CN109988314B - Hyperbranched chitosan, and preparation method and application thereof - Google Patents
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Abstract
The invention provides hyperbranched chitosan and a preparation method and application thereof. The invention adopts bifunctional reagent to graft chitin on hyperbranched polymer skeleton, and prepares hyperbranched chitosan with hyperbranched polymer skeleton as core and chitin as terminal group. By changing the type and size of the high molecular skeleton and the feeding ratio of the high molecular skeleton, the bifunctional coupling reagent and the chitin, the hyperbranched chitosan with different structures can be synthesized. The hyperbranched chitosan provided by the invention has a special hyperbranched structure, overcomes the structural limitation of the original linear chitosan, and has the environmental response characteristic of pH sensitive charge reversal because the hyperbranched chitosan has a chitosan end group with pKa of about 6.5. The hyperbranched chitosan has excellent biocompatibility and high-efficiency gene delivery performance, and has wide application prospect in the fields of gene therapy and gene editing.
Description
Technical Field
The invention relates to a medicament and a gene carrier of hyperbranched chitosan, in particular to a pH-responsive hyperbranched chitosan conjugate and application thereof in the field of gene medicament delivery, and belongs to a preparation method and technology of a novel medicament carrier in the field of medicament delivery.
Background
Gene therapy is to restore the defective genes causing diseases or to suppress the harmful genes causing diseases by introducing foreign genes into target cells, thereby restoring the normal functions of the body and achieving the purpose of treating diseases [ 1. chinese patent No. ZL201010601008.5, title of the invention: glutathione modified chitosan copolymer used as non-viral gene carrier material, preparation and application thereof. The gene therapy technology provides a new treatment method for diseases which are difficult to cure by conventional medical treatment means. The gene editing means that a target nucleotide sequence in a target DNA fragment is accurately identified, the target DNA fragment is cut by means of endoribozymes and the like to form DNA double-strand break, and gene knock-out, insertion and replacement are completed by utilizing a natural repair mechanism in a cell body. The development of molecular biology and the maturity of gene editing technology promote the clinical application of gene therapy, so that the gene therapy not only has advantages in the fields of treating hereditary congenital diseases, malignant tumors and the like, but also is more and more widely applied to the treatment of various general diseases. The key to clinical application for gene therapy and gene editing is still the development of safe and efficient gene delivery vehicles.
Chitin, as a few basic polysaccharides in nature, has been widely studied as a gene delivery vector due to its good biocompatibility and biodegradability, and strong gene compression ability, however, the insolubility of chitin under physiological pH conditions limits the further development of chitosan gene vectors. The hyperbranched polysaccharide is a polysaccharide macromolecule with a highly branched structure, a plurality of cavities are arranged in the molecule, a plurality of functionalized sugar end groups are arranged on the periphery of the molecule, and the unique branched molecular structure of the hyperbranched polysaccharide endows the hyperbranched polysaccharide with a plurality of unique functions compared with the traditional hyperbranched polymer and linear polysaccharide, can be used for supermolecular encapsulation and has wide application prospect in the field of drug controlled release. Due to the highly branched molecular structure of the hyperbranched polymer, the hyperbranched chitin is beneficial to breaking hydrogen bonds among chitosan molecular chains and increasing the solubility of the chitosan, so that the hyperbranched chitin is expected to become a safe and efficient gene carrier, however, so far, natural hyperbranched chitosan is not found in nature.
Functional molecules such as sugar and the like are conjugated to hyperbranched polymers to construct hyperbranched functional conjugates, so that many problems in clinical and basic medicine are solved. The hyperbranched chitosan is prepared by conjugating chitin at the tail end of the hyperbranched polymer, and the problems of poor solubility of the traditional chitin gene carrier and difficulty in releasing genes in cells caused by chain winding are hopefully solved. On the other hand, as the chitosan has a pKa value close to 6.5, the synthesized hyperbranched chitosan realizes charge reversal in a pH environment with a lower lesion site, has an environmental response characteristic of pH sensitive charge reversal protonation, and enhances the cellular uptake performance of the chitosan. Therefore, through the biocompatibility and pH response charge reversal performance of the chitin, the hyperbranched structure provided by the hyperbranched polymer skeleton and the biocompatibility of the chitin are utilized, the blood circulation stability, cell adhesion and uptake and intracellular gene release capacity are considered, safe and efficient gene delivery is expected to be realized, and a novel carrier is provided for gene therapy and gene editing. From the research results of the current literature, there are no reports of hyperbranched chitosan and its application in the fields of gene therapy and gene editing.
Disclosure of Invention
The invention aims to overcome the defects of the existing gene therapy and gene editing carrier and provides hyperbranched chitosan and a preparation method and application thereof.
The invention has the following inventive concept: the invention adopts bifunctional reagent to conjugate chitin periphery on hyperbranched high-molecular skeleton, and prepares hyperbranched chitosan using hyperbranched high-molecular skeleton as core and chitin as terminal group. By changing the type and size of the high molecular skeleton and the feeding ratio of the high molecular skeleton, the bifunctional coupling reagent and the chitin, the hyperbranched chitosan with different structures can be synthesized. The hyperbranched chitosan provided by the invention has a special hyperbranched structure, overcomes the structural limitation of the original linear chitosan, has pH-sensitive charge reversal environmental response characteristics due to the fact that the hyperbranched chitosan has a chitosan end group with pKa of about 6.5, can be used as a drug gene delivery carrier to promote cell uptake, and remarkably improves the cycle stability, cell adhesion and intracellular gene release performance. The hyperbranched chitosan has excellent biocompatibility and high-efficiency gene delivery performance, and has wide application prospect in the fields of gene therapy and gene editing.
The hyperbranched chitosan is realized by the following technical scheme:
firstly, dissolving a bifunctional coupling reagent in solvents such as dimethyl sulfoxide or methanol, slowly dripping 1-10% of hyperbranched polymer solution under the stirring state, reacting for 0.5-5 h at 20-50 ℃, then slowly dripping 1-5% of chitin aqueous solution or DMF or DMSO, continuing to react for 0.5-5 h at 20-50 ℃, dialyzing the reaction solution, and freeze-drying to prepare the hyperbranched chitosan.
Further, the molecular weight Mw of the chitin is 300-; the degree of deacetylation is 65-95%.
The hyperbranched polymer skeleton core is formed by crosslinking one of hyperbranched polyethyleneimine, hyperbranched polyamidoamine and hyperbranched polylysine, the hyperbranched polyethyleneimine, the hyperbranched polyamidoamine and the hyperbranched polylysine are rich in amino groups in structures and are easier to crosslink to form a hyperbranched structure, and the lower the molecular weight of the low-molecular-weight hyperbranched polyethyleneimine, the hyperbranched polyamidoamine and the hyperbranched polylysine, the more obvious the redox response is and the lower the toxicity is, but if the molecular weight is too low, the preparation of the skeleton is not easy or the hyperbranched structure is difficult to form, so that the increase of gaps in the hyperbranched structure is caused. Therefore, the molecular weight must be in the range of 1800 to 50000, preferably 20000 to 30000.
Further, the bifunctional coupling reagent is one or more of bis-succinimidyl suberate, bis-succinimidyl adipate and bis-succinimidyl succinate, the dosage of the bifunctional coupling reagent or the chitin is 0.1-1 times, preferably 0.3-0.6 times of the molar equivalent of the repeating unit of the hyperbranched polymer, and the reaction conditions are that the stirring reaction is carried out at the temperature of 20-50 ℃ for 0.5-5 hours, preferably at the temperature of 25-35 ℃ for 1-3 hours.
The invention also claims the hyperbranched chitosan prepared by the method.
The invention also discloses the application of the prepared hyperbranched chitosan as a drug gene carrier, in particular to the application in gene delivery.
Compared with the prior art, the invention has the following advantages:
1. the hyperbranched polymer is adopted as a framework, and chitin is conjugated at the end group of the framework to prepare the hyperbranched chitosan.
2. The invention obviously improves the solubility and intracellular gene release capacity of chitosan by preparing hyperbranched chitosan, and improves the controlled release capacity of the drug by pH response charge reversal performance.
Drawings
FIG. 1 is an FTIR spectrum of the hyperbranched chitosan prepared in example 1;
FIG. 2 is a diagram of hyperbranched chitosan prepared in example 11HNMR spectrogram;
FIG. 3 is a TEM photograph of the complex of hyperbranched chitosan prepared in example 1 and pGFP-N1;
FIG. 4 shows the results of transfection of pGFP-N1 with the hyperbranched chitosan prepared in example 1;
FIG. 5 shows cytotoxicity of the hyperbranched chitosan prepared in the present invention.
Detailed Description
The present invention will be described in detail below with reference to specific examples and the accompanying drawings. It is to be understood that the specific embodiments and figures described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
Dissolving 0.7g of bis (N-hydroxysuccinimide) suberate in 10mL of DMSO (solution A), dissolving 1g of PEI (MW 25000Da, PEI-25) in 20mL of ultrapure water (solution B), slowly dripping the solution B into the solution A under the stirring state, finishing dripping for 30min, and reacting for 3 h; dissolving another 1g of chitin (MW 1000Da) in 20mL of ultrapure water (solution C), slowly dropwise adding the solution C into the reaction system, dropwise adding for 30min, stirring at normal temperature for reaction for 3h, transferring the reaction solution into a dialysis bag (MWCO 1000Da), dialyzing in deionized water to remove impurities, and finally freeze-drying to obtain the hyperbranched chitosan HBPC.
Example 2
Dissolving 0.7g of bis (N-hydroxysuccinimide) suberate in 10mL of DMSO (solution A), dissolving 1g of PEI (MW 25000Da, PEI-25) in 20mL of ultrapure water (solution B), slowly dripping the solution B into the solution A under the stirring state, finishing dripping for 30min, and reacting for 3 h; dissolving another 1g of chitin (MW & lt500 & gt) in 20mL of ultrapure water (solution C), slowly dropwise adding the solution C into the reaction system, dropwise adding for 30min, stirring at normal temperature for reaction for 3h, transferring the reaction solution into a dialysis bag (MWCO & lt500 Da), dialyzing in deionized water to remove impurities, and finally freeze-drying to obtain the hyperbranched chitosan HBPC.
Example 3
Dissolving 0.7g of bis (N-hydroxysuccinimide) suberate in 10mL of DMSO (solution A), dissolving 0.25g of hyperbranched polyamidoamine (MW 6909Da) in 20mL of ultrapure water (solution B), slowly dropwise adding the solution B into the solution A under the stirring state, completing dropwise addition within 30min, and reacting for 3 h; dissolving another 1g of chitin (MW 1000Da) in 20mL of ultrapure water (solution C), slowly dropwise adding the solution C into the reaction system, dropwise adding for 30min, stirring at normal temperature for reaction for 3h, transferring the reaction solution into a dialysis bag (MWCO 1000Da), dialyzing in deionized water to remove impurities, and finally freeze-drying to obtain the hyperbranched chitosan HBPC.
Example 4
Dissolving 0.7g of bis (N-hydroxysuccinimide) suberate in 10mL of DMSO (solution A), dissolving 0.5g of hyperbranched polyamidoamine (MW: 14215Da) in 20mL of ultrapure water (solution B), slowly dropwise adding the solution B into the solution A under the stirring state, completing dropwise addition within 30min, and reacting for 1 h; dissolving another 1g of chitin (MW 1000Da) in 20mL of ultrapure water (solution C), slowly dropwise adding the solution C into the reaction system, dropwise adding for 30min, stirring at normal temperature for reaction for 1h, transferring the reaction solution into a dialysis bag (MWCO 1000Da), dialyzing in deionized water to remove impurities, and finally freeze-drying to obtain the hyperbranched chitosan HBPC.
Example 5
Dissolving 0.7g of bis (N-hydroxysuccinimide) suberate in 10mL of DMSO (solution A), dissolving 1g of hyperbranched polyamidoamine (MW 28826Da) in 20mL of ultrapure water (solution B), slowly and dropwise adding the solution B into the solution A under the stirring state, finishing dropwise adding within 30min, and reacting for 1 h; dissolving another 1g of chitin (MW 1000Da) in 20mL of ultrapure water (solution C), slowly dropwise adding the solution C into the reaction system, dropwise adding for 30min, stirring at normal temperature for reaction for 1h, transferring the reaction solution into a dialysis bag (MWCO 1000Da), dialyzing in deionized water to remove impurities, and finally freeze-drying to obtain the hyperbranched chitosan HBPC.
Example 6
Dissolving 0.64g of di (N-hydroxysuccinimide) adipate in 10mL of mixed solution of sodium hydrogen sulfonate (MSO) (solution A), dissolving 1g of PEI (MW 25000Da, PEI-25) in 20mL of ultrapure water (solution B), slowly dripping the solution B into the solution A under the stirring state, finishing dripping for 30min, and reacting for 1 h; dissolving another 1g of chitin (MW 1000Da) in 20mL of ultrapure water (solution C), slowly dropwise adding the solution C into the reaction system, dropwise adding for 30min, stirring at normal temperature for reaction for 1h, transferring the reaction solution into a dialysis bag (MWCO 1000Da), dialyzing in deionized water to remove impurities, and finally freeze-drying to obtain the hyperbranched chitosan HBPC.
Example 7
Dissolving 0.64g of di (N-hydroxysuccinimide) adipate in 10mL of mixed solution of sodium sulfate and sodium sulfate (liquid A), dissolving 0.25g of hyperbranched polyamidoamine (MW 6909Da) in 20mL of ultrapure water (liquid B), slowly dropwise adding the liquid B into the liquid A under the stirring state, completing dropwise adding within 30min, and reacting for 1 h; dissolving another 1g of chitin (MW 1000Da) in 20mL of ultrapure water (solution C), slowly dropwise adding the solution C into the reaction system, dropwise adding for 30min, stirring at normal temperature for reaction for 1h, transferring the reaction solution into a dialysis bag (MWCO 1000Da), dialyzing in deionized water to remove impurities, and finally freeze-drying to obtain the hyperbranched chitosan HBPC.
Example 8
Dissolving 0.64g of di (N-hydroxysuccinimide) adipate in 10mL of mixed solution of sodium sulfate and sodium sulfate (liquid A), dissolving 0.5g of hyperbranched polyamidoamine (MW: 14215Da) in 20mL of ultrapure water (liquid B), slowly dropwise adding the liquid B into the liquid A under the stirring state, completing dropwise adding within 30min, and reacting for 1 h; dissolving another 1g of chitin (MW 1000Da) in 20mL of ultrapure water (solution C), slowly dropwise adding the solution C into the reaction system, dropwise adding for 30min, stirring at normal temperature for reaction for 1h, transferring the reaction solution into a dialysis bag (MWCO 1000Da), dialyzing in deionized water to remove impurities, and finally freeze-drying to obtain the hyperbranched chitosan HBPC.
Example 9
Dissolving 0.64g of di (N-hydroxysuccinimide) adipate in 10mL of mixed solution of sodium sulfate and sodium sulfate (liquid A), dissolving 1g of hyperbranched polyamidoamine (MW 28826Da) in 20mL of ultrapure water (liquid B), slowly and dropwise adding the liquid B into the liquid A under the stirring state, finishing dropwise adding for 30min, and reacting for 1 h; dissolving another 1g of chitin (MW 1000Da) in 20mL of ultrapure water (solution C), slowly dropwise adding the solution C into the reaction system, dropwise adding for 30min, stirring at normal temperature for reaction for 1h, transferring the reaction solution into a dialysis bag (MWCO 1000Da), dialyzing in deionized water to remove impurities, and finally freeze-drying to obtain the hyperbranched chitosan HBPC.
Example 10
Dissolving 0.7g of bis (N-hydroxysuccinimide) suberate in 10mL of DMSO (solution A), dissolving 0.25g of hyperbranched polyamidoamine (MW 6909Da) in 20mL of ultrapure water (solution B), slowly dropwise adding the solution B into the solution A under the stirring state, completing dropwise addition within 30min, and reacting for 1 h; dissolving another 1g of chitin (MW & lt500 Da) in 20mL of ultrapure water (solution C), slowly dropwise adding the solution C into the reaction system, dropwise adding for 30min, stirring at normal temperature for reaction for 1h, transferring the reaction solution into a dialysis bag (MWCO & lt1000 Da), dialyzing in deionized water to remove impurities, and finally freeze-drying to obtain the hyperbranched chitosan HBPC.
Example 11
Dissolving 0.7g of bis (N-hydroxysuccinimide) suberate in 10mL of DMSO (solution A), dissolving 0.5g of hyperbranched polyamidoamine (MW: 14215Da) in 20mL of ultrapure water (solution B), slowly dropwise adding the solution B into the solution A under the stirring state, completing dropwise addition within 30min, and reacting for 1 h; dissolving another 1g of chitin (MW & lt500 Da) in 20mL of ultrapure water (solution C), slowly dropwise adding the solution C into the reaction system, dropwise adding for 30min, stirring at normal temperature for reaction for 1h, transferring the reaction solution into a dialysis bag (MWCO & lt1000 Da), dialyzing in deionized water to remove impurities, and finally freeze-drying to obtain the hyperbranched chitosan HBPC.
Example 12
Dissolving 0.7g of bis (N-hydroxysuccinimide) suberate in 10mL of DMSO (solution A), dissolving 1g of hyperbranched polyamidoamine (MW 28826Da) in 20mL of ultrapure water (solution B), slowly and dropwise adding the solution B into the solution A under the stirring state, finishing dropwise adding within 30min, and reacting for 1 h; dissolving another 1g of chitin (MW & lt500 Da) in 20mL of ultrapure water (solution C), slowly dropwise adding the solution C into the reaction system, dropwise adding for 30min, stirring at normal temperature for reaction for 1h, transferring the reaction solution into a dialysis bag (MWCO & lt1000 Da), dialyzing in deionized water to remove impurities, and finally freeze-drying to obtain the hyperbranched chitosan HBPC.
Example 13
Dissolving 0.64g of di (N-hydroxysuccinimide) adipate in 10mL of mixed solution of sodium hydrogen sulfonate (MSO) (solution A), dissolving 1g of PEI (MW 25000Da, PEI-25) in 20mL of ultrapure water (solution B), slowly dripping the solution B into the solution A under the stirring state, finishing dripping for 30min, and reacting for 1 h; dissolving another 1g of chitin (MW 1000Da) in 20mL of ultrapure water (solution C), slowly dropwise adding the solution C into the reaction system, dropwise adding for 30min, stirring at normal temperature for reaction for 1h, transferring the reaction solution into a dialysis bag (MWCO 500Da), dialyzing in deionized water to remove impurities, and finally freeze-drying to obtain the hyperbranched chitosan HBPC.
Example 14
Dissolving 0.64g of di (N-hydroxysuccinimide) adipate in 10mL of mixed solution of sodium sulfate and sodium sulfate (liquid A), dissolving 0.25g of hyperbranched polyamidoamine (MW 6909Da) in 20mL of ultrapure water (liquid B), slowly dropwise adding the liquid B into the liquid A under the stirring state, completing dropwise adding within 30min, and reacting for 1 h; dissolving another 1g of chitin (MW & lt500 Da) in 20mL of ultrapure water (solution C), slowly dropwise adding the solution C into the reaction system, dropwise adding for 30min, stirring at normal temperature for reaction for 1h, transferring the reaction solution into a dialysis bag (MWCO & lt1000 Da), dialyzing in deionized water to remove impurities, and finally freeze-drying to obtain the hyperbranched chitosan HBPC.
Example 15
Dissolving 0.64g of di (N-hydroxysuccinimide) adipate in 10mL of mixed solution of sodium chloride and sodium chloride (liquid A), dissolving 0.25g of hyperbranched polylysine (MW 70000Da) in 20mL of ultrapure water (liquid B), slowly dripping the liquid B into the liquid A under the stirring state, finishing dripping for 30min, and reacting for 1 h; dissolving another 1g of chitin (MW & lt500 Da) in 20mL of ultrapure water (solution C), slowly dropwise adding the solution C into the reaction system, dropwise adding for 30min, stirring at normal temperature for reaction for 1h, transferring the reaction solution into a dialysis bag (MWCO & lt1000 Da), dialyzing in deionized water to remove impurities, and finally freeze-drying to obtain the hyperbranched chitosan HBPC.
Example 16
Dissolving 0.64g of di (N-hydroxysuccinimide) adipate in 10mL of mixed solution of sodium sulfate and sodium sulfate (liquid A), dissolving 0.5g of hyperbranched polyamidoamine (MW: 14215Da) in 20mL of ultrapure water (liquid B), slowly dropwise adding the liquid B into the liquid A under the stirring state, completing dropwise adding within 30min, and reacting for 1 h; dissolving another 1g of chitin (MW & lt500 Da) in 20mL of ultrapure water (solution C), slowly dropwise adding the solution C into the reaction system, dropwise adding for 30min, stirring at normal temperature for reaction for 1h, transferring the reaction solution into a dialysis bag (MWCO & lt1000 Da), dialyzing in deionized water to remove impurities, and finally freeze-drying to obtain the hyperbranched chitosan HBPC.
Example 17
Dissolving 0.64g of di (N-hydroxysuccinimide) adipate in 10mL of mixed solution of sodium sulfate and sodium sulfate (liquid A), dissolving 1g of hyperbranched polyamidoamine (MW 28826Da) in 20mL of ultrapure water (liquid B), slowly and dropwise adding the liquid B into the liquid A under the stirring state, finishing dropwise adding for 30min, and reacting for 1 h; dissolving another 1g of chitin (MW & lt500 Da) in 20mL of ultrapure water (solution C), slowly dropwise adding the solution C into the reaction system, dropwise adding for 30min, stirring at normal temperature for reaction for 1h, transferring the reaction solution into a dialysis bag (MWCO & lt1000 Da), dialyzing in deionized water to remove impurities, and finally freeze-drying to obtain the hyperbranched chitosan HBPC.
Example 18
Dissolving 0.6g of bis (N-hydroxysuccinimide) succinate in 10mL of DMSO (solution A), dissolving 1g of PEI (MW 25000Da, PEI-25) in 20mL of ultrapure water (solution B), slowly dropwise adding the solution B into the solution A under the stirring state, completing dropwise adding for 30min, and reacting for 1 h; dissolving another 1g of chitin (MW 1000Da) in 20mL of ultrapure water (solution C), slowly dropwise adding the solution C into the reaction system, dropwise adding for 30min, stirring at normal temperature for reaction for 1h, transferring the reaction solution into a dialysis bag (MWCO 500Da), dialyzing in deionized water to remove impurities, and finally freeze-drying to obtain the hyperbranched chitosan HBPC.
Example 19
Dissolving 0.6g of di (N-hydroxysuccinimide) succinate in 10mL of DMSO (solution A), dissolving 0.25g of hyperbranched polyamidoamine (MW 6909Da) in 20mL of ultrapure water (solution B), slowly dropwise adding the solution B into the solution A under the stirring state, completing dropwise adding within 30min, and reacting for 1 h; dissolving another 1g of chitin (MW & lt500 Da) in 20mL of ultrapure water (solution C), slowly dropwise adding the solution C into the reaction system, dropwise adding for 30min, stirring at normal temperature for reaction for 1h, transferring the reaction solution into a dialysis bag (MWCO & lt1000 Da), dialyzing in deionized water to remove impurities, and finally freeze-drying to obtain the hyperbranched chitosan HBPC.
Example 20
Dissolving 0.6g of di (N-hydroxysuccinimide) succinate in 10mL of DMSO (solution A), dissolving 0.5g of hyperbranched polyamidoamine (MW: 14215Da) in 20mL of ultrapure water (solution B), slowly dropwise adding the solution B into the solution A under the stirring state, completing dropwise adding within 30min, and reacting for 1 h; dissolving another 1g of chitin (MW & lt500 Da) in 20mL of ultrapure water (solution C), slowly dropwise adding the solution C into the reaction system, dropwise adding for 30min, stirring at normal temperature for reaction for 1h, transferring the reaction solution into a dialysis bag (MWCO & lt1000 Da), dialyzing in deionized water to remove impurities, and finally freeze-drying to obtain the hyperbranched chitosan HBPC.
Performance testing
The hyperbranched chitosan HBPC prepared by the redox-responsive hyperbranched polymer skeleton composed of polyethyleneimine and a bifunctional coupling reagent has the following typical structure:
wherein R is (CH)2)n，n＝0～4。
The following performance tests are described in detail by taking the hyperbranched chitosan HBPC prepared in example 1 as an example.
1. Evaluation of Gene delivery efficiency of hyperbranched Chitosan
In this example, the hyperbranched chitosan was subjected to gene delivery efficiency evaluation, wherein the gene drug in the delivery efficiency evaluation can be illustrated by using pGFP-N1 plasmid as a reporter gene as an example, and the gene transport performance of the hyperbranched chitosan vector was evaluated. Selecting the hyperbranched chitosan prepared in example 1, preparing HBPC/pGFP-N1 complexes with different nitrogen-phosphorus ratios (N/P), obtaining complex particles with different degrees of compression for pGFP-N1, inspecting the particle size and morphology of the complex particles by a Transmission Electron Microscope (TEM), and giving a typical TEM picture of the complex particles when N/P is 4. As can be seen from FIG. 3, the composite particles have an approximately spherical morphology and a size of 40nm, facilitating cellular uptake. And (3) plating the cultured human breast cancer MCF-7 cells or human cervical carcinoma HeLa cells, culturing in an incubator until the cell fusion degree reaches 80%, absorbing the complete culture medium, washing twice by PBS, adding the HBPC/pGFP-N1 complex, and evaluating the gene delivery efficiency. When the protein was transferred under serum conditions, 400. mu.L of a medium containing 10% serum and complexes of HBPC (example 1) and pGFP-N1 (1. mu.g of DNA per well) at different N/P ratios (mass ratios) were added, and after 6 hours of culture, the medium was aspirated, and after culturing for another 48 hours with fresh medium containing 10% serum, the expression level of green fluorescent protein was observed under an inverted fluorescence microscope, and the results are shown in FIG. 4. As can be seen from fig. 4, the complex particles formed by HBPC prepared in example 1 and pGFP-N1 under the condition of N/P ═ 4 had good gene delivery efficiency in both HeLa and MCF-7 cells, significantly exceeding PEI-25.
2. Evaluation of cytotoxicity of hyperbranched Chitosan
The MTT method was used to evaluate the cytotoxicity of hyperbranched chitosan carriers in HeLa and MCF-7, respectively, and the results are shown in FIG. 5. Cells were plated in 96-well cell culture plates in 3 wells in parallel, 5X 10 wells per well4Individual cells, 5% CO at 37 ℃2Culturing in a cell culture box until the cell fusion degree reaches more than 85 percent. The culture medium was removed, washed 2 times with PBS, and then fresh culture medium and the hyperbranched chitosan prepared in example 1 were added, and after 24 hours of culture, 20. mu.L of 5mg/mL MTT solution was added to each well, and culture was continued at 37 ℃ for 4 hours, and the culture medium was removed to terminate the culture. Succinate dehydrogenase in the mitochondria of living cells reduces MTT to formazan, adds 150 μ L DMSO per well for solubilization, and continues incubation at 37 ℃ for 30 min. The absorbance of each well at a wavelength of 570nm was measured on a multifunctional microplate reader (Sunrise Tecan), the 96-well plate was shaken before detection and automatically mixed for 600s, and the microplate reader was zeroed with a cell-free medium. Cell viability was calculated according to equation 1.1:
cell survival (%) ═ a570SMP/A570CTL×100 (1.1)
Wherein A570SMPAbsorbance of cell well plates to which test vectors or complexes are added, A570CTLAbsorbance was measured for cell well plates containing medium only. According to the toxicity evaluation results of FIG. 5, it can be demonstrated that the hyperbranched chitosan gene vector prepared by the present invention has low cytotoxicity and is suitable for further in vivo studies.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (6)
1. A preparation method of hyperbranched chitosan is characterized by comprising the steps of firstly dissolving a bifunctional coupling reagent in dimethyl sulfoxide or methanol solvent, slowly dropwise adding 1-10% of hyperbranched polymer solution under stirring, reacting at 20-50 ℃ for 0.5-5 h, slowly dropwise adding 1-5% of chitosan water solution, continuing to react at 20-50 ℃ for 0.5-5 h, dialyzing the reaction solution, and freeze-drying to prepare hyperbranched chitosan;
the molecular weight Mw of the chitin is 300-3500Da, the polymerization degree is 2-20, and the deacetylation degree is 65-95%;
the hyperbranched polymer skeleton core is formed by crosslinking one of hyperbranched polyethyleneimine, hyperbranched polyamidoamine and hyperbranched polylysine, and the molecular weight Mw is 1800-50000;
the bifunctional coupling reagent is one or more of disuccinimidyl suberate, disuccinimidyl adipate and disuccinimidyl succinate, the dosage of the bifunctional coupling reagent or the chitin is 0.1-1 times of the molar equivalent of the repeating unit of the hyperbranched polymer, and the reaction condition is that the stirring reaction is carried out for 0.5-5 h at the temperature of 20-50 ℃.
2. The method for producing a hyperbranched chitosan according to claim 1, wherein the molecular weight Mw is 20000 to 30000.
3. The method for preparing hyperbranched poly-chitin according to claim 1, wherein the amount of bifunctional coupling reagent or chitin is 0.3-0.6 times of the molar equivalent of the repeating unit of hyperbranched polymer, and the reaction is carried out under stirring at 25-35 ℃ for 1-3 hours.
4. A hyperbranched chitosan prepared according to the method of claim 1.
5. Use of the hyperbranched chitosan of claim 4 in a pharmaceutical gene carrier.
6. Use according to claim 5, characterized in that the hyperbranched chitosan is used as a drug gene carrier in gene delivery.
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