CN112574344B

CN112574344B - Cationic polymer with oxidation response and charge removal functions and preparation method and application thereof

Info

Publication number: CN112574344B
Application number: CN202011450807.7A
Authority: CN
Inventors: 徐骁; 相佳佳; 陶耀业; 王建国; 申有青; 柯庆宏; 岑贝妮
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2020-12-09
Filing date: 2020-12-09
Publication date: 2021-10-15
Anticipated expiration: 2040-12-09
Also published as: CN112574344A

Abstract

The invention discloses an oxidation response charge-removing cationic polymer, a preparation method and application thereof, wherein the cationic polymer has a structure shown as a formula B-PIEMA, and is obtained by quaternizing 4-bromomethylbenzeneboronic acid or 4-chloromethylphenylboronic acid and PIEMA. Different from a common gene delivery carrier, the oxidation response charge-removing cationic polymer synthesized by the invention has a large amount of positive charges and can well wrap DNA, but can change the charges under the oxidation condition in cells after entering the cells, and quickly release the DNA for transfection; the nano-composite carrying plasmid DNA prepared by the cationic polymer can quickly escape from lysosomes, and DNA degradation is avoided. The carrier has the characteristics of high efficiency and low toxicity, and has good application prospect.

Description

Cationic polymer with oxidation response and charge removal functions and preparation method and application thereof

Technical Field

The invention relates to the fields of macromolecules and biotechnology, in particular to an oxidation response charge-removing cationic polymer, a preparation method and application as a gene delivery carrier.

Background

Gene therapy refers to the introduction of exogenous normal genes into target cells to correct or compensate for diseases caused by defective and abnormal genes, in order to achieve therapeutic goals. For tumors, especially liver cancer, which is a tumor lacking a targeted drug, gene therapy is considered as one of the most promising therapeutic approaches. Currently used delivery vectors can be divided into viral vectors and non-viral vectors.

The non-viral vector comprises cationic liposome, polymer, dendritic macromolecule and the like, and has the advantages of good safety, low immunogenicity, biocompatibility, low mass production cost and the like compared with the viral vector. However, compared with viral vectors, the lower transfection efficiency is always a bottleneck for the application of the non-viral vectors, and the non-viral vectors with good effect in vitro cannot achieve good transfection effect in vivo.

In non-viral vectors, cationic polymers are often used to neutralize the negative charge of DNA and compress it into nanoparticles to protect the DNA from degradation and to aid in its entry into cells. However, the cationic polymer/nucleic acid drug complex nanoparticles formed by positive/negative electrostatic interaction are thermodynamically stable, and the nanocomplex is difficult to dissociate to release the nucleic acid drug after entering cells, so that the nucleic acid drug is difficult to exert the drug effect. Therefore, it is necessary to design a carrier capable of rapidly releasing a nucleic acid drug in response to intracellular microenvironment to improve the drug efficacy of the nucleic acid drug.

Reactive Oxygen Species (ROS) are major molecules generated during oxidative stress of the body, and have been considered as important factors in the development and development of tumors. The ROS level in the tumor cells is often higher than that of normal cells, so that the ROS is utilized to change the charge of the nano-composite entering the tumor tissue so as to release nucleic acid, the characteristics of the tumor microenvironment can be well utilized, and the purpose of specific release of the tumor tissue is achieved.

The Chinese patent with publication number CN101597349B discloses a phenylboronic acid modified cationic polymer, the structural formula of which is shown as follows:

the phenylboronic acid group can form reversible covalent bonding with functional groups such as hydroxyl, amino and the like in biological macromolecules such as protein, polysaccharide, nucleic acid and the like, so that high-efficiency transfection of genes is realized.

The Chinese patent with the publication number of CN101659737B discloses a mercapto-terminated poly (2-methylamino ethyl) methacrylate-block-polyvinyl imidazole polymer, which has the following structural formula:

the polymer and nano-gold compound is used as a plasmid DNA carrier, is combined with the plasmid DNA, and further transfects cells, so that the polymer can be used as a carrier of tumor suppressor drugs.

However, none of the prior art discloses whether the two polymers can escape from lysosomes, avoiding DNA degradation; therefore, it is necessary to design a carrier capable of responding to intracellular microenvironment, rapidly releasing nucleic acid drugs, and avoiding DNA degradation, thereby improving the drug efficacy of nucleic acid drugs.

Disclosure of Invention

The invention provides a cationic polymer capable of oxidative response and charge removal, and a preparation method and application thereof.

The technical scheme of the invention is as follows:

an oxidation-responsive uncharged cationic polymer, said cationic polymer having the structure of formula B-pimea;

wherein n represents the degree of polymerization, and the value range is 5-500; the anion is bromide or chloride.

The B-PIEMA is charged with positive charges outside cells so as to be tightly complexed with DNA, and can react once entering the cells to weaken the positive charges, so that the B-PIEMA is rapidly dissociated from the DNA, and the expression of the DNA is promoted.

The invention also provides a preparation method of the cationic polymer, which comprises the following steps: carrying out polymerization reaction on IEMA under the action of an initiator to obtain PIEMA; quaternization of PIEMA and a compound of formula (II) to obtain a cationic polymer shown as formula B-PIEMA;

wherein n represents polymerization degree, the value range is 5-500, and the anion is bromide ion or chloride ion; and X is Br or Cl.

The initiator is 2-bromoisobutyric acid ethyl ester, azobisisobutyronitrile or ammonium persulfate.

The feeding mass ratio of the PIEMA to the compound of the formula (II) is 1: 1-2.

The invention also provides application of the cationic polymer in preparation of a gene delivery vector.

The invention also provides a nano-composite comprising the cationic polymer and the plasmid DNA.

The plasmid DNA is pEGFP plasmid DNA or luciferase plasmid DNA.

The preparation method of the nano-composite comprises the following steps: dissolving the cationic polymer shown in the formula (I) in a buffer solution to obtain a carrier solution, dissolving plasmid DNA in the buffer solution to obtain a DNA solution, adding the carrier solution into the DNA solution, oscillating and standing to obtain a solution containing the nano-composite.

The N/P molar ratio between the cationic polymer and the plasmid DNA is 2-32.

The N/P molar ratio between the cationic polymer and the plasmid DNA is 14-18.

The invention also provides application of the nano-composite in marking tumor cells.

The invention also provides application of the nano-composite in preparing antitumor drugs.

Compared with the prior art, the invention has the following beneficial technical effects:

1. the oxidation response charge-removing cationic polymer provided by the invention has a simple structure and is convenient to prepare.

2. The synthesized oxidation response charge-removing cationic polymer has a large amount of positive charges, can well wrap DNA, but can change charges under the oxidation condition in cells after entering the cells, quickly releases the DNA for transfection, has higher transfection activity, and can target a tumor microenvironment for transfection.

3. The nano-composite has strong lysosome escape capability, can escape from lysosome quickly, and avoids DNA degradation.

Drawings

FIG. 1 shows the Zeta potential under oxidizing conditions for B-PIEMA in example 3.

FIG. 2 is the electrophoresis diagram of the gel retardation experiment of the B-PIEMA/DNA nanocomplex in example 4, in which NC represents DNA plasmid alone.

FIG. 3 is a graph showing the particle size distribution and Zeta potential of the B-PIEMA/DNA nanocomposite in example 4.

FIG. 4 is a TEM image of the B-PIEMA/DNA nanocomposite obtained in example 4, wherein the nitrogen-phosphorus ratio is 16.

FIG. 5 is a cytotoxicity plot of B-PIEMA/DNA nanocomplexes of example 5.

FIG. 6 is a graph showing the cell transfection effect of B-PIEMA/DNA nanocomplexes with different N/P ratios in example 6, wherein (A) is a flow-detected fluorescence distribution graph of different cells, (B) is a graph showing the relationship between the average intensity of green fluorescence of cells and N/P, and (C) is a graph showing the relationship between the percentage of green fluorescent protein-positive cells and N/P.

FIG. 7 is a graph showing the effect of transfection of B-PIEMA/DNA nanocomplexes with N/P ratio of 16 in example 6 on control cells, wherein A is a flow-detected fluorescence distribution graph of different cells and B is a comparison of percentage of green fluorescent protein positive cells between different carriers.

FIG. 8 is a graph showing the effect of cell transfection of B-PIEMA/DNA nanocomplexes in example 6.3 of the present invention.

FIG. 9 is a graph of cell transfection of B-PIEMA/DNA nanocomplexes under different concentrations of active oxygen radical inhibitor (DPI) in example 7 of the present invention, wherein the graph (A) is a flow detection fluorescence distribution graph of different cells, the graph (B) is a relation between percentage of green fluorescent protein positive cells and N/P, and the graph (C) is a relation between average intensity of green fluorescence of cells and N/P.

FIG. 10 is a graph showing the effect of B-PIEMA/DNA nanocomplex uptake and lysosomal escape in cells under confocal laser microscopy in example 7.

FIG. 11 is a graph showing the distribution analysis of fluorescence signals of Cy5DNA and lysosome in example 7, wherein FIG. A is a graph showing the distribution analysis of fluorescence signals at 1h and FIG. B is a graph showing the distribution analysis of fluorescence signals at 6 h.

FIG. 12 is a diagram of the intratumoral injection of the B-PIEMA/DNA nanocomplex in vivo transfection in example 8.

Detailed Description

The invention is further illustrated by the following examples. It should be understood that these examples are only for illustrating the present invention and do not limit the scope of the present invention. Unless otherwise specified, various reaction raw materials, reaction equipment and the like referred to in the following examples are available from known sources, for example, from the market.

Example 1 Synthesis of B-PIEMA

1- (2-hydroxyethyl) imidazole (5.0g, 44.59mmol) was dissolved in dry dichloromethane and placed in an ice-water bath, methacryloyl chloride (5.6g, 53.57mmol) was dissolved in dry dichloromethane and slowly added dropwise using a constant pressure dropping funnel. After the dropwise addition, the ice-water bath is removed, the temperature is slowly raised, and the reaction is carried out at room temperature overnight. After the completion of the TLC detection reaction, the reaction solution was diluted with dichloromethane and washed with saturated aqueous sodium bicarbonate solution and saturated brine in this order three times, the organic phase was collected, dried over anhydrous sodium sulfate, the filtrate was collected by filtration, and the organic solvent was removed by rotary evaporation. The crude product was purified by silica gel column separation (ethyl acetate/n-hexane) to obtain 6.2g of IEMA as a colorless liquid in a yield of 77%.

IEMA (5.0g, 27.74mmol) and azobisisobutyronitrile (0.02g, 0.12mmol) were weighed, dissolved in anhydrous methanol and placed in an ampoule, argon bubbled for 30 minutes and sealed, placed in a 65 ℃ oil bath for polymerization, reacted for 24 hours, after which polymerization was terminated, precipitated three times with ice-n-hexane and dried to give 4.1g of polymer pimea having a number average molecular weight of 20kDa (n 115) at a yield of 82%.

1.0g of PIEMA and 4-bromomethylbenzeneboronic acid (1.78g, 8.28mmol) were weighed out, dissolved in N, N-dimethylformamide and reacted overnight at 50 ℃. The reaction solution was poured into an excess of tetrahydrofuran, and the precipitate was collected by filtration, dialyzed with ultrapure water, and lyophilized to obtain 1.4g of B-PIEMA (bromide ion omitted from the structural formula), with a yield of 64%.

Example 2 Nuclear magnetic characterization of B-PIEMA

The hydrogen nuclear magnetism of B-PIEMA is characterized as follows:¹H-NMR,D₂O：δ＝8.7-9.1(1H,-NCHN-),δ＝7.2-8.2(7H,ArH,-NCHCHN-),δ＝5.4-5.8(2H,ArHCH₂N-),δ＝4.2-4.7(4H,-NCH₂CH₂OOCCH-),δ＝2.3-2.6(1H,-CH₂CH-),δ＝1.6-2.2(-CH₂CH-),δ＝0.8-1.4(CH₃-)。

example 3 Charge Change of B-PIEMA under Oxidation conditions

An amount of B-PIEMA was weighed out and dissolved in HEPES buffer solution (pH 7.4,10mM) at a concentration of 3mg/mL, and hydrogen peroxide solution was added to give a final concentration of 80 mM. The pH of the solution was maintained at 7.4 throughout, the solution was incubated at 37 ℃ and the Zeta potential of the solution was measured at different times. As shown in FIG. 1, the Zeta potential of B-PIEMA decreases rapidly from 30mV to 20mV, and the charge decreases slowly with time, and then decreases to 10mV at 30 h.

Experiments prove that the charge of the B-PIEMA can be changed under the active oxygen treatment, so that the acting force with the DNA is reduced, and the DNA is released.

Example 4

4.1 preparation of B-PIEMA/DNA nanocomposites

Weighing a certain amount of B-PIEMA, dissolving the B-PIEMA in a HEPES buffer solution (the pH value is 7.4 and 10mM) to obtain a solution with the concentration of 2mg/mL (the solution is marked as B-PIEMA), diluting pEGFP plasmid DNA into a solution with the concentration of 40 mu g/mL (the solution is marked as DNA) by using the HEPES buffer solution (the pH value is 7.4 and 10mM), diluting the B-PIEMA solution into a corresponding concentration according to a set series of nitrogen-phosphorus ratios (the N/P molar ratio is 0.5, 1, 2,4, 8, 16 and 32), quickly adding the B-PIEMA solution into the DNA solution according to the volume ratio of 1:1, carrying out vortex oscillation for 10 seconds, and standing for 30 minutes at room temperature to obtain a series of nano-composite solutions with different nitrogen-phosphorus ratios.

Characterization of the physicochemical properties of the nanocomposite solution obtained above:

4.2 gel retardation test

20 mu L of prepared nano-composite solution with different nitrogen-phosphorus ratios (samples with N/P molar ratios of 0.5, 1, 2,4 and 8 are respectively selected), 10 mu L of pure pEGFP plasmid DNA is taken as a control, 6 samples are respectively loaded into gel holes prepared by 1% agarose (containing 0.5mg/mL ethidium bromide), and the gel holes are subjected to electrophoresis for 30min in 1xTAE buffer solution at 120 mV. After the electrophoresis is finished, the electrophoresis medium is placed in an ultraviolet gel imaging system for shooting, and an electrophoretogram is obtained and is shown in FIG. 2. As can be seen from FIG. 2, B-PIEMA binds DNA well and blocks DNA migration well when N/P is greater than 1.

4.3 particle size distribution and Zeta potential measurement

And (3) taking a series of prepared nano composite solutions with different nitrogen-phosphorus ratios (samples with N/P molar ratios of 2,4, 8, 16 and 32 are respectively selected) and measuring the size, the Zeta potential and the aggregation performance of the nano composite solutions by using a dynamic light scattering instrument at the temperature of 25 ℃. The results were calculated by the DTS software. Each set of experiments was repeated 3 times and averaged. As a result, as shown in FIG. 3, the particle size can be compressed to 50nm and the Zeta potential is distributed between 15 and 25 mV.

4.4 Transmission Electron microscopy of B-PIEMA/DNA

And (3) dropping a drop of the prepared nano composite with the N/P molar ratio of 16 onto a 400-mesh copper net, sucking excess liquid by using filter paper, naturally airing at room temperature, observing the shape of the sample by using a transmission electron microscope, and recording a transmission electron microscope image. As shown in FIG. 4, the nano-composite formed by electrostatic self-assembly shows spherical-like nano-particles with regular and uniform morphology and particle size of about 50nm, which is substantially consistent with the result measured by a dynamic light scattering instrument.

Example 5 cytotoxicity assays of B-PIEMA and B-PIEMA/DNA nanocomposites

The 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfonated benzene) -2H-tetrazole monosodium salt (CCK-8) method was used to test the in vitro cytotoxicity of the polymer at 7721 cells.

7721 cells were cultured in 96-well plates at a cell density of 5000 cells/well, 100. mu.L of medium (10% (v/v) fetal bovine serum) and PEI (25kDa) as a control, and cells were plated on 5% CO₂The culture was carried out in a 37 ℃ incubator at a concentration and a humidity of 95% for 24 hours.

After 24h, 100. mu.L of different concentrations of N-PDEA and nanocomposite (N-PDEA/DNA nanocomposite (Kjek gene) with a nitrogen-phosphorus molar ratio of 16) culture medium solution (the final concentrations of polymer N-PDEA were 5mg/ml, 10mg/ml, 20mg/ml, 40mg/ml and 80mg/ml respectively) was added to each well, 100. mu.L of the culture medium solution was added to the blank group, and the cells were cultured for another 48 h. After 48h incubation, the medium was discarded from each well and 100. mu.L of medium containing 10% CCK-8 reagent was added and the cells were incubated for an additional 1 h. The absorbance of the sample at 450nm was finally measured with a microplate reader. Cell viability (percentage) is expressed as absorbance values of experimental groups divided by absorbance values of blank groups. Each set of data was the average of three wells of the same set of specimens.

As shown in FIG. 5, the toxicity of B-PIEMA to 7721 cells increased with increasing concentration, the survival rate was higher than that of PEI (25KDa) at the same concentration, and the toxicity of the polymer to the cells was reduced after the polymer and DNA were combined into a nano-complex.

Example 6 transfection experiment of cellular Green fluorescent protein Gene of B-PIEMA/DNA nanocomposite

6.1, determining the preferable nitrogen-phosphorus ratio

7721 cells were cultured in 6-well plates at a cell density of 300000 cells/well in 1.75mL of medium per well and placed in 5% CO₂The culture was carried out in a 37 ℃ incubator at a concentration and a humidity of 95% for 24 hours. Then replacing the culture medium in each well with 1.75mL of serum-free culture medium, adding a series of prepared nano-composite solutions with nitrogen-phosphorus ratios (B-PIEMA/DNA nano-composite, wherein the nitrogen-phosphorus molar ratio of DNA is pEGFP plasmid (Kjeldahl gene) is respectively 2,4, 8, 16 and 32)250 mu L (containing 5 mu g of pEGFP plasmid DNA), and culturing for 4 h. The plate was then discarded from the medium containing the nanocomposite and the incubation continued for 48h with fresh medium.

After the culture was completed, the cells were digested with 0.25% pancreatin/0.03% EDTA and collected into 1.5mL centrifuge tubes, washed twice with PBS solution, and finally suspended in 500 μ LPBS solution and transferred to a flow detection tube for flow cytometry detection. The excitation wavelength of the green fluorescent protein is 488nm, and the emission wavelength is 510 nm. As shown in FIG. 6, the B-PIEMA/DNA nanocomposite with N/P of 16 has the highest cell green fluorescence positive rate and the highest average fluorescence intensity, and the gene delivery vector can effectively release DNA to promote transfection.

6.2 verification of transfection efficiency

7721 cells were cultured in 6-well plates at a cell density of 300000 cells/well in 1.75mL of medium per well and placed in 5% CO₂The culture was carried out in a 37 ℃ incubator at a concentration and a humidity of 95% for 24 hours. Then the medium in each well was replaced with 1.75mL serum-free medium, and 250. mu.L (5. mu.g plasmid DNA containing pEGFP) of the prepared nanocomposite solution (B-PIEMA/DNA nanocomposite, nitrogen to phosphorus molar ratio 16) was added and cultured for 4 h. The plate was then discarded from the medium containing the nanocomposite and the incubation continued for 48h with fresh medium.

After the culture was completed, the cells were digested with 0.25% pancreatin/0.03% EDTA and collected into 1.5mL centrifuge tubes, washed twice with PBS solution, and finally suspended in 500 μ LPBS solution and transferred to a flow detection tube for flow cytometry detection. The excitation wavelength of the green fluorescent protein is 488nm, and the emission wavelength is 510 nm. In the experiment, positive control was PEI/DNA nanocomplex and commercial transfection reagent lipofectamine3000(Thermo Fisher Scientific) and negative control was cells without any treatment. Each set of data was the average of three wells of the same set of specimens.

As shown in FIG. 7, the transfection value of B-PIEMA/DNA nanocomplexes with N/P of 16 was 78.71%, which is higher than that of positive controls PEI/DNA (24.70%) and Lipo3000 (53.63%).

6.3 confocal laser observation of Green fluorescent protein expression

7721 cells were cultured at a cell concentration of 300000/well in a glass-bottomed petri dish with a radius of 15mm, 1.75mL of medium per well, and the cells were cultured in a 37 ℃ incubator with 5% CO2 concentration and 95% humidity for 24 hours. Then, the medium in each well was replaced with 1.75mL of medium (serum content: 0%), and 250. mu.L (5. mu.g of plasmid DNA containing pEGFP) of the prepared nanocomposite solution (B-PIEMA/DNA nanocomposite, nitrogen-phosphorus molar ratio: 16, respectively) was added thereto and cultured for 4 hours. Then, the medium containing the nanocomposite was discarded from the plate and replaced with fresh medium to continue the culture for 48 hours.

After the culture was completed, the cells were observed under a confocal laser microscope. The excitation wavelength of the green fluorescent protein is 488nm, the emission wavelength is 510nm, all photos are shot under an objective lens with the power of 20 times, and the same light intensity is always used.

As shown in FIG. 8, the PEI/DNA nanocomplex transfected only a few cells, lipofectamine3000 transfected a little more cells, but the brightness of these cells was high, while the B-PIEMA/DNA nanocomplex transfected cells were more uniform, which is of great significance in gene therapy. For therapeutic genes, most cells can be transfected to produce therapeutic proteins far better than a small number of cells expressing a large amount of proteins, because these proteins often only need a small amount to cause apoptosis of the cells, achieving therapeutic effects.

Example 7 cell transfection experiment of B-PIEMA/DNA nanocomposites with active oxygen radical inhibitor (DPI)

7721 cells were cultured in 6-well plates and cellsThe density was 300000/well, 1.75mL of medium per well, and the cells were plated in 5% CO₂The culture was carried out in a 37 ℃ incubator at a concentration and a humidity of 95% for 24 hours. After 0.5 hours of incubation, the medium in each well was replaced with 1.75mL of serum-free medium containing different concentrations of DPI (these concentrations had no effect on cell viability), after which the medium in each well was replaced with 1.75mL of serum-free medium and 250 μ L of the prepared B-piama/DNA nanocomposite solution with N/P of 16 (containing 5 μ g of pEGFP plasmid DNA) was added and cultured for 4 h. The plate was then discarded and replaced with fresh medium containing DPI at the same concentration as before and the culture continued for 12 h.

After the culture was completed, the cells were digested with 0.25% pancreatin/0.03% EDTA and collected into 1.5mL centrifuge tubes, washed twice with PBS solution, and finally suspended in 500 μ LPBS solution and transferred to a flow detection tube for flow cytometry detection. The excitation wavelength of the green fluorescent protein is 488nm, and the emission wavelength is 510 nm.

The results are shown in fig. 9, where the DPI-treated gfp positive cells showed a significant decrease in both the rate and mean fluorescence intensity, demonstrating that DNA was difficult to release for efficient transfection if the cells had no reactive oxygen radicals.

Example 8 uptake and lysosomal escape experiments in cells of B-PIEMA/DNA nanocomplexes

The luciferase plasmid DNA was labeled with the Label IT Tracker Cy5 nucleic acid labeling kit (manufacturer: mirus, model: mir3725) according to the instructions. 7721 cells were cultured at a cell concentration of 80000/well in 1.5mL of medium per well in 15mm radius glass-bottom dishes, and placed in 5% CO₂The culture was carried out in a 37 ℃ incubator at a concentration and a humidity of 95% for 24 hours. Then the medium in each well was replaced with 1.5mL serum-free medium and 75. mu.L (containing luciferase plasmid) of the prepared nanocomposite solution (B-PIEMA/DNA nanocomposite, nitrogen to phosphorus molar ratio 16) was added^cy5DNA 1.5. mu.g), incubated for the indicated time (1h or 6h), respectively. Adding lysosome dye Lyso Tracker Green with final concentration of 100nM) into the culture medium, culturing for 1.5h, adding 2ul Hoechst 33342 into the culture medium,after 20min of incubation, the medium was poured off, washed 3 times with PBS solution and observed with a confocal laser microscope. The excitation wavelength of the Lyso Tracker Green is 488nm, the emission wavelength is 500-555nm, the excitation wavelength of the Hoechst 33342 is 405nm, the emission wavelength is 425-475nm, and Cy5 labeled DNA^Cy5The excitation wavelength of the DNA is 640nm, the emission wavelength is 662-737nm, all the pictures are taken under a 60-time objective lens, and the same light intensity is always used.

The results are shown in FIGS. 10 to 11: some DNA had entered the cell at 1h, and only a small amount of DNA red fluorescence overlapped with lysosome green fluorescence, and most of the DNA did not overlap with lysosome, demonstrating that the B-PIEMA/DNA nanocomplex had rapidly escaped from lysosome at 1 h.

At 6h, more red fluorescence is in the cell, most of the red fluorescence is distributed near the nucleus, and a plurality of red fluorescence can be seen in the nucleus, which shows that the B-PIEMA/DNA nano-complex has high cellular uptake rate, can rapidly enter the cell and rapidly escape from lysosomes, and releases DNA into the nucleus for efficient transfection.

Example 9 in vivo transfection experiment of B-PIEMA/DNA nanocomposites by intratumoral injection

Axillary inoculation of BALB/c female nude mice for 6 weeks at 5x10⁶7721 tumor cells, until the tumor grows to about 200mm³Then dividing the mixture into two groups at random, wherein each group comprises six groups, injecting B-PIEMA/DNA and PEI/DNA nano compound solution 60 mu L (containing luciferase plasmid DNA 15 mu g) into tumors respectively, killing nude mice after 48 hours, stripping the tumors, adding Ix cell lysate with 4 times volume, shearing and homogenizing, centrifuging 13500g for 10 minutes, taking the supernatant, adding luciferase substrate, and measuring chemiluminescence intensity by using a multifunctional microplate reader. Protein concentration was determined using BCA protein assay kit. Chemiluminescence intensities were normalized using protein concentration in units of luminescence intensity per mg of protein (RLU/mg protein).

As a result, as shown in FIG. 12, the in vivo transfection efficiency of the B-PIEMA/DNA nanocomposite with N/P of 16 was 16 times higher than that of the PEI/DNA nanocomposite, demonstrating that the B-PIEMA/DNA nanocomposite was efficiently transfected once it reached the tumor site.

Claims

1. An oxidation-responsive uncharged cationic polymer, wherein said cationic polymer has the structure shown in formula B-PIEMA;

the preparation method of the cationic polymer comprises the following steps: carrying out polymerization reaction on IEMA under the action of an initiator to obtain PIEMA; quaternization of PIEMA and a compound of formula (II) to obtain a cationic polymer shown as formula B-PIEMA;

wherein n represents polymerization degree, the value range is 5-500, and the anion of the cationic polymer is X ion; and X is Br or Cl.

2. The cationic polymer according to claim 1, wherein the charging mass ratio of PIEMA to the compound of formula (II) is 1: 1-2.

3. Use of the cationic polymer of claim 1 in the preparation of a gene delivery vector.

4. A nanocomposite comprising the cationic polymer of claim 1 and plasmid DNA, wherein the plasmid DNA is pEGFP plasmid DNA or luciferase plasmid DNA.

5. The method for preparing a nanocomposite as claimed in claim 4, comprising the steps of: dissolving the cationic polymer shown in the formula (I) in a buffer solution to obtain a carrier solution, dissolving plasmid DNA in the buffer solution to obtain a DNA solution, adding the carrier solution into the DNA solution, oscillating and standing to obtain a solution containing the nano-composite.

6. The method of claim 5, wherein the molar ratio of N/P between the cationic polymer and the plasmid DNA is 2-32.

7. The method of claim 5, wherein the N/P molar ratio between the cationic polymer and the plasmid DNA is 14-18.

8. Use of the nanocomposite according to claim 4 for labelling tumor cells for non-diagnostic purposes.

9. Use of the nanocomposite as claimed in claim 4 in the preparation of an anti-tumor medicament.