TWI361813B - Multifunctional mixed micelle of graft and block copolymers and preparation thereof - Google Patents

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a polymer type micelle having a core-shell structure, and more particularly to a multifunctional polymer type micelle having a core-shell structure, which is grafted by a graft The copolymerized polymer is formed by self-assembly of at least one double-co-polymerized polymer. First-pre-technical multi-component cells have been extensively studied in biomedical applications, and various types of multi-component cells have many advantageous properties, such as: enabling microcapsules to have special functions and enhancing cell identification for tumors. Specificity, stable microcell structure, can overcome the use defects of different materials, and can make the microcapsules appear multi-functional. [1-4]. In the field of macromolecules, the type of multi-component microcells (also known as composite microvesicles) includes a double-cluster-double-coupling copolymer polymer system, a double-cluster_triple-coupling polymer system, and three groups. The hydrazine-triple-co-polymerization polymer system φ system and the double-co-graft copolymerization polymer system [2·10]. However, there have been no reports on the composite mice of the double-cluster graft-polymerized polymer system. In the past few decades, the composite micelles have focused on the mechanism of microcells [5-10], and few studies have applied them to drug delivery [3, 4]. The complexity of complex microcell formation and the integrity of the shell core structure are the limiting bottlenecks of composite micelles in biomedical applications. SUMMARY OF THE INVENTION The present invention is directed to a novel composite microcell structure having a functional core and a hydrophilic outer shell by a graft copolymerization polymer with a mono-copolymer or a multi-agglomerate copolymerization. The polymer is self-assembled, especially a graft copolymerization. The composition of the polymer and two or more double-coupling copolymers is preferred. The size of the composite micelle is between 50 and 200 nm. The invention provides a novel composite microcell structure, comprising a functional core and a hydrophilic outer shell, which are self-assembled by a graft copolymerized polymer and one or more cluster copolymerized polymers. . Preferably, the graft copolymerized polymer is self-assembled with two or more double-co-polymerized polymers. The micelles synthesized by the present invention have a particle size of about 50-200 nm. The present invention provides a polymer type micelle having a core-shell structure, wherein the structure comprises a graft copolymerized polymer and a co-polymerized polymer, the graft copolymerized polymer comprising a main chain and a bond a hydrophobic side chain of the main chain, the co-polymerized polymer comprising a hydrophobic polymer segment and a s. hydrophilic polymer segment, wherein the hydrophobic side chain of the graft polymer aggregates, The hydrophobic segment of the copolymerized polymer is combined with the hydrophobic side chain of the graft copolymerized polymer, and the hydrophilic segment of the copolymerized polymer is exposed from the protrusion And the formation of the shell core structure. The invention further provides a preparation method of a polymer type microcell having a core-shell structure, comprising the following steps: a) dissolving a graft copolymerized polymer and a group of a copolymerized polymer in an organic solvent, wherein the The branched copolymer polymer comprises a main chain and a hydrophobic side chain bonded to the main chain of 1361813, the copolymerized polymer comprising a hydrophobic polymer segment and a hydrophilic polymer segment, b Preferably, the polymer solution of step a) is subjected to dialysis treatment against water to replace the solvent of the food machine with water. Preferably, the preparation method of the present invention further comprises c) lyophilizing the aqueous solution obtained from step b) A dried polymer type microcell is obtained. Preferably, one or more different copolymerized high φ molecules are dissolved in the organic solvent in step a). Preferably, a drug is co-dissolved in the organic solvent together with the graft copolymerized polymer and the copolymerized polymer. More preferably, the hydrophobic side chain of the graft copolymerized polymer and the hydrophobic segment of the copolymerized polymer contain the same repeating unit. Preferably, the group is a polymer-polymerized double-co-agglomerated polymer molecule comprising the hydrophobic polymer segment and the hydrophilic polymer segment. More preferably, the double-co-agglomerated polymer is methoxy-poly(ethylene ... glycol)_b-poly(D,i:-lactide) ). Preferably, the number average molecular weight of the hydrophobic polymer segment is between 500 and 50,000, and the number average molecular weight of the hydrophilic polymer segment is between 2,000 and 10,000 Å. The hydrophobic bond of the polymerized molecule is biodegradable. Preferably, the hydrophobic segment of the copolymerized polymer is polyester, polylactide, polylactic acid or polycaprolactone. More preferably, the group is co-polymerized 1361813 The hydrophobic segment of the polymer is polylactide. Preferably, the hydrophilic segment of the graft copolymerized polymer is a polyacrylic acid ester, or an acid-base/ion strength sensitive polymer, wherein the acid-base/ion strength sensitive polymer is polyacrylic acid, poly Mercaptopropionic acid, poly(butenedioic aicd), polyhistidine, or poly(vinyl imidazole). Preferably, the hydrophilic segment of the copolymerized polymer is a poly bond, φ polyethylene glycol (p〇ly (ethylene glycol)), methoxy-p〇ly (ethylene) Glycol)), or poly(2-ethyl-2-oxazoline) (P〇ly (2-ethyl-2-oxazoline)). Preferably, the main chain of the graft copolymerized polymer comprises a first repeating unit having hydrophilicity, and the hydrophobic side chain is bonded to the first repeating unit. More preferably, the first repeating unit has a carboxyl structure and the hydrophobic side chain is biodegradable. Preferably, the main chain of the graft copolymerized polymer is polyacrylic acid, polyacrylic acid, polybutyric acid, polyhistamine or decyl vinylimidazole. More preferably, the main chain of the graft copolymerized polymer is polyacrylic acid. Preferably, the hydrophobic side chain of the graft copolymerized polymer is polyester, polylactide, polylactic acid or polycaprolactone. More preferably, the hydrophobic side chain of the graft copolymerized surface molecule is polylactide. Preferably, the graft copolymerized polymer backbone further comprises a second repeating unit, and the second repeating unit is different from the first repeating unit, wherein the second repeating unit is responsive to temperature changes to cause the microcell The nuclear collapse. More preferably, the second repeating unit of the main chain of the graft copolymerized polymer is formed by a monomer of ν·isopropyl 1361813-based N-isopropyl acrylamide. The main chain of the graft copolymerized polymer is a copolymerized polymer of N-isopropylacrylamide and methacrylic acid. Preferably, the polymer type microcell has a particle size of 5 〇 2 〇〇 nm. Preferably, the two-co-polymerized two molecules have a terminal functional molecule attached to one end of the hydrophilic polymer segment, and the terminal functional molecule is a receptor that can interact with the surface of the tumor cell (recept〇 r) Binding ligand φ (Hgand). More preferably, the ligand is a galactose residue. Preferably, the bis-co-polymerized polymer has a terminal functional molecule attached to one end of the hydrophilic rain molecular segment, and the terminal functional molecule is a fluorescent group. More preferably, the fluorophore group is fluor〇reseein isQthiQeyanate. Preferably, the bis-co-polymerized polymer has a terminal functional molecule attached to one end of the hydrophilic polymer segment, and the terminal functional molecule is a dye. More preferably, the dye is a near infrared ray dye. Preferably, the structure comprises a plurality of different copolymerized copolymerized polymers, and each of the copolymerized copolymers comprises a hydrophobic polymer segment and a hydrophilic knives. More preferably, each of the plurality of different coalescence copolymer molecules is a bi-cluster copolymerized polymer comprising a hydrophobic polymer bond segment and a hydrophilic polymer bond segment. Preferably, the hydrophobic polymer segment of the different co-polymerized polymer has an identical repeating unit. Preferably, the hydrophilic polymer segment of the different co-polymerized polymer has an identical repeating unit. 1361813 Preferably, the hydrophilic polymer segments of the different co-polymerized polymers have different repeating units. Preferably, the plurality of different agglomerated copolymerized polymers are linked to different terminal functional molecules at the ends of the hydrophilic high molecular segment. More preferably, one of the terminal functional molecules is a ligand that binds to a receptor on the surface of a tumor cell. Most preferably, the ligand is a galactose residue. Optionally, one of the terminal functional molecules is a fluorescent group, such as a fluorescent isothiocyanate. Optionally, one of the terminal functional molecules is a dye, such as a near infrared ray dye. Embodiments In the present invention, a multi-component microcell has been prepared from a graft copolymerized polymer and a bi-polymer polymeric polymer, and the microparticles are controlled by the difference in self-bounding cell concentration (CMC). The size of the path. The composite cell of this particular core-shell structure can be widely applied by careful design and manipulation of each component. # •中—Use is applied to anticancer drug carriers. The application of internal medicines to release a very important cancer treatment drug delivery route. This pathway increases the toxicity of the drug to cells in the target area and reduces the side effects of the drug on normal cells or tissues. In general, 'intracellular induction of drug release from the carrier can be caused by changes in the enzymes in the lysosome or by the acidity of the small body acid. Many materials have been developed and synthesized for application. See the drug delivery inside. However, because the material is strongly charged or has a strong charge or is used in the body, it is recognized by the mononuclear macrophage system (MP S ). It is difficult to reach the tumor tissue and achieve drug-controlled release. 1361813 used. Therefore, in order to treat cancer by intracellular drug delivery, it is very important that the hydrophilic molecule structure is on the surface of the microcell or the _• subcarrier. One embodiment of the present invention prepares a novel composite type microcapsule having a multifunctional shell/core, which is composed of an environmentally responsive graft copolymer: poly(N-isopropyl propyl amide-CO-A) Poly(N-isopropyl acrylamide·co-methacryl acid-gp〇ly(D,I-lactide)) (abbreviated as φ P) (NIpAAm-co-MAAc)-g-PLA), co-polymer with two pairs of conjugates: methoxy t (ethyl alcohol) - b-poly (£), 1-lactic acid vinegar) (meth〇xy p〇 Ly(ethylene glycol)-bp〇ly(D,L-lactUe)) (abbreviated as mpEG_pLA) and poly(2-ethyl-2-oxazoline-b-polylactide) (poly (2-ethyl-) 2-oxazoline)-b-poly(ZD,Z_lactide)) (abbreviated as PEOz-PLA) together constitutes 'this nanostructure can completely conceal the highly negatively charged graft copolymer in the core and make the core structure versatile . This versatile complex type of microcapsules is used for intracellular drug delivery to show drug release lines. • It has a strong correlation with the function of the micelles. In addition, the concealment of the composite microcapsule also affects cytotoxicity; composite micelles and grafted copolymers (P(NIPAAm-C〇-MAAC)_g-PLA, [NIPAAm]/[MAAc]/[pLA Compared with the microvesules formed by 84 · 5·9 . 2.5 mol/mol), the composite micelles showed higher drug activity and lower material toxicity. This embodiment not only proposes a novel micro-cell structure composed of a double-agglomeration-graft copolymerization polymer system and provides - a graft copolymerization polymer and a double-coupling copolymer inter-turned knife or double-dual joint The concept of multifunctional microcapsules prepared by copolymerizing polymers is applied to drug delivery. 1361813 Another embodiment of the present invention provides a multifunctional microcapsule for application to cancer cell target, distribution development, and anticancer drug delivery by an environmentally-responsive graft copolymer P (NIPAAm-co) -MAAc)-g-PLA, a double-cluster copolymer mPEG-PLA, and a bifunctional bi-co-linked copolymer galactose

(galactosamine)-PEG-PLA (abbreviated as Gal-PEG-PLA) is composed of fluorescein isothiocyanate-PEG-PLA φ (FITC-PEG-PLA). The multifunctional microcell can accurately identify liver cancer cells by a receptor ("hepatoma cell")_Gai (multifunctional microcell) receptor-mediated tumor targeting mechanism. Changes in the acid-base environment after endocytosis disrupt the internal structure of the multifunctional micro-cells, resulting in drug release and, in turn, increased toxicity to liver cancer cells. The development function of the multifunctional microcell can be clearly observed in the cell using a Confocal iaser scanning micr〇sc〇py (CLSM). The polymer type microcapsules of the present invention can be widely applied to cancer diagnosis, cancer identification, and cancer treatment by careful design and precise manipulation of molecular composition. The invention may be understood by the following examples, which are intended to be illustrative only and not to limit the scope of the invention. Example 1: Materials Outside / methacrylic acid (methacrylic acid, abbreviated as MAAc) was ordered from Lancaster. Methyl p-toluenesulfonate (MeOTs), stannous octoate (Sn(〇ct)2), 2-hydroxyethyl methacrylate Methacrylate (abbreviated as HEMA), pyrene and azobisisobutyronitrile (abbreviated as AIBN) were placed on Aldrich. N-Isopropyl acrylamide (NIPAAm) and ethyl oxazoline (2-ethyl-2-oxazoline) were ordered from TCI. Oxime polyethylene glycol (mPEG, Φ weight average molecular weight (Mw) = 5 〇〇〇 Da) was ordered from Sigma. The racemic lactide was recrystallized twice with tetrahydrofuran (THF) before use. N-isopropyl acrylamide and azobisisobutyronitrile were recrystallized with n-hexane and acetonitrile before use, respectively. Mercaptoacrylic acid and hydroxyethyl methacrylate were purified by distillation under reduced pressure before use. Ethyloxazoline and p-toluenesulfonate were dehydrated by hydrogenation of calcium hydride (CaH2) before use, followed by distillation under reduced pressure. # P(NIPAAm-co_MAAc)-g-PLA (Grafted I, G1) Graft Copolymer Preparation Purified racemic lactic acid vinegar (£), Z_lact: ide) (4 g), HEMA ( 0.20 g) and toluene (5 mL) in water were placed in a round bottom double neck bottle and connected to a water system to remove oxygen. The monomer was completely dissolved at 10 ° C and a catalyst Sn(OCt) 2 (1 wt%) was added, and a cationic ring-opening polymerization reaction was carried out under a nitrogen atmosphere. After the reaction was carried out for 16 hours, the reaction was terminated by the addition of 0.1 N methanol K〇H (methanolic KOH). The product was reprecipitated by hexane and the salt was removed from the Shixi rubber column.

For classes and impurities, PLA-EMA (Μη = 2000) is available. Quantitative pla-EMA 13 1361813 (0.35 g), NIPAAm monomer (1.15 g), MAAc monomer (0.16 g) and initiator AIBN (0.023 g) were placed in a double neck round bottom flask with acetone (15 mL) The reaction was dissolved and nitrogen was passed through. The radical polymerization reaction was carried out at 7 (TC for 24 hours. After the completion of the reaction, reprecipitation was carried out with H2 hydrazine and diethyl ether respectively to remove unreacted two times. After drying, graft copolymer P (NIPAAm-co-MAAc) was obtained. -g-PLA (grafted j, G1) ([NIPAAm].[MAAc]:[PLA] = 84 : 5.9 : 2.5 mol/mol) 〇mPEG-PLA (cluster I, B1) double-co-polymer Preparation of purified racemic lactide (1 g), mpEG (10 g, Mw = 5000 Da) and toluene (4 mL) in water, placed in a round bottom flask, attached to a vacuum tube system Deoxidation by water removal. Ion ring-opening polymerization was carried out under nitrogen atmosphere under the complete dissolution of rc monomer (Sn(OCt)2 (1 wt%). The reaction was carried out for 16 hours and then added to 〇. 1 n methanol. The reaction is terminated by KOH. The product is crystallized at a low temperature via a mixture of dichlorodecane and diethyl ether, and dried to obtain a double-co-linked copolymer mPEG-PLA (Group I, Bl) ([EG ]:[LA] = 113:7 mol/mol) PEOz-PLA (Group Π, Β 2) Double-cluster Co-combination The preparation of PEOz-PLA is a modification of the old reaction procedure [GH Hsiue] , C. Ch. Wang, CL Lo, CH Wang , J. p. Li, JL Yang, Int. J. Pharm. 20 06, 317, 69] 'The procedure is as follows. The starter Me〇Ts (0.232 mg) is placed in a round bottom double neck bottle, connected to Under the vacuum tube system, water and oxygen were removed, then acrylonitrile (30 mL) was added and the reaction temperature was controlled at 1 Torr. 14 1361813 The water-removed monomer ethyl chewing gum (10 mL) was refluxed for 30 hours. After the reaction was terminated, 0.1 N sterol KOH was added to terminate the reaction. The product was purified by diethyl ether reprecipitation. The ruthenium tube column was used to remove salts and impurities, and dried to obtain PE〇z_〇H. PE0z (2 g) and racemic lactic acid were added. The lactide (0.426 g) was subjected to ion ring-opening polymerization with Sn(〇Ct)2 (1 Wt%). The reaction temperature was controlled to reflux at 13 〇 for 6 hours. After the reaction was completed, 〇. i N methanol KOH was terminated. The reaction was purified by diethyl ether reprecipitation to obtain a double-co-linked copolymer pe〇z_PLa (Group II, B2) ([E〇z]: [la] = 52.5: 9.7 mol/mol). The chemical structure and the number average molecular weight were identified using a nuclear magnetic resonance spectrometer ih_nmr (AMX_5〇〇, Bruker). The degree of polymerization distribution index of the copolymerized ruthenium molecule is measured by a gel permeation chromatography (Gpc). The mobile phase of GPC is N, Ndimethylf〇rmamide (DMF), and the flow rate is! Mi/min, the measured temperature is 4 〇β〇. Copolymerization The critical micelle concentration (CMC) of the 咼 knife is based on the "hydrophobic dye" 胄光 spectrometer at 393 nm. Observe the highest concentration of the sample and the lowest concentration of the sample at the 3 3 0-3 40 nm of the southernmost fluorescence intensity, and the ratio of the intensity (a) spoon 337" / 〗 33 5) plot the concentration logarithmic coordinates The concentration at the bottom of the curve and the inflection point of the two lines of the parental cross point is defined as the critical cell concentration.纟i lists the identification results of each copolymerized polymer. 15 1361813 Table 1

Mn [Da] Hydrophilic segment Mn [Da] Lipophilic segment polymerization degree distribution index CMC [g/mL] Grafting I 9970 6150 1.24 1.27 χ ΙΟ'6 Cluster I 5000 500 1.05 8.39 χ ΙΟ·5 Group II 2200 700 1.16 1.70 χ 6 '6 Preparation of composite nano-cells A fixed concentration of graft 1 copolymer (0.13 M) was dissolved in DMSO φ, and different concentrations of the group I copolymer were added. After uniformly mixing, they were placed in a dialysis bag (MWCO 6000 to 8000, SpectrumLabs, Inc.), and the pure water was replaced once every 2 to 3 hours at room temperature for 48 hours to prepare a G1B1 composite type nanocapsule. The product was collected for analysis after lyophilization (Het〇_H〇lten A/S, Denmark). Copolymerization of a fixed concentration of graft j

The compound (0.13 M) and the group I copolymer (〇·2〇M) were dissolved in DMSO. 'Additional concentrations of the group conjugated co-polymer were added separately, and then uniformly mixed and placed in a dialysis bag (MWCO 6000 to 8000, SpectrumLabs, Inc.), replacing pure water once every 2 to 3 hours at room temperature for 48 hours to prepare G1B1B2 composite nano-cells. After lyophilization, the product was collected for analysis. ...Fig. 1 The average particle size and particle size distribution of the A-type G 1B 1 complex type microvesicles under different bundled I double-co-polymerization compositions. The concentration of graft I graft copolymer in gibi composite micelles was fixed at 1〇 〇mg/L. Fig. 1B is a graph showing the average particle size and particle size distribution of the G1B1B2 composite type microcells under the composition of different agglomerated u-double-coupling copolymers. The concentration of grafted I grafted copolymer in the g 1B composite microsphere preparation process was fixed at 1 〇〇 mg/L. The concentration of the double-co-linked copolymer was fixed at 5.625 mg/L. 1361813 Table 2 lists the concentrations of the composite micelles of Figures 1A and 1B which are grafted I, ligated I and conjugated II. Table 2 G1 (mg/L) B1 (mg/L) B2 (mg/L) G1 : B1 : B2 = 33.9 : 55.7 : 10.4 mol/mol 10.0 5.625 1.125 G1 : B1 = 5〇: 5〇mol/mol 10.0 3.15 -- φ Tri-component G1B1B2 complex type nano-cells (G1 : B1 : B2 = 33.9 : 55.7 : 10.4 mol/mol) are self-assembled into a microcell structure by the aggregation and arrangement of hydrophobic polymer segments PLA. The fixed cell concentration (〇. 1 mg/mL) was dispersed in phcisphate buffer saline (PBS) and its particle size was 182.3 ± 1.5 nm by dynamic light scattering (DLS). (polydispersity index, PDI) is 0.038 ± 0.014, with a uniform particle size and a narrow distribution. The interfacial potential of the composite micelles in PBS (0.1 mg/mL) was measured by Doppler microelectrophoresis (Zetasizer 3 000HS, Malvern) to determine the double-band copolymer to the composite micelle. The degree of concealment of the kernel. The grafted I mice formed by graft copolymerization of the polymer were compared, and the interface potential was measured as _丨5.5 ± 〇 9 mV. The interfacial potential of the composite micelle is _7·8±1.3 mV, and the high-negative MAAc of the core has been obscured by the shell structures mPEG and PEOz. The most direct evidence for the core structure of the composite micelles can be stained with MAAc via an acetate shaft (2 wt./〇) with a transmission electron microscope (teM; Hitachi H-6O0 microscope, accelerating voltage = 100 kV). 17 1361813 Observed, as shown in Figure 2. The dyed area of Figure 2 is distributed in the core, that is, the core is composed of the technology I, and the unstained area of the shell is composed of mPEG and PEOz. In addition, the surface type of the composite micelle can be observed by atomic microscopy (AFM). The results show that the G1B1B2 composite microparticles are uniform in size and spherical, and the particle size is consistent with the results obtained by dynamic light scattering. The effect of the double-cluster co-polymer on the formation of complex micelles was investigated by GIB 1 complex type micelles composed of different φ ratios and G1B1B2 complex type micelles. The three groups of copolymerized polymers have different critical cell concentration: the critical cell concentration of the group I is much larger than that of the group II and the graft 1 (Table 1). When a fixed concentration of graft I and a different concentration of the group I are mixed to prepare a composite type of micelle, the particle size of the G IB 1 composite type cells of different ratios is about 〇6〇nm (as shown in FIG. 1A). 'And less than the grafted I microcells and the clustered I microcells.粒径 1 b 1 The particle size distribution of the composite micelles is narrowly distributed. When the group is added to a solid concentration of G1B1 (the critical microcell concentration of the cluster II is close to the grafting I critical microcell concentration but less than the clustered I critical microcell concentration), the particle size of the G1B 1B2 composite type microcell is The addition of Tuanlian II is irrelevant (as shown in Figure 1B) and is smaller than each single nucleus or G1B2 composite haplotype (330.2 ± 0.9 nm; PDI = 0.072 ± 0.011), but with G1B1 complex type similar. However, when the molar content of the group was more than 0.54, the micelle could not be formed. These results show that the copolymerized polymer of the high critical microcell concentration determines the particle size of the composite nanocell. That is to say, in the present system, the presence of the group I can regulate and control the formation of the composite micelle, and control and reduce the particle size of the micelle. (N-iS〇pr〇pylaCrylamide), 18 1361813 (PNIPAAm) is a water-soluble and hydrophilic polymer and is temperature-sensitive. Its low temperature critical solution temperature (LCST) is between 28-35 °C. If the temperature is lower than the temperature of the low temperature critical solution, the polymer is in a dissolved state; and when the temperature is higher than the temperature of the low temperature critical solution, it is in an aggregated state. If a PNIPAAm is introduced into a hydrophilic material such as MAAc, the aggregation ability of the PNIPAAm isopropyl group is destroyed, thereby increasing the phase transition temperature of the copolymer. In the present invention, the MAAc having dissociation ability enhances the phase transfer temperature of the material while imparting an acid-base responsiveness. In an acidic environment, P(NIPAAm-co-MAAc) produces aggregation and precipitation because MAAc protonation reduces the hydrophilicity of P(NIPAAm-co-MAAc) and lowers the critical temperature of the low temperature solution to 32 °C. This acid-base responsiveness is correlated with temperature responsiveness. It has been shown in previous studies that grafted I micelles have the property of producing microstructural changes in an acidic and high temperature environment (C. L. Lo, K. M. Lin, G.. H. Hsiue, J. Controlled Release 2005, 104, 477). Figure 3 shows the structural change of G1B1B2 composite micelles (G1:B1: Β2 = 33·9:55.7: 10.4 φ mol/mol) in an acidic environment (pH 4.0) as a function of temperature. The composite micro-cells were destroyed by a sputum as a hydrophobic dye, and the ////3 change of the ruthenium molecule was observed by a fluorescence spectrometer. The sputum solution was used as a control group. Because the molecular symmetry of the ruthenium molecule or the π-electron cloud is affected by the polarity and environmental perturbation, the first emission spectrum (/7) will change greatly, but it is different from the other four. There is no obvious change in the emission spectrum of the wavelength, so it is a commonly used dye in the discussion of the formation mechanism of the microcells, and ////3 represents the change of the environment (K. Kalyanasundaram, J. K. Thomas J. Am. Chem. Soc. 199.7, 99, 2039). /7 / /j 19 1961813 Low' means that the polarity of the environment is lower; the higher the plant / h, the higher the polarity of the environment. As shown in Figure 3, the heart value of the ruthenium solution was reduced from 181 to 174 as the heat consumption increased with temperature. For composite micelles in pH 4.0 environment, when the temperature is higher than 37, the value is rapidly increased from [Μ to 1.46, indicating that the environment is weakened by the internal polarity of the composite cells. Higher solution. In the present invention, the difference between the structure of the composite type cell and the structure after the damage is analyzed by time 二次f flight φ secondary i〇n mass spectr〇metry (T〇F SIMS). The structure of the composite microcells was analyzed before and after the structural damage, and the chemical composition of the surface was analyzed by positive and negative ions, respectively. Analysis by secondary ion mass spectrometry showed that grafting was revealed due to structural damage. The structure of the composite micelles was stained with uranyl acetate (2 wt❶/.) before and after structural damage, and the structural differences were observed by transmission electron microscopy (TEM). As shown in Figures 4A and 4B, the GIB 1B2 complex type microcells (G1: B1: B2 = 33.9: 55.7: 10.4 mol/mol) had no obvious core-nuclear structure after structural failure. Example 2: This example uses a graft copolymer p(NipAAm-co-MAAc)-g-PLA and a double-cluster copolymer mpEG_PLA to form a composite microcapsule to form a hydrophobic anticancer drug. Red per (d〇xorubicin, abbreviation

Dox). When the blood circulates, the drug can be coated in the core without causing side effects. The composite microcapsule-coated anticancer drug D〇x was prepared by the same method of dialysis 1361813. The steps are similar to those in Example 1. P(NIPAAm-co-MAAc)-g-PLA (graft I) copolymer (20 mg) and mPLA-b-PEG (coupling I) copolymer (2 mg) were dissolved in DMSO/DMF (2 In a solution of mL/8 mL), a mixture of mixed Dox-HCl (20 mg) and triethylamine (TEA, 0.3 mL) was added to make Dox hydrophobic to facilitate microcapsule core carry. After stirring for 2 hours, dialysis was carried out with a dialysis membrane (MWCO 6000-8000), and water was changed every 2 hours for a total of 72 hours. The product in the dialysis bag was collected and lyophilized, and stored in a refrigerator at 4 °C. The drug coating amount was evaluated by dissolving the above-mentioned composite drug cells in DMSO, and after the structure was dissolved, the drug and the polymer were separated by an ultrafiltration device (MWCO 1000), and Dox was measured at 485 nm with UV/Vis. Absorption. Insert the absorbance value into the Dox calibration line to convert the actual amount of drug in the composite micelle. The drug content is estimated as follows: drug content (% w/w) = (drug weight of complex micelles) / (drug weight of composite micelles + weight of polymer in composite micelles) X 10 0. The composite microcapsule-coated anticancer drug Dox was observed by AFM to exhibit a spherical shape with a particle size of about 1.65 nm. Discussion on drug release behavior. The compound drug cells were dosed in a buffer solution of different pH values (50 mg/L), sampled at 37 ° C or 25 ° C for a fixed time with an ultrafiltration device (MWCO 10000), and an ultraviolet spectrometer ( UV/Vis) Analyze drug concentration. The amount of drug released at a given time was calculated against the drug coverage rate to investigate the effect of the acid-base response behavior of the compound drug cells on drug release. The pH value of the buffer solution was pH 7.4 phosphate buffer solution and pH 5.0 succinate buffer solution, respectively. UV/Vis measured its absorbance at 485 nm. 21 丄361813 cell killing test. Cell cytotoxicity test (gr〇wth inhibiti〇n assay) ^ 疋 别 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入 加入Survival rate, comparison of drugs, grafted drug cells and complex drug cells to the toxicity of HeLa cancer cells. Average standard deviation & = 6). HeLa human cervical cancer cells were cultured in a Dulbecc〇is m〇dified Eagle, s medium (DMEM) medium in a 37 °c, 5% c〇2 incubator until the cells grew to a certain number. Cultured in 96 well plates, the number of cells was 5 X 103 cel 1/mL·. After about 8 hours of cell attachment, the medium was removed and the drug containing different concentrations, the grafted I drug micelles, and the complex drug cell culture medium were added. After 48 hours, analysis was performed by colorimetric assay (MTT assay). Further, the step of performing the material toxicity analysis of the drug-free grafted copolymer microcapsules and the composite type microcapsules is the same as the above method. Endocrine assessment. The distribution of drug and complex drug micelles in the cells was observed using a Confocal Laser Scanning Microscopy (CLSM). Place a cover glass on a 6-well culture plate. Place a reinforced ring on the cover glass to mark the observation range. Plant i x i 〇 5 cells per well. After the cells were attached, d〇x with a drug concentration of 1 〇 /mL and a composite drug cell culture medium were added. The medium was removed at a fixed time, washed with PBS, and added to LysoTracker DND-26 2 mL (50-70 nM in DMEM medium) for another 0.5 hours. The cells were washed with pbS and 0.10/〇 Triton X-100 PBS. Fixation of the cell test piece was carried out in a PBS solution of paraformaldehyde (4 wt%) for 30 minutes, and then washed with 22 1361813 pbs, and then 80% glycerol (giycer〇i) in PBS was added dropwise to the reinforcing circle. 15 μί and attached to the slide to protect from light. The conjugate focal length microscope objective is set to 40 Χ; the Dox excitation laser light wavelength is 485 nm, the emission light wavelength is 590 nm; the LysoTracker excitation laser light wavelength is 5〇4 nm, and the emission light wavelength is 511 nm. The image obtained by the scan overlaps with the phase difference microscope image. To determine where the Dox is released. The composite drug cells were measured by UV/Vis and their drug content was about 19% corrected. The drug release test uses a dialysis membrane to separate the drug from the micelles. Figure 5 shows the release of the drug from the complex drug micelles in different pH environments. The compound drug micelles showed significant drug release differences in the pH 5.0 buffer solution compared to the pH 7 4 buffer solution. The drug release behavior was also significantly different at 25 ° C and 37 ° C under pH 5 buffer solution. At pH 5 〇 buffer solution and 25 ° C, the drug release rate of a rapid drug release characteristic was about 25 wt. % in the first 2 hours. Then, the drug release behavior does not increase with time, because the core of the composite microcapsule has a strong hydrogen bond force with the outer shell, and the hydrogen bond causes the microcapsule to compress and the partial drug is discharged by hanging. This resulted in the release of 25% of the drug at the beginning. However, as time passed to % hours, the drug was still stored in the core of the composite drug microcapsule, indicating that the microkernel core structure did not cause structural damage due to argon bond forces. On the other hand, in the pH 5.0 buffer solution and 37t, there was a drug release amount of about 5〇% in the initial 2 hours, which was caused by the destruction of the cell structure. The drug release result strongly showed that the cell structure damage controlled drug release. The cytotoxic killing test was used to understand the inhibitory effect of the composite drug microcells on the growth of cancer cells and the ability to kill cancer cells. In addition to the test, the D〇x.HC1 was tested with the compound 23 1361813. The grafted j drug micelles were also co-ordered for observation as a control group. Different concentrations of the composite drug cell D〇X HC1 and the graft 1 drug micelle were co-cultured with 2×10 4 HeLa cells, respectively, to observe cell survival. As shown in Figure 6, Dox.HCl has a higher toxic ability in 48 hours of culture, and its 1 (:5 〇 is about 8 gg/m, L. and the complex drug micelles and grafts! Drug micelles The iCm is 3 pg/mL and 6 pg/mL, respectively. The reason is that D〇x · HC1 small molecule is _ diffuse into the cell and directly kills cancer cells, so its action speed is faster; Rice drug microcells and grafting 1The drug micelles enter the cells by pinocytosis, and change the micelles by environmental acidification in the end 〇smal space compartment or the lysosomal compartment (iys〇smal c〇mpartments). After the structure, the drug was released. In addition, the drug release amount of the composite nanomedicine microcapsules and the grafted I drug microvesicles was about 05% and 60% of the total amount under the 48-hour culture condition. Compared with d〇x The HC1 drug has a low effect on cells, so its cell toxic ability is poor. On the other hand, because the grafted I drug microcapsule has a strong negative charge, its ability to enter cells is poor, so its cell killing effect is better than that. The composite nanomedicine micelles have poor ability with D〇x HC1. In order to separate the cytotoxic sources, they are derived from carriers or drugs, and the drug-free composite micelles and grafted cells are also HeLa cells were tested for toxicity. In 48 hours of culture, the cytotoxicity of the composite micelles and the grafted micelles increased with increasing concentration. The IC50 of the composite micelles was 2.5 mg/mL; Then it is i 5 mg/mL. It shows that the composite microcytotoxicity is lower than I microcells, this is due to the strong negative charge obscured by mPEG. In addition, the concentration of the carrier converted from the drug concentration shows that the cytotoxicity caused by the 24 1361813 micro-cells is higher than that of the chelated nano-cells. The grafted I microcells have poor applicability in cancer therapy and intracellular drug delivery. After the introduction of the double-cluster copolymer into the microcells, the microcell structure and the concealed negatively charged material such as MAAc can be stabilized, which not only increases the phagocytosis and decreases Cytotoxicity can also overcome the limitations of polyionic polymers (P〇ly i〇nS) in the use of biomedicine. Intracellular distribution and drug release behavior of complex drug micelles and drugs were observed by co-culture of HepG2 cells (human liver cancer cells) with complex music cells or drugs by a co-focus microscope. LysoTr acker can be used to observe the distribution of acid organelles or acidified organelles to determine the distribution and drug release of the composite drug cells in the cells. Dox. HC1 is distributed in the nucleus in either 1 hour or 8 hours and in HepG2 culture. LysoTracker is distributed in the cytoplasm and nucleus, which is caused by d〇x. HC1 acidified cells. When the composite drug micelles were cultured for an initial period of 1 hour, the coated Pox was released into the cytoplasm, and the green light shown by the LysoTracker was also distributed in this region. At 8 hours of culture, Dox was distributed not only in the cytoplasm but also in the nucleus, while LysoTracker was only distributed in the cytoplasm. This shows that after the compound drug micelles enter the cells via pinocytosis, acidification of the endo-somai compartments or lysosomal compartments causes destruction of the cell structure and release of the drug. The drug was only distributed in the cytoplasm in the initial i-hour culture. After 8 hours, Dox enters the nucleus and the microtubules continue to release the drug, so Dox is also distributed in the cytoplasm. Similar results can also be observed in Chinese hamster cells 25 1361813 (CH0-K1). In this example, the composite music microcells can be rapidly structurally destroyed by intracellular acid assay to release zk^ and enemies, and also have a hydrophilic outer shell for concealing highly charged materials and adding materials. Solubility. Example three:

In the same step as in Example 1, the group M (mPEG5〇〇〇pLAi(4) has a molecular weight distribution of i.15, the critical cell concentration is 16 mg/L) and the group IV (mPEG5_-PLAl750, the molecular weight distribution is 1.2 〇, the critical micro The synthesis of the copolymerized polymer with a cell concentration of 5-4 mg/L is to open the ring of mpE(j (Mn 5〇〇〇) disk racemic lactate (AL_laetide) with tin catalyst as the initiator. These polymers have the same chemical structure, but different composition ratios. This example uses double-coupling copolymers with different molecular weight lengths and different critical cell concentrations (union, m- or m) Recombinant micelles were prepared separately from _grafted copolymers (Examples - prepared Graft) to understand the effect of the di-tuplex on the morphology and structure of the composite micelles. Firstly, the graft copolymer and the double-cluster co-polymer are dissolved together in a mixed solvent of DMSO/DMF (4/1 v/v) to prepare a polymer solution. The use of the mixed solvent can be prepared in this solvent. Composite microcells of particle size. The concentration of grafted copolymer in solvent is fixed at i〇mg/mL The composition ratio of the graft copolymer to the double-co-polymerization is 1:9. The composite micelles are prepared by the dialysis method in the same manner as in the first step. The three groups of the two-composite composite micelles are prepared (respectively Twigs and lumps combined with haplotypes, grafted 26 1361813! and ganglian Π! composite type of microcells, and ligated with gang iv composite micelles) TEM observation of its core structure and particle size The following three conclusions can be obtained for the size: (1) For all the composite micelles in this example, the structure of the core structure is known to be a grafted copolymer, and the outer shell structure is composed of a hydrophilic polymer mPEG. (7) The composite microcell core radius (8) decreases as the molecular weight of the double-clustered copolymer hydrophobic segment PLA increases.

(Rplasoq > RPLA1(m > RPLAm()). (3) The size of the composite micelle increases with the molecular weight increase of the hydrophobic group bond PLA of the di-group (tetra) polymer. Short-segment PLA can form The smaller size of the composite microcysts The type 5 microcells are tested for stability in the presence of serum or serum proteins. This test uses graft 1 (251!1〇1%) and agglomerate 1乂 ( 75111〇1%) composite microcapsules. The composite micelles were suspended in physiological saline (2 mg/mL) containing 4 wt% bovine serum albumin (BSA) and fixed at 37 °C. The dynamic light scattering instrument was used to observe the change of the particle size of the composite micelles under the time. The dynamic light scattering measurement mode was set to C0NTIN, and the particle size of the composite cell solution before mixing with BSA was observed by dynamic light scattering. The grafted I microcells were used as a control group. The particle size change ratio was obtained by dividing ί,· by G, ί, ./ία. From the results, it was found that the composite micelles showed a stable state within 72 hours, which was due to The mPEG of the microcapsule blocks the adsorption of serum proteins to avoid aggregation. This phenomenon shows that the composite micelles have long-term blood. Cycle characteristics. Example 4: Double-coupling co-polymer Gal-PEG34〇〇-PLA830 27 1361813 (Gal-PEG-PLA, [Gal]:[PEG]:[LA] = 8.4.:7.6:84 Mol/mol) and the developed double-coupling copolymer FITC-PEG34〇〇-PLA830 (FITC-PEG-PLA, [FITC]: [PEG]: [LA] = 4:8:88 mol/mol) It is prepared by the thiol-maleimide coupling reaction. Synthesis of FITC-PEG-PLA double-cluster copolymer. PLA-NH2. First N-Boc-L-alanine alcohol (alaninol) is treated with potassium/naphthalene to form a metal oxide (N-Boc-L-alaninol-OK). Dissolve racemic lactide (D, Z-lactide) (2 g) N-Boc-L-alaninol-OK (0.35 g) was used as a starting agent in toluene (2 mL) for 12 hours at 100 ° C. After the reaction was completed, the reaction was terminated with acetic acid, and then reprecipitated with diethyl ether. The polymer PLA-NHBoc was obtained, and then PLA-NHBoc (2.1 g) was dissolved in a mixed solution of formic acid (20 mL) and gas (20 mL) at room temperature for 9 hours and then reprecipitated with diethyl ether. Product (1.5 g) Mixing solution with triethylamine (20 mL) and chloroform (20 mL) at room temperature After deprotonation 'and then with ether reprecipitation can be obtained when processing PLA-NH2 # 8 hours. PLA-SH. Dissolve PLA-NH2 (2 g) in acetonitrile (1 〇 mL), add an excess of 2-iminothiolane hydrochloride (0.458 g), and add an appropriate amount of TEA- The buffer (concentration 50 mM, Ph 8 · 0) was reacted at room temperature for 15 hours. Unreacted 2-iminothiolane was removed by dialysis with 5 mM HCl solution and 1 mM HCl solution several times, and lyophilized or vacuum dried to obtain PL A-SH. Maleimide-PEG-NH2 «N-Methoxycarbonylmaleimide (0.2 g) was dissolved in DMSO (JOmL) 28 1361813 and then added at room temperature. The amine (polyamine xyethylene biS (amine)) (1 g) was reacted for 6 hours in an aqueous solution. After completion of the reaction, recrystallization was carried out at a concentration of methylene chloride and diethyl ether (丨/丨.v / v) at 2 ° C to obtain maleimide-PEG_NH2. PLA-PEG-NH2. PLA_SH (0·8 g) was dissolved in 0_1 M Tris/acrylonitrile (1/3 v/v) (aq, pH 6 5) 〇5, and then maleine _PEG_NH2 was added at room temperature ( 2 g) Tris solution (10 mL) was reacted for 6 hours. After the completion of the reaction, dialysis was carried out with physiological saline and deionized ion water, followed by freeze drying. The dried product was dissolved in methylene chloride and reprecipitated with diethyl ether to remove PLA-SH to obtain NH2-PEG-PLA. FITC-PEG_PLA. NH2-PEG-PLA (1 g) was dissolved in methanol (40 mL) and then reacted with FITC (〇丨$g) at room temperature for 24 hours. After dialysis with 0.5 M NaCl aqueous solution and deionized water to remove unreacted materials, THFc_PEG-PLA was obtained after drying in cold or vacuum drying.

Synthesis of Gd-PEG-PLJ double-cluster copolymer. Ga Bu Malay imine. N-methoxycarbonyl maleimide (〇68g) was dissolved in DMS〇(1() mL) Φ and then added to the aqueous solution of galactosamine hydrochloride (〇·5 g) at room temperature. Reaction for 6 hours. After the reaction is completed, reprecipitation with diethyl ether gives Gal-maleimine. pla-PEG-SH. PLA-PEG-NH2 (1 g) was dissolved in acetonitrile (15 can), and an excess of 2-iminothiolane hydrochloride (0.1 g) was added to add 'the appropriate amount of TEA-buffer (concentration: 50 mM, pH 8.0) was reacted at room temperature for 15 hours. The unreacted 2-iminothione was dialyzed by a 5 mM HCl solution and a 1 mM HCl solution, and then lyophilized or vacuum dried to obtain PLA-PEG-SH. Gal-PEG-PLA. 29 1361813 PLA-PEG-SH (1 g) was dissolved in methanol (15 mL) and then reacted with Gai-maleimide (0.1 g) at room temperature for 24 hours. After dialysis with 0.5 M NaC1 aqueous solution and deionized water to remove unreacted materials, freeze-drying or vacuum drying to obtain Gai-PEG_PLAe, Example 5:

The multifunctional composite drug micelle of this example was prepared by a dialysis method. First, the anticancer drug Dox was dissolved in a DMSO/DMF (4/1 v/v) mixed solvent, and the HC1 was neutralized with an excess of triethylamine (1.2 mol). Then graft the m〇l%), the group IV (20 mol%), Gal_pEG_pLA (15 mail, and FITC-PEG-PLA (15 mol%) dissolved in the drug solution and dialyzed (MWCO 6000-8000 After dialysis, freeze-drying or vacuum drying can be used to multi-functional composite drug micelles. After the composite drug micelles are dissolved in DMSO, the drug content can be measured by ultraviolet light reading instrument to be about 31 (four). The TEM observation of the versatile composite drug microvesicles stained with uranium acetate was carried out. The results showed that the functional composite drug micro-cells had a complete shell-core structure with a particle size of about 16 〇 nm. The vascular penetration into the tissue must be through the open gap, vascular caveolae, or vascular hole defect, etc., and the pore gap is between 38〇 and 78〇nm. The multifunctional compound drug prepared in this example The cell size is less than 2〇〇nm and is suitable for overflowing the blood vessel through the above-mentioned pore gap. It is known from the literature that the particle size of the PEG-covered microcapsule affects the distribution of particles in the in vivo device, and the particle size is less than 200 nmT. Avoid filtering effects by the spleen. Particle size It also affects the ability of intracellular sputum. Particles larger than 500 nm require cage-independent endocytosis, while particles less than 200 nm can be non-specific cage-related procedures (non_specific clathrin). -dependent process) enters the cell. Therefore, the uni-monthly compound I1 prepared by the present example has a particle size of about 16 〇nm, which is in accordance with the particle size limit required for physiological conditions. The microcytes affect the release of the drug from the microcells in response to environmental responsiveness. Figure 8 is a graph showing the drug release behavior of the multifunctional composite drug micelles in pH 7 4 • and ρη 5·〇 buffer solution. At pH 7 4 and 37 C裒 brother's Xia Gonggong compound drug has a 1 5 wt·% initiai burst at the initial stage. After that, almost no drug is released from the carrier to maintain a stable total drug release. At pH 5 〇 and 37, the drug release profile can be clearly distinguished into two parts. A rapid burst release in the first two hours releases about 35 wt.% of the drug, and then the drug continues to release the drug at a sustained rate. The release rate of the drug reached 70 wt.%. The release behavior of the drug confirmed that the multifunctional compound drug has acid-base responsiveness, and changing the acid value of the ring environment can destroy the core structure of the cell and release the anticancer drug d〇. x. In this example, a multifunctional multifunctional micelle (without drug) was prepared by grafting I, agglomerated IV, FITc_PEG pLA, and Gal-PEG-PLA, and the preparation method was the same as above. Grafting 1 in multifunctional haplotypes mainly plays the role of environmental response in order to achieve the effect of drug controlled release. The versatile composite type of microcells mainly control the structural integrity of the core of the microcells and obtain the uniformity of the granules. The FITC-PEG-PLA double-cluster copolymerized polymer of the camping light development function mainly provides direct evidence to show the cumulative position of the multifunctional composite micelle. Targeted function 31 1361813

The Gal-PEG-PLA double-co-polymerized polymer mainly recognizes the receptor asaloglycoprotein on the surface of liver cancer cells to achieve the function of active tumor targeting. In this example, a multifunctional multifunctional drug microcapsule is co-cultured with cancer cells at different concentrations to determine the growth inhibitory effect of the multifunctional composite drug microtubule on cancer cells and the ability to kill cancer cells. D〇x Hci was used as a control group. After co-cultivation for 24 hours and 72 hours, it was found that 1〇50 of Dox.HCI φ (concentration causing half cell death) was about 1.2 pg/mL. The IC5 of the multi-functional complex drug microtubule cultured at 24 hours was about 25 pg/mL. The ic50 under 72 hours of culture was about 4 pg/niL, which is close to Dox.HCI. On the other hand, the versatile composite micelle (without drug) had an icq of 792 pg/mL at 72 hours. This result shows that cytotoxicity results from drug release from multifunctional versatile drug micelles. In order to evaluate the functionality of the multifunctional composite drug microcells for biomarker applications, the drug release and accumulation positions of the multifunctional drug-type microcapsules were observed by a conjugated focal microscope with HeLa cells for 6 hours. For most microcarrier vectors, the microcarrier carrier drug release trigger mechanism occurs in the phagosome and is then released into the cytoplasm. The results of the conjugated focus microscope showed that HeLa cells showed green fluorescence (fitC) in the cytoplasm, indicating the cumulative position of the multifunctional composite drug micelles. Red fluorescent (Dox) accumulates in the cytoplasm and nucleus. The route of delivery of the multifunctional multifunctional drug micelles in this example can be clearly visualized by FITC-labeled micelles. Example 6: 32 1361813 In order to evaluate the specificity of the multifunctional multifunctional drug micelles for tumors, the multifunctional drug celllets prepared by Example 5 and the compound drug micelles of Example 2 are shared with human liver cancer HepG2 cells. Culture to observe cell poisoning. Mushroom mushroom #尨炉活. HepG2 cells were cultured in DMEM medium in a constant temperature incubator at 37 °C, 5% C〇2, until the cells were grown to a certain number, and then cultured in 96 wel丨 culture plates, the number of cells was 2 χΐ〇4 / • weU. After about 8 hours of cell attachment, the medium was removed and cultured at 4C or 37C by adding a multi-functional complex type drug cell and a complex drug cell culture medium containing different concentrations. After cocultivation for 2 hours, the culture solution containing the drug micelles was washed away, and fresh culture solution was added at 37. The armpits were cultured for 24 and 48 hours. Upon completion, the cells were stained with trypanblue and the survival rate of HepG2 cells was calculated by phase contrast microscopy. In addition, the same experiment was repeated in the presence of galactose in the same concentration (15 mM) for behavioral analysis of the inhibitor. _ The results are shown in Fig. 9. The cytotoxicity of the multifunctional composite drug microcapsules and the composite drug microtubules at 37 °c under the co-culture of μ hours is greater than 4 C, and the multifunctional composite drug micelles Have a higher cell toxic rate. Since the pinocytosis of the cells stops at 4 °C, the drug entering the nucleus is due to the binding of Gal to the receptor asial〇glycoprotein, which is overexpressed on the cell surface, and then enters the cells at 37<>>c culture. In the first two hours, it was 37. (In the case of culture, the multifunctional sputum drug micro-cell can enter the cell by the combination of pinocytosis and Gal surface receptor, so its cytotoxicity is greater than the cytotoxicity treated at 4 ° C for the first two hours 33 1361813 Killing. The higher the toxicity of the cells, the higher the toxicity of the cells. The galactose-added system competes with the multifunctional compound drug cells, and the culture conditions and time are the same as above. Figure 10. The cell viability of the versatile composite drug cell or the composite drug cell is greater than that of the system without galactose, regardless of the 24-hour or 48-hour culture. The compound drug micelle does have the function of a tumor target. From Example 5 and Example 6, it can be seen that the multifunctional compound drug cell has been successfully prepared by dialysis and can be used for cancer diagnosis and cancer drug delivery. The results showed that the multifunctional composite drug micelles exhibited a spherical shape and a particle size of about 160 nm, which met the particle size limitation required for physiological conditions. The tumor target analysis and conjugate focal microscope were observed. The results show that the multifunctional composite drug microvesicles can exhibit strong cytotoxicity after entering the cells by recept〇r_mediated endocytosis. The present invention aims to provide a new concept, that is, to prepare one while having a long time Blood circulation, tumor identification, and ideal celllets combined with cancer diagnosis and controlled release of cancer drugs. If the FITC of the multifunctional compound drug cell is replaced by a near-infrared dye such as 5 5 , it can be applied to animals. To evaluate the in vivo distribution of multi-functional composite drug micelles and the therapeutic effect of cancer. Brief Description of the Drawings FIG. 1A shows the G1Bi complex type micelles in different examples of the double-cluster copolymers in the first example of the present invention. The average particle size and particle size distribution index of the molar ratio, wherein the concentration of the graft copolymerization of the G1B1 complex type microcells during the preparation of the 34 1361813 is fixed at 10.0 mg / L. Figure 1B shows the present invention In Example 1, the average particle size and particle size distribution index of G1B1B2 complex type microcells under different molar ratios of double-clustered copolymers I, in which G1B1B2 complex type microcells The concentration of the grafted polymer graft I was fixed at 10.0 mg/L, and the concentration of the double-clustered copolymer group I was fixed at 5.625 mg/L. Figure 2 is a G1B1B2 composite type in the first example of the present invention. A photomicrograph of a transmissive φ electron microscope (TEM) of a microcell (labeled on a scale of 500 nm). Figure 3 shows that the G1B1B2 composite type microcapsules are compounded in a fixed acidic environment (pH 4.0) at different temperatures in the first example of the present invention. The intensity ratio of the vibration bond in the fluorescence spectrum of the pyrene molecule of the type cell is (/;//;). Fig. 4A is a transmission electron microscope of the G1B1B2 complex type microcapsule before the structural failure in the first example of the present invention ( TEM) image (marked on a scale of 2 nanometers). Fig. 4B is a transmission electron microscope (TEM) image of the G1B1B2 composite type microcapsule in the first example of the present invention after the structure is broken (marking scale is 2 奈 nanometer). Fig. 5 is a diagram showing the drug release of the composite drug micelles in different acidic and neutral environments in Example 2 of the present invention at 25 ° C and 37 t, respectively. Fig. 6 is a graph showing the growth inhibitory ability of the composite drug microcells for human cervical cancer HeLa cells in Example 2 of the present invention, wherein the grafted I drug micelles formed by free and graft copolymerized polymers were used as a control group. Fig. 7 is a through-electron microscopy (TEM) image of a multifunctional multifunctional drug cell of Example 5 of the present invention. 35 1361813 Figure 8 is a diagram showing the drug release profile of the multifunctional multifunctional drug (IV) (IV) in an acidic (pH 5.0) and neutral (pH 7 4) environment at 37 °C. Fig. 9 is a graph showing 24 and 48 hour cell growth inhibition of human hepatocellular carcinoma HepG2 cells by the multifunctional multifunctional drug microcapsules of Example 6 of the present invention. The compound drug micelle of Example 2 was used as a control group. Fig. 10 is a graph showing the cytostatic inhibition of human hepatocellular carcinoma HepG2 cells at a high concentration of galactose (15 mM) and .48 hours in the sixth embodiment of the present invention. The compound drug microrun of the second example was used as a control group.

36 1361813 References: [1] X. Gao, Y. Cui, R. M. Levenson, L. W. K. Chung, S. Nie, Nat. Biotechnol. 2004, 22, 969.

[2] N. Kang, Μ. E. Perron, R. E. Prud, Homme, Y. Zhang, G. Gaucher, J. C. Leroux, Nano lett. 2005, 5, 315.

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Claims (1)

1361813 (revised in December 2011) Ihm pound original j application patent Fan Park announcement fl 1. A polymer-type microcapsule with a shell-core structure, wherein the structure comprises a graft copolymerized polymer and a copolymerized polymer, the graft copolymerization is high The molecule comprises a main chain and a hydrophobic side chain bonded to the main chain, the co-polymerized polymer comprising a hydrophobic polymer segment and a hydrophilic polymer segment, wherein the graft copolymerization is high Hydrophobic side chain aggregation of the molecule, the hydrophobic segment stack of the copolymerized polymer is combined with the hydrophobic side chain of the graft copolymerized polymer to form a core structure, and the cluster is copolymerized A hydrophilic segment of the polymer protrudes from the core structure and is exposed outside the core structure to form a shell structure surrounding the core structure. 2. The macromolecule of the invention of claim 3, wherein the hydrophobic side chain of the graft copolymerized polymer and the hydrophobic segment of the copolymerized polymer comprise the same repeating unit. 3. The polymer type micelle according to the third aspect of the patent application, wherein the group = polymerized polymer double-co-polymerized polymer, the cerium comprises the hydrophobic group n as a sub-segment and the hydrophilic polymer segment . Water:: The polymer type micelle of the patent range f 3, wherein the number average molecular weight of the molecular segment is between 500 and 2500, and the number average molecular weight of the molecule is between 2 and _ 1〇〇〇〇. 5. The molecular type of micronuclei as claimed in the scope of patent application, wherein the group 38 1361813 (revised in December 2011) is a biodegradable absorption of the hydrophobic segment of the co-polymerized polymer. 6. The polymeric micelle according to claim 5, wherein the hydrophobic segment of the copolymerized polymer is polyester, polylactide, polylactic acid or polycaprolactone. 7. The polymeric micelle according to item 6 of the patent application, wherein the hydrophobic segment of the copolymerized polymer is polylactide. 8. The polymeric microcapsule of claim 3 The hydrophilic segment of the double-co-polymerized polymer is a polyacrylate or an acid-base/ion strength sensitive polymer, wherein the acid-base/ion strength sensitive polymer is a polyacrylic acid or a polyfluorene group. Acetic acid, poly(butenedioic aicd), polyhistidine, or poly(vinyl imidazole) 〇9. a polymer type microcell, wherein the hydrophilic segment of the double-co-polymerized polymer is a polyether, a poly(ethylene glycol), or a methoxy-poly (ethylene glycol) )), or poly(2-ethyl-2-oxazoline). 10. The macromolecule of the third aspect of the patent application, wherein the double-co-polymerized polymer is methoxypolyethylene glycol-b-polylactide 39 1361813 (as amended in December 2011) ( Metlioxy-poly(ethylene glycolhb-poly^Hactide)). 11. The polymeric micelle according to claim 1, wherein the main chain of the graft copolymerized polymer comprises a first repeating unit having hydrophilicity. And the hydrophobic side chain is bonded to the first repeating unit. 12. The polymeric cell of claim 11, wherein the first repeating unit has a carboxyl structure and the hydrophobic side chain is biodegradable. 13. The polymeric micelle according to claim 12, wherein the main chain of the graft copolymerized polymer is polyacrylic acid, polymethyl methacrylate, polyhistamine, Or polyethylene-based taste. 14. The polymeric micelle according to item n of the patent application, wherein the main chain of the graft copolymerized polymer is polyacrylic acid. 15. The polymeric micelle according to claim 12, wherein the hydrophobic side chain of the graft copolymerized polymer is polyester polylactide, polylactic acid or polycaprolactone. 16. For example, please select the polymer type microcells in the fifteenth patent range, which are grafted and copolymerized. The hydrophobic side chain of the same knife is polylactic acid vinegar.丨1361813 (Revised in December 2011) 17. The polymeric micelle according to claim 11, wherein the graft copolymerized polymer backbone further comprises a second repeating unit, and the second The repeating unit is different from the first repeating unit, and the second repeating unit collapses the core of the micelle in response to a change in temperature. 18. The polymeric micelle according to claim 17, wherein the second repeating unit of the main chain of the graft copolymerized polymer is formed by N-isopropyl acrylamide monomer. By. 19. The polymer type cell according to claim 18, wherein the main chain of the graft copolymerized polymer is a copolymerized polymer of N-isopropylacrylamide and methacrylic acid. 20_ The polymeric micelle according to the third aspect of the patent application, wherein the high molecular type microcell has a particle size of 50-200 nm. 21. The macromolecule of the third aspect of the patent application, wherein the double-co-polymerized polymer has a terminal functional molecule attached to one end of the hydrophilic polymer segment and the terminal functionality A molecule is a ligand (iigand) that binds to a receptor on the surface of a cancer cell. 22. The macromolecule cell of claim 21, wherein the ligand is a galactose residue 〇^361813 I, » (as amended in December 2011) 23. a macromolecular type of microcell having a terminal functional molecule attached to one end of the hydrophilic polymer segment and a terminal functional molecule which is a fluorescent group (fluorescence group) ° 24. The macromolecular type cell of claim 23, wherein the fluorophore group is fluoreseein isothiocyanate 〇 25. as claimed in claim 3 The macromolecular type microcell, wherein the double-trapped copolymerized southern molecule has a terminal functional molecule attached to one end of the hydrophilic polymer segment, and the terminal functional molecule is a dye (dye) . 26. The polymeric micelle according to claim 25, wherein the dye is a near-infrared dye. 27. The macromolecule of the invention of claim 2, wherein the structure comprises a plurality of different copolymerized macropolymers, and each of the copolymerized polymers comprises a hydrophobic polymer segment and A hydrophilic polymer segment. 28. The polymeric micelle according to claim 27, wherein each of the plurality of different agglomerated polymers is a double-co-polymerization 42 1361813 ... (as amended in December 2011) 'It contains a hydrophobic polymer bond segment and a hydrophilic polymer bond segment. 29. The polymeric micelle of claim 28, wherein the hydrophobic polymer backbone of the different co-polymerized polymer has an identical repeating unit. 30. The polymeric micelle according to claim 28, wherein the hydrophilic polymer segment of the different copolymerized polymer has the same repeating unit. 31. The polymeric micelle according to claim 28, wherein the hydrophilic polymer segment of the different copolymerized polymer has different repeating units. 32. The polymeric micelle according to claim 28, wherein the plurality of different agglomerated copolymerized polymers have different terminal-functional molecules attached to the ends of the hydrophilic polymer segment. 33. The polymeric cell of claim 32, wherein one of the terminal functional molecules is a ligand that binds to a receptor of a cancer cell surface. 34. The polymeric micelle according to claim 33, wherein the 43 1361813 (revised December 2011) ligand is a galactose residue. 35. As claimed in the patent scope h tS >>, 32 of the molecular type micelles, wherein one of the terminal functional molecules is a fluorescent group. 36. For example, the molecular type of microtubules of the 35th item of the patent application, wherein the fluorescent group is a fluorescent isothiocyanate. 37. The polymeric micelle of claim 32, wherein one of the terminal functional molecules is a dye. 38. The polymeric micelle according to claim 37, wherein the dye is a near-infrared dye. < 39. A composite microcell structure comprising a functional core and a hydrophilic shell. The grandchild is self-assembled by a graft copolymerized polymer and one or more linked copolymerized polymers. 4. A composite type microcell structure as claimed in claim 39, which is self-assembled by a graft copolymerized polymer and two or more double-co-polymerized polymers. 41. The composite microcell structure of claim 39, which has a particle size of about 50-200 nm. 1361813 (Revised December 2011) 42. A method for preparing a polymer type microcapsule having a core-shell structure, comprising the steps of: a) dissolving a graft copolymerized polymer and a group of copolymerized polymer in an organic In the solvent, wherein the graft copolymerization polymer comprises a main chain and a hydrophobic side chain bonded to the main chain, the co-polymerization polymer comprises a hydrophobic ruthenium molecular segment and a high hydrophilicity Molecular segment, b) The polymer solution of step a) is subjected to dialysis treatment against water to replace the organic solvent with water. 43. The preparation method of claim 42, further comprising c) lyophilizing the aqueous solution obtained in the step b) to obtain a dried polymer type micelle. 44. The preparation method of claim 42, wherein in the step a), one or more different copolymerized copolymerized polymers are dissolved in the organic falling agent. 45. The preparation method of claim 42, wherein an anticancer drug is co-dissolved in the solvent with the graft copolymer polymer and the copolymerized polymer. 45