CN109734921B - Polyethyleneimine-b-polylactic acid block copolymer, and preparation method and application thereof - Google Patents
Polyethyleneimine-b-polylactic acid block copolymer, and preparation method and application thereof Download PDFInfo
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Abstract
The polyethyleneimine-b-polylactic acid segmented high-molecular copolymer is prepared and used as a novel carrier for simultaneously encapsulating insoluble antitumor chemicals and nucleic acid medicaments, and application type research is carried out on the copolymer. The invention adopts a cross-linking-condensation method to connect the amino group of polyethyleneimine with the terminal carboxyl group of polylactic acid, and the prepared novel carrier (PEI-PLA) is safe and nontoxic, has better cell membrane penetrating capability, has high drug loading capacity for insoluble antitumor chemicals, and can be well compounded with nucleic acid drugs. The PEI-PLA copolymer is used for simultaneously encapsulating the insoluble antitumor chemical and the nucleic acid drug to prepare the nanoparticles, so that the PEI-PLA high molecular polymer has a good tumor cell inhibition effect, and has a good development prospect.
Description
Technical Field
The invention belongs to the field of biomedical materials, relates to a polymer material, and particularly relates to a preparation method, screening and application of a polyethyleneimine-b-polylactic acid block copolymer.
Background
At present, the main means for clinically treating malignant tumors include operations, radiotherapy, chemotherapy and the like. Most chemotherapy is a systemic treatment, and the medicine is distributed in most organs and tissues along with blood circulation, so that the chemotherapy is more suitable for middle and late-stage tumors which tend to spread or have metastasized. However, the high toxicity of chemotherapeutic drugs to normal tissues remains a major reason limiting the widespread use of chemotherapy. Nucleic acid drugs, as a novel therapeutic model, can inhibit tumor metastasis, reduce drug resistance and drug toxicity, and have been the main subjects of cancer treatment. In particular, the discovery of RNA interference (RNAi) technology has allowed the use of siRNA to specifically "silence" the expression of disease-causing genes in humans, thereby allowing treatment of diseases that are currently "incurable by conventional therapy". Recently, the synergistic effect of antitumor drugs and nucleic acid drugs in cancer treatment has attracted many researchers' attention. The nano drug delivery system is supposed to be utilized to deliver the anti-tumor chemical drug and the nucleic acid drug to tumor tissues together, on one hand, the double-loaded particles can improve the chemotherapy effect by influencing drug resistance channels, on the other hand, the nucleic acid in the double-loaded particles can silence related oncogenes, and the chemical drug can directly act on tumor cells, so that the double-inhibition effect on the proliferation of the tumor cells can be achieved. The combined delivery of anti-neoplastic agents and nucleic acid drugs has broad prospects in the treatment of cancer.
In order to exert the best synergistic effect, the anti-tumor chemotherapeutic drug and the nucleic acid drug need to act on the same tumor cell, but both drugs have limitations, which is a main problem preventing the co-loading of the two drugs. Most chemotherapy drugs are fat-soluble and non-specific, have toxic and side effects on normal tissues, and have limited effect on killing tumor cells. Nucleic acid has short half-life period in vivo circulation, poor stability in plasma and easy degradation by nuclease, and in addition, the nucleic acid is hydrophilic and negatively charged and is difficult to permeate fat-soluble and negatively charged cell membranes, so the nucleic acid cannot enter tumor cells to exert effects. The adverse factors limit the co-delivery of the anti-tumor drugs and the nucleic acid drugs, so how to solve the problems becomes a research hotspot, and the nano drug delivery system has been accepted more and more so far due to the advantages of biodegradability, low immunity, high targeting property, diversified preparation forms and the like. The nano-carrier can be combined with drug molecules, so that the drug molecules are positioned on a target organ through active or passive targeting, toxicity or other adverse reactions to normal tissues are reduced, and the drug molecules can be prevented from being removed from blood circulation. In summary, the nano drug delivery system is utilized to jointly encapsulate the anti-tumor drugs and the nucleic acid drugs, and further modify the anti-tumor drugs and the nucleic acid drugs, so that the toxic and side effects of the chemotherapeutic drugs can be reduced, the nucleic acid drugs are protected from being phagocytosed by organisms, and the nano drug delivery system can play a role in passive targeting by virtue of the special EPR (enhanced peptide delivery) effect of the nano drug delivery system.
Based on the theory and research foundation, the aim of the method is to synthesize a polyethyleneimine-b-polylactic acid block (PEI-PLA) high molecular polymer, encapsulate insoluble antitumor chemical drugs and nucleic acid drugs together, design a tumor targeted nano drug delivery system, and research and discuss the application value of the system in lung cancer treatment. Polyethyleneimine (PEI) is a hydrophilic cationic polymer, is the most commonly used drug carrier in gene delivery, has the advantages of high transfection efficiency, proton pump effect and the like, and is widely concerned in the field of preparations. However, the high cation concentration of PEI leads to poor stability and toxicity of the drug in vivo. Therefore, it is the key to research to reduce its toxicity in the circulation in vivo and to maintain its penetration in tumor cells by modification. Polylactic acid (PLA) is a high-molecular polymer with strong lipophilicity, and a paclitaxel micelle taking PEG-PLA as a carrier is sold in the market abroad, so that the effect is good. Researches show that the paclitaxel micelle can reduce the toxic and side effects of the medicine in normal tissues, improve the medicine loading rate and increase the stability of the medicine. The PEI-PLA polymer serving as an amphiphilic block copolymer can be independently loaded to form micelles, and the specific method is that a hydrophobic end of PLA is coated with an insoluble antitumor drug, and a hydrophilic end of PEI with positive electricity is loaded with siRNA with negative electricity.
The invention uses PEI and PLA to synthesize a novel block high molecular polymer, which is used for realizing the co-loading of an anti-tumor chemical drug and a nucleic acid drug, and obviously improves the transfection efficiency of genes and the drug loading capacity of the anti-tumor drug. Research results show that the synthesized carrier material has low toxicity, the PEI-PLA high molecular polymer can form stable nanoparticles, and the in vivo and in vitro pharmacodynamic evaluation of the nanoparticles shows that the composite nanoparticles can be efficiently absorbed by tumor cells and have good double inhibition efficiency on the tumor cells.
Disclosure of Invention
The technical problem to be solved by the invention is a novel high-molecular polymer for co-delivering anti-tumor chemical drugs and nucleic acid drugs, which has low cytotoxicity, high drug loading rate and transfection efficiency.
In addition, there is a need to provide a novel method for preparing a high molecular polymer for co-delivering an anti-tumor drug and a nucleic acid drug.
In order to solve the technical problem, the invention provides the following technical scheme:
one aspect of the technical scheme of the invention provides a high molecular polymer PEI-PLA formed by polyethyleneimine modified polylactic acid, which has a specific structural formula as follows: the number of PEIm-PLAN,
the specific structure of PEIm-PLAN is as follows:
in the formula, m and n represent the degrees of polymerization of a polyethyleneimine chain (i.e., a moiety represented by PEI) and a polylactic acid chain (i.e., a moiety represented by PLA), respectively. Wherein the polymerization degree m of the polyethyleneimine chain is 2 to 200, preferably 4 to 80, and more preferably 6 to 20; wherein the degree of polymerization n of the polylactic acid chain is 10 to 300, preferably 7 to 300, more preferably 14 to 70.
The molecular weight of the polyethyleneimine chain part is 500-50000Da, such as 1000-20000Da, such as 1500-5000 Da; wherein the molecular weight of the polylactic acid chain part is 500-20000Da, such as 1000-10000Da, such as 1000-5000 Da.
In the present invention, the sizes of the polyglutamic acid chain portion and the polylactic acid chain portion may be expressed in terms of both molecular weight and polymerization degree, as long as they do not contradict each other. The lower right hand corner, when expressed in terms of degree of polymerization, is an unitless integer having an m of from 2 to 200 and an n of from 10 to 300, e.g. PEI15-PLA10(ii) a When expressed in terms of molecular weight, the lower right hand corner is a number of 500-50000 or 3k-50k (for the polyethyleneimine chain moiety) or a number of 500-20000 or 5k-20k (for the polylactic acid chain moiety), for example PEI10k-PLA10kAlso, for example, PEI10000-PLA10000。
Polylactic acid is a hydrophobic chain, a hydrophobic inner core can be formed after the polylactic acid is connected with polyethyleneimine, the polyethyleneimine is used as a hydrophilic outer shell, the block copolymer can be self-assembled in an aqueous solution to form a micelle structure, the hydrophobic inner core can wrap a poorly-soluble chemical drug, and meanwhile, the polyethyleneimine with a positively charged hydrophilic outer shell can be compounded with a nucleic acid drug with a negative charge. The system improves the stability of the carrier, and the polylactic acid is connected with the polyethyleneimine, so that the lipophilicity of the material can be increased, the affinity to cells is improved, and the intracellular delivery of the medicament is further increased.
The invention also provides a method for preparing the high molecular polymer, which comprises the following steps:
1) dissolving PLA-COOH in dimethyl sulfoxide (DMSO), adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as condensing agents, and activating at 20-40 ℃ for 2-4 h;
2) adding PEI into the solution, adding a small amount of triethylamine, and continuing to react for 12-48h at the temperature of 20-40 ℃;
3) after the reaction is finished, dialyzing for 24-48h by using a dialysis bag with the molecular weight cut-off of 100-3000, and freeze-drying to obtain the high molecular polymer PEI-PLA.
In the preparation method of the PEI-PLA high molecular polymer, the preferable ratio of polylactic acid to DMSO is 1g polyethyleneimine dissolved in 1-25ml DMSO, and the more preferable ratio of polylactic acid to DMSO is 1g polyethyleneimine dissolved in 1-10ml DMSO; the volume ratio of the added triethylamine to the DMSO is 1:20-200, and the preferred volume ratio of the added triethylamine to the DMSO is 1: 100-200; the preferred molar ratio of EDC to polylactic acid is 1:1-30, and the more preferred molar ratio of EDC to polylactic acid is 1: 5-10; the preferred molar ratio of NHS to polylactic acid is 1:1-30, more preferred molar ratio of NHS to polylactic acid is 1: 5-10; the preferred molar ratio of polylactic acid to polyethyleneimine may be from 1:1 to 10, and more preferably, the molar ratio of polylactic acid to polyethyleneimine may be from 1:1 to 5.
In a further aspect of the present invention, there is provided the use of the above-mentioned aliphatic hydrocarbon grafted low molecular weight polyethyleneimine as a drug delivery vehicle, particularly an in vivo or in vitro drug delivery vehicle, or the use thereof for preparing an in vivo or in vitro drug delivery vehicle, such as for delivering poorly soluble anti-tumor chemotherapeutic drugs, plasmid DNA, siRNA, and the like. In one embodiment, when the PEI-PLA is used as a drug delivery vehicle, the drug loading of the entrapped paclitaxel is 1-5%, the size of the delivered plasmid DNA is 1-30Kb, and the size of the delivered siRNA is 15-30 bp. When the high molecular polymer is used for encapsulating the anti-tumor chemical drug, the anti-tumor chemical drug is prepared by a dialysis method, the mass ratio of the high molecular polymer to the anti-tumor chemical drug is 50:1-5:1, and more preferably, the mass ratio of the high molecular polymer to the anti-tumor chemical drug is 15:1-5: 1. When the high molecular polymer is used for coating the anti-tumor chemical drug, the ratio of DMSO to the high molecular polymer is 1mLDMSO to dissolve 5-50mgPEI-PLA, and more preferably, the ratio of DMSO to the high molecular polymer is 1mLDMSO to dissolve 20-30 mgPEI-PLA. When the high molecular polymer is used for coating the anti-tumor chemical drug, the volume ratio of the added DMSO to the water is 1:5-100, and more preferably, the volume ratio of the added DMSO to the water is 1: 5-20. When the high molecular polymer is used for coating the anti-tumor chemical drug, the selected dialysis bag has the molecular weight cut-off of 3000-10000, and more preferably, the selected dialysis bag has the molecular weight cut-off of 5000-8000. The mass ratio of the high molecular polymer to the nucleic acid has a significant effect on the transfection efficiency. Preferably, the mass ratio of the high-molecular polymer to the nucleic acid is selected from 1:1 to 100:1, and more preferably, the mass ratio of the high-molecular polymer to the nucleic acid is selected from 1:1 to 50: 1. Under the condition of room temperature, the PEI-PLA high molecular polymer and the indissolvable chemical can be self-assembled to form micelles in organic solvents such as DMSO, chloroform, methanol or acetone; the nucleic acid can be compounded in HBS buffer solution, HBG buffer solution, RPMI-1640 cell culture solution or DMEM cell culture solution according to a certain mass ratio to form a compound. The nanoparticles prepared by the high molecular polymer provided by the invention can be administrated by intravenous injection, lung intratracheal instillation, aerosol inhalation, local intratumoral injection and other methods.
The preparation method and the application of the PEI-PLA block copolymer have the following beneficial effects:
1) the polyethyleneimine is combined on free carboxyl of polylactic acid molecules by adopting a one-step synthesis method, so that the preparation method is simple; 2) the block copolymer is used for simultaneously encapsulating the antitumor drug and the gene, and other auxiliary materials such as cosolvent solubilizer and the like are not needed, so that the toxic and side effects are small; 3) the nanoparticles prepared by PEI-PLA have high gene transfection efficiency and high anti-tumor drug loading; 4) the nanoparticles prepared by PEI-PLA have high uptake rate to tumor cells.
Drawings
FIG. 1 is a synthetic route to example 1.
FIG. 2 is a diagram showing the preparation of high molecular weight polymer PEI-PLA obtained in example 11H-NMR spectrum.
FIG. 3 is a transmission electron microscope photograph of the high molecular polymer PEI-PLA prepared in example 3.
FIG. 4 is a graph showing cytotoxicity results of the carrier material prepared in Experimental example 1.
FIG. 5 is a co-carried paclitaxel and siRNA prepared according to Experimental example 2surThe effect of the nanoparticles in inhibiting cell proliferation in vitro is shown.
FIG. 6 shows the co-loading Oregon GreenPTX and siRNA prepared according to Experimental example 3CY3The nano-particle is used for taking a result picture of A549 lung cancer cells.
Detailed Description
Examples
Example 1: synthesis of PEI-PLA
Weighing 1300mgPLA1.3k-COOH was placed in a 50mL round bottom flask and dissolved by adding 10mL of LDMSO. After the solution was sufficiently dissolved, 958.5mg of EDC (molar ratio to polylactic acid: 1:5) and 575.5mg of NHS (molar ratio to polylactic acid: 1:5) were added and reacted at 25 ℃ for 2 hours to activate PLA. After the activation reaction is finished for 2h, 200 mu L of triethylamine (the volume ratio of the triethylamine to the DMSO is 1:50) and 600mg of PEI are added into the reaction solution1.8k(the molar ratio of the PEI-PLA to the PLA is 3:1), continuously reacting for 24h at 25 ℃, dialyzing for 48h in deionized water by using a dialysis bag (with the molecular weight cut-off of 2500) after the reaction is finished to obtain the PEI-PLA, and freeze-drying the final product. The reaction formula is shown in figure 1. Taking appropriate amount of lyophilized product, dissolving in heavy water (D)2O), determination of hydrogen nuclear magnetic resonance spectrum (1H-NMR), the measurement frequency was 600MHz, and the results are shown in FIG. 2.
EXAMPLE 2 preparation of paclitaxel-Loading (PTX) nanoparticles Using PEI-PLA synthesized from PEI and PLA at different feed ratios and measurement of particle size, potential and drug-loading
Respectively weighing 200, 300, 400 and 500mgPLA1.3k-COOH was placed in a 50mL round bottom flask and dissolved by adding the appropriate amount of DMSO. After the solution was sufficiently dissolved, 590, 885, 1180, 1475mg EDC (molar ratio to polylactic acid 1:20), 354, 531, 708, 885mg NHS (molar ratio to polylactic acid 1:20) were added, and the mixture was reacted at 25 deg.CAnd 2h, activating the PLA. After the activation reaction is finished for 2 hours, adding a small amount of triethylamine and 278mgPEI into the reaction solution1.8k(the molar ratio of the PEI-PLA to the PLA is 1:1, 2:1, 3:1 and 4:1 respectively), continuously reacting for 24 hours at 25 ℃, and dialyzing for 48 hours in deionized water by using a dialysis bag (the molecular weight cutoff is 2.500Da) after the reaction is finished to obtain the PEI-PLA with different degrees of substitution.
50mg of the PEI-PLA and 5mg of PTX were weighed, respectively dissolved in 2mL of LDMSO, and mixed well, and added dropwise into 20mL of water with a burette under stirring. After fully stirring, putting the mixture into a dialysis bag with the molecular weight cutoff of 7000, dialyzing for 24h, filtering the mixture by using a 0.45 mu m filter membrane, and freeze-drying the filtered mixture to obtain different PEI-PLA nanoparticles (PEI-PLA/PTX) carrying PTX. After the nanoparticles are dissolved in distilled water, the solution is diluted by 10 times, and the particle size distribution and the zeta potential of the composite particles are respectively measured by a laser particle sizer.
Weighing 10mg of different PEI-PLA nanoparticles carrying PTX, adding 100ml of acetonitrile, and dissolving completely in 1 min. Taking the subsequent filtrate after filtration with 0.45um microporous membrane, treating with the same method, and determining according to the following chromatographic conditions and determination method to obtain paclitaxel concentration in nanoparticles, and calculating the drug loading according to the following formula.
Octadecylsilane chemically bonded silica is used as a filler for chromatographic conditions and system applicability tests; methanol-water-acetonitrile (23:41:36) is used as a mobile phase, and the detection wavelength is 227 nm. Injecting 10 μ L of system applicability test solution into liquid chromatograph under related substance item, wherein the separation degree of paclitaxel peak, impurity I peak and impurity II peak should be greater than 1.0.
The determination method comprises precisely weighing appropriate amount (about equivalent to 60mg of paclitaxel) of the nanoparticles, placing in a 50ml measuring flask, adding acetonitrile to dissolve and dilute to scale, shaking, filtering, precisely measuring 5ml of subsequent filtrate, placing in a 50ml measuring flask, adding acetonitrile to dilute to scale, shaking, using as sample solution, precisely measuring 10 μ L, injecting into a liquid chromatograph, and recording chromatogram; taking paclitaxel reference substance, adding acetonitrile to dissolve, and quantitatively diluting to obtain solution containing 0.12mg per 1ml, and determining by the same method. The results of the particle size potential and the drug loading are shown in Table 1.
Table 1 shows that the particle size, the potential and the drug loading capacity of the paclitaxel-loaded nanoparticles prepared by synthesizing PEI-PLA high molecular polymers with different feeding ratios
Example 3 PEI-PLA/PTX/siRNAsurPreparation of composite nano-particle and observation of shape and size
A PEI-PLA high molecular polymer was synthesized as in example 1, and 25mg of PEI-PLA and 2.5mg of PTX were weighed out and dissolved in 1mL of LDMSO to be sufficiently mixed, and the mixture was added dropwise to 10mL of water with stirring using a burette. After fully stirring, putting the mixture into a dialysis bag with the molecular weight cutoff of 7000, dialyzing for 24h, filtering the mixture by using a 0.45 mu m filter membrane, and freeze-drying the filtered product to obtain the PEI-PLA nanoparticle (PEI-PLA/PTX) carrying the PTX. Survivin siRNA (siRNA) with DEPC Watersur) Dissolving the dry powder into a solution of 20 pmol/mu L, mixing the PEI-PLA/PTX aqueous solution and 20 pmol/mu L survivin siRNA solution in equal volume according to the mass ratio of the polymer to the siRNA of 20:1, whirling for 10s, and incubating for 20min at room temperature to obtain the PEI-PLA/PTX/siRNAsurComposite nanoparticles. Diluting the composite nanoparticles by 10 times with water, dripping the diluted composite nanoparticles on the surface of a wax block, sucking excessive liquid after 5 minutes, carrying out negative dyeing by using 2% tungsten phosphate dye solution, drying for 15 minutes, and observing the shape and size of the composite nanoparticles under a Transmission Electron Microscope (TEM), wherein the result is shown in figure 3.
Experimental example:
experimental example 1: in vitro cytotoxicity Studies of Carrier materials
The monolayer cultured 4T1 breast cancer cells were digested with 0.25% trypsin, and single cell suspensions were prepared in RPMI 1640 medium at 4X 10 per well3Each cell was seeded in a 96-well plate at 100. mu.L per well volume and cultured at 37 ℃ for 24 hours under 5% CO 2. After 24h, the medium was aspirated off and the medium was added separatelyPEI and PEI-PLA high molecular polymer in example 1 in different concentrations were cultured in RPMI 1640 medium of 200. mu.L for 24h and 48h, respectively, and then the medium was aspirated, 20. mu.L of MTT (5mg/mL concentration) was added to each well, and after 4h of continuous culture, the solution was aspirated, 150. mu.L of LDMSO was added, and shaking was carried out on a shaker for 10 min. And (3) calculating the cell viability by taking the holes without medicine as control holes, inspecting the cytotoxicity of the PEI-PLA carrier material, and arranging 6 repeated holes in each hole. The absorbance (A) was measured at 490nm using a microplate reader, and the survival rate,% cell survival, was calculated as followsS/AbX 100%. Wherein A isS、AbAbsorption values of the administered group and the blank group, respectively. The toxicity of the carrier material to tumor cells was examined by calculating the cell viability, and the results are shown in table 2 and fig. 4.
TABLE 2 cytotoxicity of support materials
Experimental example 2: functional siRNAsurNanoparticle in vitro anti-tumor cell proliferation effect
Human lung cancer A549 cells in logarithmic growth phase at 5 × 103The density of each well is laid in a 96-well plate, 37 ℃ and 5% CO2And culturing for 24 h. After 24h, the same dose of blank carrier material (PEI-PLA), paclitaxel, survivin RNA, paclitaxel-loaded nanoparticles (PEI-PLA/PTX) and survivin RNA-loaded nanoparticles (PEI-PLA/siRNA) prepared according to example 3 were addedsur) And double-loading composite nanoparticles (PEI-PLA/PTX/siRNA)sur) Adding into corresponding wells, culturing for 48h, and setting 6 multiple wells in each group. After 48h the medium was aspirated, 20. mu.L of MTT reagent was added to each well, incubation was continued for 4h, and then 150. mu.L of DMSO solution was added to each well. The absorbance (A) was measured at 490nm using a microplate reader, and the cell viability (cell viability)% -, A, was calculated according to the following formulaS/AbX 100%. Wherein A isS、AbAbsorption values of the administered group and the blank group, respectively. And (3) investigating the killing capacity of the nanoparticles to the tumor cells by calculating the cell survival rate. The results showed that PEI-PLA/PTX/siRNA was comparable to each of the other groups, as shown in Table 3, FIG. 5surThe cell viability of the group was only 14.70%, with a significant difference (P)<0.05*). Therefore, PEI-PLA/PTX/siRNAsurHas good inhibition effect on A549 cell proliferation.
TABLE 3 in vitro inhibition of cell proliferation by co-loading nanoparticles
Experimental example 3: laser confocal observation of PEI-PLA/Oregon Green PTX/siRNACY3Cellular uptake profile of composite nanoparticles
A549 cells in logarithmic growth phase at 10 × 104The density of each well is laid in 12-well plate, 37 deg.C, 5% CO2Incubated under conditions overnight. PEI-PLA/Oregon Green PTX/siRNA is prepared by taking siRNA marked by Oregon Green PTX and CY3 as model drugsCY3And (3) compounding nanoparticles, transfecting the nanoparticles into corresponding holes at a siRNA dose of 100 pmol/hole, and observing the cell entering conditions in different time periods by using laser confocal after 4 hours. From the research result of the ingestion experiment (figure 6), the cell-entering condition of the composite nanoparticles is increased (the yellow part is the common cell-entering condition of the two medicines) along with the prolonging of time, and the cell-entering is basically complete at 4h, which shows that the PEI-PLA high molecular polymer can effectively deliver the paclitaxel and the siRNA into the cells.
Claims (9)
1. A preparation method of a high molecular polymer formed by modifying polylactic acid with polyethyleneimine is characterized in that the high molecular polymer has the following general formula: the number of PEIm-PLAN,
wherein PEI represents a polyethyleneimine chain, and PLA represents a polylactic acid chain; m and n represent the polymerization degrees of the polyethyleneimine chain and the polylactic acid chain, respectively; wherein the polymerization degree m of the polyethyleneimine chain is 2-200;
the preparation method of the high molecular polymer comprises the following steps:
1) dissolving polylactic acid chain end carboxylation in dimethyl sulfoxide, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide as condensing agents, and activating for 2-4h at 20-40 ℃;
2) adding polyethyleneimine into the solution obtained in the step 1), adding a small amount of triethylamine, and continuously reacting for 12-48h at the temperature of 20-40 ℃;
3) after the reaction is finished, dialyzing for 24-48h by using a dialysis bag with the molecular weight cutoff of 100-3000 by adopting a dialysis method, and freeze-drying to obtain a high molecular polymer;
wherein,
the ratio of polylactic acid to dimethyl sulfoxide is that 1g of polylactic acid is dissolved in 1-25ml of dimethyl sulfoxide;
the molar ratio of the added 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the polylactic acid is 1: 1-30;
the molar ratio of the added N-hydroxysuccinimide to the polylactic acid is 1: 1-30;
the molar ratio of polylactic acid to polyethyleneimine is 1: 1-10.
2. A method for producing a high-molecular weight polymer according to claim 1, wherein the molecular weight of the polyethyleneimine moiety is 500-50000 Da; wherein the molecular weight of the polylactic acid chain portion is 500-20000 Da.
3. The production method according to claim 1, wherein the polylactic acid and dimethyl sulfoxide are dissolved in a ratio of 1g of polylactic acid to 1 to 10ml of dimethyl sulfoxide.
4. The production method according to claim 1, wherein the molar ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the polylactic acid is 1: 5-10.
5. The method according to claim 1, wherein the molar ratio of N-hydroxysuccinimide to polylactic acid is 1: 5-10.
6. The process according to claim 1, wherein the molar ratio of polylactic acid to polyethyleneimine is 1:1 to 5.
7. The use of the high molecular weight polymer obtained by the preparation method of claim 1 in the preparation of a co-delivery carrier for an anti-tumor drug and nucleic acid, characterized in that the high molecular weight polymer encapsulates the anti-tumor drug and is prepared by a dialysis method, the mass ratio of the high molecular weight polymer to the anti-tumor drug is 50:1 to 5:1, the ratio of dimethyl sulfoxide to the high molecular weight polymer is 1mL of dimethyl sulfoxide dissolved with 5 to 50mg of PEI-PLA, the volume ratio of the added dimethyl sulfoxide to water is 1:5 to 100, the cut-off molecular weight of the selected dialysis bag is 3000-10000, and the mass ratio of the high molecular weight polymer to the nucleic acid is selected from 1:1 to 100: 1.
8. The use of claim 7, wherein said anti-neoplastic agent is selected from the group consisting of paclitaxel and docetaxel and said nucleic acid is selected from the group consisting of RNA and DNA.
9. The use according to claim 8, wherein said RNA is selected from the group consisting of small interfering RNA;
the size of the DNA is 1-30 Kb; the size of the small interfering RNA is 15-30 bp.
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