항체aCTLA-4 Antibody aCTLA-4항체+DegradaBALLaCTLA-4 Antibody + DegradaBALL IL-21/aCTLA-4항체+DegradaBALLIL-21 / aCTLA-4 Antibody + DegradaBALL 320320 8989 2121 위암Stomach cancer SGC-7901SGC-7901 종양크기(mm 3; 28일)Tumor size (mm 3 ; 28 days) 12851285 IL-24IL-24 IL-24+DegradaBALLIL-24 + DegradaBALL 423423 121121 췌장암Pancreatic cancer HS766THS766T 종양크기(mm 2; 28일)Tumor size (mm 2 ; 28 days) 8181 IL-13IL-13 IL-13+DegradaBALLIL-13 + DegradaBALL 1414 3.23.2 두경부암Head and Neck Cancer KCCT873KCCT873 종양크기(mm 2; 30일)Tumor size (mm 2 ; 30 days) 138138 IL-13IL-13 IL-13+DegradaBALLIL-13 + DegradaBALL 2929 1111 IL-13/CpGIL-13 / CpG IL-13+DegradaBALL/CpGIL-13 + DegradaBALL / CpG IL-13+DegradaBALL/CpG+DegradaBALLIL-13 + DegradaBALL / CpG + DegradaBALL 1919 4.24.2 1.71.7

실시예 7. 본 발명 전달체의 부작용 감소효과 확인Example 7. Confirmation of the side effect reduction effect of the present invention delivery vehicle

사이토카인의 투여로 인한 다양한 부작용이 존재하는데, 그 중, IL-2 투여로 인한 부작용으로 혈관 누출 증후군(capilary leak syndrome (CLS) 또는 vascular leak syndrome (VLS))이 잘 알려져 있다. 이에, 본 발명 전달체가 담지하는 물질의 부작용을 감소시키는 효과가 존재하는지 확인하고자 하였고, IL-2 사이토카인을 담지한 다공성 실리카 입자인 BALLkine-2를 이용하여 실험을 진행하였다.There are a variety of side effects due to the administration of cytokines, among which the side effects due to the administration of IL-2 (capilary leak syndrome (CLS) or vascular leak syndrome (VLS)) is well known. Thus, the present invention was to determine whether there is an effect of reducing the side effects of the carrier supported by the present invention, the experiment was carried out using BALLkine-2, porous silica particles carrying IL-2 cytokine.

도 83의 투여일정과 CLS분석 프로토콜에 따라 실험을 진행하였는데, 구체적으로, 3일간 IL-2(15 μg)와 BALLkine-2(15 μg)을 C57BL/6 마우스에 투여한 후, 4일 째 분석하였다. 완충액(i.v 또는 s.c), DegradaBALL(i.v 또는 s.c), IL-2(i.v 또는 s.c) 또는 BALLkine-2(i.v 또는 s.c)를 투여한 마우스 폐 조직의 H&E 염색 대표 이미지(도 84)를 참조하면, BALLkine-2 투여군에서 IL-2(i.v 또는 s.c) 투여군 대비 혈관 내부와 주변의 염증 세포 수를 감소시킴을 확인할 수 있다(막대는 200 μm을 나타냄). 또한, 폐부종을 평가하기 위해 젖은 폐를 분리해 무게를 측정하고, 폐를 60 ℃ 오븐에서 3일 동안 건조하고 무게를 측정하였는데(도 85), BALLkine-2 투여군에서 IL-2(i.v 또는 s.c) 젖은 폐 대비 건조한 폐의 무게비(젖은 폐 무게/건조한페 무게)가 유의미하게 낮음을 확인하였다. 또한, 상기 VLS 모델에서 꼬리 정맥을 통해 에반스 블루 염료를 희생하기 2시간 전 마우스에 주입하고, 희생 후 간(도 86)과 폐(도 87)를 분리하고 VLS의 심화정도를 에반스 블루의 혈관 외 유출을 통해 측정하여 분석하였는데, BALLkine-2 투여군에서 IL-2(i.v 또는 s.c) 투여군 대비 그 유출의 정도가 현저히 낮음을 확인하였다. 또한, 3일째에 마우스의 혈액학적 분석을 위해 혈액을 채취하여 백혈구, 호중구 및 림프구를 분석하였는데, 모두 BALLkine-2 투여군에서 IL-2(i.v 또는 s.c) 투여군 대비 그 수가 유의하게 높음을 확인하였다(도 88, 데이터는 완충액 및 DegradaBALL 처리군 당 3~4마리, BALLkine-2 군 당 4~7마리 마우스의 평균±표준 편차로 나타냄; 완충액(i.v 또는 s.c) 군과 유의한 차이( #p<0.05; ##p<0.01; ###p<0.001), IL-2(i.v 또는 s.c) 군과 유의한 차이( *p<0.05; **p<0.01; ***p<0.001).The experiment was carried out according to the administration schedule of FIG. 83 and the CLS analysis protocol. Specifically, three days after the administration of IL-2 (15 μg) and BALLkine-2 (15 μg) to C57BL / 6 mice, the analysis was performed on the fourth day. It was. Referring to a representative H & E staining image of mouse lung tissue administered buffer (iv or sc), DegradaBALL (iv or sc), IL-2 (iv or sc) or BALLkine-2 (iv or sc) (FIG. 84), In the BALLkine-2 administration group, the number of inflammatory cells in and around the blood vessels was reduced compared to the IL-2 (iv or sc) administration group (the rod indicates 200 μm). In addition, wet lungs were separated and weighed to evaluate pulmonary edema, and lungs were dried and weighed for 3 days in an oven at 60 ° C. (FIG. 85), IL-2 (iv or sc) in the BALLkine-2 administration group. It was confirmed that the weight ratio (wet lung weight / dry weight) of the dry lung to the wet lung was significantly lower. In addition, in the VLS model, mice were injected 2 hours before sacrificing Evans blue dye through the tail vein, and after sacrifice, the liver (Fig. 86) and the lung (Fig. 87) were separated, and the degree of VLS intensified by Evans Blue et al. It was measured and analyzed through the outflow, it was confirmed that the degree of the outflow was significantly lower in the BALLkine-2 administration group than the IL-2 (iv or sc) administration group. In addition, on day 3, blood was collected for hematological analysis of mice, and leukocytes, neutrophils, and lymphocytes were analyzed, and all of them were significantly higher in the BALLkine-2 group than in the IL-2 (iv or sc) group. 88, the data buffer and DegradaBALL process 3-4, shown as the mean ± SD of BALLkine-2 4 ~ 7 mice per group per group; buffer (iv or sc) significantly as the group difference (# p <0.05 ; ## p <0.01;### p <0.001), significant difference from IL-2 (iv or sc) group ( * p <0.05; ** p <0.01; *** p <0.001).

실제 암의 면역치료에 있어서도 혈관 누출 증후군을 감소시킬 수 있는지 확인하기 위해, 도 89의 프로토콜에 따라 종양 보유 마우스 모델에서 혈관 누출 증후군(VLS) 분석을 진행하였다. 폐부종을 평가하기 위해 젖은 폐를 분리해 무게를 측정하고, 폐를 60 ℃ 오븐에서 3일 동안 건조하고 무게를 측정하였는데(도 90), BALLkine-2 투여군에서 IL-2(i.v 또는 s.c) 투여군 대비 그 무게비가 유의하게 낮았고, 정상군과 유사한 수치를 나타내었다. 또한, VLS 모델에서 꼬리 정맥을 통해 에반스 블루 염료를 희생하기 2시간 전 마우스에 주사한 후, 간(도 91)과 폐(도 92)를 분리하고, 에반스 블루의 혈관 외 유출을 통해 측정한 VLS를 분석하였는데, BALLkine-2 투여군에서 IL-2(i.v 또는 s.c) 투여군 대비 그 유출의 정도가 현저히 낮음을 확인하였다.Vascular Leakage Syndrome (VLS) analysis was performed in a tumor bearing mouse model according to the protocol of FIG. 89 to determine if vascular leakage syndrome could be reduced even in actual cancer immunotherapy. To evaluate pulmonary edema, wet lungs were separated and weighed, and lungs were dried and weighed for 3 days in an oven at 60 ° C. (FIG. 90), compared to IL-2 (iv or sc) -administered groups in the BALLkine-2 administration group. The weight ratio was significantly lower and showed similar values to the normal group. In addition, in the VLS model, mice injected 2 hours before sacrificing Evans blue dye through the tail vein, and then separated the liver (Figure 91) and lung (Figure 92) and measured VLS through extravasation of Evans Blue It was confirmed that the BALLkine-2 administration group was significantly lower than the IL-2 (iv or sc) administration group.

상기의 결과는, 본 발명 다공성 실리카 입자에 항체 또는 사이토카인과 같은 면역반응 물질을 담지하여 생체 내 전달하는 경우, 각 항체 또는 사이토카인이 갖는 본래의 부작용을 현저히 감소시키는 결과를 낳음으로써, 본래의 약리적 치료효과를 증대시킬 수 있음을 입증하는 유의미한 결과로 판단된다.The above results result in significantly reducing the inherent side effects of each antibody or cytokine when the porous silica particles of the present invention carry an immunoreactive substance such as an antibody or cytokine and deliver them in vivo. The results suggest that the pharmacological treatment can be enhanced.

Claims (12)

항체(antibody) 또는 사이토카인(cytokine)을 담지한 다공성 실리카 입자를 포함하고, It comprises porous silica particles carrying an antibody or cytokine (cytokine), 상기 다공성 실리카 입자는 직경 5nm 미만의 기공을 갖는 실리카 입자를 120℃ 내지 180℃에서 24시간 내지 96시간 동안 팽창제와 반응시켜 상기 직경 5nm 미만의 기공을 팽창시키는 단계; 및 상기 기공이 팽창된 실리카 입자를 400℃ 이상의 온도에서 3시간 이상 하소하는 단계를 포함하여 제조되며,The porous silica particles react with silica particles having pores less than 5 nm in diameter at 120 ° C. to 180 ° C. for 24 to 96 hours to expand the pores less than 5 nm in diameter; And calcining the pores of expanded silica particles at a temperature of 400 ° C. or higher for at least 3 hours. 상기 다공성 실리카 입자의 평균 직경은 150 nm 내지 1000nm이고, 그 BET 표면적은 200m 2/g 내지 700m 2/g이고, 그 g당 부피는 0.7ml 내지 2.2ml이며,The average diameter of the porous silica particles is 150 nm to 1000 nm, the BET surface area is 200m 2 / g to 700m 2 / g, the volume per g is 0.7ml to 2.2ml, 상기 다공성 실리카 입자는 하기 수학식 1의 흡광도의 비가 1/2이 되는 t가 24 이상인 것인 면역반응 물질 전달체:The porous silica particles have an immunoreactive substance carrier in which t is 24 or more such that the ratio of absorbance of Equation 1 is 1/2: [수학식 1] [Equation 1] A t/A 0 A t / A 0 (식 중, A 0는 상기 다공성 실리카 입자 1mg/ml 현탁액 5ml를 직경 50kDa의 기공을 갖는 원통형 투과막에 넣고 측정된 다공성 실리카 입자의 흡광도이고,Wherein A 0 is the absorbance of the porous silica particles measured by placing 5 ml of the 1 mg / ml suspension of the porous silica particles into a cylindrical permeable membrane having pores having a diameter of 50 kDa, 상기 투과막 외부에는 상기 투과막과 접하며, 상기 현탁액과 동일한 용매 15ml가 위치하고, 상기 투과막 내외부는 37℃에서 60rpm 수평 교반되며,Outside of the permeable membrane is in contact with the permeable membrane, 15ml of the same solvent as the suspension is located, the inside and outside of the permeable membrane is stirred 60 rpm at 37 ℃ horizontal, 상기 현탁액의 pH는 7.4이고,The pH of the suspension is 7.4, A t는 상기 A 0의 측정시로부터 t시간 경과 후에 측정된 다공성 실리카 입자의 흡광도임).A t is the absorbance of the porous silica particles measured after t hours have elapsed since the measurement of A 0 ). 청구항 1에 있어서, 상기 항체는 인터류킨(interleukins) 또는 인터페론(interferons) 단백질에 특이적으로 결합하는 항체인 전달체.The carrier of claim 1, wherein the antibody is an antibody that specifically binds to an interleukins or interferons protein. 청구항 1에 있어서, 상기 항체는 IgG 이거나; PD-1, CTLA-4, TIM-3, BTLA, VISTA, LAG-3, PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD27, CD137, HVEM, GITR, VEGFR, VEGF, EGFR, EGF, IL-1, IL-6, IL-23, TGF-beta, CTGF, TSLP, TNF-alpha, Notch 및 OX40으로 이루어진 군에서 선택된 적어도 하나의 단백질에 특이적으로 결합하는 항체인 전달체.The method of claim 1, wherein the antibody is an IgG; PD-1, CTLA-4, TIM-3, BTLA, VISTA, LAG-3, PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD27, CD137, HVEM, GITR, VEGFR, VEGF, A carrier that is an antibody that specifically binds to at least one protein selected from the group consisting of EGFR, EGF, IL-1, IL-6, IL-23, TGF-beta, CTGF, TSLP, TNF-alpha, Notch and OX40. 청구항 1에 있어서, 상기 항체는 PD-1, PD-L1 및 CTLA-4로 이루어진 군에서 선택된 적어도 하나의 단백질에 특이적으로 결합하는 항체인 전달체.The carrier of claim 1, wherein the antibody is an antibody that specifically binds to at least one protein selected from the group consisting of PD-1, PD-L1, and CTLA-4. 청구항 1에 있어서, 상기 사이토카인은 인터류킨(interleukins) 또는 인터페론(interferons)인 전달체.The carrier of claim 1, wherein the cytokine is interleukins or interferons. 청구항 1에 있어서, 상기 사이토카인은 IL-7, IL-10, IL-12, IL-13, IL-15, IL-21, IL-23, IL-24, IL-27, G-CSF, GM-CSF, HGF, EGF, VEGF, LTF, TGF-β 및 IL-2로 이루어진 군에서 선택된 적어도 하나인 전달체.The method according to claim 1, wherein the cytokine is IL-7, IL-10, IL-12, IL-13, IL-15, IL-21, IL-23, IL-24, IL-27, G-CSF, GM -A carrier which is at least one selected from the group consisting of CSF, HGF, EGF, VEGF, LTF, TGF-β and IL-2. 청구항 1에 있어서, 상기 사이토카인은 IL-2, IL-12, IL-15, IL-21, IL-24 및 IL-13으로 이루어진 군에서 선택된 적어도 하나인 전달체.The carrier of claim 1, wherein the cytokine is at least one selected from the group consisting of IL-2, IL-12, IL-15, IL-21, IL-24, and IL-13. 청구항 1에 있어서, 상기 다공성 실리카 입자는 외부 표면 또는 기공 내부가 중성의 pH에서 전하를 띠는 것인 전달체.The carrier of claim 1, wherein the porous silica particles are charged at an external pH or inside the pore at a neutral pH. 청구항 1에 있어서, 상기 다공성 실리카 입자는 외부 표면 또는 기공 내부에 친수성 또는 소수성 작용기를 갖는 것인 전달체.The carrier of claim 1, wherein the porous silica particles have hydrophilic or hydrophobic functional groups on the outer surface or inside the pores. 청구항 1에 있어서, 항체(antibody)를 담지한 다공성 실리카 입자; 및 사이토카인(cytokine)을 담지한 다공성 실리카 입자를 포함하는 전달체.The method according to claim 1, Porous silica particles carrying an antibody (antibody); And a porous silica particle carrying a cytokine. 청구항 1 내지 10 중 어느 한 항의 전달체를 포함하는 면역요법(immunotherapy)용 조성물.An immunotherapy composition comprising the carrier of any one of claims 1 to 10. 청구항 1 내지 10 중 어느 한 항의 전달체를 포함하는 암 또는 면역질환의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for the prophylaxis or treatment of cancer or immune disease comprising the carrier of any one of claims 1 to 10.
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