[go: up one dir, main page]

WO2025239786A1 - Glutathione-sensitive monomer - n-acrylocystine, polymer comprising n-acrylocystine, their synthesis and the use of the polymer as a carrier for intracellular, controlled drug delivery - Google Patents

Glutathione-sensitive monomer - n-acrylocystine, polymer comprising n-acrylocystine, their synthesis and the use of the polymer as a carrier for intracellular, controlled drug delivery

Info

Publication number
WO2025239786A1
WO2025239786A1 PCT/PL2025/050044 PL2025050044W WO2025239786A1 WO 2025239786 A1 WO2025239786 A1 WO 2025239786A1 PL 2025050044 W PL2025050044 W PL 2025050044W WO 2025239786 A1 WO2025239786 A1 WO 2025239786A1
Authority
WO
WIPO (PCT)
Prior art keywords
polymer
monobiss
cancer
drug
reaction
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.)
Pending
Application number
PCT/PL2025/050044
Other languages
French (fr)
Inventor
Şerife DAĐDELEN
Marcin MAĆKIEWICZ
Jan Romański
Marcin KARBARZ
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.)
Uniwersytet Warszawski
Original Assignee
Uniwersytet Warszawski
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 claimed from PL449234A external-priority patent/PL449234A1/en
Application filed by Uniwersytet Warszawski filed Critical Uniwersytet Warszawski
Publication of WO2025239786A1 publication Critical patent/WO2025239786A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/57—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C323/58—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton
    • C07C323/59—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton with acylated amino groups bound to the carbon skeleton
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • 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
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L33/00—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides or nitriles thereof; Compositions of derivatives of such polymers
    • C08L33/24—Homopolymers or copolymers of amides or imides
    • C08L33/26—Homopolymers or copolymers of acrylamide or methacrylamide

Definitions

  • the subject matter of the invention is a novel comonomer, N-acryloylocystine (monoBISS), a novel polymer comprising N-acryloylocystine, their synthesis and the use of thus obtained glutathione-sensitive pNIPA gel as a drug carrier.
  • Combination therapies with the use of several drugs are becoming increasingly popular. By acting on cancer cells at different stages of the cell cycle, combination therapy increases the likelihood of eliminating all cancer cells and preventing the development of drug resistance. For example, one drug can interfere with DNA replication, while another interferes with protein synthesis, effectively attacking malignant cells and reducing the chances of developing drug resistance. Optimizing combination therapy is aimed at achieving synergistic effects of several drugs, thanks to which the combined effect of the drug mixture exceeds the effect of individual drugs (Y.
  • DDS drug delivery systems
  • Polymeric drug delivery systems provide an effective approach to the treatment of a variety of diseases, including neurological disorders, cardiovascular diseases, and cancer. These systems enable a targeted and controlled release of therapeutic agents, thereby limiting the side effects of drugs (Robert M Sharkey, David M Goldenberg, Targeted therapy of cancer: new prospects for antibodies and immunoconjugates, Cancer Journal for Clinicians, 56 (2006), pp. 226-43).
  • cancer which remains one of the leading causes of human death, is of particular concern (J Ferlay et al., Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods, International Journal of Cancer, 15;144 (2019), pp. 1941-1953).
  • IARC International Agency for Research on Cancer
  • 19.3 million cancers were diagnosed worldwide in 2023, and the estimated increase by 2040 is 30.2 million.
  • nano-DDS drug delivery nanosystems
  • Nanogels a type of nano-DDS, are materials comprising a three-dimensional polymer network, with size in the submicrometer range. They exhibit unique properties such as the ability to absorb large amounts of water and undergo significant volume change in response to external factors such as temperature, pH (ionization-sensitive comonomers), ionic strength (charged comonomers), light (photosensitive comonomers), and field (S. Dagdelen et al., Redox-responsive degradable microgel modified with superparamagnetic nanoparticles exhibiting controlled, hyperthermia- enhanced drug release, Journal of Material Science, 58 (2023), pp. 4094-4114).
  • the volumetric phase transition i.e.
  • micro/nanogels can be targeted to specific sites in the body by functionalizing them with receptors capable of specifically recognizing target objects, such as cancer cells. Modified micro- and nanogels can be transported by blood, skin, or inhalation, to diseased tissues, minimizing interactions with healthy tissues (S. S. Makhathini, S. et al., Biomedicine Innovations and Its Nanohydrogel Classifications, Pharmaceutics, 14 (2022)).
  • Temperature-responsive polymers including alkylacrylamides, vinyl ethers, vinyl caprolactam, and monomers of alkylene oxides such as ethylene oxide and propylene oxide are often used to obtain the above drug delivery systems.
  • Poly(N-isopropylacrylamide) (pNIPA) is often used in the synthesis of drug carriers, due to its lower critical dissolution temperature (LCST) close to human body temperature (32 °C), making it a good candidate for drug delivery applications (A. Krishnan, S. Roy, S. Menon, Amphiphilic block copolymers: From synthesis including living polymerization methods to applications in drug delivery, European Polymer Journal, 172 (2022)).
  • LCST pNIPA can be adjusted by copolymerizing it with hydrophilic or hydrophobic comonomers.
  • Crosslinking agents such as N,N'-methylenebisacrylamide (BIS) are commonly used in these systems.
  • Polymeric drug delivery systems especially nano-DDS, which include nanogels, provide promising opportunities for targeted and controlled release of therapeutic agents. This is a key aspect leading to better treatment outcomes and reduced drug side effects, especially for a variety of diseases, including cancer.
  • the adjustable properties and speed of response of these systems to external stimuli provide them with versatility and great potential in the preparation of precise drug delivery strategies.
  • the object of the present invention is N-acryloy Icy stine (monoBISS) of formula (I):
  • a further object of the invention is a polymer comprising the above-mentioned N- acryloylocystine as a comonomer.
  • the polymer of the invention is characterized in that it is a polymer based on N- isopropylacrylamide and N-acryloylocystine, cross-linked with N,N'-methylenebisacrylamide and having a structure of formula (II): characterized in that it is a polymer based on N- isopropylacrylamide and N-acryloylocystine, cross-linked with N,N'-methylenebisacrylamide and having a structure of formula (II): or a polymer based on N-isopropylacrylamide and N-acryloylocystine, cross-linked with N,N'- bisacryloylocystine (BISS) and having a structure of formula (III):
  • Another object of the invention is a method for preparing N-acryloylocystine monomer (monoBIS S), characterized in that it comprises the following steps: a) acryloyl chloride is added to the basic solution of L-cystine, especially in NaOH, b) the reaction mixture is acidified, c) the reaction mixture is stirred, cooled and the supernatant is collected, d) the collected supernatant is acidified and then saturated with NaCl and extracted with ethyl acetate, e) the organic phase is evaporated and the residue is purified by column chromatography on silica gel to afford the product.
  • step a) 1.65 M NaOH is used and the reaction is carried out for 30 minutes at 0°C.
  • step e) the purification by column chromatography on silica gel is conducted using 20% methanol, 2% CF3COOH in chloroform.
  • Another object of the invention is a method for preparing a polymer, characterized in that a precipitation polymerization method in an inert gas atmosphere is used, comprising the following steps: a) N-isopropylacrylamide (NIPA) is reacted with a cross-linking agent in an aqueous solution, wherein N,N'-methylenebisacrylamide (BIS) or N,N'-bisacryloylocystine (BISS) is used as the crosslinking agent, b) N,N,N,N-tetram ethylethylenediamine and ammonium persulfate (APS) are added and a polymerization reaction is conducted, c) an aqueous solution of N-acryloylcystine (monoBISS) is added and the polymerization reaction
  • step a) the reaction is carried out at a temperature of 80°C and deoxidized for 0.5 hour with argon.
  • step c) a 10 mol% solution of N-acryloylocy stine (monoBISS) is used and the reaction is carried out for 4 hours.
  • N-acryloylocy stine monoBISS
  • the gel is purified by placing it in a dialysis membrane with a cut-off molecular weight of 10,000 Da and removing the unreacted substrates.
  • a further object of the invention is a polymer as defined above, for use in pharmacy.
  • the polymer for use of the invention is a drug carrier, especially for use in the treatment or prevention of cancer.
  • the polymer for use of the invention is a drug carrier for use in the treatment or prevention of lung cancer, laryngeal cancer, oral cancer, breast cancer or ovarian cancer.
  • the present invention provides a novel comonomer, N-acryloylcystine (hereinbelow referred to as monoBISS), which is then used to synthesize, using precipitation polymerization, an intelligent drug carrier, a pNIPA gel sensitive only to glutathione.
  • Precipitation polymerization is a method used to produce gels with desired properties. In this method, the polymerization reaction takes place in a solvent system in which the monomers are soluble, but the resulting polymers are no longer, leading to the formation of polymer particles by precipitation. The monomers are dissolved in a suitable solvent, then a polymerization initiator is added to initiate the reaction.
  • the resulting polymers cross-link and form micro/nanogel particles that precipitate out of the solvent.
  • the resulting micro/nanogels are typically well formed, meaning that they have a defined shape and structure and typically exhibit a monodispersive size distribution - a narrow range of particle sizes. This is preferred in drug delivery, as obtaining micro/nano gels of the same size and constant properties allows for controlling the kinetics of drug release.
  • the physicochemical properties of the obtained gels can be controlled by the concentration of monomers, polymerization temperature, initiator concentration, mixing rate or reaction time.
  • the novel micro/nanogel can be a polymer based on monomers, such as: N-vinyl caprolactam (VCL), diethylene glycol methyl ether methacrylate (DEGMEA), poly(ethylene glycol) methyl ether methacrylate (EGMEA), N- isopropyl acrylamide (NIPA) and vinyl pyrrolidone (VP) and may be cross-linked by crosslinking agents such as: N,N-methylenebisacrylamide (BIS) or degrading N,N-bis(acryloyl)cystamine (BAC).
  • VCL N-vinyl caprolactam
  • DEGMEA diethylene glycol methyl ether methacrylate
  • EGMEA poly(ethylene glycol) methyl ether methacrylate
  • NIPA N- isopropyl acrylamide
  • VP vinyl pyrrolidone
  • crosslinking agents such as: N,N-methylenebisacrylamide (BIS) or degrading N,N-
  • monoBISS due to the presence of carboxyl and a-amino acid groups in monoBISS, such gels are pH-sensitive and stable. Thus, the use of monoBISS facilitates the synthesis of micro/nanogels, there is no need to use additional comonomers responsible for stability and pH- sensitivity.
  • carboxyl and amino groups in monoBISS allows for covalent binding of various anti cancer drugs and control of their release. The most important thing is that the drug can be released from the gel after cutting the S-S bonds, which is possible in the presence of glutathione, which is found in human cells. Glutathione leads to the reduction of S-S bonds to thiol groups.
  • Glutathione is a tripeptide that is usually found in elevated concentrations in most cancer cells. It is worth noting that the concentration of glutathione in the cytoplasm is much higher (in the range of 0.5-10 mM) compared to extracellular fluids (2-20 pM) and reaches up to 1000 times higher concentrations.
  • cancers such as lung cancer, laryngeal cancer, oral cancer, breast cancer, and ovarian cancer show higher glutathione levels (up to 40 mM) compared to healthy cells. Cancer cells often show higher levels of intracellular glutathione compared to healthy cells, a mechanism that promotes survival of these cells and resistance to chemotherapy. These in vitro studies were performed on MCF-7 breast cancer cells with a GSH concentration of 40 mM and on MCF-10A healthy cells with a GSH concentration of approximately 1 mM.
  • the aim of the present invention is therefore to provide a carrier for the specific intracellular delivery of cysteine-modified therapeutic substances. It is essential that such a substance has a free amino or carboxyl group that can be used to form a peptide bond with a monoBISS monomeric unit in a gel carrier network.
  • a negatively charged drug may readily bind to positively charged comonomer groups, while a positively charged drug may be effectively loaded into negatively charged comonomer groups, and vice versa.
  • This strategy allows the selective and controlled loading and release of drugs from micro/nanogels, which provides potential benefits for various biomedical applications.
  • the present invention allows to increase the effectiveness of drug delivery and cancer treatment.
  • N,N'-bis(acryloyl)cystamine BAC
  • BISS N,N'- bis(acryloyl)cystine
  • the polymer of the invention is interchangeably referred to as a gel or microgel.
  • the particle sizes of the microgel are in the range of 100 nm to 100 pm.
  • the process of preparing a polymer of suitable particle size may be modulated by manipulating key parameters such as mixing speed, temperature, and total microgel concentration. By selecting these parameters appropriately, gel variants with particle sizes of less than 100 nm, corresponding to nanogels, can also be synthesized.
  • BISS comprising the S-S bonds acts as a crosslinking agent, giving the monoBISS gel, among other things, degradable properties.
  • the unique distinguishing features of the present solution consist in the multifunctionality and water solubility of the monoBISS comonomer, which constitutes the main functional component of the gel drug carrier.
  • monoBISS allows the synthesis of gels in aqueous environments without the need for organic solvents.
  • monoBISS is distinguished by multifunctional properties due to the presence of carboxyl and a-amino acid groups, and -S-S- bridges in its structure. These functional groups provide pH-sensitivity, stability, possibility of modification with therapeutic substances comprising amino or carboxyl groups.
  • a selective response to glutathione provides a controlled release of the drug, particularly in the tumor environment, minimizing undesired side effects.
  • the presence of carboxyl and amino groups in monoBISS allows for covalent binding of two various anti cancer drugs with gels of the invention and control of their loading. Thanks to the invention, it is possible to use the synergistic effect of two drugs to achieve more effective toxicity to cancer cells. It is important that the drug can be released from the microgels after the reduction of S-S bonds and this is only possible in the presence of the cutting S-S bonds, glutathione, which is found in cells and its concentration in cancer cells is usually elevated.
  • Figure 3A shows the hydrodynamic diameter (Dh) of the p(NIPA-MonoBISS) microgel as a function of temperature and pH.
  • the ionic strength was kept constant at 10 mM.
  • Figure 3B shows a graph of the dependence of Dh on temperature for microgels suspended in solutions with different salt concentrations (NaCl).
  • Figure 4 shows the DOX release profiles
  • Figure 5 shows a diagram of synthesis of p(NIPA-BIS-monoBISS) gels.
  • Figure 6 shows a diagram of synthesis of p(NIPA-BISS-monoBISS) gels.
  • FIG. 2a A diagram of the synthesis of the monomer of the invention - N-acryloylcystine (monoBISS) is shown in Figure 2a.
  • L-cystine 2.65 g, 11 mmol
  • NaOH 1 M
  • acryloyl chloride 404 pl, 5 mmol
  • pH 6 with 6 M HC1.
  • the excess L-cystine precipitate was filtered out and washed with water several times.
  • NIPA N-isopropylacrylamide
  • BISS N-bis(acryloyl)cystine
  • microgels were synthesized in an aqueous solvent that provides polymerization environment.
  • the synthesis of the microgel can be carried out at different molar fractions of the crosslinking agent or the main monomer.
  • a precipitation polymerization method was used for the synthesis of microgels. Polymerization was carried out in a three-necked flask equipped with a reflux condenser, magnetic stirrer (set at 250 rpm during the entire polymerization process), inlet and outlet of inert gas.
  • p(NIPA-BIS-monoBISS) microgel synthesis A diagram of p(NIPA-BIS-monoBISS) microgel synthesis is shown in Figure 5.
  • the main NIPA monomer and BIS cross-linking agent (2 mol%) were dissolved in 16 mL deionized water and placed in a flask. The solution was heated to 80°C and deoxidized for 0.5 hour with argon. Then 5 pl of N,N,N,N-tetramethylethylenediamine (TEMED) and 9 mg of an initiator - ammonium persulfate (APS) dissolved in 2 ml of deionized water were added to initiate polymerization. Further, an aqueous monoBISS solution (10 mol%, 2 ml, pH 4) was added to the reactor. The reaction lasted 4 hours under argon. The total monomer concentration was 100 mM.
  • TEMED N,N,N,N-tetramethylethylenediamine
  • APS initiator - ammonium persulfate
  • the resulting emulsion was stirred overnight at room temperature using a magnetic stirrer.
  • the microgels were then purified by placing them in a dialysis membrane with a cut-off molecular weight of 10,000 Da (Spectra/Por® 7 dialysis membrane), thereby removing the unreacted substrates. Dialysis lasted 5 days, the water was changed daily.
  • the concentration of NIPA, BIS and monoBISS was 100 mM at 2% mole fraction of BIS and 10% mole fraction of monoBISS.
  • the APS concentration was 5 mM.
  • the main NIPA monomer and BISS cross-linking agent (2 mol%) were dissolved in 16 mL deionized water and placed in a flask. The solution was heated to 80°C and deoxidized for 0.5 hour with argon. Then 5 pl of N,N,N, N-tetram ethylethylenediamine (TEMED) and 9 mg of an initiator - ammonium persulfate (APS) dissolved in 2 ml of deionized water were added to initiate polymerization. Further, an aqueous monoBISS solution (10 mol%, 2 ml, pH 4) was added to the reactor. The reaction lasted 4 hours under argon. The total monomer concentration was 100 mM.
  • TEMED N,N,N, N-tetram ethylethylenediamine
  • APS initiator - ammonium persulfate
  • the resulting emulsion was stirred overnight at room temperature using a magnetic stirrer.
  • the microgels were then purified by placing them in a dialysis membrane with a cut-off molecular weight of 10,000 Da (Spectra/Por® 7 dialysis membrane), thereby removing the unreacted substrates. Dialysis lasted 5 days, the water was changed daily.
  • the concentration of NIPA, BISS and monoBISS was 100 mM at 2% mole fraction of BIS and 10% mole fraction of monoBISS.
  • the APS concentration was 5 mM.
  • Microgel characterization was performed using techniques such as transmission electron microscopy (TEM), scanning electron microscopy (SEM), and dynamic light scattering (DLS) (see Figure 2). Based on the results of electron microscopy, the morphology of the microgels was examined. The resulting microgels were spherical, with a diameter of about 120 nm after drying. Microgels showed sensitivity to temperature and underwent phase transformation from swollen to shrunken at a temperature of about 34°C. The hydrodynamic mean of the particles measured at 37°C was 370 ⁇ 15 nm, while at 60°C it was 210 ⁇ 10 nm ( Figure 2c). The measured polydispersity index was less than 0.2 at all tested temperatures.
  • TEM transmission electron microscopy
  • SEM scanning electron microscopy
  • DLS dynamic light scattering
  • Microgels were pH-sensitive due to the presence of carboxyl and amino groups in the microgel derived from monoBISS. To estimate the charge of the microgel particles, the changes in the zeta potential of the microgel at different pH values were measured. The results are shown in Figure 2c. At pH 2, the carboxyl and amino groups were protonated so that the microgels exhibited a positive charge coming from the protonated amino groups. The zeta potential measured at pH 2 was 7 ⁇ 2.3 mV. As pH increased, the zeta potential decreased as the carboxyl groups and protonated amino groups were deprotonated. The zeta potential measured at pH 9 was -15 ⁇ 1.3 mV. The negative charge came from the negatively charged carboxyl groups.
  • the next step was to examine the effect of pH on the size and stability of microgels. These dependencies are shown in Figure 3 A.
  • the presence of carboxyl and amino groups present in the p(NIPA-MonoBISS) microgel makes it sensitive to pH changes and provides it with stability in high ionic strength solutions, which, from a physiological point of view, is very important in drug delivery.
  • the microgels were stable over the entire range of measured pH and temperature. This was due to the presence of both carboxyl and amino groups in the polymer network of microgels. At low pH, the positive charge from protonated amino groups was responsible for stability, while at alkaline pH, the negative charge from carboxyl groups provided stability to the microgel.
  • the swelling of the microgel is caused by the repulsion of mononymous negative charges.
  • the hydrodynamic diameter measured at 37°C human body temperature
  • the hydrodynamic diameter measured at 37°C was 370, 390, and 415 nm at pH 6, 8, and 9, respectively.
  • repulsion occurs between positively charged amino groups.
  • the charge of positively charged amino groups cause swelling of the microgel, but not as much as in the case of carboxyl groups. This is because the amount of amine groups is half that of the carboxyl groups in the monoBIS S monomer.
  • the hydrodynamic diameter measured at 37°C (human body temperature) was 290 nm at pH 2. It should also be mentioned that the swelling of the gel is influenced by the osmotic pressure, which is caused by the charge on the polymer network.
  • microgels were temperature sensitive and shrunk as temperature increased. In addition, they showed very good stability at salt concentrations up to 0.4 M NaCl due to the presence of a negative charge from ionized carboxyl groups of monoBISS. At 0.6 M NaCl, microgels became unstable as temperature increased. The temperature at which the microgels began to lose stability and started to aggregate (increase in size) decreased as the concentration of NaCl increased.
  • the gel at 0.6 M NaCl began to lose stability at 48°C, while the gel at 0.8 M NaCl began to lose stability at 43°C.
  • the reason for the instability and aggregation of gels is the shielding of their charges by salt ions and thus the reduction of electrostatic repulsion between molecules. This effect is more pronounced as the salt concentration increases.
  • the gel is stable at a salt concentration corresponding to the saline concentration, i.e. 0.15 M.
  • Combustion analysis was used to determine the elemental composition of the obtained gels, which allowed for the quantitative analysis of the carbon, hydrogen, nitrogen and sulfur content (A simplified oxygen-flask combustion procedure for polymer analysis, David R. Burfield and Swee- Cheng Ng, Journal of Chemical Education 1984 61 (10), 917, DOI: 10.1021/ed061p917).
  • the elemental composition was analyzed: monoBISS, p(NIPA-BIS-monoBISS) gel and p(NIPA-BISS-monoBISS) gel.
  • the synthesized microgels were subjected to combustion analysis by the Schbniger method, where they were combusted in a controlled oxygen environment. During combustion, the organic constituents of the microgels oxidized to form gaseous byproducts that were collected and analyzed by gas chromatography to quantify the sulfur content, which is very important due to glutathionesensitive bonds in the comonomer and microgel.
  • the calculated sulfur content is 15.76%, and the experimental value ranges from 15.53% to 17.74%.
  • P(NIPA-BIS-monoBISS) microgels assuming reaction yield of 100%, should comprise 4.56% sulfur, and the actual results ranged from 3.89% to 4.00%.
  • the drug loading capacity (DLC) was about 4.53% and was determined by equation (1): wherein total DOX mass used in the loading process is the excess DOX mass that has not been bound to the microgel, and ' is the mass of the microgel loaded with the drug.
  • the loading efficiency (LE) wasdetermined using equation (2) and was about 58.9%:
  • microgel of the invention was then tested as a drug carrier under in vitro conditions.
  • both non-cancerous human MCF-10A breast epithelial cells and MCF-7 breast cancer cells were treated with the drug alone, the microgel alone, and the DOX-comprising microgel.
  • the studies showed that the resulting gel was not toxic at all of the tested gel concentrations.
  • a decrease in cell viability was observed, but cell growth remained at an acceptable level (above 80%), indicating good cell survival.
  • Both free DOX and p(NIPA-monoBISS) microgel with DOX showed similar cytotoxicity (Figure 4c).
  • the IC50 values for free DOX and microgel with DOX were 0.197 pM and 2.188 pM, respectively. It is worth noting that for MCF-10A cells, the IC50 value for free DOX was 0.35 pM, while the determination of the IC50 value was not possible for the microgel with DOX at the tested concentrations, which means a lower release of the drug from the carrier in healthy cells, and therefore the microgel with the drug was not toxic to healthy cells.
  • Doxorubicin was chosen as a model drug for testing the potential ability of microgels to release the drug under reducing conditions. Covalent bonds between carboxyl groups in monoBISS and amino groups in DOX were used to bind the drug to the microgel.
  • DOX loading experiments were conducted as follows: l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (20 mg), N- hydroxysuccinimide (10 mg), and DOX (2 mg) were dissolved in 4 mL of dimethylformamide. The monoBISS gel (4 mL) was then added to the solution, under stirring at room temperature.
  • 1.0 mL of microgel suspension with doxorubicin was transferred to a dialysis bag (MWCO, 10 kDa), which was immersed in 9 mL of phosphate buffer (pH 7.4) or acetate buffer (pH 5.0) with or without GSH (40 mM). During drug release, the buffer was stirred at 250 rpm at 37°C. At desired intervals, 1 ml of external buffer was drawn for analysis by UV-Vis and the concentration of the released DOX was determined by measuring the absorbance at 480 nm.
  • MCF-10A cells healthy human mammary epithelial cells
  • MCF-7 cells human breast cancer cells
  • ATCC American Type Culture Collection
  • the MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay was used to assess cell viability. After a 24-hour incubation period, the cells were exposed to a culture medium comprising DOX, p(NIPA-monoBISS) microgel, and p(NIPA-monoBISS) microgel with DOX and incubated for additional 72 hours.
  • MCF-7 cells were cultured in Dulbecco Modified Eagle Medium (DMEM, BioWest) comprising 1% of mixture of penicillin and streptomycin (Bio West), 10% of fetal bovine serum (FBS, Gibco) and 1% of L-glutamine (BioWest).
  • DMEM Dulbecco Modified Eagle Medium
  • FBS fetal bovine serum
  • BioWest fetal bovine serum
  • MCF-10A cells were cultured in a DMEM medium comprising 5% of equine serum, 10 ng/mL of epithelial growth factor, 5 pg/mL of hydrocortisone, and 10 pg/mL of human insulin.
  • the mechanism of action of the present technology is based on the unique properties of the monomer, N-acryloylocy stine (monoBISS), and its use in the manufacture of drug delivery gels. Recent studies have shown a better response with combination therapy compared to monotherapy, and co-administration of multiple drugs minimizes the risk of disease recurrence when no single mutation causes cross-resistance to both drugs.
  • the present invention makes it possible to provide a combination of drugs for cancer treatment and to obtain a synergistic effect.
  • the coexistence of carboxyl and amino groups in the comonomer allows for the covalent loading of two different drugs into the gel, reducing toxicity to healthy cells and increasing toxicity to cancer cells.
  • monoBISS is water soluble which allows the synthesis of gels in aqueous environment without the need for toxic organic solvents.
  • the present invention makes it possible not only to reduce toxicity but also to improve the manufacturing process.
  • monoBISS is multifunctional, comprises carboxylic and a-amino acid groups and -S-S- bridges, which provide many key functions in drug delivery applications.
  • the carboxylic and amino groups provide pH-sensitivity to the gels of the invention. In addition, they facilitate the modification of gels with targeting agents, increasing the specificity for cancer cells.
  • the -S-S- bridges play a key role in drug release as they can only be ruptured by glutathione present in high concentrations in certain cancer cells such as lung cancer, laryngeal cancer, oral cancer, breast cancer, and ovarian cancer, ensuring the selectivity of drugs released at the cancer site.
  • the presence of carboxyl and amino groups in monoBIS S allows for covalent binding of the anti cancer drugs to the gels of the invention, allowing for precise control over the loading and release of the drug.
  • This mechanism provides a novel approach to targeted drug delivery, offering better efficacy and reduced side effects compared to traditional methods.
  • the uniqueness of this technology allows to overcome the limitations of existing solutions by providing a safer, more effective and targeted approach to cancer treatment.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Polymers & Plastics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Inorganic Chemistry (AREA)
  • Medicinal Preparation (AREA)

Abstract

The subject matter of the invention is N-acryloylocystine (monoBISS) of formula (I), a polymer comprising N-acryloylocystine, a method for their preparation and the use as a drug carrier.

Description

Glutathione-sensitive monomer - N-acrylocystine, polymer comprising N-acrylocystine, their synthesis and the use of the polymer as a carrier for intracellular, controlled drug delivery
The subject matter of the invention is a novel comonomer, N-acryloylocystine (monoBISS), a novel polymer comprising N-acryloylocystine, their synthesis and the use of thus obtained glutathione-sensitive pNIPA gel as a drug carrier.
Over the past three decades, there has been a growing interest in the targeted delivery of bioactive substances in the treatment of a variety of diseases. Systemic therapy is commonly used in patients with advanced cancer. However, traditional drug delivery methods and chemotherapy pose a major challenge due to their lack of selectivity, limited efficacy, ineffective biodistribution, lack of tumor targeting, potential toxicity to healthy tissues, multi-drug resistance and numerous side effects (S. Senapati, A.K. et al., Controlled drug delivery vehicles for cancer treatment and their performance, Signal Transduction and Targeted Therapy, 3 (2018), p. 7; Peer D., et al., Nanocarriers as an emerging platform for cancer therapy, Nature Nanotechnology, 2 (2007), pp. 751-760; He Y., etal., Co-delivery of erlotinib and doxorubicin by pH-sensitive charge conversion nanocarrier for synergistic therapy, Journal of Controlled Release, 10 (2016), pp. 80-92). Combination therapies with the use of several drugs are becoming increasingly popular. By acting on cancer cells at different stages of the cell cycle, combination therapy increases the likelihood of eliminating all cancer cells and preventing the development of drug resistance. For example, one drug can interfere with DNA replication, while another interferes with protein synthesis, effectively attacking malignant cells and reducing the chances of developing drug resistance. Optimizing combination therapy is aimed at achieving synergistic effects of several drugs, thanks to which the combined effect of the drug mixture exceeds the effect of individual drugs (Y. Wang et al., Doxorubicin/cisplatin co-loaded hyaluronic acid/ chitosan-based nanoparticles for in vitro synergistic combination chemotherapy of breast cancer, Carbohydrate Polymers, 225 (2019)). Recent studies have shown a higher success rate with combination therapy compared to monotherapy, and co-administration of several drugs minimizes the risk of cancer recurrence when a single mutation is not resistant to both drugs (Y. Ma, P. K. Newton, Role of synergy and antagonism in designing multidrug adaptive chemotherapy schedules, Physical Review E, 103 (2021)). Although the benefits of combination therapy are obvious, an individual approach to the patient should be considered (R.P Riechelmann et al., Potential drug interactions and duplicate prescriptions among cancer patients, The Journal of the National Cancer Institute, 99 (2007), pp. 592-600). Drug combinations can have therapeutic benefits, but can also lead to increased toxicity and unwanted side-effects. Side effects of combination therapy make it difficult to determine the appropriate dose of individual drugs. If two medicinal products have a similar adverse reaction profile, accumulation of adverse reactions may result in more severe clinical manifestations and grade 3 to 4 toxicity. New strategies have emerged to overcome these challenges, such as the use of drug delivery systems (DDS), which release drug as a result of response to the tumor microenvironment, such as low pH or high glutathione concentration. These innovative approaches aim to increase the efficacy and safety of combination therapy by delivering bioactive substances specifically to the desired site while minimizing systemic toxicity and adverse effects (A.M. Vargason, A.C. Anselmo, S. Mitragotri, The evolution of commercial drug delivery technologies, Biomedical Engineering, 5 (2021), pp. 951-967; Qingqing Huo et al., Integrated metalloproteinase, pH and glutathione responsive prodrug-based nanomedicine for efficient target chemotherapy, J Biomed Nanotechnol. 15 (2019), pp. 1673-1687).
Current research focuses on improving combination therapy through innovative drug delivery systems and targeted strategies aimed to optimize treatment outcomes while mitigating potential risks and improving patients' overall well-being and prognosis. Polymeric drug delivery systems provide an effective approach to the treatment of a variety of diseases, including neurological disorders, cardiovascular diseases, and cancer. These systems enable a targeted and controlled release of therapeutic agents, thereby limiting the side effects of drugs (Robert M Sharkey, David M Goldenberg, Targeted therapy of cancer: new prospects for antibodies and immunoconjugates, Cancer Journal for Clinicians, 56 (2006), pp. 226-43). Among said diseases, cancer, which remains one of the leading causes of human death, is of particular concern (J Ferlay et al., Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods, International Journal of Cancer, 15;144 (2019), pp. 1941-1953). As one in five people in the world develop cancer during their lifetime, cancer prevention has become a key public health challenge in the 21st century. According to the International Agency for Research on Cancer (IARC), 19.3 million cancers were diagnosed worldwide in 2023, and the estimated increase by 2040 is 30.2 million.
In conclusion, drug delivery nanosystems (nano-DDS) are very promising among the various drug delivery systems due to their ability to modify the pharmacokinetics and biodistribution of drugs. They act as reservoirs for the prolonged release of the drug, prevent the rapid degradation and clearance of drugs, and increase the concentration of the drug at the target site, thus reducing the doses of drugs and minimizing their side effects (J.K. Patra et al., Nano-based drug delivery systems: Recent developments and future prospects, Journal of Nanobiotechnology, 16 (2018), p. 71).
Nanogels, a type of nano-DDS, are materials comprising a three-dimensional polymer network, with size in the submicrometer range. They exhibit unique properties such as the ability to absorb large amounts of water and undergo significant volume change in response to external factors such as temperature, pH (ionization-sensitive comonomers), ionic strength (charged comonomers), light (photosensitive comonomers), and field (S. Dagdelen et al., Redox-responsive degradable microgel modified with superparamagnetic nanoparticles exhibiting controlled, hyperthermia- enhanced drug release, Journal of Material Science, 58 (2023), pp. 4094-4114). The volumetric phase transition, i.e. the transition of hydrogels from the swollen state to the shrunked state, allows the control of the availability of functional groups of the polymer network. The reaction of micro/nanogels to external stimuli compared to macroscopic gels is much faster and can lead to the rapid release of active substances from the micro/nanogels network. Additionally, micro/nanogels can be targeted to specific sites in the body by functionalizing them with receptors capable of specifically recognizing target objects, such as cancer cells. Modified micro- and nanogels can be transported by blood, skin, or inhalation, to diseased tissues, minimizing interactions with healthy tissues (S. S. Makhathini, S. et al., Biomedicine Innovations and Its Nanohydrogel Classifications, Pharmaceutics, 14 (2022)).
Temperature-responsive polymers including alkylacrylamides, vinyl ethers, vinyl caprolactam, and monomers of alkylene oxides such as ethylene oxide and propylene oxide are often used to obtain the above drug delivery systems. Poly(N-isopropylacrylamide) (pNIPA) is often used in the synthesis of drug carriers, due to its lower critical dissolution temperature (LCST) close to human body temperature (32 °C), making it a good candidate for drug delivery applications (A. Krishnan, S. Roy, S. Menon, Amphiphilic block copolymers: From synthesis including living polymerization methods to applications in drug delivery, European Polymer Journal, 172 (2022)). LCST pNIPA can be adjusted by copolymerizing it with hydrophilic or hydrophobic comonomers. Crosslinking agents such as N,N'-methylenebisacrylamide (BIS) are commonly used in these systems.
Polymeric drug delivery systems, especially nano-DDS, which include nanogels, provide promising opportunities for targeted and controlled release of therapeutic agents. This is a key aspect leading to better treatment outcomes and reduced drug side effects, especially for a variety of diseases, including cancer. The adjustable properties and speed of response of these systems to external stimuli provide them with versatility and great potential in the preparation of precise drug delivery strategies.
There is still a need to provide an improved carrier, suitable for intracellular, controlled drug delivery. The present invention solves this problem.
The object of the present invention is N-acryloy Icy stine (monoBISS) of formula (I): A further object of the invention is a polymer comprising the above-mentioned N- acryloylocystine as a comonomer.
Preferably, the polymer of the invention is characterized in that it is a polymer based on N- isopropylacrylamide and N-acryloylocystine, cross-linked with N,N'-methylenebisacrylamide and having a structure of formula (II): characterized in that it is a polymer based on N- isopropylacrylamide and N-acryloylocystine, cross-linked with N,N'-methylenebisacrylamide and having a structure of formula (II): or a polymer based on N-isopropylacrylamide and N-acryloylocystine, cross-linked with N,N'- bisacryloylocystine (BISS) and having a structure of formula (III):
Another object of the invention is a method for preparing N-acryloylocystine monomer (monoBIS S), characterized in that it comprises the following steps: a) acryloyl chloride is added to the basic solution of L-cystine, especially in NaOH, b) the reaction mixture is acidified, c) the reaction mixture is stirred, cooled and the supernatant is collected, d) the collected supernatant is acidified and then saturated with NaCl and extracted with ethyl acetate, e) the organic phase is evaporated and the residue is purified by column chromatography on silica gel to afford the product.
Preferably, in the method of the invention, in step a) 1.65 M NaOH is used and the reaction is carried out for 30 minutes at 0°C.
Preferably, in the method of the invention, in step b) the pH of the reaction mixture is adjusted to pH = 6 with 6 M HC1.
Preferably, in the method of the invention, in step d) the pH of the solution is adjusted to pH = 3.2 with 6 M HCl.
Preferably, in the method of the invention, in step e) the purification by column chromatography on silica gel is conducted using 20% methanol, 2% CF3COOH in chloroform. Another object of the invention is a method for preparing a polymer, characterized in that a precipitation polymerization method in an inert gas atmosphere is used, comprising the following steps: a) N-isopropylacrylamide (NIPA) is reacted with a cross-linking agent in an aqueous solution, wherein N,N'-methylenebisacrylamide (BIS) or N,N'-bisacryloylocystine (BISS) is used as the crosslinking agent, b) N,N,N,N-tetram ethylethylenediamine and ammonium persulfate (APS) are added and a polymerization reaction is conducted, c) an aqueous solution of N-acryloylcystine (monoBISS) is added and the polymerization reaction is continued, d) the resulting solution is mixed and the precipitated gel is separated from the reaction mixture.
Preferably, in the method of the invention, in step a) the reaction is carried out at a temperature of 80°C and deoxidized for 0.5 hour with argon.
Preferably, in the method of the invention, in step c) a 10 mol% solution of N-acryloylocy stine (monoBISS) is used and the reaction is carried out for 4 hours.
Preferably, in the method for preparing a polymer of the invention, in step d) the gel is purified by placing it in a dialysis membrane with a cut-off molecular weight of 10,000 Da and removing the unreacted substrates.
A further object of the invention is a polymer as defined above, for use in pharmacy.
Preferably, the polymer for use of the invention is a drug carrier, especially for use in the treatment or prevention of cancer.
Preferably, the polymer for use of the invention is a drug carrier for use in the treatment or prevention of lung cancer, laryngeal cancer, oral cancer, breast cancer or ovarian cancer.
The present invention provides a novel comonomer, N-acryloylcystine (hereinbelow referred to as monoBISS), which is then used to synthesize, using precipitation polymerization, an intelligent drug carrier, a pNIPA gel sensitive only to glutathione. Precipitation polymerization is a method used to produce gels with desired properties. In this method, the polymerization reaction takes place in a solvent system in which the monomers are soluble, but the resulting polymers are no longer, leading to the formation of polymer particles by precipitation. The monomers are dissolved in a suitable solvent, then a polymerization initiator is added to initiate the reaction. As the polymerization progresses, the resulting polymers cross-link and form micro/nanogel particles that precipitate out of the solvent. The resulting micro/nanogels are typically well formed, meaning that they have a defined shape and structure and typically exhibit a monodispersive size distribution - a narrow range of particle sizes. This is preferred in drug delivery, as obtaining micro/nano gels of the same size and constant properties allows for controlling the kinetics of drug release. The physicochemical properties of the obtained gels can be controlled by the concentration of monomers, polymerization temperature, initiator concentration, mixing rate or reaction time. By using the above synthesis method and the new monomer monoBISS for the synthesis of gels, it is possible to obtain micro/nanogels responsive to the presence of glutathione, which may be preferred for obtaining targeted drug delivery systems or applications that use systems responsive to environmental stimuli. The novel micro/nanogel can be a polymer based on monomers, such as: N-vinyl caprolactam (VCL), diethylene glycol methyl ether methacrylate (DEGMEA), poly(ethylene glycol) methyl ether methacrylate (EGMEA), N- isopropyl acrylamide (NIPA) and vinyl pyrrolidone (VP) and may be cross-linked by crosslinking agents such as: N,N-methylenebisacrylamide (BIS) or degrading N,N-bis(acryloyl)cystamine (BAC). The monoBISS comonomer of the invention, unlike other monomers comprising S-S bridges, is water soluble and thus allows synthesis in an aqueous medium. Additionally, due to the presence of carboxyl and a-amino acid groups in monoBISS, such gels are pH-sensitive and stable. Thus, the use of monoBISS facilitates the synthesis of micro/nanogels, there is no need to use additional comonomers responsible for stability and pH- sensitivity. Importantly, the presence of carboxyl and amino groups in monoBISS allows for covalent binding of various anti cancer drugs and control of their release. The most important thing is that the drug can be released from the gel after cutting the S-S bonds, which is possible in the presence of glutathione, which is found in human cells. Glutathione leads to the reduction of S-S bonds to thiol groups. Glutathione is a tripeptide that is usually found in elevated concentrations in most cancer cells. It is worth noting that the concentration of glutathione in the cytoplasm is much higher (in the range of 0.5-10 mM) compared to extracellular fluids (2-20 pM) and reaches up to 1000 times higher concentrations. Among the different types of cancers, cancers such as lung cancer, laryngeal cancer, oral cancer, breast cancer, and ovarian cancer show higher glutathione levels (up to 40 mM) compared to healthy cells. Cancer cells often show higher levels of intracellular glutathione compared to healthy cells, a mechanism that promotes survival of these cells and resistance to chemotherapy. These in vitro studies were performed on MCF-7 breast cancer cells with a GSH concentration of 40 mM and on MCF-10A healthy cells with a GSH concentration of approximately 1 mM.
The aim of the present invention is therefore to provide a carrier for the specific intracellular delivery of cysteine-modified therapeutic substances. It is essential that such a substance has a free amino or carboxyl group that can be used to form a peptide bond with a monoBISS monomeric unit in a gel carrier network.
In addition, the presence of carboxyl and amino groups in monoBISS allows for the introduction of two different drugs into the micro/nanogel. This is a unique opportunity to achieve loading of two types of drugs, both drugs can be effectively encapsulated in a micro/nanogel matrix. Using the reactivity of the functional groups, it is possible to ensure the effective coupling of each drug molecule with micro/nanogels, which allows the development of multifunctional micro/nanogel systems capable of simultaneous delivery of multiple therapeutic agents. This chemical approach allows the formation of robust bonds that will reduce the toxicity of drugs to healthy cells. Furthermore, the electrostatic interaction between micro/nanogels and drugs serves as an alternative mechanism for drug loading. In particular, a negatively charged drug may readily bind to positively charged comonomer groups, while a positively charged drug may be effectively loaded into negatively charged comonomer groups, and vice versa. This strategy allows the selective and controlled loading and release of drugs from micro/nanogels, which provides potential benefits for various biomedical applications. By taking advantage of the existence of higher glutathione concentrations in the specific cancer cells listed above, the present invention allows to increase the effectiveness of drug delivery and cancer treatment.
In addition, an alternative to commercially available N,N'-bis(acryloyl)cystamine (BAC) - a water-insoluble gel cross-linking agent that degrades in the presence of glutathione - is N,N'- bis(acryloyl)cystine (BISS). Unlike BAC, BISS is not only degradable, but it is water soluble, pH- sensitive, and gives stability to gels in a high ionic strength environment.
In the present description, the polymer of the invention is interchangeably referred to as a gel or microgel. The particle sizes of the microgel are in the range of 100 nm to 100 pm. However, the process of preparing a polymer of suitable particle size may be modulated by manipulating key parameters such as mixing speed, temperature, and total microgel concentration. By selecting these parameters appropriately, gel variants with particle sizes of less than 100 nm, corresponding to nanogels, can also be synthesized.
According to the present invention, BISS comprising the S-S bonds acts as a crosslinking agent, giving the monoBISS gel, among other things, degradable properties.
The unique distinguishing features of the present solution consist in the multifunctionality and water solubility of the monoBISS comonomer, which constitutes the main functional component of the gel drug carrier. In contrast to existing glutathione-sensitive monomers, monoBISS allows the synthesis of gels in aqueous environments without the need for organic solvents. In addition, monoBISS is distinguished by multifunctional properties due to the presence of carboxyl and a-amino acid groups, and -S-S- bridges in its structure. These functional groups provide pH-sensitivity, stability, possibility of modification with therapeutic substances comprising amino or carboxyl groups. In addition, a selective response to glutathione provides a controlled release of the drug, particularly in the tumor environment, minimizing undesired side effects. The presence of carboxyl and amino groups in monoBISS allows for covalent binding of two various anti cancer drugs with gels of the invention and control of their loading. Thanks to the invention, it is possible to use the synergistic effect of two drugs to achieve more effective toxicity to cancer cells. It is important that the drug can be released from the microgels after the reduction of S-S bonds and this is only possible in the presence of the cutting S-S bonds, glutathione, which is found in cells and its concentration in cancer cells is usually elevated.
The object of the invention is illustrated in the embodiments and in the drawing, where:
Figure 1 shows the spectra obtained for the MonoBISS monomer:
(aj'HNMR,
(b)13CNMR.
Figure 2 shows:
(a) MonoBISS synthesis scheme,
(b) TEM (A) and SEM (B) microphotographs of p(NIPA-MonoBISS) gel particles, and
(c) changes in the hydrodynamic diameter of the p(NIPA-MonoBIS S) microgel as a function of temperature. The Zeta potential of the nanogel as a function of pH was measured at room temperature.
Figure 3A shows the hydrodynamic diameter (Dh) of the p(NIPA-MonoBISS) microgel as a function of temperature and pH. The ionic strength was kept constant at 10 mM.
Figure 3B shows a graph of the dependence of Dh on temperature for microgels suspended in solutions with different salt concentrations (NaCl).
Figure 4 shows the DOX release profiles
(a) from microgels with DOX at pH 5.0 and pH 7.4 and in the presence or absence of GSH (40 mM),
(b) for MCF-10A and MCF-7 cell lines after 72 hours of incubation in p(NTPA-monoBISS) gels with the drug. Cell nuclei were stained with Hoechst stain; the next image shows DOX. MTT assay results on MCF-7 cell line.
(c) results on MCF-10A cell line,
(d) results after 72 hours of treatment of cells with p(NIPA-monoBISS) gels, free drug DOX and p(NIPA-monoBISS) with DOX.
A one-way ANOVA was used to check statistical significance. Differences with respect to the control sample were marked with an asterisk *, while ** are differences between groups. The difference was considered significant for P <0.05.
Figure 5 shows a diagram of synthesis of p(NIPA-BIS-monoBISS) gels.
Figure 6 shows a diagram of synthesis of p(NIPA-BISS-monoBISS) gels.
Synthesis of monoBISS monomer
A diagram of the synthesis of the monomer of the invention - N-acryloylcystine (monoBISS) is shown in Figure 2a. To an ice-cold stirred solution of L-cystine (2.65 g, 11 mmol) in 1.65 M NaOH (25 mL), acryloyl chloride (404 pl, 5 mmol) was added dropwise for 30 minutes at 0° C. After another 30 minutes, the pH of the reaction mixture was adjusted to pH = 6 with 6 M HC1. After 20 minutes of stirring and cooling, the excess L-cystine precipitate was filtered out and washed with water several times. The collected filtrates and washings were then adjusted to pH = 3.2 with 6 M HC1, then the NaCl solution was saturated with NaCl and repeatedly extracted with ethyl acetate. The combined organic phases were evaporated and the residue was purified by column chromatography on silica gel (20% methanol, 2% CF3COOH in chloroform) to give the product as a white solid (890 mg, 33%).
Results of 1 HNMR and 13CNMR for the synthesized monoBISS are shown in Figures 1 a and 1 b, respectively.
HRMS (ESI): calculated for C9Hi4N2O5S2Na [M+Na]+: 317.02363, measured: 317.02320 1H NMR (300 MHz, D2O) 5: 6.35-6.10 (m, 2H), 5.80-5.69 (m, 1H), 4.80-4.70 (m, 1H), 4.15-4.05 (m, 1H), 3.37-3.26 (m, 2H), 3.09-2.95 (m, 2H)
13C NMR (75 MHz, D2O) 5: 174.0, 171.8, 168.5, 129.2, 128.6, 52.7, 52.0, 38.1, 37.3
Synthesis of microgel
Further, a new microgel based on N-isopropylacrylamide (NIPA) and monoBISS was synthesized, cross-linked with N,N'-methylenebisacrylamide (BIS) or N,N'-bis(acryloyl)cystine as a cross-linking agent (BISS).
The microgels were synthesized in an aqueous solvent that provides polymerization environment. The synthesis of the microgel can be carried out at different molar fractions of the crosslinking agent or the main monomer. A precipitation polymerization method was used for the synthesis of microgels. Polymerization was carried out in a three-necked flask equipped with a reflux condenser, magnetic stirrer (set at 250 rpm during the entire polymerization process), inlet and outlet of inert gas. a) p(NIPA-BIS-monoBISS) microgel synthesis A diagram of p(NIPA-BIS-monoBISS) microgel synthesis is shown in Figure 5.
The main NIPA monomer and BIS cross-linking agent (2 mol%) were dissolved in 16 mL deionized water and placed in a flask. The solution was heated to 80°C and deoxidized for 0.5 hour with argon. Then 5 pl of N,N,N,N-tetramethylethylenediamine (TEMED) and 9 mg of an initiator - ammonium persulfate (APS) dissolved in 2 ml of deionized water were added to initiate polymerization. Further, an aqueous monoBISS solution (10 mol%, 2 ml, pH 4) was added to the reactor. The reaction lasted 4 hours under argon. The total monomer concentration was 100 mM. The resulting emulsion was stirred overnight at room temperature using a magnetic stirrer. The microgels were then purified by placing them in a dialysis membrane with a cut-off molecular weight of 10,000 Da (Spectra/Por® 7 dialysis membrane), thereby removing the unreacted substrates. Dialysis lasted 5 days, the water was changed daily. The concentration of NIPA, BIS and monoBISS was 100 mM at 2% mole fraction of BIS and 10% mole fraction of monoBISS. The APS concentration was 5 mM. a) p(NIPA-BISS-monoBISS) microgel synthesis (A diagram of p(NIPA-BIS-monoBISS) microgel synthesis is shown in Figure 6.
The main NIPA monomer and BISS cross-linking agent (2 mol%) were dissolved in 16 mL deionized water and placed in a flask. The solution was heated to 80°C and deoxidized for 0.5 hour with argon. Then 5 pl of N,N,N, N-tetram ethylethylenediamine (TEMED) and 9 mg of an initiator - ammonium persulfate (APS) dissolved in 2 ml of deionized water were added to initiate polymerization. Further, an aqueous monoBISS solution (10 mol%, 2 ml, pH 4) was added to the reactor. The reaction lasted 4 hours under argon. The total monomer concentration was 100 mM. The resulting emulsion was stirred overnight at room temperature using a magnetic stirrer. The microgels were then purified by placing them in a dialysis membrane with a cut-off molecular weight of 10,000 Da (Spectra/Por® 7 dialysis membrane), thereby removing the unreacted substrates. Dialysis lasted 5 days, the water was changed daily. The concentration of NIPA, BISS and monoBISS was 100 mM at 2% mole fraction of BIS and 10% mole fraction of monoBISS. The APS concentration was 5 mM.
Characterization of microgel
Microgel characterization was performed using techniques such as transmission electron microscopy (TEM), scanning electron microscopy (SEM), and dynamic light scattering (DLS) (see Figure 2). Based on the results of electron microscopy, the morphology of the microgels was examined. The resulting microgels were spherical, with a diameter of about 120 nm after drying. Microgels showed sensitivity to temperature and underwent phase transformation from swollen to shrunken at a temperature of about 34°C. The hydrodynamic mean of the particles measured at 37°C was 370 ± 15 nm, while at 60°C it was 210 ± 10 nm (Figure 2c). The measured polydispersity index was less than 0.2 at all tested temperatures. Microgels were pH-sensitive due to the presence of carboxyl and amino groups in the microgel derived from monoBISS. To estimate the charge of the microgel particles, the changes in the zeta potential of the microgel at different pH values were measured. The results are shown in Figure 2c. At pH 2, the carboxyl and amino groups were protonated so that the microgels exhibited a positive charge coming from the protonated amino groups. The zeta potential measured at pH 2 was 7 ± 2.3 mV. As pH increased, the zeta potential decreased as the carboxyl groups and protonated amino groups were deprotonated. The zeta potential measured at pH 9 was -15 ± 1.3 mV. The negative charge came from the negatively charged carboxyl groups. It is also possible to obtain other gel properties using a different method of synthesis and a different gel composition. Depending on the expected results, precipitation polymerization, semi- periodic polymerization, microemulsion polymerization, and distillation polymerization may be used for the synthesis of micro/nanogel particles.
The next step was to examine the effect of pH on the size and stability of microgels. These dependencies are shown in Figure 3 A. The presence of carboxyl and amino groups present in the p(NIPA-MonoBISS) microgel makes it sensitive to pH changes and provides it with stability in high ionic strength solutions, which, from a physiological point of view, is very important in drug delivery. The microgels were stable over the entire range of measured pH and temperature. This was due to the presence of both carboxyl and amino groups in the polymer network of microgels. At low pH, the positive charge from protonated amino groups was responsible for stability, while at alkaline pH, the negative charge from carboxyl groups provided stability to the microgel. At a higher pH, the swelling of the microgel is caused by the repulsion of mononymous negative charges. For example, the hydrodynamic diameter measured at 37°C (human body temperature) was 370, 390, and 415 nm at pH 6, 8, and 9, respectively. On the other hand, at a lower pH, repulsion occurs between positively charged amino groups. The charge of positively charged amino groups cause swelling of the microgel, but not as much as in the case of carboxyl groups. This is because the amount of amine groups is half that of the carboxyl groups in the monoBIS S monomer. For example, the hydrodynamic diameter measured at 37°C (human body temperature) was 290 nm at pH 2. It should also be mentioned that the swelling of the gel is influenced by the osmotic pressure, which is caused by the charge on the polymer network.
The effect of the ionic strength on the size of the p(NIPA-MonoBISS) microgels was then investigated. The dependence of the hydrodynamic diameter of microgels on temperature was determined for six selected salt concentrations (0.01 M - 0.8 M). Microgels were temperature sensitive and shrunk as temperature increased. In addition, they showed very good stability at salt concentrations up to 0.4 M NaCl due to the presence of a negative charge from ionized carboxyl groups of monoBISS. At 0.6 M NaCl, microgels became unstable as temperature increased. The temperature at which the microgels began to lose stability and started to aggregate (increase in size) decreased as the concentration of NaCl increased. For example, the gel at 0.6 M NaCl began to lose stability at 48°C, while the gel at 0.8 M NaCl began to lose stability at 43°C. The reason for the instability and aggregation of gels is the shielding of their charges by salt ions and thus the reduction of electrostatic repulsion between molecules. This effect is more pronounced as the salt concentration increases. Importantly, the gel is stable at a salt concentration corresponding to the saline concentration, i.e. 0.15 M.
Combustion analysis Combustion analysis was used to determine the elemental composition of the obtained gels, which allowed for the quantitative analysis of the carbon, hydrogen, nitrogen and sulfur content (A simplified oxygen-flask combustion procedure for polymer analysis, David R. Burfield and Swee- Cheng Ng, Journal of Chemical Education 1984 61 (10), 917, DOI: 10.1021/ed061p917).
The elemental composition was analyzed: monoBISS, p(NIPA-BIS-monoBISS) gel and p(NIPA-BISS-monoBISS) gel.
The synthesized microgels were subjected to combustion analysis by the Schbniger method, where they were combusted in a controlled oxygen environment. During combustion, the organic constituents of the microgels oxidized to form gaseous byproducts that were collected and analyzed by gas chromatography to quantify the sulfur content, which is very important due to glutathionesensitive bonds in the comonomer and microgel.
The above analysis revealed the following results:
For monoBISS, the calculated sulfur content is 15.76%, and the experimental value ranges from 15.53% to 17.74%.
For p(NIPA-BISS-monoBISS) microgel, the calculated sulfur content, assuming reaction yield of 100%, is 4.94%, and the experimental value ranges from 3.81% to 3.89%.
P(NIPA-BIS-monoBISS) microgels, assuming reaction yield of 100%, should comprise 4.56% sulfur, and the actual results ranged from 3.89% to 4.00%.
Loading of the drug into the microgel
Then, the loading of the drug into the microgel and its usefulness as a carrier of doxorubicin (an anti-cancer drug) were investigated. The drug loading capacity (DLC) was about 4.53% and was determined by equation (1): wherein total DOX mass used in the loading process is the excess DOX mass that has not been bound to the microgel, and ' is the mass of the microgel loaded with the drug. The loading efficiency (LE) wasdetermined using equation (2) and was about 58.9%:
After loading the drug into the microgel, the effect of pH and GSH on the effectiveness of DOX release from the p(NIPA-MonoBISS) microgel was investigated. As can be seen in Figure 4a, the release of the drug was dependent on the presence of the reducing agent-GSH, but was not dependent on the pH value. As can be seen in Figure 4a, in samples comprising GSH, the cumulative release of DOX was about 70% after 30 hours at both pH values. Without GSH, the cumulative release is about 10% after 30 hours at both pH values. However, when 40 mM GSH was added to the buffers, the cumulative release increased to about 66% after 40 hours. The results clearly indicate that most of the drug is released in the presence of 40 mM GSH, i.e. under conditions typical for most cancer cells.
The microgel of the invention was then tested as a drug carrier under in vitro conditions. During in vitro studies, both non-cancerous human MCF-10A breast epithelial cells and MCF-7 breast cancer cells were treated with the drug alone, the microgel alone, and the DOX-comprising microgel. As shown in Figure 4, the studies showed that the resulting gel was not toxic at all of the tested gel concentrations. At higher gel concentrations, a decrease in cell viability was observed, but cell growth remained at an acceptable level (above 80%), indicating good cell survival. Both free DOX and p(NIPA-monoBISS) microgel with DOX showed similar cytotoxicity (Figure 4c). For MCF-7 cells, the IC50 values for free DOX and microgel with DOX were 0.197 pM and 2.188 pM, respectively. It is worth noting that for MCF-10A cells, the IC50 value for free DOX was 0.35 pM, while the determination of the IC50 value was not possible for the microgel with DOX at the tested concentrations, which means a lower release of the drug from the carrier in healthy cells, and therefore the microgel with the drug was not toxic to healthy cells.
Doxorubicin (DOX) was chosen as a model drug for testing the potential ability of microgels to release the drug under reducing conditions. Covalent bonds between carboxyl groups in monoBISS and amino groups in DOX were used to bind the drug to the microgel. DOX loading experiments were conducted as follows: l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (20 mg), N- hydroxysuccinimide (10 mg), and DOX (2 mg) were dissolved in 4 mL of dimethylformamide. The monoBISS gel (4 mL) was then added to the solution, under stirring at room temperature. The suspension was then placed in dialysis bags (10 kDa) and dialyzed in PBS buffer (100 mL, pH 7, 0.1 M) for one day to remove the excess gel-unbound drug at room temperature. The concentration of the drug outside the dialysis membrane was analyzed by UV-Vis (Lambda 35, Perkin-Elmer, USA) at 468 nm. Loaded drug content (LC) and encapsulation efficiency (EE) were determined according to the following formulas: LC (%) = (weight of loaded drug)/(total weight of gel); EE (%) = (weight of loaded drug)/(initial weight of used drug).
In vitro drug release
In order to study in vitro drug release, 1.0 mL of microgel suspension with doxorubicin was transferred to a dialysis bag (MWCO, 10 kDa), which was immersed in 9 mL of phosphate buffer (pH 7.4) or acetate buffer (pH 5.0) with or without GSH (40 mM). During drug release, the buffer was stirred at 250 rpm at 37°C. At desired intervals, 1 ml of external buffer was drawn for analysis by UV-Vis and the concentration of the released DOX was determined by measuring the absorbance at 480 nm.
Assays on cells
MCF-10A cells (healthy human mammary epithelial cells) and MCF-7 cells (human breast cancer cells) were obtained from American Type Culture Collection (ATCC). The MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay was used to assess cell viability. After a 24-hour incubation period, the cells were exposed to a culture medium comprising DOX, p(NIPA-monoBISS) microgel, and p(NIPA-monoBISS) microgel with DOX and incubated for additional 72 hours. The wells were then prepared for UV-Vis and 50 pL of MTT solution was added to each well, and the resulting formazan crystals were dissolved in 200 pL of 2-propanol. Absorbance readings were obtained at 570 nm using an ELX800 absorption microplate reader (BioTek Instruments). The IC50 values for the DOX and the DOX-loaded microgel were determined using the GraphPad Prism 7 (the GraphPad software) by non-linear regression analysis, using the best fitting Hill slope curve. Results were presented as mean ± standard deviation (SD) from two independent experiments (n=6). A confocal microscope (Fluoview Olympus FVlOi) was used to assess the effectiveness of cell penetration by drug carriers. All cell cultures were maintained at 37°C in an atmosphere enriched with 5% CO2. MCF-7 cells were cultured in Dulbecco Modified Eagle Medium (DMEM, BioWest) comprising 1% of mixture of penicillin and streptomycin (Bio West), 10% of fetal bovine serum (FBS, Gibco) and 1% of L-glutamine (BioWest). MCF-10A cells were cultured in a DMEM medium comprising 5% of equine serum, 10 ng/mL of epithelial growth factor, 5 pg/mL of hydrocortisone, and 10 pg/mL of human insulin.
In addition to cytotoxicity studies, the effect of carriers on selected cells was investigated under a confocal microscope. All cells were treated with a drug-loaded microgel at a concentration of 0.01 pM. Cell nuclei were pre-treated with Hoechst dye to stain the nucleus, emitting blue-cyanic light while DOX emitted red light. As shown in Figure 4b, 2D images of healthy cells (MCF-10A) and cancer cells (MCF-7) after 72 hours of interaction with p(NIPA-monoBISS) microgels with DOX show clear differences. In tumor cells, DOX was mainly present in the nuclei, while in healthy cells, although DOX was also present in the nuclei, the intensity was clearly lower. In addition, staining was also visible in the cytoplasm, which indicated a different distribution of the drug in healthy cells compared to cancer cells. The significantly greater accumulation of DOX in the nuclei of MCF-7 cells can be explained by the greater toxicity of the microgel with drug for tumor cells than for healthy cells. These results indicate that microgels increase the effectiveness of cancer treatment with DOX while protecting healthy cells. Based on stability studies, it can be concluded that p(NIPA-monoBISS) microgels are stable in the intercellular space. Probably the mechanism of transport of the microgel to the cell lies in the process of endocytosis. In the reducing environment of the cell, the polymer network of the gel is degraded, leading to drug release and subsequent cell death. Table 1 shows the synergistic drug combinations, molecular formulas, and pKa values of some anticancer drugs.
Table 1. Synergistic drug combinations, molecular formulas, and pKa values of some anti cancer drugs
The mechanism of action of the present technology is based on the unique properties of the monomer, N-acryloylocy stine (monoBISS), and its use in the manufacture of drug delivery gels. Recent studies have shown a better response with combination therapy compared to monotherapy, and co-administration of multiple drugs minimizes the risk of disease recurrence when no single mutation causes cross-resistance to both drugs. The present invention makes it possible to provide a combination of drugs for cancer treatment and to obtain a synergistic effect. The coexistence of carboxyl and amino groups in the comonomer allows for the covalent loading of two different drugs into the gel, reducing toxicity to healthy cells and increasing toxicity to cancer cells. In contrast to existing glutathione-sensitive co-mon omers, monoBISS is water soluble which allows the synthesis of gels in aqueous environment without the need for toxic organic solvents. The present invention makes it possible not only to reduce toxicity but also to improve the manufacturing process. Moreover, monoBISS is multifunctional, comprises carboxylic and a-amino acid groups and -S-S- bridges, which provide many key functions in drug delivery applications. The carboxylic and amino groups provide pH-sensitivity to the gels of the invention. In addition, they facilitate the modification of gels with targeting agents, increasing the specificity for cancer cells. The -S-S- bridges play a key role in drug release as they can only be ruptured by glutathione present in high concentrations in certain cancer cells such as lung cancer, laryngeal cancer, oral cancer, breast cancer, and ovarian cancer, ensuring the selectivity of drugs released at the cancer site. In addition, the presence of carboxyl and amino groups in monoBIS S allows for covalent binding of the anti cancer drugs to the gels of the invention, allowing for precise control over the loading and release of the drug. This mechanism provides a novel approach to targeted drug delivery, offering better efficacy and reduced side effects compared to traditional methods. The uniqueness of this technology allows to overcome the limitations of existing solutions by providing a safer, more effective and targeted approach to cancer treatment.

Claims

Claims
1. N-acryloy Icy stine (monoBISS) of formula (I):
2 A polymer comprising N-acryloy Icystine as defined in claim 1 as a comonomer.
3 The polymer according to claim 2, characterized in that it is a polymer based on N- isopropylacrylamide and N-acryloylocystine, cross-linked with N,N'-methylenebisacrylamide and having a structure of formula (II): or a polymer based on N-isopropylacrylamide and N-acryloylocy stine, cross-linked with N,N'- bisacryloylocystine (BISS) and having a structure of formula (III):
4 A method for preparing N-acryloylocystine monomer (monoBIS S), characterized in that it comprises the following steps: a acryloyl chloride is added to the basic solution of L-cystine, especially in NaOH, b the reaction mixture is acidified, c the reaction mixture is stirred, cooled and the supernatant is collected, d the collected supernatant is acidified and then saturated with NaCl and extracted with ethyl acetate, e the organic phase is evaporated and the residue is purified by column chromatography on silica gel to afford the product.
5 The method according to claim 4, characterized in that in step a) 1.65 M NaOH is used and the reaction is carried out for 30 minutes at 0°C.
6 The method according to claim 4, characterized in that in step b) the pH of the reaction mixture is adjusted to pH = 6 with 6 M HC1.
7 The method according to claim 4, characterized in that in step d) the pH of the supernatant is adjusted to pH = 3.2 with 6 M HC1.
8. The method according to claim 4 characterized in that in step e) the purification by column chromatography on silica gel is conducted using 20% methanol, 2% CF3COOH in chloroform.
9. A method for preparing a polymer, characterized in that a precipitation polymerization method in an inert gas atmosphere is used, comprising the following steps: a) N-isopropylacrylamide (NIPA) is reacted with a cross-linking agent in an aqueous solution, wherein N,N'-methylenebisacrylamide (BIS) or N,N'-bisacryloylocystine (BISS) is used as the crosslinking agent, b N,N,N,N-tetram ethylethylenediamine and ammonium persulfate (APS) are added and a polymerization reaction is conducted, c an aqueous solution of N-acryloylcystine (monoBISS) is added and the polymerization reaction is continued, d the resulting solution is mixed and the precipitated gel is separated from the reaction mixture.
10 The method according to claim 9, characterized in that in step a) the reaction is carried out at a temperature of 80°C and deoxidized for 0.5 hour with argon.
11 The method according to claim 9, characterized in that in step c) a 10% mol solution of N- acryloylocystine (monoBISS) is used and the reaction is carried out for 4 hours.
12 The method for preparing a polymer according to claim 9, characterized in that in step d) the gel is purified by placing it in a dialysis membrane with a cut-off molecular weight of 10,000 Da and removing the unreacted substrates.
13 The polimer according to claims 2-3, for use in pharmacy.
14 The polymer for use according to claim 13, characterized in that it is a drug carrier, especially for use in the treatment or prevention of cancer.
15 The polymer for use according to claim 13, characterized in that it is a drug carrier for use in the treatment or prevention of lung cancer, laryngeal cancer, oral cancer, breast cancer or ovarian cancer.
PCT/PL2025/050044 2024-05-16 2025-05-16 Glutathione-sensitive monomer - n-acrylocystine, polymer comprising n-acrylocystine, their synthesis and the use of the polymer as a carrier for intracellular, controlled drug delivery Pending WO2025239786A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
PLP.448586 2024-05-16
PL44858624 2024-05-16
PLP.449234 2024-07-15
PL449234A PL449234A1 (en) 2024-05-16 2024-07-15 Glutathione-sensitive monomer - N-acryloylcystine, N-acryloylcystine-containing polymer, their synthesis and use of the polymer as a carrier for intracellular, controlled drug delivery

Publications (1)

Publication Number Publication Date
WO2025239786A1 true WO2025239786A1 (en) 2025-11-20

Family

ID=96391544

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/PL2025/050044 Pending WO2025239786A1 (en) 2024-05-16 2025-05-16 Glutathione-sensitive monomer - n-acrylocystine, polymer comprising n-acrylocystine, their synthesis and the use of the polymer as a carrier for intracellular, controlled drug delivery

Country Status (1)

Country Link
WO (1) WO2025239786A1 (en)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MACKIEWICZ MARCIN ET AL: "Nanohydrogel withN,N'-bis(acryloyl)cystine crosslinker for high drug loading", INTERNATIONAL JOURNAL OF PHARMACEUTICS, ELSEVIER, AMSTERDAM, NL, 18 March 2017 (2017-03-18), XP085102102, ISSN: 0378-5173, DOI: 10.1016/J.IJPHARM.2017.03.031 *

Similar Documents

Publication Publication Date Title
Lv et al. Doxorubicin-loaded amphiphilic polypeptide-based nanoparticles as an efficient drug delivery system for cancer therapy
Xiao et al. Multifunctional unimolecular micelles for cancer-targeted drug delivery and positron emission tomography imaging
Guo et al. Matrix metalloprotein-triggered, cell penetrating peptide-modified star-shaped nanoparticles for tumor targeting and cancer therapy
Sadekar et al. Comparative biodistribution of PAMAM dendrimers and HPMA copolymers in ovarian-tumor-bearing mice
Ni et al. Recent research progress on polyphosphazene-based drug delivery systems
Song et al. Erythrocyte-biomimetic nanosystems to improve antitumor effects of paclitaxel on epithelial cancers
Yoon et al. Bioreducible hyaluronic acid conjugates as siRNA carrier for tumor targeting
Zhang et al. Galactosylated reduction and pH dual-responsive triblock terpolymer Gal-PEEP-a-PCL-ss-PDMAEMA: a multifunctional carrier for the targeted and simultaneous delivery of doxorubicin and DNA
US9149535B2 (en) Polymers and the preparation of nanogel drug cocktails
JP2011524446A (en) Chitosan oligosaccharide fatty acid graft product modified with polyglycol, its preparation method and use thereof
CN110229323B (en) Reduction-sensitive and reversibly cross-linked polymersomes with asymmetric membrane structure and their application in the preparation of drugs for the treatment of liver cancer
Lei et al. Co-delivery of paclitaxel and gemcitabine via a self-assembling nanoparticle for targeted treatment of breast cancer
US20160120814A1 (en) Method for Preparing Protein Cage, and In Situ Method for Preparing Hydrophobic Additive-supported Core-shell Structured Polymer-protein Particles
CN103110954A (en) Cholesterol-modified biodegradable polycation carrier as well as preparation method and application thereof
Zhang et al. A polymeric nanocarrier with a tumor acidity-activatable arginine-rich (R 9) peptide for enhanced drug delivery
Zhang et al. Zinc finger-inspired nanohydrogels with glutathione/pH triggered degradation based on coordination substitution for highly efficient delivery of anti-cancer drugs
Liao et al. Dual-drug delivery based charge-conversional polymeric micelles for enhanced cellular uptake and combination therapy
CN109762099B (en) Polymer-antitumor drug conjugate and preparation method and application thereof
Liu et al. Drug pH-sensitive release in vitro and targeting ability of polyamidoamine dendrimer complexes for tumor cells
Nakka et al. Dual drug-loaded tumor-targeted polymeric nanoparticles for enhancing therapeutic response in pancreatic ductal adenocarcinoma
CN105001426B (en) A kind of polyaminoacid graft copolymer with tumor-targeting and preparation method thereof
CN108339124B (en) Preparation method and application of a dual-stage brain-targeting polymer micelle drug delivery system
CN106389388A (en) PH redox double-sensitive PAMAM targeting nano drug delivery carrier and preparation method thereof
CN110804178B (en) A kind of nano-drug loading system with glutathione responsiveness and its preparation method and application
Shi et al. Fabrication and characterization of a folic acid-bound 5-fluorouracil loaded quantum dot system for hepatocellular carcinoma targeted therapy