TW200817456A - Multifunctional mixed micelle of graft and block copolymers and preparation thereof - Google Patents

Abstract

The present invention discloses a novel mixed micelle structure with a functional inner core and hydrophilic outer shells self-assembled from a graft macromolecule and one or more block copolymer, and preferably from a graft copolymer and two or more diblock copolymers. The micelle synthesized in the present invention has a size of about 50-200 nm, which can be used as a cancer diagnosis agent and a cancer drug delivery carrier.

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. Prior art multi-component micelles 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 the specificity of cell lines for tumor recognition. It can stabilize the structure of the microcells, overcome the defects in the use of different materials, and make the microcapsules appear versatile. In the polymer field, the type of multi-component micelles (also known as composite micelles) includes a double-cluster-double-coupling copolymer polymer system, a double-cluster_triple-coupling polymer system, and three groups. _ three-group co-polymerization polymer system, and double-coupling graft copolymerization polymer system [2_10]. However, to date, no literature has reported a composite cell of a double-agglomerated-graft copolymerized polymer system. And in the past few decades, the composite micelles have focused on the micropore-making machine []'s few studies have applied its drug delivery [3,4]. The complexity of the formation of complex micelles and the integrity of the structure of the core nucleus are the limiting bottlenecks of the integrated micelles in biomedical applications. SUMMARY OF THE INVENTION The present invention discloses a novel composite microcell structure having a functional core of 200817456 and a hydrophilic outer shell, which is copolymerized with a polymer or a copolymerized polymer by a graft copolymerization polymer. The polymer self-assembles into a composition in which a graft copolymerized polymer and two or more double-co-polymerized polymers are preferred. The size of the composite micelle is between 50 and 2 nanometers. The present invention provides a novel composite microcell structure comprising a functional core and a hydrophilic outer shell which are self-assembled from 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 microcell synthesized by the invention has a particle size of about 50-200 nm. The present invention provides a polymer-type microcell having a core-shell structure, wherein the structure comprises a graft copolymerized polymer and a cluster. Polymerized polymer The graft copolymerized polymer comprises a main chain and a hydrophobic side chain bonded to the main chain, and the copolymerized polymer comprises a hydrophobic polymer segment and a hydrophilic polymer chain a segment in which a hydrophobic side chain of the graft copolymerized polymer is aggregated, and a hydrophobic segment of the Schottky copolymerized polymer is stacked on the hydrophobic side chain of the graft copolymerized polymer, i The hydrophilic segment of the copolymerized polymer is exposed from the outside to form a shell-core structure. The present invention further provides a preparation method comprising the following steps: a polymer-type microcell-co-polymerized polymer having a core structure, a dissolved macromolecule containing a host and a) a graft copolymerized polymer and a In the organic solvent, wherein the graft copolymerization 6 200817456 is bonded to the hydrophobic side chain of the main chain, the copolymerized polymer contains

The organic solvent is replaced with water.

Preferably, one or more different agglomerates are copolymerized in step a), and a molecule is dissolved in the organic solvent in step a). Che Yujia, a music substance and the graft copolymerized polymer and the copolymerized copolymer polymer are dissolved in the organic solvent. In the rut, the hydrophobic side chain of the ruthenium graft copolymerized polymer and the hydrophobic segment of the conjugated ruthenium molecule contain the same repeating unit. Preferably, the group is polymerized with a polymer-based double-agglomerated copolymerized polymer, which comprises the hydrophobic polymer segment and the hydrophilic polymer segment. More preferably, the bis-co-polymerized polymer is methoxy-p〇ly (ethylene glycol-b, poly(jD, ZMactide). 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 segment of the polymeric polymer is biodegradable and absorbable. Preferably, the hydrophobic segment of the co-polymerized polymer is polyester, polylactic acid vinegar, polylactic acid or polycaprolactone. More preferably, the copolymer is co-polymerized. 200817456 The hydrophobic segment of the polymer is polylactide. Che's father's 'the hydrophilic segment of the graft copolymerized polymer is polyacrylate' or acidity/ion strength sensitive polymer, wherein the acid-base/ion strength sensitive polymer is polyacrylic acid, polymethyl Acrylic acid, polybutylene diacid (polyhistidine), or polyimidazole (plyly (vinyl imidazole)). Preferably, the hydrophilic segment of the copolymerized polymer is polyether, polyethylene glycol (polyethylene glycol), methoxypolyethylene glycol (meth〇xy-p〇ly ( Ethylene glyc〇1)), or poly(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, polymethacrylic acid, polybutyric acid, polyhistamine or polyvinylimidazole. More preferably, the main chain of the graft copolymerized polymer is polymethacrylic acid. Preferably, the hydrophobic side chain of the graft copolymerized polymer is polyester, long-term milk § Jiufu S曰 lactic acid or poly-caprolactone. More preferably, the graft is copolymerized and the hydrophobic side chain of the 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 first repeating unit is responsive to a temperature change 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 isopropyl 200817456 N-is〇propyl acryiamide. Most preferably, the main chain of the graft copolymerized polymer is a copolymerized polymer of N-isopropylacrylamide and methacrylic acid. Preferably, the macromolecule has a particle size of 5〇-2〇〇 nm. Preferably, the double-co-polymerized polymer has 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. More preferably, the ligand is a galact〇se residue. 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 fiuorescence group. More preferably, the fluorophore group is fluor〇resicin 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 agglomerated copolymerized polymers, and each of the copolymerized copolymers comprises a hydrophobic polymer segment and a hydrophilic hydrazine molecular segment. More preferably, each of the plurality of different agglomerated copolymerized polymers is a bi-cluster copolymerized polymer comprising a hydrophobic polymer segment and a hydrophilic polymer 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. 9.200817456 Preferably, the hydrophilic polymer segment of the aggregate-polymerized polymer has different repeating units. Preferably, the plurality of different agglomerated copolymerized polymers are linked to different terminal functional molecules at the proximal end 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 by graft copolymerization of a polymer/molecule copolymerized ruthenium molecule, and neutrino is controlled by a difference in critical microcell concentration (CMC). The size of the path. The complex type of microvesicles in the special core-nuclear structure can be applied to anticancer drug carriers by carefully designing and manipulating each component and having a wide range of applications/medium-use. Intracellular drug release is a very important cancer therapeutic drug delivery route. This pathway can increase the toxicity of the drug to cells in the target area to reduce the side effects of the drug on normal cells. In general, the intracellular induction of drug release from the carrier can be caused by changes in the enzymes in the 4 enzymes or changes in the pH of the phagosome. The 0-day access to the ancient 曰 巳 巳 多 材料 材料 材料 材料 材料 材料 材料 材料 材料 材料 多 多Intracellular drug delivery. However, due to the strong chargeability or sparseness, it is recognized by the mononuclear macrophage system (Μ) when it is used in the body, and it is difficult to reach the tumor tissue to achieve drug-controlled release. Therefore, in order to treat cancer by intracellular drug delivery, it is very important that the hydrophilic molecular structure is on the surface of the microcell or particle carrier. One embodiment of the present invention prepares a multifunctional shell. /nuclear novel composite type microcells, which are grafted copolymers with environmental response: poly(N-isopropyl decylamine-CO-mercaptoacrylic acid)_g_polylactic acid vinegar (poly(N-) Isopropyl acrylamide-co-methacryl acid)-g-poly(AL-lactide) (abbreviated as P(NIPAAm-co-MAAc)-g-PLA) 'with two-paired co-polymer: oxime-based poly(B Glycol)-b-poly(LU-lactide)(meth〇xy p〇ly(ethylene glyC〇l)-bP〇ly(D,L-lactide) (abbreviated as mPEG_pLA) and poly(octaethyl) -2- oxazoline-b_poly(AZ-lactide) (p〇ly (2-ethyl-2-〇Xaz〇line)-bp〇ly(Az]actide)) (abbreviated as PE0z_PLA) 'This nanostructure can completely conceal the highly negatively charged grafted copolymer in the inner core and make the core structure versatile. This multifunctional composite type microcapsule is applied to cell (four) transport to show drug release behavior and cell The functionality is strongly correlated. In addition, the concealment of the composite microcapsules is also strong and the composite micelles and grafted copolymers (P(NIPAA(4)._MAAe)_g_PLA, Chen AAm]/[MAzhen/[ Pla]] 1.9 ·2·5 mol/mo1) Compared with the microvessels formed, the composite microcapsules showed higher drug activity and lower sputum toxicity, and the present example not only proposed a brand new one. By the double-agglomeration-grafting of the sputum, the micro-cell I structure consisting of a genus, and the k-grafting 妓 _ _ / / ♦ 咼 咼 咼 与 与 咼A multi-functional microcapsule concept prepared by co-polymerizing a polymer or a multi-dual group of qiqiupu people/z, a knife/knife for application to a drug delivery. 200817456 Another embodiment of the present invention is to prepare one more Functional micelles are applied to cancer cell markers, distribution imaging, and anticancer drug delivery. A graft copolymer having the environmental response P (NIPAAm-co-MAAc) -g-PLA, a group of double-linked copolymers mPEG_PLA, and two functional groups linked copolymer bis-galacto amidoglucosan

(galactosamine)-PEG-PLA (abbreviated as Gal-PEG-PLA) is composed of fluorescein isothiocyanate-PEG-PLA (FITC-PEG-PLA). The versatile microcell can accurately identify liver cancer cells by the asic〇glycoprotein _Gal (multifunctional microcell) receptor retraction targeting mechanism. The acidity of the cell after the cell sputum changes the internal structure of the versatile micro-cell to cause drug release, and thereby increase its toxicity to liver cancer cells. The development function of the multifunctional microcell can be utilized, and the distribution of the intracellular cells is clearly observed by the Confocal laser scanning microscopy (CLSM). By careful design and precise control of molecular composition, the polymer type microcapsules of the present invention can be widely applied to cancer diagnosis, cancer identification, and cancer treatment. 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: Material racemic lactide (£U_Lactide) and methacrylic acid (maAc) ordered from Lancaster 〇 甲 笨 12 200817456 Methyl p-toluenesulfonate (MeOTs) , stannous octoate (Sn(Oct) 2), 2-hydroxyethyl methacrylate (HEMA), pyrene and azobisisobutyronitrile (2) 2, azobisisobutyronitrile, abbreviated as ΑΙΒΝ) δ gambling from Aldrich. N-Isopropyl acrylamide (NIPAAm) and ethyl oxazolidine (2-ethyl-2-〇xaZ〇line) were ordered from TCI. Oxyloxy polyethylene glycol (mPEG, , weight average molecular weight (Mw) = 5000 Da) was ordered from Sigma. The racemic lactide was recrystallized twice with tetrahydrofuran (THF) before use. N-isopropylacrylamide and azobisisobutyronitrile were recrystallized with hexane and acetone before use, respectively. Methylpropanol acid and hydroxyethyl methacrylate were purified by distillation under reduced pressure before use. Ethyl oxazolidine and p-toluene acid methyl ester were dehydrated by calcium hydride (CaH2) before use and then distilled under reduced pressure. Preparation of P(NIPAAm-co-MAAc)-g_PLA (Grafted I, G1) Graft Copolymer The purified racemic lactide (AZ_lactide) (4 g), hema (0.26 g) and Water toluene (5 mL) was placed in a round bottom double neck flask and connected to a vacuum tube system to remove water and remove oxygen. Completely dissolve the monomer at 10 (rc) and add catalyst

Sn(OCt)2 (1 wt%) was subjected to a cationic ring-opening polymerization reaction under a nitrogen atmosphere. After the reaction was carried out for 16 hours, the reaction was terminated by the addition of 〇]N methanol K〇H (methan〇iic KOH). The product was purified by reprecipitation from hexane, and the column was removed to remove salts and impurities to obtain PLA-EMA (Mn = 2 Å). Quantitative pla_ema 13 200817456 (〇·3 5 g), NIPAAm monomer (1.15 g), MAAc monomer (〇·16 g) and initiator AIBN (0.023 g) were placed in a double neck round bottom flask with acetone added. (15 mL) Dissolve the reactants and pass nitrogen. The radical polymerization was carried out at 70 ° C for 24 hours. After completion of the reaction, reprecipitation was carried out with h20 and diethyl ether respectively to remove unreacted two. After drying, graft copolymer P (NIPAAm-co-MAAc)-g-PLA (graft I, G1) was obtained ([NIPAAm] :[MAAc]:[PLA] = 84 : 5·9 : 2·5 mol/mol). Preparation of mPEG-PLA (cluster I, B1) di-co-linked co-polymers. Purified racemic lactide (1 g), mPEG (10 g, Mw = 5 000 Da) and deuterated benzene (4 mL) was placed in a round bottom double neck bottle and connected to a vacuum tube system to remove water and remove oxygen. The catalyst Sn(OCt)2 (1 wt%) was added under complete dissolution of the i 3 〇 ° C monomer, and ion-opening polymerization was carried out under a nitrogen atmosphere. After the reaction was carried out for 16 hours, the reaction was terminated by the addition of 〇·1 N methanolic KOH. The product is crystallized at a low temperature by a mixture of methylene chloride and diethyl ether, and dried to obtain a double-co-linked copolymer mPEG-PLA (Group I, Bl) ([EG]: [LA] = 113:7 mol/mol). Preparation of PEOz-PLA (Bundle II, B2) double-co-polymerization The synthesis of PEOz-PLA is to modify the old reaction procedure [GH Hsi^, C.Ch.Wang, CLLo, CHWang, JPLi , JLYang, Int J. Pharm. 20 06, 3 17, 69], the steps are as follows. The starter Me〇Ts (0.232 mg) was placed in a round bottom double neck flask, and connected to a vacuum tube system to remove water and remove oxygen and then add acrylonitrile (30 mL) and control the reaction temperature at 1 (10). Hey. Add 14 200817456 The monomeric ethyloxazoline (10 mL), which has been dehydrated, is refluxed for 3 hours. After the reaction was terminated, the reaction was terminated by the addition of 〇.丨N sterol K〇H. The product was reprecipitated by diethyl ether, and the gel column was used to remove salts and impurities, and dried to obtain pe〇z_〇h. PE0z (2 §) and racemic lactide (0.426 g) were ion-opened and polymerized with Sn(OCt)2 (1 Wt / °). The reaction temperature was controlled to reflux at 130 ° C for 1 6 hours. After the reaction is completed, the reaction is terminated by adding 〇·丨n sterol KOH, and purified by B-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 number average molecular weight of the copolymerized polymer were identified by the nuclear magnetic resonance spectrometer iH-NMR (AMX_5〇〇, Bruke〇. The I 5 degree distribution index of the copolymerized knives was obtained by using a gel permeation chromatograph ( GPC) The GPC mobile phase is dimethyl decylamine (N, N_dimethylf〇rmamide, DMF for short), the flow rate is i ml/min, and the measurement temperature is 4 〇. The critical microcell concentration of the copolymerized polymer (critical The micelle c〇ncentmiQn, 'CMC) was identified as a hydrophobic dye with a fluorescence spectrometer at 393 nm in the excitation spectrum. The highest concentration of the sample and the lowest concentration of the sample at 330-340 nm were observed. The intensity, and the ratio of the intensity (about 133 7.5/133 5) is plotted against the logarithmic scale of the concentration. The concentration at the bottom of the curve and the inflection point are defined as the critical cell concentration. Table 1 lists each Identification results of copolymerized polymers. 15 200817456 Table 1 Mn [Da] Hydrophilic segment Mn [Da] Lipophilic segment polymerization degree distribution CMC [g/mL] Grafting I 9970 6150 1.24^ 1.27 χ ΙΟ-6 Group Union I 5000 500 1.0^^~~ 8.39 x 10° Mission II 5200 700 1.70 x 10-6 Preparation of composite nano-cells A fixed concentration of grafted I copolymer (〇·13 M) was dissolved in DM SO, and different concentrations of the group I copolymer were added. After uniformly mixing, put into a dialysis bag (MWCO 6000 ~ 8000, SpectrumLabs, Inc.), and replace the pure water once every 2 to 3 hours at room temperature for 48 hours to prepare G1B1 composite type nano-cells. Freeze-drying (Het〇_H〇lten a/S,

After collecting the product for analysis. Copolymerization of a fixed concentration of grafted rhodium

The compound (0·13 Μ) and the group I copolymer (0.20 μ) were dissolved in DMSO, and different concentrations of the group conjugated co-polymer were added, uniformly mixed and placed in a dialysis bag (MWC0 6000 8000 to 8000, SpectrumLabs). , Inc.), replacing the pure water once every 2 to 3 hours at room temperature for a total of 48 hours to prepare a G1B 1B 2 composite type nanocell. After lyophilization, the product was collected for analysis. Fig. 1A is a graph showing the average particle size and particle size distribution of G1B1 composite micelles under different bundled I doublet copolymerization compositions. The concentration of grafted I grafted copolymer was fixed at 1〇 〇 mg/L during the preparation of G1Bi composite micelles. 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 π double-clustered copolymers. The concentration of grafted I grafted co-polymer was fixed at 1〇 〇 mg/L during the preparation of G1B1B2 composite cells, and the concentration of the double-linked copolymer was fixed at 5.625 mg/]L. 16 200817456 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 v O / 1.125 G1 : B1 = 50 : 50 mol/mol 10.0 3.15 Two-component G1B1B2 complex type nano-cells (G1: Β1: Β2 = 33·9: 55·7: 10.4 mol/mol) self-assembled into a microcell structure by aggregation and arrangement of hydrophobic polymer segments pla . The fixed cell concentration (〇1 mg/mL) was dispersed in physiological buffer saline (PBS) and its particle size was observed by dynamic light scattering (DLS) to be 182.3 ± 1.5 nm, and the particle size distribution index ( The 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 (〇·1 mg/mL) was measured by Doppler microelectrophoresis (Zetasizer 3 00OHS, Malvern) to determine the double-cluster copolymer for the composite micro The degree of concealment of the core. The grafted I microcells formed by graft copolymerization of the polymer were compared, and the interface potential was measured as _丨5 · 5 soil 〇. 9 mV. The interfacial potential of the composite micelle is -7·8 soil ι·3 melon, and the high-negative MAAc of the inner core has been obscured by the outer shell structure mPEG and pe〇z. The most direct evidence for the core structure of the composite micelles can be stained with MAAc via uranyl acetate (2 wt %) by a transmission electron microscope (TEM; Hitachi H-600 microscope, accelerating voltage = 100 kV) 17 Observed in 200817456, as shown in Figure 2. The dyed area of Figure 2 is distributed in the inner core, that is, the inner core is composed of graft I, and the unstained area of the outer 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 particle size of the G1 b 1 b2 composite microspheres is uniform 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 G1B1 complex type micelles composed of different proportions and G1B1B2 complex type micelles. The two groups of copolymerized ruthenium molecules have different critical cell concentration: the critical cell concentration of the group I is much larger than that of the group 接枝 and graft I (Table 1). When a fixed concentration of graft I is mixed with different concentrations of the group I to prepare a composite type of micelle, the particle size of the G1B 1 composite type micelles of different ratios is about 〇6〇nm (as shown in FIG. 1A). And less than the grafted z-cells and the clustered j-cells. The particle size distribution of the G1Bl complex type microcells is narrowly distributed. When agglomerated π is added to a fixed concentration of G1B1 (the critical microcell concentration of the cluster is close to the critical microcell concentration of the graft I, but less than the critical microcell concentration of the cluster I), the particle size and cluster of the G1B1B2 composite type microcell The addition of II is irrelevant (as shown in Figure 1B), and is smaller than each single constituent cell or G1B2 complex type cell (33〇·2 soil·9 nm; PDI = 〇〇72 ± 0.011), but complexed with G1B1 The type of micelles are similar. However, when the molar content of the π-joining π is more than 〇·54, the micelle cannot 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 1 can regulate and control the formation of the composite type of cells, and control and reduce the particle size of the micelles. (Ν-isopropyl isopropylamine) (p〇ly (N_is〇p(7) 18 200817456 abbreviation (PNIPAAm)) is a water-soluble and hydrophilic polymer with temperature-sensitive properties. Its low temperature critical solution temperature (LCST) The temperature is between 28 and 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 a state of aggregation. If the PNIPAAm is introduced into a hydrophilic material such as MAAc, Then, the aggregation ability of the isopropyl group of PNIPAAm is destroyed, thereby increasing the phase transition temperature of the copolymer. However, the present invention increases the phase transition temperature of the material with MAAc having dissociation ability, and at the same time imparts acid-base responsiveness. Next, P(NIPAAm-co-MAAc) will produce aggregation and precipitation, because MAAc protonation reduces the hydrophilicity of P(NIPAAm-co-MAAc) and lowers the temperature of the critical solution to 32 °C. Alkali responsiveness is associated with temperature responsiveness. In previous studies it was shown that grafted I microcells have the property of producing microstructural changes in acidic and high temperature environments (CL Lo, KM Lin, GH Hsiue, J. Controlled Release 2005, 104, 477) Figure 3 shows that G1B1B2 composite micelles (G1: B1: B2 = 33·9: 55·7: 10·4 mol/mol) change with temperature in an acidic environment (pH 4.0) The structure of the composite micro-cells is destroyed by the use of hydrazine as a hydrophobic dye, and the spectroscopy is observed by a fluorescence spectrometer. The hydrazine solution is used as a control group. Because of its molecular symmetry Or the π electron cloud is affected by the polarity and environmental perturbation, which will cause a great change in the first emission spectrum (/;), but no significant difference compared to the other four different wavelengths of the emission spectrum. The change is therefore a commonly used dye in the discussion of the mechanism of cell formation, and /; / / 3 represents the change of the environment in which K. Kalyanasimdaram JK Thomas, J. Am. Chem. Soc. 1997, 99, 2039)./// /3 The more 19 • 200817456

, 7 / / L over the face, that is, the value of the / / 3 value of the solution is due to heat • 74. For composite micelles, the lower the polarity of the environment, the lower the polarity of the environment where the enthalpy is located. As shown in Figure 3, the consumption decreases from 1 · 8 1 to 1 in the environment with increasing temperature. 1.25 When the temperature is higher than 37X: When the value of 八//3 is rapidly increased from 1.46, it means that the environment is composed of composite type micro A solution with a low polarity inside the cell due to contact with the environment with high polarity after destruction of the cell. In the present invention, the difference between the structure of the composite type cell and the structure after the damage is analyzed by a time-of-flight secondary ion mass spectrometer (time_〇f_fHght 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 revealed that graft I was revealed due to structural damage. The structure of the composite micelles was stained with acetic acid axis (2 wt%) and the structure was observed by a transmission electron microscope (TEM) before and after structural damage. As shown in Figures 4A and 4B, the G1B1B2 complex type cell (Gl: Β1 : Β2 = 33·9 : 55.7 : 10.4 mol/mol) is broken in structure.

After the bad, there is no obvious core structure. Example 2: This example uses a graft copolymer p (NIPAAm_co-MAAc)-g_PLA and a double-cluster copolymer mPEG-PLA to self-assemble to form a composite microcapsule for coating a hydrophobic anticancer drug cranberry ( Doxorubicin, referred to as Dox). When the blood circulates, the drug can be coated on the core without causing side effects. The composite microcapsule-coated anticancer drug D〇x was prepared in the same manner as Dialysis 200817456. 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 1 0 0. The composite microcapsule-coated anticancer drug Dox was observed to have a spherical shape by AFM, and the particle size was about 165 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 UV 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 200817456 Gan, Yue has been tested by the mother. Cytotoxic killing experiment (gr〇wth inhib out (10)assay), J 疋 knife to add drugs, graft 1 drug micelles and complex drug micelles, the drug concentration is in Bu (10) _L, calculate the cell and tongue rate at different times and concentrations To compare the effects of drugs, grafted I drug micelles and compound drug cells on the toxicity of HeLa cancer cells. The mean standard deviation (n = 6) was cultured in a culture medium of 37,5 % c〇2 in human cultured cervical cancer cells with Dulbecc〇, sm〇dified Eagle, s(5) and 匕(5) (referred to as DMEM). After the cells have grown to a certain number, they are cultured in a 96 well culture plate, and the number of cells is 5 x lO3 ceii/mL. After about 8 hours of cell attachment, the medium is removed and the drug containing different concentrations is added and grafted! Microcells and complex drug cell culture medium. After 48 hours, analysis was performed by colorimetric assay (MTT assay). In addition, drug-free grafted polymer microcapsules and composite micelles were also analyzed for material toxicity. The procedure is the same as above. Endocytosis evaluation. The distribution of drug and complex drug cells in the cells is observed by Confocal Laser Scanning Microscopy (CLSM). Placed in a 6-well culture plate. Cover the slides on the coverslips and stick the reinforcement circle to indicate the observation range. Plant 1 x per well; [ 〇 5 cells. After the cells are attached, add Dox with compound concentration 1 〇pg /mL and compound drug culture medium. .to The medium was removed at a fixed time, washed with PBS, and then added to LysoTracker DND-26 2 mL (50_70 nM in DMEM medium) for another 0.5 hour. The cells were washed with PBS and 0.1% Triton X-100 PBS. Fix in a PBS solution of paraformaldehyde (4 wt%) for 30 minutes, then 22 200817456

After PBS washing, 8q% glycerol was dripped in the reinforced circle (giyeer 〇M PBS solution 15 and attached to the slide to protect from light. The co-focus microscope objective lens was set to 40X; Dox excitation laser light wavelength was 485 nm, The wavelength of the emitted light is 590 nm; the wavelength of the laser light is 5〇4 nm and the wavelength of the emitted light is 5llnm. The image obtained by the sweeping cat overlaps with the phase difference microscope image to judge the position of Dox release. The composite drug cell, UV/Vis; The amount of the drug is about 19 wt.%. The drug release test uses a dialysis membrane to separate the drug from the micelle. Figure $ shows the release of the drug from the complex drug micelle at different pH environments. The 5.0 drug buffer had a significant difference in drug release compared to the pH 74 buffer solution, and the drug release behavior was significantly different between hc and 3rc in pH 5. 〇 buffer solution. 2 hours before the initial pH in buffer solution and 25 f It has a rapid drug release characteristic, and the drug release amount is about 25 Wt. %. Then the drug release behavior does not increase with time (4), because the composite microcapsule core and the outer shell have a dihydrogen The force of the bond, and the hydrogen bond is caused by the compression of the microcapsule, so that the part (4) is subjected to the sowing pressure, and W, _ causes 25% of the drug to be released at the beginning. However, when the time increases to %: the drug is still stored in the compound drug micro Cell kernel, indicating the microkernel kernel

; The structure does not cause structural damage due to the chlorine bond force. On the other hand, in the pH buffer solution and 37 ° C, the initial 2 ^, 昧 g 卩 ancient times there are about 5 〇 Wt% of the drug, which is caused by the destruction of the cell structure. Drug release results strongly indicate that cell structure disruption controls drug release behavior. . The cytotoxic killing test is used to understand the inhibitory effect of the complex drug microtubules on the growth of cancer cells and the ability to kill cancer cells. In the study (1), D〇x · HC1 and grafted I drug micelles were also observed together as a control group, except that the mice were tested with the compound of 2008/07456. Different concentrations of the composite drug cells, Dox.HCl, and the grafted I drug micelles were separately cultured with 2xl〇4 HeLa. As shown in Figure 6, Dox. HCl has a higher toxic ability in 48 hours of culture with an IC5 〇 of about 0.8 Kg/mL. The IC5G of the composite drug micelles and the grafted I drug micelles were 3 pg/mL and 6 pg/mL, respectively. The reason is that d〇x · HC1 small molecule enters the cell by diffusion and directly kills the cancer cell, so its action speed is faster; while the composite nano drug cell and the grafted I drug cell are by cell After the drinking effect enters the cells, the drug is released after changing the cell structure by environmental acidification in the end 〇smal space compartment or the lysosomal compartment (iyS〇s〇 〇 c〇mpartments). In addition, under the 48-hour culture condition, the drug release S of the composite nano-medicine micelles and the grafted drug-cells reached about 65% and 60% of the total amount. Compared with the d〇x.HCi drug, the amount of action on cells is low, so its cell toxic ability is poor. On the other hand, since the v-grafted I drug microcapsule has a strong negative charge, its ability to enter cells is poor, so its cell cytotoxic effect is inferior to that of the composite nano drug cell and HC1. The source of cytotoxicity is derived from the carrier or drug, and the drug-free composite micelles and grafts are also tested for toxicity of HeLa cells. In 48 hours of culture, the cytotoxicity of the composite micelles and the grafted I micelets increased significantly with increasing concentration. The IC5〇 of the compound micelles was 2.5 mg/mL, while the grafted J mice cells were 15 can. The apparently complex microcytotoxicity is lower than that of the grafted I mice, which is obscured by mPEG due to strong negative charge. In addition, it can be seen from the concentration of the drug-converted carrier that the cytotoxicity caused by the micro-cells is higher than that of the composite nano-cells. That is, grafted I micelles are less useful in cancer treatment and intracellular drug delivery. After the introduction of the double-cluster copolymer into the microcell, the microcell structure and the concealed negatively charged material such as MAAc can be stabilized, which not only increases the phagocytosis and reduces the cytotoxicity, but also overcomes the polyionic polymer (p〇lyi〇ns). Restrictions on 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 drugs by cells or drugs by conjugated focal microscope. The LysoTracker can be used to observe the distribution of acidic organelles or acidified organelles to determine the distribution of drug-like cells in the cells and drug release. D〇x.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 ϋ〇χ · HC1 acidified cells. When the composite drug micelles were cultured for an initial period of 1 hour, the coated Dox was released into the cytoplasm, and the green light displayed by the LyS〇Tracker was also distributed in this region. At 8 hours of culture, D〇x was not only distributed in the cell blast, but also in the nucleus, and LyS〇Tracker was only distributed in the sputum. This indicates that the complex drug microvesicles enter the cells via pinocytosis, and the acidification of the cells by endocytic sputum to 〇smalmal C〇mpartments or lysosomal c〇mpartments causes destruction of the cell structure and The drug is released, so the drug is only distributed in the cytoplasm in the initial 1 hour culture. After 8 hours, D〇x enters the nucleus, and the micelles continue to release the drug, so Dox is also distributed in cell f. Similar results can also be observed in Chinese hamster cells 25 200817456 (CHO-K1). In this example, the compound & drug microcapsules can be rapidly structurally disrupted by intracellular acid assays to release the drug. They also have a hydrophilic outer shell to mask the electrical material and increase the solubility of the material. Example 3: The same procedure as in Example 1, 圑 ΠΙ (mPEG5〇〇〇_pLAi_, molecular weight distribution of 1.15, critical cell concentration of 16 mg / L) and ganglian ^ (mPEGwoo-PLAim, molecular weight distribution of 12 〇, The critical microcell concentration is 5.4 mg/L. The synthesis of the copolymerized polymer is the ring-opening polymerization of mpEG (Mn 5〇(9)) and racemic lactide (AZ-lactide) with tin catalyst as the initiator. And got it. These polymers have the same chemical structure but different composition ratios.

This example utilizes a double-cluster copolymer having a different molecular weight length and a different critical cell concentration (union I, agglomerate π or agglomerated Iv), respectively, with a graft copolymer (Graft prepared in Example 1). Preparation of composite micelles to understand the effect of double-co-linked copolymers on the morphology and structure of composite micelles. f First--grafted copolymers and double-linked copolymers are co-dissolved in A high concentration of cut solution was prepared in a mixed solvent of DMSO/DMF (4/1 v/v). The use of a mixed solvent is such that a smaller particle size composite micelle can be prepared in this solvent. The concentration of the grafted copolymer in the solvent is fixed at a composition ratio of the grafted copolymer to the double-cluster copolymer of $丄:9. The complex human cells were prepared by the dialysis method in the same manner as in the example. The prepared two groups of composite micelles (grafting Ϊ and 团 I I complex type micelles, grafting 26 200817456 I and ganglian III composite type micelles, and grafting and gangming ιν complex type Microtubules The following three conclusions can be obtained by observing the core structure and particle size of TEM: (1) For all composite micelles, the core structure is dyed to know that its composition is grafted copolymer. The outer shell structure is composed of hydrophilicity and knife mPEG. (2) The core radius of the composite microcell (R) decreases as the molecular weight of the hydrophobic cluster segment of the double-co-polymer is increased (Rpla· > RPLA1G88 > RPLA175G). (3) The particle size of the composite micelle increases as the molecular weight of the hydrophobic group segment of the double-clustered copolymer increases. The short segment PL·A can form a composite micelle of a smaller particle size. Compound sputum cells were tested for stability in the presence of serum or serum proteins. In this test, a composite type of microcells composed of graft I (25 mol0/〇) and agglomerated IV (75 m〇1%) was used. The composite microcapsules were suspended in a physiological saline solution containing 4 wt·% bovine serum albumin (BSA), and the composite vesicles were observed by dynamic light scattering at a fixed time of 37 °C.佐之麦化' G. The moving light scattering measurement mode is set to CQNTIN. The particle size of the composite type cell solution which was not mixed with B S 为 was measured by dynamic light scattering. Grafted I micelles served as a control group. The particle size change ratio is obtained by dividing G. ‘G·/heart. From the results, it was found that the composite type microvesules exhibited a stable state within 72 hours. This was due to the adsorption of mPEG-blocking serum proteins of the microcapsule shell to avoid aggregation. This phenomenon shows that the composite micelle has long-term blood circulation characteristics. Example 4· The dual-co-polymeric polymer with the function of Gal-PEG3_-PLA83() 27 200817456 (Gal-PEG-PLA,[Gal]:[PEG]:[LA] = 8·4·:7·6 : 84 mol/mol) and the developed double-coupling copolymer fitc-peg34()()-pla830 (FITC-PEG-PLA, [FITC; h[PEG;h[LA] = 4:8:88 Mol/mol) is prepared by a thiol-maleimide coupling reaction. F7TC - Synthesis of EG-PZ 乂 double-co-polymer. PLA-NH2. First, N-Boc-L-alanine (alaninol) was treated with potassium/naphthalene to form a metal oxide oxide (N_Boc-L-alaninol-OK). The racemic lactide (Z), L-lactide) (2 g) was dissolved in toluene (2 mL) with N-Boc-L-alaninol-OK (0.35 g) as the starting agent at 100 ° The reaction was carried out for 1 hour at C. After completion of the reaction, the reaction was terminated with acetic acid, followed by reprecipitation with diethyl ether to obtain a polymer PLA-NHBoc. Then, PLA-NHBoc (2.1 g) was dissolved in a mixed solution of formic acid (20 mL) and chloroform (20 mL) at room temperature for 9 hours and then reprecipitated with diethyl ether. The product (1.5 g) was treated with a mixed solution of triethylamine (20 mL) and chloroform (20 mL) at room temperature for 8 hours to remove protons, and then reprecipitated with diethyl ether to obtain pla-nh2. PLA-SH. Dissolve PLA-NH2 (2 g) in acetonitrile (10 mL), add an excess of 2-iminothiolane hydrochloride (0.45 8 g), and add the appropriate amount of TEA-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 PLA-SH. Maleidin-PEG-NH2. N-Methoxycarbonylmaleimide (0·2 g) was dissolved in DMSO (10 mL) 28 200817456 and then added polyoxyethylene bis (amine) at room temperature. (1 g) The reaction was carried out for 6 hours in an aqueous solution. After the completion of the reaction, recrystallization was carried out at -20 ° C with a mixed solution of dichlorosilane and diethyl ether (1 / Γ v / v) 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) (15 mL), then maleimide-PEG-NH2 was added at room temperature. (2 g) Tris solution (10 mL) was reacted for 6 hours. After completion of the reaction, the cells were dialyzed against physiological saline and deionized water, respectively, and then lyophilized. The dried product was dissolved in dichloromethane and reprecipitated with diethyl ether to remove PLA-SH. NH2-PEG-PLA〇FITC-PEG-PLA〇NH2-PEG-PLA (1 g) was dissolved in methanol (40 mL). The reaction was carried out by adding FITC (0.15 g) at room temperature in the dark for 24 hours. The unreacted material was removed by dialysis against 0.5 M aqueous NaCl solution and deionized water, and then freeze-dried or vacuum-dried to obtain FITC-PEG-PLA. Synthesis of five G-PLd double-cluster copolymers. Gal-maleimide. N-methoxycarbonylmaleimide (0.68 g) was dissolved in DMS0 (10 mL), and then added to aqueous solution of galactosamine hydrochloride (0.5 g) at room temperature for 6 hours. After the reaction is completed, reprecipitation with diethyl ether gives Gal-maleimide. PLA-PEG-SH. PLA-PEG-NH2 (1 g) was dissolved in acetonitrile (15 mL), and an excess of 2-iminothiolane hydrochloride (0.1 g) was added, and an appropriate amount of TEA was added. -buffer (concentration: 50 mM, ρ Η 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 200817456 PLA-PEG-SH (1 g) was dissolved in methanol (15 mL) and then added with Gal_maleimide (〇·1 g) for 24 hours at room temperature. After dialysis with 0.55 M NaCl aqueous solution and deionized water to remove unreacted materials, freeze-drying or vacuum drying to obtain Gal-PEG_PLA. Example 5: The versatile composite drug micelle of this example was prepared by dialysis. First, the anticancer drug Dox was dissolved in a DMSO/DMF (4/1 v/v) mixed solvent and neutralized with an excess of triethylamine (1.2 mol). Graft t (5〇mol%), iv (20 m〇1%), Gal_pEG_pLA 〇5 m〇i%), and FITC_PEG_PLA (15 mol%) were dissolved in the drug solution and dialyzed (MWCO 60) 〇〇-8〇00). After dialysis, freeze-drying or vacuum drying can be used to multi-functional composite drug micelles. After dissolving the complex drug micelles in DMSO, the drug content can be measured by ultraviolet spectrometry to be about 3 Torr. Figure 7 shows the TEM observation of the versatile composite drug microvesicles after uranyl acetate (2 staining. The results show that the functional composite drug micro-cells have a complete shell-core structure, and the particle size is about 16 〇 nm. Because the giant molecules penetrate from the tumor blood vessels to the tissue through the open space of the blood vessels, the cavernous invagination of the blood vessels, or the defects of the blood vessel holes, etc., the gap between the holes is about 38〇 to the same as m. The composite compound drug has a particle size of 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 internal organs. Below 2000 nm, you can sleep in the magnetic, wind, and f, and protect from the light. The particle size also affects the cell's internal sputum ability. The particle size is greater than $(10). Particles need to be cage-independent 30 200817456 clathrin-independent end〇cyt〇sis, while particles less than 200 nm in size can be nnn-specific Clathrin-dependent process Enter the cell. Therefore, this is The prepared multifunctional composite drug microcell has a particle size of about 16 〇 nm, which is in accordance with the particle size limitation required for physiological conditions. The multifunctional versatile drug microcapsule affects the release of the drug from the microcell. Fig. 8 is a diagram showing the drug release behavior of the sputum 7.4 7.4 匕 α α 马 夕 功 7.4 7.4 7.4 7.4 7.4 7.4 7.4 7.4 。 。 。 7.4 7.4 174 174 174 174 174 174 174 174 174 174 174 174 174 174 174 174 174 174 In the early stage, the compound drug cell had a 15% correction of the phenomenon of initiai burst. After that, almost no drug was released from the carrier to maintain the total release of the drug. At pH 5 () and grasp Under the environment, the drug release curve can be clearly divided into two parts. A rapid burst release in the initial two hours releases about 35 wt.% of the drug, and then the drug continues to release the drug stably, and the drug release amount is 7 约 after about 14 hours. %. The release behavior of this drug confirmed that the versatile compound drug has acid responsiveness, and changing the acid value of the environment can destroy the core structure of the microcapsule and release the anticancer drug. Iv, FITC_PEG_PLA, and

Gal-PEG-PLA co-prepares multifunctional multifunctional micelles (without drug) in the same manner as described above. graft! In the versatile compound type (4), it mainly plays the role of gluteal 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 microcells with the same particle size distribution. The FITC-PEG-PLA double-co-polymerized polymer with fluorescent development mainly provides direct evidence to show the cumulative position of the multifunctional composite micelle. With the function of the mark 31 200817456

The Gal PEG_PLA double-co-polymerized polymer mainly recognizes the receptor asiatic glyCopr〇tein on the surface of liver cancer cells to achieve the function of active tumor targeting. In this example, the multifunctional composite drug microcapsules are co-cultured with cancer cells at different concentrations, and the growth inhibitory effect on the cancer cells and the ability to kill cancer cells are turned off by the multifunctional multiplexed (tetra) microcapsules. As a control group. After co-cultivation for 24 hours and 72 hours, it was found that 1C5 〇 (concentration causing half of cell death) of d〇x.hci was about 1⁄2^/mL. The multi-functional composite drug microcells have an IC5Q of about 25 in 24 hours.

Pg/mL. The 1〇5〇 under 72 hours of culture was about 4^/mL, which was close to d〇x hci. On the other hand, the versatile composite micelle (without drug) had an ICw of 792 pg/mL at 72 =. This result shows that cytotoxicity results from drug release from multifunctional versatile drug micelles. In order to evaluate the functionality of the multifunctional drug-type drug cells in the biomarker application, the conjugated focal microscope was used to observe the drug release and cumulative position of the multi-functional composite drug cells and HeLa cells cultured for 6 hours. For the cytoplasmic vector, the microcarrier carrier drug release trigger mechanism occurs in the phage phage and then is released to the cytoplasm. The results of observation by a common focus microscope showed that HeLa cells showed green fluorescence (FITC) in the cytoplasm, indicating the cumulative position of the multifunctional composite drug micelles. Red luminescence (D〇x) = accumulated in the cytoplasm and nucleus. In this example, the route of delivery of the multifunctional composite type of music cells can be clearly visualized by FITC-labeled micelles. Example 6: 32 200817456 In order to evaluate the versatility of composite drug micelles for tumor specificity, the multifunctional drug-type microvesicles prepared by Example 5 and the compound drug cells of Example 2 are shared with human liver cancer HePG2 cells. Culture to observe cell poisoning.磨瘤# from the 绎 live. HepG2 cells were cultured in DMEM medium in a 37 C, 5% C〇2 incubator until the cells were grown to a certain number, and the cells were cultured in 96 well plates at a cell count of 2 x 104 / well. After about 8 hours of cell attachment, the medium was removed and cultured at 4 ° C or 37 ° C 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 medium was added to culture at 37 t: 24 and 48 days. After Yuancheng, the cells were stained with trypanbiue. The survival rate of HepG2 cells was calculated by phase contrast microscopy. In addition, the same experiment was repeated for the behavioral analysis of the inhibitor in the presence of bismuth galactose (150 mM). As a result, as shown in Fig. 9, under the 24-hour co-cultivation, the cytotoxicity of the multifunctional compound drug cell and the complex drug cell at 37 ° C was more than 4. (: 'And the versatile compound drug cells have a higher cell cytotoxicity rate. Since the pinocytosis of the cells stops at 4 ° C, the drug that enters the nucleus is a receiver that is overexpressed by Gal and cell surface. The asialoglycoprotein binds and then enters the cells when cultured at 37 ° C. However, in the case of culture at 37 ° C for two hours, the multifunctional composite drug cells can enter the cells by the pinocytosis and the binding of the Gal surface receptor. Internal 'so its cytotoxicity is greater than the first two hours to treat 4 cytotoxicity 33 200817456

Killing sex. And the higher the toxicity of 4 nA 1 long cells over time. Add galactose (Galactose) to the system + ritual sugar to fight against the library, and raise the sputum. You can use this kind of sputum-type drug cell to carry out the reaction and raise the condition and time. On the 24th or 48th hour ▲,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, ..., system, 冼 by r, β6 / ι 夕 此 此 此 此 此 此 此 此 此 此 此 此 此 此 此 此 此 此 此 此 此 此 此s ® gong nrr example 6 knows that 'multifunctional composite drug micro-cells have been prepared, and can be used for cancer diagnosis and cancer drug delivery M results. It can be seen that the multifunctional composite drug micro-cells are spherical and grain: size is about 16 〇 nm, the particle size limit required for physiological conditions. The analysis of the tumor and the observation of the total light microscopic microscope showed that the multifunctional composite drug microvesicles could be strongly cytotoxic by entering the cells by the receptor-reducing pinocytosis (recep-endocytGsis). The present invention is directed to providing a two-new concept, i.e., an ideal cell for simultaneous long-term blood circulation, tumor identification, and combined cancer diagnosis and controlled release of cancer drugs. However, if the multi-functional compound drug cell: FITC is replaced by a near-infrared dye such as Cy5.5, it can be applied to animals to evaluate the in vivo distribution of multi-functional compound drug cells and the therapeutic effect of cancer. . BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A shows the average particle size and particle size distribution index of G1B1 complex type microcells in the different double-cluster copolymer group enthalpy in the first example of the present invention, wherein the G1B1 complex type Graft copolymerization during cell preparation 34 200817456 The concentration of graft I was fixed at 1〇·〇mg/L. 1B shows the average particle size and particle size distribution index of GIB 1B2 composite type microcells in different double-cluster copolymer group I in the first example of the present invention, wherein the preparation process of G1B1B2 composite type micelles The concentration of the grafted copolymer I was fixed at 10.0 mg/L, and the concentration of the double-clustered copolymer I was fixed at 5.625 mg/L. Fig. 2 is a photograph of a transmission electron microscope (TEM) of a G1B1B2 composite type microcell in Example 1 of the present invention (marking scale is 500 nm). Fig. 3 is a graph showing the intensity ratio of a vibration bond in the fluorescence spectrum of a pyrene molecule of a composite type cell of a GIB 1B2 composite type microcell in a fixed acidic environment (pH 4.0) at a different temperature in the first embodiment of the present invention (/7// 3). Fig. 4A is a transmission electron microscope (TEM) image of the G1B1B2 composite type microcapsule in the first example of the present invention before the structure is broken (marking scale is 2 奈 nanometer). 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 Example 2 of the present invention under different acidic and neutral environments at 25 ° C and 37 ° C, respectively. Figure 6 is a graph showing the growth inhibition ability of the composite drug microcapsules for human cervical cancer HeLa cells in Example 2 of the present invention, wherein the grafted I drug micelles formed by free D〇x and graft copolymerized polymers are 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 200817456 Fig. 8 is a multi-functional composite of drug-releasing drug in the environment of 11±(ΡΗ 5·〇) and towel (ρΗ 7.4) in 37C. A 48-hour cell growth inhibition map of a drug-type cell. Take the actual control group. Figure 9 is a composite drug cell of 24 and 2 of the liver cancer HepG2 cells of Example 6 of the present invention as Figure 10 is an example of the present invention. Human hepatocellular carcinoma HepG2 cells are in the form of a conjugated drug cell. 24 and 48 hours of cell growth deduction ^. The monthly package of galactose content ((9) secret) was used as a control group. | Figure. Example 2 of the compound drug micro 36 200817456 References: [1] X. Gao, Y. Cui? R. M. Levenson, L. W. K. Chung, S. Nie, Nat. BiotechnoL 2004, 22? 969.

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Claims (1)

200817456 X. Patent application scope 1. A polymer type microcell having a core-shell structure, wherein the structure comprises a graft copolymerized polymer and a conjugated copolymer polymer, and the graft copolymer polymer comprises a main a chain and a hydrophobic side chain bonded to the main chain, the copolymerized polymer comprising a hydrophobic polymer segment and a hydrophilic polymer segment, wherein the hydrophobicity of the graft copolymerized polymer The side chain is aggregated, and 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 from The protrusion is exposed to the outside to form a core structure. 2. The polymeric micelle according to claim 1, 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 microcell of the first aspect of the patent application, wherein the group and the co-polymerized polymer double-co-polymerized polymer comprise the hydrophobic polymer segment and the hydrophilic polymer chain segment. 4. The polymeric micelle according to item 3 of the patent application, wherein the number of the hydrophobic polymer segments has an average molecular weight of 500-2500, and the number average molecular weight of the hydrophilic polymer segments is 2000_10000 . 5. The polymeric micelle according to item 3 of the patent application, wherein the hydrophobic segment of the copolymerized polymer is biodegradable and absorbable. 38 200817456 6·如申# Patent|& The fifth type of polymer type microcells, wherein the hydrophobic bond of the polymer of the group is poly-, polylactic acid, polylactic acid or poly-caprol S Purpose. 7. The macromolecular type microcell of claim 6, wherein the hydrophobic segment of the copolymerized polymer is polylactide. 8. The polymeric micelle according to item 3 of the patent application, wherein the hydrophilic segment of the graft copolymerized polymer is a polyacrylate, or an acid-base/ion strength sensitive polymer, wherein the acid/base/ The ionic strength sensitive polymer is polyacrylic acid, polymethacrylic acid, polybutylene diacid (p〇ly (butenedi〇ic aicd), polyhistidine, or poly(vinyl). Imidazole)) 〇9. The polymeric micelle according to item 3 of the patent application, wherein the hydrophilic segment of the copolymerized polymer is polyether, poly(ethylene glycol), hydrazine Oxy-poly(ethylene glycol) or poly(2-ethyl-2-oxazoline). 10. The polymer type microcell of the third aspect of the patent application, wherein the double-co-polymerized polymer is methoxy-poly(ethylene glycol)-b -poly(AZ-lactide)). 39. The polymer type cell of the first aspect of the invention, wherein the main chain of the graft copolymerized polymer comprises a hydrophilic first repeating unit, and the hydrophobic side chain is bonded to the first A repeating unit. 12. The polymeric micelle of claim U, wherein the first repeating monocyclic 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, polymethacrylic acid, polybutyric acid, polyhistamine, or polyethylene. Imidazole. 14. The polymer type micelle according to claim 13, wherein the main chain of the graft copolymerized polymer is polyacrylic acid. 1 5 . The polymeric cell of claim 12, wherein the hydrophobic side chain of the molecule is polyester, polylactide, polylactic acid or polycaprolactone. I6· The polymer type micelle according to item 15 of the patent application, wherein the hydrophobic side chain of the a/, 3 polymer is polylactide. The polymer type microcapsule of claim 11, wherein the 40 200817456 branched polymer backbone further comprises a second repeating unit, and the first repeating unit is different from the first repeating unit, The second repeat unit collapses the core of the micelle in response to a change in temperature. 18. The polymer type micelle according to claim 17, wherein the second repeating unit of the main chain of the graft copolymerized polymer is formed by a monomer of N-isopropyl acrylamide. By. 1 9. The macromolecular type microcell of claim 8, wherein the main chain of the graft copolymerization host is a copolymerized polymer of N-isopropylacrylamide and methacrylic acid. 20. The polymeric cell of the invention of claim i, wherein the high molecular size cell size is 50-200 nm. 2 1. The polymer type microcell of claim 3, wherein the double-co-polymerized polymer has a terminal functional molecule attached to one end of the hydrophilic polymer segment, and the terminal function A sex molecule is a ligand that binds to a receptor (recept〇r) on the surface of a tumor cell. 22. The polymeric cell of claim 21, wherein the ligand is a galactose residue. 23. The polymeric micelle according to claim 3, wherein the double 41 200817456 copolymerized polymer has a terminal functional molecule attached to one end of the hydrophilic polymer segment, and the terminal The functional molecule is a fluorescent group 〇24. The polymer type microcell of claim 23, wherein the fluorophore group is fluorescein isothiocyanate. . 25. The polymer type microcapsule according to claim 3, wherein the double-co-polymerized polymer has a terminal functional molecule attached to the hydrophilic polymer segment, and the terminal The functional molecule is a dye (dye) 〇26. The polymer type micelle according to claim 25, wherein the dye is a near-infrared dye. 2. The polymeric micelle according to the scope of claim 2, wherein the structure comprises a plurality of different copolymerized macropolymers, and each of the copolymerized polymers comprises a hydrophobic polymer segment With a hydrophilic polymer segment. 28. The polymeric micelle according to claim 27, wherein each of the plurality of different copolymerized polymers is a double-co-polymerized polymer comprising a hydrophobic polymer segment Paragraph with a hydrophilic polymer chain 42 200817456. 29. The polymeric micelle of claim 28, wherein the hydrophobic polymer segment of the different co-polymerized polymer has an identical repeating unit. 30. The polymeric cell of claim 28, wherein the hydrophilic polymer segment of the different co-polymerized polymer has an identical 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 macromolecule cell of claim 28, wherein the plurality of different 15 co-polymerized polymers have different terminal functional molecules attached to the end of the hydrophilic polymer segment. 3 3 · The polymeric cell of the third aspect of the patent scope, wherein one of the terminal functional molecules is a ligand that binds to a receptor on the surface of a tumor cell. 34. The polymeric cell of claim 33, wherein the ligand is a galactose residue. 43 200817456 Please refer to the macromolecule of the 32nd item of the patent range, wherein one of the terminal functional molecules is a fluorescent group. 36. As claimed in the patent range, the 35th photo group is a fluorescent isothiocyanate ^ The polymer type micelle of the item, wherein the 37. is a polymer type cell of the 32nd item in the functional molecule of the end of the patent application, wherein the one is a dye. 38. If the patent application scope is the near-infrared dye. 37-type polymer type microcapsules The 39-type complex type microcell structure comprises a functional outer shell, which is obtained by self-assembly of a copolymerized polymer and a polymerized polymer. The sexual core is combined with a hydrophilic group or a plurality of groups. 40. The composite type microspheres of the compounding type 39 are self-assembled by two- or two double-coupling copolymers. . , its southern molecule 4 1 · If the scope of the patent application is I, the size is about 50_200 nm. 39 composite micro-cell structure, granule 44 200817456 42. The preparation method of the polymer type microcell having a shell-core structure comprises the following steps: 匕a) A graft copolymerized polymer is combined with a group The polymerized polymer is dissolved in an organic solvent, wherein the graft copolymerized polymer comprises a hydrophobic side chain bonded to the main chain and a hydrophobic side chain bonded to the main chain. a segment and a hydrophilic polymer segment, ^ b) replacing the polymer solution of step a) with water as a dialysis processor to replace water. 4: The preparation of the formula according to the scope of claim 42 contains an aqueous solution obtained from the step b): a progressive polymer type microcapsule. 7 Donggan, h and obtained dry: 4 · as in the scope of patent application No. 42 - one or more different groups - in / solvent. K. The knife is broken and dissolved in the organic 45. As in the scope of the patent application, the 42nd and the grafting of the "Eight Workers' Method" - the drug-drinking drink, the mouth-supplying molecule and the agent solvent. Hemi-molecules are dissolved together in the 45