CN100553684C - MRI is visible, the preparation method of liver targeting, degradable nano-gene carrier - Google Patents

Abstract

The invention provides a new method for preparing a core-shell type degradable nanometer gene carrier system visible to a magnetic resonance imager (MRI), targeted to the liver. The synthesis and assembly of the gene carrier system of the present invention include: 1) synthesis of the core of the gene carrier system; 2) synthesis of the shell of the gene carrier system; 3) assembly of the core and the shell. The core layer of the gene carrier system designed in the present invention is nanoparticles, the particle size is 70-85nm, the Zeta potential is positive and greater than +17mV, and it is assembled from the target gene and degradable polycation; the degradable shell layer has liver Targeting group and polymer magnetic resonance imaging contrast agent, which stay longer in the liver than commercial small molecule contrast agents, with a relaxation rate of 7.77mM ^-1 s ^-1 , and a particle size of the core-shell assembly at 95- 105nm, Zeta potential is negative, the system has excellent storage stability and dispersion, and is suitable as a carrier for gene transfection experiments in vivo.

Description

MRI-visible, liver-targeted, degradable nano-gene carrier preparation method

technical field

The invention is related to the design, synthesis and assembly method of the carrier used in gene therapy, especially a method for preparing MRI-visible, liver-targeted, core-shell type and degradable nanometer gene carrier.

Background technique

Gene therapy is a biomedical high technology that introduces normal genes or genes with therapeutic effects into living target cells in a certain way to correct gene defects or exert therapeutic effects, so as to achieve the purpose of treating diseases. Vector technology is the core technology of gene therapy, and it is the bottleneck restricting whether gene therapy can enter clinical practice. In the early days, scientists mainly used viruses as vectors to implement gene therapy for diseases. However, due to the clinical safety problems of viral vectors, non-viral (synthetic) gene vectors have become more and more attractive in gene therapy. However, in the research and application of non-viral gene vectors At present, there are still the following problems:

1) Since the Zeta potential on the surface of the gene carrier is positive, the gene carrier will produce non-specific binding with the inner wall of the blood vessel, serum components (such as hemoglobin) and other irrelevant tissues after entering the body, making the delivery efficiency of the gene very low;

2) The gene carrier with larger particles will induce the body to produce an immune response, and at the same time activate the liver, causing the gene carrier to be quickly cleared in the circulatory system before it reaches the diseased cells or tissues;

3) The gene carrier system lacks selectivity for specific tissues, organs or cells, cannot form an effective distribution in the body, acts on diseased tissues, organs or cells, and also produces serious toxic side effects on normal tissues, organs or cells;

4) The non-degradability of the gene carrier makes it difficult to metabolize in the body and is prone to cumulative toxicity;

5) After the gene carrier enters the body, there is no effective way to monitor the distribution of the gene carrier in the body in real time. At present, the most commonly used method for drug research is radioisotope labeling. This method needs to kill a large number of experimental animals, and the experimental process is cumbersome. , the research cost is high.

Contents of the invention

The object of the present invention is to overcome the above-mentioned defects and provide a new method for preparing a degradable nano-gene carrier system visible to a magnetic resonance imager (MRI) and targeted to the liver.

Realize the technical scheme of the present invention as follows:

The above-mentioned purpose of the present invention is achieved by a novel "core-shell" structural gene carrier system, wherein,

1) The core of the carrier system is composed of nanoparticles formed by degradable polycations and the target gene. The particle size of the nanoparticles is 70-85nm, and the Zeta potential is positive and greater than +17mV;

2) The degradable shell has a liver targeting group and a polymer magnetic resonance imaging contrast agent, and the residence time in the liver is longer than 24 hours, which is longer than that of the commercial small molecule contrast agent, (commercial small molecule contrast agent The residence time of the agent in the liver is 6 hours), the relaxation rate is 7.77mM ^-1 s ^-1 , which is better than the relaxation rate of 5.95mM ^-1 s ^-1 of the commercial small molecule contrast agent, and the Zeta potential is negative and less than -10mV;

3) The opposite Zeta potential of the shell layer and the core layer makes it easy for the shell layer to assemble on the periphery of the core layer. By adjusting the ratio of the shell layer to the core layer, the Zeta potential of the nano-gene carrier system can be negative, preventing the gene carrier from entering the body. Produce non-specific binding with the inner wall of blood vessels, serum components (such as hemoglobin) and other irrelevant tissues to improve gene delivery efficiency;

4) The liver-targeting group in the shell enables the carrier system to be targeted to the liver, and at the same time enables the carrier system to have a larger hydration volume, and the system has excellent storage stability and dispersibility;

5) The magnetic resonance imaging contrast agent of the shell provides an effective method for real-time monitoring of the distribution of the gene carrier in the body with a magnetic resonance imaging instrument, without killing the experimental animals, the experimental process is simple, and the research cost is low; the shell and core The layers are all degradable and will not produce cumulative toxicity;

6) The particle size of the shell and core layer assembly is 95-105nm, and the Zeta potential is negative, which will not induce the body to produce an immune response, and can circulate in the circulatory system for a long time;

Implementation steps of the present invention are as follows:

1) Synthesis of degradable gene transfection reagent and its assembly with the target gene—construction of the core of the gene carrier system

As shown in Figure 1, the polyethylenimine (PEI) prepolymer with a molecular weight of 6000-10000 is mixed with the alcohol solution of polyacrylic acid ester, sodium alkoxide is added, and the reaction is stirred at room temperature for 2-6 days, and the low boiling point solvent is removed, purified, Drying, packaging, and gene transfection reagents were obtained, and the structure was determined by nuclear magnetic resonance and its degradation performance was studied;

The polyacrylic acid ester is selected from 1,4-butanediol dipropylene ester, ethylene glycol dipropylene ester, glycerol tripropylene ester, pentaerythritol tetrapropylene ester, etc., preferably with 1,4-butanediol dipropylene ester. The alcohol used for dissolving is methyl alcohol, and described sodium alkoxide can select sodium methylate for use;

The above-mentioned gene transfection reagent and the target gene are assembled in a buffer solution under sterile conditions so that the particle size is 70-85nm, and the Zeta potential is positive and greater than +17mV, and the nucleus of the gene carrier system is obtained.

a. Synthesis of polyacrylic esters

Weigh a certain amount of polyhydric alcohol and dissolve it in an anhydrous aprotic solvent containing an organic base, add dropwise acryloyl chloride in an anhydrous aprotic solvent solution under low-temperature stirring, stir for 4 hours, filter, and distill under reduced pressure to collect the required fractions. Store in a desiccator for later use.

b. Synthesis of degradable gene transfection reagent

Mix the alcohol solution of polyethyleneimine (PEI) prepolymer and the alcohol solution of polyacrylic acid ester, add sodium alkoxide, stir and react at room temperature for 2-6 days, remove the low boiling point solvent, purify, and dry to obtain degradable gene transfection Reagents, as shown in Figure 1; use NMR to determine the structure and study its degradation performance.

c. Assembly of degradable gene transfection reagent and target gene—construction of gene carrier system core

Mix the above-mentioned degradable gene transfection reagent with the target gene in a certain proportion under sterile conditions, and shake for 15 minutes to make the particle size 70-85nm, and the Zeta potential is positive and greater than +17mV, and the gene carrier system is obtained. the nuclear layer.

2) Synthesis of shell layer of gene carrier system - synthesis of targeting polymer contrast agent

With polyamino acid as the main chain, the ligand of the liver targeting group (the ligand of Gd ³⁺ ) is coupled to make its Zeta potential negative and less than -10mV.

In phosphate buffer solution, D-lactobionic acid or hepatic targeting peptide was linked to α,β-poly[(2-aminoethyl)-L-asparagine by EDC condensation method, and the sugar content was determined by quantitative ¹³ C NMR, The monoactive ester of diethylenetriaminepentaacetic anhydride was linked to the above carrier, its structure was determined by ¹ H NMR, and the Zeta potential was determined by dynamic light scattering. According to the different feeding amount of D-lactobionic acid or liver-targeting peptide, the molar content of D-galactose residue or liver-targeting peptide residue in the shell layer of the obtained gene carrier system is 5%-30%.

The carrier was complexed with Gd ³⁺ , the structure was determined by nuclear magnetic resonance, the Gd content was determined by inductively coupled plasma spectroscopy, the relaxation rate was measured by magnetic resonance imaging, and its contrast enhancement performance and liver targeting were studied.

3) Assembly of core and shell——construction of "core-shell" structural gene carrier system

The above-mentioned core and shell are mixed in a certain way and incubated for a certain period of time to form a "core-shell" structural gene carrier system.

The core and shell are mixed in a certain way and incubated for a certain period of time to form a "core-shell" structural gene carrier system. The ratio of the core and the shell is regulated, so that the particle size of the core-shell assembly nanoparticle is 95-105nm, the particle size distribution is uniform, and the Zeta potential is less than 0. The particle size, particle size distribution and Zeta potential of the core-shell assembly nanoparticles were determined by dynamic light scattering.

The particle size, particle size distribution and Zeta potential of core, core and shell assembly nanoparticles were determined by dynamic light scattering.

The method of the invention has simple experimental process and low research cost; both the shell layer and the core layer are degradable and will not produce cumulative toxicity. The zeta potential of the shell layer of the prepared gene carrier system is negative and less than -10mV, the residence time in the liver is longer than 24 hours, and the relaxation rate is higher than that of commercial small molecule contrast agents.

Since the core of the carrier system is composed of nanoparticles formed by degradable polycations and the target gene, the particle size of the nanoparticles is 70-85nm, and the Zeta potential is positive and greater than +17mV; the opposite Zeta potential of the shell layer and the core layer makes the shell The layer is easily assembled on the periphery of the nuclear layer. By adjusting the ratio of the shell layer to the nuclear layer, the Zeta potential of the nano-gene carrier system can be negative, preventing the gene carrier from interacting with the inner wall of the blood vessel, serum components (such as hemoglobin) and others after entering the body. Unrelated tissues produce non-specific binding to improve gene delivery efficiency; the particle size of the core and shell assembly of the gene carrier system is 95-105nm, which will not induce the body to produce an immune response and will not activate the liver to cause the gene carrier to reach the lesion Cells or tissues that are previously cleared rapidly in the circulatory system can circulate in the circulatory system for a long time.

The gene carrier system is selective to the liver, can form an effective distribution in the body, and will not cause serious toxic and side effects to normal tissues, organs or cells; An effective method for real-time monitoring of the distribution of gene carriers in the body by making an instrument. The gene carrier system is visible to the magnetic resonance imager (MRI). After the gene carrier enters the body, MRI can be used to monitor the distribution of the gene carrier in real time in the body without killing the experimental animals; The degradable shell has a liver targeting group and a polymer magnetic resonance imaging contrast agent, and the residence time in the liver is greater than 24 hours, which is longer than that of the commercial small molecule contrast agent, (the commercial small molecule contrast agent is in the The liver residence time is 6 hours), the relaxation rate is better than that of commercial small molecule contrast agents, and the Zeta potential is negative and less than -10mV.

Description of drawings

The synthetic route diagram of Fig. 1 gene transfection reagent;

The ¹ H NMR spectrum of the gene transfection reagent in Fig. 2, wherein the horizontal axis represents the chemical shift;

The ¹ HNMR online research spectrogram of the degradation performance of Fig. 3 gene transfection reagent;

The assembly process of Fig. 4 gene transfection reagent and gene (that is, the formation of gene carrier system nucleus);

Figure 5 is the particle size and particle size distribution spectrum of the core of the gene carrier system, where the horizontal axis represents the particle size in nm; the vertical axis represents the intensity, and the curve represents the particle size distribution;

Figure 6 Zeta potential spectrum of the gene carrier system nucleus, where the horizontal axis represents the Zeta potential in mV; the vertical axis represents the intensity;

Figure 7 is a schematic diagram of the structure of the shell of the gene carrier system;

Figure 8 Zeta potential spectrum of the shell of the gene carrier system, where the horizontal axis represents the Zeta potential in mV; the vertical axis represents the intensity;

Fig.9 Relaxation rate of polymer targeted contrast agent and commercial contrast agent (gadopentetate meglumine) as the shell layer; the horizontal axis is the concentration of gadolinium in the contrast agent, and the vertical axis is the relaxation time; the slope of the curve represents the relaxation Rate;

The assembly process of Fig. 10 core and shell;

Figure 11 The particle size and particle size distribution spectrum of the assembly of the core and the shell, where the horizontal axis represents the particle size, in nm; the vertical axis represents the intensity; the curve represents the particle size distribution;

Figure 12 Zeta potential spectrum of the core and shell assembly, where the horizontal axis represents Zeta potential in mV; the vertical axis represents intensity.

Detailed ways

Embodiment 1: the synthesis of 1,4-butanediol diacrylate

Weigh 90g (1mol) of 1,4-butanediol and dissolve it in 200mL of refined N,N-dimethylformamide (DMF), add 1.2mol of anhydrous pyridine, and add 1.2mol of acryloyl chloride dropwise under stirring at low temperature. 200mL of refined DMF solution, stirred at low temperature for 4h, continued to react at room temperature for 4h, filtered, and distilled under reduced pressure to collect the required fractions to obtain 1,4-butanediol diacrylate, which was stored in a desiccator away from light for future use.

Embodiment 2: Synthesis of degradable gene transfection reagent (1)

Weigh 100g of polyethyleneimine prepolymer with a molecular weight of 10,000 and dissolve it in 200mL of absolute anhydrous methanol, add 9.9gl, 4-butanediol diacrylate and 2 drops of sodium methoxide, stir and react at room temperature for 5 days in the dark, and depressurize The solvent was removed, the residue was dissolved in distilled water, and the pH value of the solution was adjusted to neutral with 0.05mol/L hydrochloric acid, filtered, low-temperature dialysis for 3 days, freeze-dried, and vacuum-packed to obtain a degradable gene transfection reagent. Study its degradation performance, the results are shown in Figure 2 and Figure 3. The obtained gene transfection reagent was obviously degraded from the second day.

Example 3: Assembly of degradable gene transfection reagent and target gene—construction of gene carrier system core

Prepare the degradable gene transfection reagent obtained in Example 2 into a 7 mg/mL ultrapure aqueous solution, filter and purify it with a 0.22 μm syringe filter for later use; prepare a 0.1 mg/mL plasmid ultrapure aqueous solution in the same way; add the plasmid solution to the gene In the transfection reagent solution, the molar ratio of the nitrogen in the gene transfection reagent solution to the phosphorus in the plasmid solution is 10:1 (i.e. N/P=10); shake for 15 minutes to obtain a particle size of 70-85nm, see Figure 5 ; Zeta potential is positive and greater than +17mV, see Figure 6; the nuclear layer of the gene carrier system is obtained.

The particle size, particle size distribution, and Zeta potential of the obtained nanoparticles were tested by Zetasizer Nano-ZS type dynamic light scattering (DLS) of MARLVEN Company, the test temperature was 25°C, and the incident laser wavelength was 633nm. Atomic force microscopy (AFM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) can also be used to measure the particle size, particle size distribution, and morphology of nanoparticles. See

Example 5: Synthesis of the Shell—Synthesis of Targeted Polymer Contrast Agent (hereinafter referred to as PV)

1) α, the synthesis of β-poly (L-asparagine) (hereinafter referred to as PI)

Add 25 grams (0.19mol) of L-aspartic acid powder and 12.5 grams of 85% phosphoric acid in a 500ml round bottom flask, mix well, and react under reduced pressure at 185°C on a rotary evaporator for 3 hours until no more water vapor is produced. until. Add 100mL of DMF and stir to dissolve the solid matter, then slowly add the obtained solution dropwise into a beaker filled with 500mL of water under stirring, to obtain a white precipitate, let it stand, pour off the supernatant, filter, and wash the solid with water until neutral. Vacuum drying at 60° C. for 24 hours yielded 17 g of powdery solid with a yield of 93%.

2) Synthesis of α, β-poly[(2-aminoethyl)-L-asparagine] (hereinafter referred to as PII)

Weigh 2.5 g of PI (26 mmol) and dissolve it in 30 mL of DMF, add it dropwise to 150 mL of ethylenediamine under rapid stirring, react at room temperature for 3 hours, filter, concentrate under reduced pressure to 30 mL, and add diethyl ether dropwise to the residue to obtain a viscous solid. The solid was dissolved in 20 mL of methanol, precipitated with ether, and dried in vacuo to obtain 3.5 g of light yellow powdery solid (PII), with a yield of 86%.

3) Synthesis of D-galactose residue-containing polymer (hereinafter referred to as PIII)

Weigh 2 g of lactobionic acid (5.58 mmol) and dissolve in 40 mL of phosphate buffer at pH=5.7, and cool in an ice bath. Add 20mL phosphate buffer solution of 2.6g (22.3mmol) N-hydroxysuccinimide and 4.3g (22.3mmol) EDC.HCl solid, stir in ice bath for 20 minutes, add polymer PII2.9g (18.6mmol), ice After bath reaction for 1 hour, the reaction was continued at room temperature for 36 hours. After 72 hours of dialysis of the reactant mixed solution bag, the solution was concentrated under reduced pressure to dryness to obtain 4.4 g of a light yellow solid with a yield of 91%.

4) Synthesis of diethylenetriaminepentaacetic acid monoactive ester (Tyr-DTPA-OSu) containing tyrosine residues

a) Synthesis of diethylenetriaminepentaacetic acid (DTPA) dual active ester

Weigh 8.6g DTPA bis-anhydride, 6.69g N-hydroxysuccinimide and 100mg 4-dimethylaminopyridine (DMAP) dissolved in 100ml N,N-dimethylformamide (DMF) and stir at room temperature for 24 hours. The product was precipitated with a 1:1 ethanol-ether mixture, washed with methanol, soaked in ether overnight, and dried to obtain 10.7 g of a white powdery solid with a yield of 76%.

b) Synthesis of diethylenetriaminepentaacetic acid monoactive ester (Tyr-DTPA-OSu) containing tyrosine residues

Weigh 6.3541g (27.4mmol) tyrosine methyl ester hydrochloride and dissolve it in 30ml DMF, add 4ml triethylamine, stir and react for 20 minutes, filter out the white solid produced, transfer the filtrate to the dropping funnel, slowly add In the 40ml DMF solution of DTPA biactive ester (16.0975g, 27.4mmol), after stirring reaction under ice-salt bath condition for 12 hours, continue to react at room temperature for 24 hours. The product was precipitated with a 1:1 ethanol-ether mixture, washed with methanol, soaked in ether overnight, and dried to obtain 11.4 g of a milky white powdery solid with a yield of 60%.

5) Coupling of Tyr-DTPA-OSu with polymer PIII (hereinafter referred to as PIV)

透析袋透析72小时后，45℃下将溶液减压浓缩至干得淡黄色固体8.6g，产率87％。Add 150ml aqueous solution of 3.7g PIII into a 500ml round bottom bottle, cool in an ice bath, slowly add 52ml DMF solution containing 9.8mmol Tyr-DTPA-OSu dropwise, stir and react in an ice bath for 6 hours, then continue to react at room temperature for 24 hours. Mix the solution at 4°C with 3500

After the dialysis bag was dialyzed for 72 hours, the solution was concentrated to dryness under reduced pressure at 45° C. to obtain 8.6 g of light yellow solid with a yield of 87%.

6) Synthesis of liver-targeted polymer contrast agent (hereinafter referred to as PV)

Weigh 0.1407g (0.19mmol) of PIV and 0.0831g (0.22mmol) of GdCl ₃ .6H ₂ O into a 100ml round bottom bottle, add 10ml of distilled water to dissolve the solid, adjust the pH value of the solution to 6 with dilute hydrochloric acid, and stir the reaction at room temperature After 6 hours, the reaction mixture was dialyzed at -4°C for 72 hours, and the dialysate was freeze-dried to obtain PV. The galactose residue content was 20.22% (mol%) as measured by carbon nuclear magnetic resonance.

7) Determination of relaxation rate and Zeta potential

Zeta potential was measured by dynamic light scattering method, and the shell Zeta potential was negative and less than -10mV.

Polymer contrast medium (PIV) and commercial contrast medium (gadopentetate dimeglumine) were formulated into solutions with four concentrations, and the concentrations of gadolinium ions were 0.24mmol/L, 0.12mmol/L, 0.06mmol/L, 0.03mmol/L, respectively. L. Put the solution in a 1.5ml centrifuge tube and measure the _T value of the five contrast agents with different gadolinium ion concentration solutions by the inversion recovery method on a 0.3T magnetic resonance instrument, by the formula (T ₁ w is the relaxation time of pure water, T ₁ m is the actual measured relaxation time of different concentrations of contrast agents):

${\mathrm{<span\; class="notranslate">\; C</span>}}_{\mathrm{<span\; class="notranslate">\; Gd</span>}}<span\; class="notranslate">\; \&CenterDot;</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{{\mathrm{<span\; class="notranslate">\; <figure-callout\; id="1"\; label="T"\; filenames="C20061011765500101.png,C20061011765500111.png"\; state="\{\{state\}\}">T</figure-callout></span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}}<span\; class="notranslate">\; +</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{{\mathrm{<span\; class="notranslate">\; T</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}\mathrm{<span\; class="notranslate">\; w</span>}}}<span\; class="notranslate">\; =</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{{\mathrm{<span\; class="notranslate">\; T</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}\mathrm{<span\; class="notranslate">\; m</span>}}}$

Relaxation rate r ₁ =1/T ₁ , using computer fitting to find the slope, can calculate the relaxation rate of targeted polymer contrast agent and commercial contrast agent gadopentetate meglumine respectively.

The relaxation rate was measured by a magnetic resonance imager, and the results are shown in Figure 9, where the slope of the curve represents the relaxation rate. The relaxation rate of the commercial contrast agent (gadopentetate dimeglumine) is 5.95mM ^-1 s ^-1 , and the relaxation rate of the polymer contrast agent synthesized by the present invention is 7.77mM ^-1 s ^-1 , as shown in FIG. 10 .

Embodiment 6 Assembly of gene carrier core layer and shell layer

Mix the aqueous solution of the core and the shell in a 1.5ml reagent bottle and incubate for 0.5 hours to form a "core-shell" structural gene carrier system. The ratio of the core and the shell is adjusted to a mass ratio of 1:2, so that the particle size of the core-shell assembly nanoparticle is 95-105 nm, the particle size distribution is uniform, and the Zeta potential is less than 0. The particle size, particle size distribution and Zeta potential of the core-shell assembly nanoparticles were measured by dynamic light scattering, see Figures 11 and 12.

The above-mentioned embodiments are only used to illustrate the present invention, but are not limited thereto. It should be understood that there may be various modifications or replacements without departing from the spirit of the present invention.

Claims (9)

1. A method for preparing a magnetic resonance imager visible, liver-targeted, core-shell type, degradable nano-gene carrier system, comprising the following steps: 1), the synthesis of degradable gene transfection reagent; 2), the degradable gene transfection reagent and the target gene are assembled into the nuclear layer of the gene carrier system; 3) The synthesis of the shell layer of the gene carrier system is the synthesis of the targeting polymer contrast agent; 4) Assembly of the core and shell of the gene carrier system. 2. The preparation method according to claim 1, wherein the degradable gene transfection reagent is formed by reacting polyethylenimine and degradable linking monomers at normal temperature, and the degradable linking monomers Contains ester bonds. 3. The preparation method according to claim 2, wherein the polyethyleneimine is a prepolymer with a molecular weight of 6000-10000. 4. preparation method as claimed in claim 2, is characterized in that, described degradable connection monomer is selected from 1,4-butanediol dipropylene ester, ethylene glycol dipropylene ester, glycerol tripropylene ester , Pentaerythritol tetrapropenyl ester. 5. The preparation method according to claim 4, characterized in that, the degradable linking monomer is 1,4-butanediol dipropylene ester. 6 . The preparation method according to claim 1 , wherein the shell of the gene carrier system contains a magnetic resonance imaging contrast agent of a liver-targeting group. 6 . 7. The preparation method according to claim 6, wherein the magnetic resonance imaging contrast agent of the gene carrier system shell is composed of α, β-poly[ (2-aminoethyl)-L-asparagine] as the main chain, and contains Gd ³⁺ ions, and the ligand of Gd ³⁺ is diethylenetriaminepentaacetic acid monoactive ester containing L-tyrosine residues . 8. The preparation method according to claim 7, characterized in that the molar content of D-galactose residues or hepatic targeting peptide residues in the shell layer of the gene carrier system is 5%-30%. 9. preparation method as claimed in claim 7, is characterized in that, the main chain of the magnetic resonance imaging contrast agent of described gene carrier system shell layer is the α that contains D-galactose residue, β-poly[(2- Aminoethyl)-L-asparagine].

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