Integrated nano system for liver-targeting co-delivery of genes/drugs and preparation method

Abstract

The present disclosure discloses an integrated nano system for liver-targeting co-delivery of genes/drugs and a preparation method, belonging to the field of biomedicines. In the disclosure, a plurality of functions are integrated in a carrier having good biocompatibility and safety, a nucleic acid/drug-loading copolymer portion having a pH-stimulating response function is formed by poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA) grafted with poly(3-azido-2-hydroxypropylmethacrylate) (PGMA-N3), and a fluorescence-based imaging component Rhodamine B (RhB) and galactose are used as targeting ligands. The drug delivery system provided by the present disclosure is safe, is capable of taking a synergistic effect of gene/drug therapy, and is expected to play a great role in clinical application.

Claims

1. A liver-targeting pH-sensitive tracing copolymer nano micelle system, comprising a copolymer formed by monomer N,N-dimethylaminoethyl methacrylate DMAEMA having pH sensitivity and hydrophobic monomer 3-azido-2-hydroxypropyl methacrylate PGMA-N3, wherein one end of the copolymer is modified with a fluorescent molecule having a fluorescence tracing function, and the other end is modified with galactose or galactosamine.

2. The liver-targeting pH-sensitive tracing copolymer nano micelle system according to claim 1, capable of loading nucleic acid for gene therapy, or drug for chemotherapy.

3. The liver-targeting pH-sensitive tracing copolymer nano micelle system according to claim 1, wherein the copolymer has a structural formula as shown in Formula 1: ##STR00002## wherein A is one or more identical or different fluorescent molecules having a fluorescence tracing function selected from Rhodamine B, fluorescein isothiocyanate and BODIPY; wherein m and n are each integers representing the degree of polymerization.

4. The liver-targeting pH-sensitive tracing copolymer nano micelle according to claim 3, wherein the copolymer has a structural formula as shown in Formula 1: ##STR00003## wherein A is one or more identical or different fluorescent molecules having a fluorescence tracing function selected from Rhodamine B, fluorescein isothiocyanate and BODIPY.

5. The liver-targeting pH-sensitive tracing copolymer nano micelle system according to claim 3, wherein m is 25, and n is 50; and the fluorescent molecule is Rhodamine B, the mole ratio of Rhodamine B to DMAEMA to GMA-N3 is 1:25:50, and the ratio of galactose or galactosamine to GMA-N3 is 1:1.

6. The liver-targeting pH-sensitive tracing copolymer nano micelle system according to claim 2, wherein the drug for chemotherapy is a hydrophobic anti-cancer drug and an anti-tumor drug.

7. The liver-targeting pH-sensitive tracing copolymer nano micelle system according to claim 2, wherein the nucleic acid for gene therapy comprises plasmid DNA or small molecule siRNA.

8. The liver-targeting pH-sensitive tracing copolymer nano micelle system according to claim 1, wherein the average particle size of the nano micelle is 155 nanometer.

9. The liver-targeting pH-sensitive tracing copolymer nano micelle system according to claim 2, wherein the average particle size of the nano micelle is 155 nanometer.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1: particle size distribution of copolymer micelle Gal-micelles and nucleic acid/drug-loading micelle DOX@Gal-micelles/pGFP. a) a transmission electron microscope graph of copolymer micelle Gal-micelles; b) a transmission electron microscope graph of nucleic acid/drug-loading micelle DOX@Gal-micelles/pGFP; c) a dynamic light scattering particle size distribution graph of copolymer micelle Gal-micelles; and d) a dynamic light scattering particle size distribution graph of copolymer micelle Gal-micelles.

(2) FIG. 2: relative survival rates of human hepatoma cells HepG2 and Huh7 cells and human embryonic kidney cells HEK293 cells after being cultured for 48 h in the presence of different concentrations of copolymer micelles Gal-micelles.

(3) FIG. 3: laser scanning confocal experiment result graphs of human hepatoma cells HepG2 and Huh7 cells and human embryonic kidney cells HEK293 cells after being incubated with copolymer micelle Gal-micelles in the absence or presence of galactose competition.

(4) FIG. 4: flow cytometry results of human hepatoma cells HepG2 and Huh7 cells and human embryonic kidney cells HEK293 cells, a) blank control, b) an experimental result that copolymer micelle Gal-micelles is endocytosed by cells in the presence of galactose competition, and c) an experimental result that copolymer micelle Gal-micelles is endocytosed by cells in the absence of galactose competition.

(5) FIG. 5: laser scanning confocal experimental result graphs of human hepatoma cells HepG2 and Huh7 cells and human embryonic kidney cells HEK293 cells after being incubated with drug/gene-loading copolymer micelle DOX@Gal-micelles/pGFP in the absence or presence of galactose competition.

(6) FIG. 6: flow cytometry results of human hepatoma cells HepG2 and Huh7 cells, a) blank control, b) an experimental result that nucleic acid/drug-loading copolymer micelle DOX@Gal-micelles/pGFP is endocytosed by cells in the presence of galactose competition, and c) an experimental result that nucleic acid/drug-loading copolymer micelle DOX@Gal-micelles/pGFP is endocytosed by cells in the absence of galactose competition.

(7) FIG. 7: volume change graphs and final representativeness experiment graphs of subcutaneous solid tumor mice in different treatment groups, wherein the different treatment groups are respectively: a) a normal saline-injected group, b) a blank copolymer micelle Gal-micelles-injected group, c) an adriamycin and Bcl-2 siRNA-loading targeting-free micelle DOX@Glc-micelles/siRNA-injected group, d) an adriamycin-loading copolymer micelle DOX@Gal-micelles-injected group, e) a Bcl-2 siRNA-loading copolymer micelle Gal-micelles/siRNA-injected group, f) an adriamycin and siRNA complex-injected group, and g) an adriamycin and Bcl-2 siRNA-loading target copolymer micelle DOX@Gal-micelles/siRNA-injected group.

(8) FIG. 8: final tumor number and volume and final representativeness experiment graphs of in situ mice in different treatment groups, wherein the different treatment groups are respectively: a) a normal saline-injected group, b) a blank copolymer micelle Gal-micelles-injected group, c) an adriamycin and Bcl-2 siRNA-loading targeting-free micelle DOX@Glc-micelles/siRNA-injected group, d) an adriamycin-loading copolymer micelle DOX@Gal-micelles-injected group, e) a Bcl-2 siRNA-loading copolymer micelle Gal-micelles/siRNA-injected group, f) an adriamycin and siRNA complex-injected group, and g) an adriamycin and Bcl-2 siRNA-loading target copolymer micelle DOX@Gal-micelles/siRNA-injected group.

DETAILED DESCRIPTION

(9) Next, embodiments of the present disclosure will be described in detail in conjunction with Embodiments, those skilled in the art will understand that the following Embodiments are only for illustrating the present disclosure but not considered as defining the scope of the present disclosure. If specific conditions are not noted in Embodiments, these Embodiments are performed according to conventional conditions or manufacturer's recommendation conditions. If manufacturers of used agents or instruments are not noted, all the agents or instruments are commercially available conventional products.

(10) Embodiment 1: Preparation of Bromo-Rhodamine B Initiator

(11) 15.52 g (25.00 mmol) of glycol and 1.01 g (10.00 mmol) of triethylamine are weighed and placed in a 100 mL conical flask to be stirred, and ice-water bath is carried out to 0 C., and then 1.20 mL (10.00 mmol) of 2-bromoisobutyryl bromide is dropwise added in nitrogen atmosphere. Then, the temperature is slowly raised to room temperature, and magnetic stirring is carried out for 3 h. 100 mL of deionized water is added into reacted solution for quenching, and extraction is carried out with dichloromethane (100 mL3). The collected organic phase is extracted with deionized water (100 mL3). A proper amount of anhydrous magnesium sulfate is added in the extracted organic phase, and drying is carried out for 12 h. The oily crude product is obtained by spin distillation after filtration, and a colorless and sticky product hydroxyethyl bromoisobutyrate is obtained by reduced pressure distillation (85 C., 30 mTorr).

(12) 4.81 g (10.00 mmol) of Rhodamine B, 2.90 g (15.00 mmol) of 1-ethyl-(3-dimethylaminopropyl) carbonyl diimine hydrochloride and 3.22 g (15.00 mmol) of compound hydroxyethyl bromoisobutyrate are weighted and dissolved into 40 mL of anhydrous dichloromethane to be stirred, and ice-water bath is carried out to 0 C. Then 1.82 g (15 mol) of 4-dimethylaminopyridine is added, and then the temperature is slowly raised to room temperature and react for 12 h. Reaction solution is extracted with 0.1 M HCl (50 mL3) and then washed for three times respectively with saturated sodium bicarbonate solution and saturated salt solution. The organic phase is dried with anhydrous magnesium sulfate and filtered, and then the solvent is removed by spin distillation. And the bromo-Rhodamine B initiator is obtained by separation on a silica gel column (a volume ratio of eluent to dichloromethane/methanol is 10:1). The specific method can refer to a document (Marcromolecules, 2011, 44, 2050-2057).

(13) Embodiment 2: Preparation of 3-Azido-2-Hydroxypropyl Methacrylate GMA-N3

(14) 3.71 g (57.00 mmol) of sodium azide and 3.81 g (45.20 mmol) of sodium bicarbonate are dissolved into 60mL of tetrahydrofuran/water (5:1v/v) and stirred. Then 5.42 g (37.80 mmol) of glycidyl methacrylate is slowly added to the mixture to react for 48 h at room temperature. The solvent is removed by spin distillation after insoluble salt substances are filtered out, and the obtained concentrate is extracted twice with dichloromethane. Then the obtained organic phase is dried with anhydrous magnesium sulfate and filtered, and the solvent is removed by spin distillation. Then 3-azido-2-hydroxypropyl methacrylate is obtained by separation on a silica gel column (a volume ratio of eluent to n-hexane/ethyl acetate is 9:1). A specific method can refer to a document (Polymer Chemistry, 2015, 6, 3875-3884; Soft Matter, 2009, 5, 4788-4796).

(15) Embodiment 3: Preparation of Rhodamine B Modified Copolymer (RhB-PDMAEMA25-c-PGMA50-N.sub.3)

(16) 70.0 g (0.10 mmol) of the initiator bromo-Rhodamine B, 484.0 mg (2.75 mmol) of monomer DMAEMA and 1.01 g (5.50 mmol) of compound 3-azido-2-hydroxypropyl methacrylate are correctly weighed and added into a 25mL round-bottom flask and dissolved with 2 mL of tetrahydrofuran. Then the argon is introduced for 30 min to remove oxygen in the flask, wherein, the stabilizer in the monomer DMAEMA needs to be removed in advance, namely, the DMAEMA crude product is enabled to rapidly pass through an alkaline alumina column. 18.9 mg (0.10 mmol) of CuBr and PMDETA (28 L, 0.10 mmol) are added in turn under the protection of nitrogen. Then the flask is sealed, and reaction is carried out for 8 h at room temperature under the protection of nitrogen. After the reaction is ended, tetrahydrofuran (10 mL) is added into reaction solution. The reaction solution in the flask is sufficiently stirred, and then passes through a neutral alumina column to remove a copper ligand in the mixed solution. The obtained liquid is collected and the solvent is removed by spin distillation, then sticky liquid in the flask is dropwise and slowly added into petroleum ether (500 mL) for repeated precipitation for three times. The obtained precipitate is vacuum-dried to obtain Rhodamine B modified copolymer RhB-PDMAEMA25-c-PGMA50-N.sub.3.

(17) For other proportions of copolymers, different concentrations of monomers DMAEMA are respectively added at the starting of reaction to obtain copolymer RhB-PDMAEMA-c-PGMA-N.sub.3 having different proportions of monomers, including RhB-PDMAEMA10-c-PGMA50-N.sub.3 and RhB-PDMAEMA50-c-PGMA50-N.sub.3.

(18) Embodiment 4: Preparation of Propargyl-Modified Deacetylated Galactose

(19) 6.21g (15.90 mmol) of peracetyl galactose is dissolved into 75 mL of anhydrous dichloromethane, and then 1.0 mL (18.00 mmol) of propargyl alcohol is added. Cooling is carried out to 0 C. and stirring is carried out for 5 min. Then 3.0 mL (24.30 mmol) of boron trifluoride etherate is dropwise added within 15 min. Stirring is continued at 0 C. for 10 min, and then reaction is carried out for 10 h at room temperature. The reaction is ended by saturated potassium carbonate solution. Then the organic phase is extracted with dichloromethane and washed for three times with saturated salt solution. The organic phase is dried with anhydrous magnesium sulfate and filtered, and the solvent is removed by spin distillation, so propargyl-modified peracetyl galactose is obtained.

(20) 2.01 g (5.20 mmol) of propargyl-modified peracetyl galactose is dissolved into 50 mL of 0.30 mol/L methanol solution of sodium methoxide to react at room temperature. Point panel detection is carried out until raw materials disappear. Then H.sup.+ exchange resin is added to adjust the reaction solution to be neutral, and filtration is carried out. Then then solvent is removed by spin distillation, and propargyl-modified deacetylated galactose is obtained by separation on a silica gel column (a volume ratio of dichloromethane to methanol is 10:1). A specific method can refer to a document (Bioconjugate Chemistry, 2012, 23, 1166-1173).

(21) Embodiment 5: Preparation of Galactose Modified Copolymer RhB-PDMAEMA25-c-PGMA50-Gal

(22) 350.0 g of propargyl-modified deacetylated galactose in Embodiment 4 and 200.1 mg of Rhodamine B modified copolymer RhB-PDMAEMA25-c-PGMA50-N.sub.3 in Embodiment 3 are dissolved into 5 mL of DMF, then 98.0 mg (0.61 mmol) of copper sulfate and 242.0 mg (12 mmol) of sodium ascorbate are dissolved into 5 mL of water to be further dropwise added into the above reaction solution. And the reaction solution is stirred for 48 h at room temperature. The reaction solution is filtered and then dialyzed in aqueous solution (dialysis bag molecular cut off: 8-10 kDa), and then the copolymer of interest RhB-PDMAEMA25-c-PGMA50-Gal is obtained.

(23) Embodiment 6: Preparation of Copolymer RhB-PDMAEMA-c-PGMA-Gal in Other Different Proportions

(24) The copolymers RhB-PDMAEMA-c-PGMA-N.sub.3 having different proportions of monomers obtained in Embodiment 3 replace RhB-PDMAEMA25-c-PGMA50-N.sub.3 in Embodiment 5, and other operations are the same as those in Embodiment 5, so as to obtain Rhodamine B modified galactose residue-containing copolymer RhB-PDMAEMA-c-PGMA-Gal having different proportions of monomers, including RhB-PDMAEMA10-c-PGMA50-Gal and RhB-PDMAEMA50-c-PGMA50-Gal.

(25) Embodiment 7: Preparation of Copolymer Micelle

(26) RhB-PDMAEMA25-c-PGMA50-Gal in Embodiment 5 is dissolved with DMSO and stirred for 5 h. Dialysis is carried out in double distilled water for 48 h, and water is changed once every 6 hours, so as to obtain micelle solution. Then the micelle solution is lyophilized using a freeze-drier to prepare copolymer micelle Gal-micelles.

(27) Embodiment 8: Preparation of Other Micelles

(28) RhB-PDMAEMA-c-PGMA-Gal in Embodiment 6 respectively replaces RhB-PDMAEMA25-c-PGMA50-Gal in Embodiment 7, and other operations are the same as those in Embodiment 7. Corresponding micelles are respectively prepared.

(29) Embodiment 9: Preparation of Drug-Loading Micelle

(30) RhB-PDMAEMA25-c-PGMA50-Gal in Embodiment 5 and the adriamycin hydrochloride standard product are respectively dissolved with DMSO. Then triethylamine is added in adriamycin hydrochloride standard product DMSO solution. Two solutions are mixed and stirred for 5 h. The mixed solution is dialyzed in double distilled water for 48 h, and water is changed once every 6 hours, so as to prepare adriamycin-loading micelle solution.

(31) Later, the polymer micelle solution is filtered through a 0.45 m membrane to remove unloaded adriamycin, and adriamycin-loading micelle DOX@Gal-micelles is prepared by lyophilization in a freeze-drier.

(32) The prepared micelle is sufficiently dissolved in DMSO. The absorbance of the solution at 483 nm is measured using a microplate reader, and corresponding concentrations of adriamycin in the drug-loading micelle are obtained according to standard curves of adriamycin DMSO solutions.

(33) Drug load of the obtained micelle is equal to adriamycin mass in micelle/micelle mass, and is 12.60.03%; encapsulation efficiency is equal to adriamycin mass in micelle/starting administration mass, and is 58.03.4%.

(34) Embodiment 10: Preparation of Gene/Drug-Loading Copolymer Micelle

(35) The drug-loading DOX@Gal-micelles in Embodiment 9 is dissolved into PBS buffer (140 mM NaCl, 2.7 mM KCl, 10 Mm Na.sub.2HPO4, 1.8 mM KH.sub.2PO.sub.4, pH7.4) to be prepared into 3.5 mg/mL stock solution. Plasmid pGFP or small molecule siRNA (Bcl-2 siRNA) is diluted into 20 g/mL stock solution with double distilled water. The complex of a copolymer and nucleic acid is prepared according to the mole ratio 30:1 of the content of nitrogen in a monomer DMAEMA in the copolymer and the content of phosphorus in nucleic acid. Based on it, these two solutions are mixed evenly, and then stayed for 30 min at room temperature, so as to obtain copolymer micelle loading both genes and a drug, namely DOX@Gal-micelles/pGFP or DOX@Gal-micelles/siRNA.

(36) Embodiment 11: Characterization of Copolymer Micelle

(37) Particle size distribution of Gal-micelles and DOX@Gal-micelles/pGFP prepared in Embodiments 7, 8 and 10 is measured by a dynamic light scattering technology, and changes in particle sizes before and after gene/drug is loaded are compared.

(38) As shown in FIG. 1, through dynamic light scattering particle size detection, Gal-micelles can form a micelle having a particle size of about 155 nm, and its particle size is still about 155 nm after genes and drugs are loaded. However, neither of RhB-PDMAEMA10-c-PGMA50-Gal nor RhB-PDMAEMA50-c-PGMA50-Gal can form uniform micelles.

(39) Stability is one of the most important properties of a nano carrier. A nano particle applied to the field of biomedicines must be stably dispersed in salt solution or a medium within a certain concentration range. The copolymer nano micelle prepared in this experiment is dispersed in aqueous solution, PBS buffer (pH=7.4) and a medium containing 10% fetal bovine serum to measure the change in its particle size. There is no distinct particle size change within 7 days, indicating that this copolymer nano micelle has good stability.

(40) Embodiment 12: Cytotoxicity Experiment of Copolymer Micelle Gal-Micelles

(41) The copolymer micelle Gal-micelles in Embodiment 7 is added into culture solution for a cell culture experiment, and the relative survival rate of cells is measured using 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazole bromine (MTT). A specific experiment method can refer to: Chem. Eur. J. 2016, 22, 15216-15221.

(42) Human hepatoma cells HepG2 and Huh7 cells and human embryonic kidney cells HEK293 cells are respectively planted on a 96-well plate in a density of 510.sup.3 per well. The cells are stably grown in the well plate after being incubated for 48 h, and then the copolymer micelles Gal-micelles in Embodiment 7 are respectively added, with concentrations of 0, 10, 25, 50, 100, 150, 200, 400 and 500 g/mL. After materials and the cells are co-incubated for 48 h, the medium is removed, and washing is carried out for three times with PBS. Then 100 L of medium containing 0.5 mg/mL MTT without addition of phenol red is added into each well, and then 100 L of DMSO is added into each well. The absorbance values (=490 nm) of all wells in the 96-well plate that has been developed are detected using a microplate reader. 6 groups of parallel experiments are repeatedly made for each sample, wherein, cell groups which are not acted on by the materials are defined as having 100% cell activity, and wells having only DMSO solution rather than cells are defied as blank control groups for correcting the absorbance value in each well.

(43) Cell survival is calculated as follows:
Cell survival rate%=(OD.sub.sample/OD.sub.control)100%

(44) In the formula, OD.sub.sample is an absorbance value of an experiment sample group, and OD.sub.control is an absorbance value of a cell group which is not acted on by the materials.

(45) FIG. 2 is a result graph of Gal-micelles cytotoxity, showing that the concentration of copolymer micelle in Embodiment 7 ranges from 0 to 500 g/mL, and the survival rates of three cells are all more than 80%, which indicates that this copolymer micelle has low toxicity and good biocompatibility.

(46) Similarly, the micelles of the present disclosure prepared in other Embodiments are verified, and the results show that the survival rates of the cells are high, which indicates these micelles are extremely small in toxicity or are non-toxic.

(47) Embodiment 13: the Laser Scanning Confocal Experiment Proves that the Copolymer Micelle Gal-Micelles can Perform Specific Target Recognition on an Asialoglycoprotein Receptor on the Surfaces of Hepatoma Cells HepG2 and Hun7 Cells

(48) The blank copolymer micelle Gal-micelles obtained in Embodiment 8 is added to culture solution to carry out cell culture experiment with HEK293 cells, and then cell nucleus is stained with 4,6-diamidino-2-phenyl indole. Through observation under a laser scanning confocal microscope, it can be seen that only a few of Gal-micelles enter HEK293 cells.

(49) The blank copolymer micelle Gal-micelles obtained in Embodiment 7 is added into the culture solution to carry out cell culture experiment with HepG2 and Huh7 cells, and then cell nucleus is stained with 4,6-diamidino-2-phenyl indole. The laser scanning confocal microscope experiment results are as shown in FIG. 3, fluorescence of Rhodamine B from the micelle can be clearly observed in the HepG2 Huh7 cells, and this fluorescence is almost not observed in COS7 and HEK293 cells. Moreover, under the environment in the presence of galactose competition, the fluorescence of Rhodamine B cannot be observed in HepG2 and Huh7 cells either. This experiment proves that Gal-micelles can be recognized through the galactose-asialoglycoprotein receptor and enter the HepG2 and Huh7 cells.

(50) Embodiment 14: Flow Cytometry Detection Proves that the Copolymer Micelle Gal-Micelles can Perform Specific Target Recognition on Asialoglycoprotein Receptors on the Surfaces of Hepatoma Cells HepG2 and Huh7 Cells

(51) In the flow cytometry detection experiment, various cells are cultured in a DMEM medium containing 10% new-born bovine serum (containing 100 U/ml penicillin and 100 g/mL streptomycin), and are placed in a 37 C., 5% CO.sub.2 incubator for growth. Twenty-four hours before transfection, cells at a logarithmic phase are taken, which are digested with 0.02% EDTA and 0.25% trypsin-containing digestive juice and then incubated into a 24-well plate in a density of 510.sup.4 cells per well. 1 mL of complete culture solution is added into each well, and the culture plate is placed in the incubator to be cultured for 24 h. Wherein, galactose having a final concentration of 1 mmol/L is added in one group to continue to culture for 24 h. Then culture solution is removed when the density of cells in each well reaches 70%, and Gal-micelles nano micelle is added into 490 L of serum-free and antibiotic-free culture solution to be evenly mixed, and then the mixture is respectively added in different wells. Sample solution in the 24-well plate is sucked after culturing for 5 h at 37 C., and 1 mL of complete medium is added in each well to continue to culture for 20 h. Cells are digested with pancreatin and centrifuged for 3 min at the rotating speed of 1000rpm. Then the supernate is discarded, and gathered cells are resuspended with PBS and then blown off. This centrifugation process is repeated for three times to remove remaining medium and micelle solution, so as to reduce interference on fluorescence detection. Finally, cells are dispersed with PBS and placed in a flow type tube, and fluorescence intensity of cells in each group is detected using a flow cytometry.

(52) In the experiment, HEK293, HepG2 and Huh7 cells are cultured by using two manners, including culturing with a galactose-containing medium (surface receptor is saturated by galactose in advance) and culturing with galactose-free medium (surface asialoglycoprotein receptor is not affected).

(53) After the three types of cells are acted on by the same Gal-micelles nano micelle, the fluorescence intensity of the Rhodamine B molecules in cells is detected using a flow cytometry.

(54) As shown in FIG. 4, for HEK293 cells, under different environments, the speeds of the Gal-micelles nano micelle entering the cells basically have no difference, there has almost no difference compared with the control groups which are not treated by the Gal-micelles nano micelle. It is because there is only the low-expressed asialoglycoprotein receptor on the surfaces of HEK293 cells. Thus, the micelle cannot rapidly enter the cells through surface galactose-asialoglycoprotein receptor targeting mediated endocytosis. However, for HepG2 and Huh7 cells, under different environments, the speeds of Gal-micelles nano micelles entering the cells are distinctly different. Under the condition that surface asialoglycoprotein receptor is saturated in advance, the Gal-micelles nano micelle cannot recognize HepG2 and Huh7 cells through surface-bound galactose, and can only enter tumor cells through non-targeting endocytosis. Thus, the fluorescence intensity of Rhodamine B is extremely low. However, for cells whose surface receptors are over-expressed, Gal-micelles nano micelle can rapidly bind to asialoglycoprotein receptors on the surfaces of the tumor cells through surface-bound galactose, and then enter the tumor cells through receptor mediated endocytosis.

(55) The experiment proves that Gal-micelles nano micelle can perform specific target recognition on the receptors on the surfaces of the HepG2 and Huh7 cells, and are successfully endocytated into cells.

(56) Embodiment 15: Laser scanning confocal microscope pictures prove that gene nucleic acid/drug-loading copolymer micelle DOX@Gal-micelles/pGFP has a strong nucleic acid and drug co-delivery ability

(57) The nucleic acid/drug-loading micelle DOX@Gal-micelles/pGFP obtained in Embodiment 10 is added into culture solution to respectively carry out a cell culture experiment with HEK293, HepG2 and Huh7 cells, then cell nucleus is re-stained with 4,6-diamidino-2-phenyl indole. The laser scanning confocal microscope experiment results are as shown in FIG. 5, it can be seen that fluorescence of adriamycin DOX, green fluorescent protein GFP and Rhodamine B in the micelle is obviously present in HepG and Hu7 cells; however, under the environment in the presence of galactose competition, the above three types of fluorescence are almost not observed. The experiment results indicate that DOX@Gal-micelles/pGFP can selectively enter HepG2 and Huh7 cells and can co-deliver drugs and nucleic acid to the two types of cells, and successfully carries out gene transfection.

(58) Embodiment 16: Flow Cytometry Detection Proves that DOX@Gal-Micelles/pGFP Micelle can Perform Target Recognition on Asialoglycoprotein Receptors on the Surfaces of the HepG2 and Huh7 Cells, and Successfully Carries Out Drug Delivery and Bene Transfection

(59) In a flow cytometry detection experiment, various cells are cultured in a DMEM medium containing 10% new-born bovine serum (containing 100 U/ml penicillin and 100 g/mL streptomycin), and are placed in a 37 C., 5% CO.sub.2 incubator for growth. Cells at a logarithmic phase are taken 24 h before transfection, are digested with 0.02% EDTA and 0.25% trypsin-containing digestive juice and then incubated into a 24-well plate in a density of 510.sup.4 cells per well, 1 mL of complete culture solution is added into each well, and a culture plate is placed in the incubator to be cultured for 24 h. Wherein, galactose having a final concentration of 1 mmol/L is added in one group to continue to culture for 24 h, then the culture solution is removed when the density of cells in each well reaches 70%. DOX@Gal-micelles/pGFP nano micelle is added into 490 L of serum-free and antibiotic-free culture solution to be evenly mixed, and then the mixture is respectively added in different wells. Sample solution in the 24-well plate is sucked after culturing for 5 h at 37 C., and 1 mL of complete medium is added in each well to continue to culture for 20 h. Cells are digested with pancreatin and centrifuged for 3 min at the rotating speed of 1000 rpm. Supernate is discarded, and gathered cells are resuspended with PBS and then blown off. This centrifugation process is repeated for three times to remove remaining medium and micelle solution, so as to reduce interference on fluorescence detection. Finally, cells are dispersed with PBS and placed in a flow type tube, and fluorescence intensity of cells in each group is detected using a flow cytometry.

(60) In the experiment, HEK293, HepG2 and Huh7 cells are cultured by using two manners, including culturing with a galactose-containing medium (surface receptor is saturated by galactose in advance) and culturing with galactose-free medium (surface asialoglycoprotein receptor on the surface is not affected).

(61) As shown in FIG. 6, after three types of cells are acted on by the same DOX@Gal-micelles/pGFP nano micelle, the fluorescence intensity of different fluorescent molecules in cells is detected using a flow cytometry. The results show that for HEK293 cells, under different environments, the speeds of the DOX@Gal-micelles/pGFP nano micelle entering the cells basically have no difference, there is almost no difference compared with control groups which are not treated by the DOX@Gal-micelles/pGFP nano micelle. It is because there is only low-expressed asialoglycoprotein receptors on the surfaces of HEK293 cells. Thus, the micelle cannot rapidly enter the cells through surface galactose-asialoglycoprotein receptor targeting mediated endocytosis. However, for HepG2 and Huh7 cells, under different environments, the speeds of DOX@Gal-micelles/pGFP nano micelles entering the cells are distinctly different. Under the condition that surface asialoglycoprotein receptor is saturated in advance, the DOX@Gal-micelles/pGFP nano micelles cannot recognize HepG2 cells through surface-bound galactose, and can enter tumor cells only through non-targeting endocytosis. Thus, the fluorescence intensity of Rhodamine B and green fluorescent protein is extremely low. For cells whose surface receptors are over-expressed, DOX@Gal-micelles/pGFP nano micelles can rapidly bind to asialoglycoprotein receptors on the surfaces of the tumor cells through surface-bound galactose, and then enter the tumor cells through receptor-mediated endocytosis, so as to successfully deliver adriamycin and perform gene transfection.

(62) The experiment proves that DOX@Gal-micelles/pGFP nano micelle can perform specific target recognition on the receptors on the surfaces of the HepG2 and Huh7 cells, and can successfully deliver drugs and genes.

(63) Embodiment 17: DOX@Gal-Micelles/siRNA Inhibits Growth of Tumor in a Subcutaneous Solid Tumor Mouse Model

(64) For establishment of the subcutaneous solid tumor mouse model, cells are inoculated in right axilla in the density of 0.1 mL 110.sup.8 cells/mL PBS to the 5-week-old male BALB/c naked mice having the average weight of 15-18 g. Two weeks later, when the size of the tumor exceeds 30 mm.sup.3 (volume=0.5tumor lengthtumor width.sup.2), mice are divided into 7 groups, with 5 mice in each group. Normal saline, blank copolymer micelle Gal-micelles, adriamycin and Bcl-2 siRNA-loading non-targeting micelle DOX@Glc-micelles/siRNA, adriamycin-loading copolymer micelle DOX@Gal-micelles, Bcl-2 siRNA-loading copolymer micelle Gal-micelles/siRNA, adriamycin and siRNA complex DOX/siRNA, and adriamycin and Bcl-2 siRNA-loading targeting copolymer micelle DOX@Gal-micelles/siRNA are respectively injected, with three doses one week, totally for three weeks. The volume of the tumor tissue is recorded every 2-3 days, and the tumor tissue is isolated from the body and recorded on the last day.

(65) As shown in FIG. 7, DOX@Gal-micelles, Gal-micelles/siRNA and DOX@Gal-micelles/siRNA all have the good effect of inhibiting tumor growth, and the inhibition efficiency of the DOX@Gal-micelles/siRNA group is higher than a sum of efficiencies of the single DOX@Gal-micelles group and the single Gal-micelles/siRNA group. According to data of tumor volume of each group obtained on the last experiment day, tumor inhibition efficiency is calculated (tumor inhibition efficiency=(1-average tumor volume of experiment group/average tumor volume of normal saline control group)100%), the inhibition efficiency of the DOX@Gal-micelles/siRNA group is 84%, and the inhibition efficiencies of DOX@Gal-micelles and Gal-micelles/siRNA are respectively 42% and 40%. It can be concluded that DOX@Gal-micelles/siRNA can maximally inhibit tumor growth.

(66) Embodiment 18: DOX@Gal-Micelles/pGFP Inhibits Growth of Tumor in an In Situ Tumor Mouse Model

(67) For establishment of the in situ tumor mouse model, 5-week-old male BALB/c naked mice having the average weight of 15-18 g are taken, and their abdomens are cut to expose liver lobes, and left outer lobes of livers are squeezed out. Cells are inoculated in right axilla in the density of 0.1 mL 110.sup.8 cells/mL PBS, and suturing is carried out to close the abdomens. Two weeks later, mice are divided into 7 groups, with 5 mice in each group. Normal saline, blank copolymer micelle Gal-micelles, adriamycin and Bcl-2 siRNA-loading non-targeting micelle DOX@Glc-micelles/siRNA, adriamycin-loading copolymer micelle DOX@Gal-micelles, Bcl-2 siRNA-loading copolymer micelle Gal-micelles/siRNA, adriamycin and siRNA complex, and adriamycin and Bcl-2 siRNA-loading targeting copolymer micelle DOX@Gal-micelles/siRNA are respectively injected, with three-time doses one week, totally for three weeks. On the last day, the liver tissue is isolated from the body to observe the metastasis of cancer cells in liver, and the number and volumes of the tumors in liver are recorded.

(68) As shown in FIG. 8, the DOX@Gal-micelles/siRNA group has the minimum tumor number and the most smallest tumor volume among all the experiment groups, and has the highest tumor growth inhibition efficiency.