A multifunctional shell material HPSO and a preparation method and use thereof

Abstract

The application provides a multifunctional shell material HPSO and a preparation method and application thereof, and belongs to the field of biological medicine. The structure of the multifunctional shell material HPSO is shown in formula I. After the HPSO is combined with a cationic polymer gene carrier, the positive charge of the cationic polymer gene carrier can be shielded, so that the gene carrier has a negative charge. The gene carrier with the negative charge can be used for in-vivo treatment through intravenous injection, and the problem of strong toxic side effects such as hemolysis caused by the combination of the existing cationic polymer gene carrier and negative proteins in blood is overcome. Meanwhile, the transfection efficiency of the cationic polymer gene carrier is not reduced after being combined with the shell material. In addition, the non-viral shell material HPSO can enhance the immune activation effect. The shell material HPSO has a good application prospect in the preparation of a gene carrier for in-vivo treatment. Formula I

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a multifunctional shell material HPSO, its preparation method, and its uses. Background Technology

[0002] Gene therapy refers to a novel approach that delivers therapeutic genes to target organs or tissues via vectors to repair damaged or missing genes and exert therapeutic effects. Current gene therapy strategies primarily include the introduction of tumor suppressor genes, suicide genes, siRNA, drug resistance genes, immune genes, and the CRISPR-Cas9 system. Therefore, the key to gene therapy lies in how to efficiently and with low toxicity deliver exogenous genes into target cells. The lack of highly efficient and low-toxicity gene vectors is one of the significant factors limiting the development of gene therapy. The design of gene vectors has become one of the cutting-edge research areas.

[0003] Currently, widely used gene vectors are mainly divided into viral vectors and non-viral vectors. Viral vectors include retroviruses, adenoviruses, and adeno-associated viruses. The simplest viral core is nucleic acid (DNA or RNA), surrounded by a layer of regularly arranged protein subunits called the capsid. The morphological subunits that make up the capsid are called capsomeres, and the particle composed of nucleic acid and capsid proteins is called the nucleocapsid. Viral vectors can effectively integrate exogenous genes into the host chromosome, thereby achieving persistent expression of the target sequence. In terms of infectivity, they can effectively infect various cell types such as neurons, hepatocytes, cardiomyocytes, tumor cells, endothelial cells, and stem cells, thus achieving good gene therapy effects. For some cells that are difficult to transfect, such as primary cells, stem cells, and undifferentiated cells, the use of lentiviral vectors can greatly improve the transduction efficiency of the target gene, and greatly increase the probability of the target gene integrating into the host cell genome, enabling relatively convenient and rapid long-term and stable expression of the target gene. Although viral vectors have high gene transfection efficiency, they have two serious safety issues: immunogenicity and gene insertion mutations.

[0004] In recent years, with the rapid development of pharmaceutics, materials science, and other disciplines, a large number of synthetic and natural materials have emerged, and non-viral vectors have attracted much attention. These include cationic liposomes or lipid complexes and cationic polymers. Their advantages as vectors include the ability to deliver large-sized nucleic acids; well-defined chemical structures; and ease of commercialization. However, non-viral vectors have low gene transfection efficiency, which cannot meet the needs of gene therapy. Therefore, it is necessary to chemically modify non-viral vectors to improve their transfection efficiency.

[0005] In gene delivery research, cationic materials are widely used due to their ability to efficiently compress and deliver negatively charged nucleic acids through electrostatic interactions. However, their strongly positively charged surfaces can easily cause blood compatibility issues, non-specific tissue accumulation, and systemic toxicity under intravenous administration conditions, severely limiting clinical translation. Therefore, coating a cationic core with a negatively charged or electrically neutral shell has become an important strategy in the design of non-viral vectors. However, this "charge shielding" process often weakens charge-driven cellular uptake and endocytosis efficiency, leading to decreased transfection efficiency and creating a typical contradiction between in vivo safety and transfection efficiency. Summary of the Invention

[0006] To address the aforementioned problems, the present invention aims to provide a multifunctional shell material HPSO, its preparation method, and its applications.

[0007] This invention provides a multifunctional shell material HPSO, the structure of which is shown in Formula I: Formula I Where x is 57 and y is 58.

[0008] Furthermore, the aforementioned multifunctional shell material HPSO is prepared from raw materials in the following weight ratio: 10-20 parts of oxaliplatin oxide, 1-10 parts of linker molecule, and 50-100 parts of HA-PEG; The structure of the connecting molecule is as follows: ; The structure of the HA-PEG is as follows: x is 57, y is 58; The structure of the oxaliplatin oxide is as follows: .

[0009] Furthermore, the aforementioned multifunctional shell material HPSO is prepared from raw materials in the following weight ratio: 10 parts of oxaliplatin oxide, 4-5 parts of linker molecules, and 50 parts of HA-PEG.

[0010] Furthermore, the preparation method of the oxaliplatin oxide includes the following steps: (a) Dissolve oxaliplatin in a solvent, add hydrogen peroxide to react, and obtain a reaction solution; (b) Dissolve and recrystallize the precipitate in the reaction solution to obtain oxaliplatin oxide; Preferably, In step (a), the solvent is water; And / or, in step (a), the concentration of hydrogen peroxide is 3% to 10%; And / or, in step (a), the mass-to-volume ratio of oxaliplatin to hydrogen peroxide is 1 g: (1~10) mL; And / or, in step (a), the reaction temperature is 20~40°C and the reaction time is 20~40h; And / or, in step (b), the solvent used for dissolution is anhydrous methanol; And / or, in step (b), the solvent for recrystallization is diethyl ether.

[0011] Furthermore, the preparation method of the HA-PEG includes the following steps: (A) Dissolve hyaluronic acid in a solvent, and then add EDCI and NHS for activation; (B) Add PEG 2000 -NH2 reacts; (C) After the reaction, dialyze and freeze-dry to obtain HA-PEG; Preferably, In step (A), the solvent is MES buffer; And / or, in step (A), the mass ratio of hyaluronic acid, EDCI and NHS is 100:1~10:1~10; And / or, in step (B), the PEG 2000 The mass ratio of -NH2 to hyaluronic acid is 1:1~5; And / or, in step (B), the reaction temperature is 20~40℃ and the reaction time is 30~60h.

[0012] This invention also provides a method for preparing the aforementioned multifunctional shell material HPSO, which includes the following steps: (1) The linker molecule is dissolved in a solvent, and oxaliplatin oxide, DMAP and DCC are added, and the reaction proceeds. (2) Add DMAP, HA-PEG and DCC to the reaction system obtained in step (1) and react; (3) Dialyze and freeze-dry the reaction product obtained in step (2) to obtain the final product.

[0013] Furthermore, In step (1), the solvent is formamide; And / or, in step (1), the mass ratio of oxaliplatin oxide, DMAP and DCC is 10:1~5:1~5; And / or, in step (1), the reaction is first carried out at 0~4℃ for 10~60 minutes, and then at 20~40℃ for 30~60 hours; And / or, in step (2), the mass ratio of HA-PEG, DMAP and DCC is 50:1~5:1~5; And / or, in step (2), the reaction is first carried out at 0~4℃ for 10~60 minutes, and then at 20~40℃ for 30~60 hours.

[0014] The present invention also provides the use of the aforementioned multifunctional shell material HPSO in the preparation of gene vectors.

[0015] Furthermore, the multifunctional shell material HPSO is used as the shell material for gene vectors; Preferably, the gene vector is a cationic polymer nonviral gene vector.

[0016] The present invention also provides a gene vector comprising the aforementioned multifunctional shell material HPSO.

[0017] This invention presents the first fully functional shell material, HPSO, for efficient tumor-targeted nanodrug delivery and immune activation. HPSO is composed of hyaluronic acid, polyethylene glycol, and oxidized oxaliplatin. HPSO can form stable nanoparticles with cationic carriers through electrostatic adsorption, enabling efficient gene delivery to tumor cells.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a multifunctional shell material, HPSO. When combined with a gene-carrying cationic polymer gene vector, HPSO shields its positive charge, giving the gene vector a negative charge. This negatively charged gene vector can be administered intravenously for in vivo therapy, overcoming the problem of hemolysis and other severe toxic side effects caused by the binding of existing cationic polymer gene vectors to negatively charged proteins in the blood. Furthermore, the transfection efficiency of the cationic polymer gene vector is not reduced after combining with this shell material. In addition, the non-viral shell material HPSO of this invention can enhance immune activation. HPSO has promising applications in the preparation of gene vectors for in vivo therapy.

[0019] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0020] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0021] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of HPSO, the non-viral shell material of this invention.

[0022] Figure 2 This is the Fourier transform spectrum of HPSO, the non-viral shell material of this invention.

[0023] Figure 3 The structural characterization results of HPSOC are shown in the following figures: a) Particle size of HPSOC; b) Potential characterization results of HPSOC; c) Agarose gel retardation experiment results. In the figures, 1:2, 1:5, 1:10, 1:15, and 1:20 represent the mass ratio of plasmid to PA (n=3).

[0024] Figure 4 The following figures illustrate the results of HPSOC cell transfection efficiency evaluation: a) shows the transfection efficiency of HPSOC in lung cancer LLC cells, with a scale bar of 100 μm; b) shows the statistical results of HPSOC transfection efficiency in LLC cells; n=3.

[0025] Figure 5 The following are the results of the immune activation evaluation of HPSOC: a) shows the expression results of DC cell maturation markers; b) shows the detection results of cytokines secreted by DC cells after activation; c) shows the detection results of cytokines secreted by T cells after activation. Detailed Implementation

[0026] The raw materials and equipment used in the specific embodiments of this invention are all known products, obtained by purchasing commercially available products. The main materials are as follows: Oxa: Oxaliplatin; HA: Hyaluronic acid; EDCI: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; NHS: N-hydroxysuccinimide; PEG 2000 -NH2: methoxy polyethylene glycol 2000-amino; DCC: N,N'-dicyclohexylcarbodiimide; DMAP: 4-dimethylaminopyridine.

[0027] In the specific embodiments of the present invention, the reactions involved, unless otherwise specified, are reactions at room temperature; "overnight" in the specific embodiments of the present invention refers to 10-12 hours; "room temperature" in the specific embodiments of the present invention refers to 20-40°C.

[0028] Example 1: Preparation of HPSO, a non-viral outer shell material of the present invention Step 1: Synthesis of O-Oxa Oxa (0.5 g, 1.26 mmol) was dissolved in 2 mL of water, and then 3.0 mL of 3% H2O2 solution was added dropwise. The mixture was then reacted at room temperature for 24 h. During this time, some product precipitated. The precipitated product was filtered and dissolved in 20 mL of anhydrous methanol. Then, 100 mL of diethyl ether was added to the solution and the mixture was heated for recrystallization. After filtering with filter paper and cooling to room temperature, the crystalline product was obtained, which was oxidized oxaliplatin (O-Oxa).

[0029] Step 2: Synthesis of HA-PEG Prepare a MES buffer solution with a pH of 5.5–6.0 in advance. Dissolve HA (100 mg, 0.2524 mmol) in 10 ml of MES buffer. After complete dissolution, add EDCI (5.8 mg, 0.03029 mmol) and NHS (3.5 mg, 0.03029 mmol) for catalysis. After activation for 2 hours, add PEG. 2000 -NH2 (the average molecular weight of PEG is 2000) (50 mg, 0.02524 mmol), reacted at room temperature for 48 h, then dialyzed in pure water using a dialysis bag with a molecular weight cutoff of 3500 Da. After dialysis, the PEG was freeze-dried to obtain HA-PEG. 2000 The obtained HA-PEG 2000 (HP) Score.

[0030] Step 3: Synthesis of HPSO A one-pot synthesis was performed. First, O-Oxa and s-linker were activated in formamide using a DCC / DMAP system. Then, without further processing, HP and a DCC / DMAP system were added for a second reaction, followed by dialyzing and lyophilization. Specifically, 4.9 mg of s-linker (0.0233 mmol) was dissolved in 5 mL of formamide. Then, O-Oxa (10 mg, 0.0233 mmol) and DMAP (1.71 mg, 0.014 mmol) were added under ice bath conditions. After 10 minutes, DCC (2.89 mg, 0.014 mmol) was added dropwise. The reaction was continued under ice bath conditions for 30 minutes, then the ice bath was removed, and the reaction was allowed to proceed at room temperature for 48 h. After 48 h, DMAP (1.71 mg, 0.014 mmol) and HA-PEG were added... 2000 50 mg of the sample was added to the reaction mixture, followed by the dropwise addition of DCC (2.89 mg, 0.014 mmol). The reaction was carried out in an ice bath for 30 minutes, then the ice bath was removed, and the reaction was allowed to proceed at room temperature for 48 hours. Finally, the product was dialyzed directly in pure water (using a dialysis bag with a molecular weight cutoff of 3500 Da), and then freeze-dried to obtain the shell material HPSO.

[0031] The structure of the s-linker is as follows: .

[0032] After characterization, the structure of the shell material HPSO obtained by this invention is shown below: HPSO Where x+y=115, x=57, y=58.

[0033] The following specific experimental examples demonstrate the beneficial effects of the present invention.

[0034] Example 1: Structural characterization of HPSO, the non-viral shell material of the present invention. 1. Experimental Methods Using nuclear magnetic resonance hydrogen spectroscopy (NMR) 1 The structure of the non-viral shell material HPSO prepared in Example 1 of this invention was confirmed by 1H NMR. Simultaneously, the structures of the raw material HA, product HP, HPS, and HPSO were analyzed by Fourier transform infrared spectroscopy. The preparation method of HPS is as follows: 4.9 mg of s-linker (0.0233 mmol) was dissolved in 5 mL of formamide, and DMAP (1.71 mg, 0.014 mmol) and HA-PEG were added. 2000 50 mg of the sample was added to the reaction mixture, followed by the dropwise addition of DCC (2.89 mg, 0.014 mmol). The reaction was carried out in an ice bath for 30 minutes, then the ice bath was removed, and the reaction was allowed to proceed at room temperature for 48 hours. Finally, the product was dialyzed directly in pure water (using a dialysis bag with a molecular weight cutoff of 3500 Da), and then freeze-dried to obtain HPS.

[0035] 2. Experimental Results The proton NMR spectrum is as follows: Figure 1 As shown, the Fourier transform infrared spectrum is as follows: Figure 2 As shown. By Figure 1 and Figure 2 It can be seen that the present invention successfully synthesized HPSO.

[0036] Test Example 2: Verification of the multifunctionality of the non-viral shell material HPSO of the present invention 1. Experimental Methods 1.1 Preparation of cationic gene vector PA The cationic gene vector PA was prepared according to the method described in Example 1 of Chinese Patent No. CN112210077B. The specific method is as follows: Arginine and polyethyleneimine with a molecular weight of 1800 were prepared according to a molar ratio of arginine to primary amino groups in polyethyleneimine of 1:2. First, 0.1 mol of arginine was dissolved in 9 mL of MES buffer, and 0.6 mg of EDCI / NHS was added as a catalyst. Catalysis was carried out at 25 °C for 4 h, with a mass ratio of EDCI to NHS of 1:2. Subsequently, 12.5 mmol of polyethyleneimine with a molecular weight of 1800 (1 mol of polyethyleneimine with a molecular weight of 1800 contains 16 mol of primary amino groups) and the catalyzed arginine were dissolved together in 12 mL of N-N-dimethylformamide to obtain a mixed solution. 0.5 mL of triethylamine was added to the mixed solution as a catalyst. The above reaction system was then placed at 25 °C and stirred for 72 h to carry out the substitution reaction. After the reaction was completed, the reaction system was transferred into a dialysis bag with a molecular weight cutoff of 1000 and dialyzed in double-distilled water for 3 days. The dialyzed solution was filtered through a 0.45 μm filter membrane and then freeze-dried to obtain a white powder product, which is the arginine-modified polyethyleneimine (PA) of this invention. The molar substitution ratio of primary amine hydrogen in the arginine-modified polyethyleneimine is 29%.

[0037] 1.2 Verification of the multifunctionality of the non-viral shell material HPSO of this invention The cationic gene vector PA and PX330 plasmid (pDNA) were mixed at a mass ratio of 5:1 and incubated for 20 minutes to form a PA@pDNA complex. Then, HPSO (HPSO, PA and pDNA in a mass ratio of 20:5:1, 20 / 5 / 1 μg) or HA (HA, PA and pDNA in a mass ratio of 20:5:1, 20 / 5 / 1 μg) prepared in Example 1 of this invention was added to the PA@pDNA complex, and the mixture was incubated for 25 minutes to form HPSOC or HAC, respectively.

[0038] This invention simulates a tumor microenvironment with high HAase expression. By modifying the conditions, a certain amount of hyaluronidase (HAase) was added to HPSOC, and the degradation products were analyzed. The specific experimental steps were as follows: HPSOC was added to 200 μL of a 100 μg / ml hyaluronidase (HAase) PBS solution (HPSOC concentration was 5 mg / ml), and then incubated at 37°C for 24 h to evaluate the hyaluronidase responsiveness. The obtained product was HPSOC + HAase.

[0039] HPSOC was added to 200 μL of 3% H2O2 (HPSOC concentration was 5 mg / ml), and then incubated at 37℃ for 24 h. The product obtained was HPSOC + H2O2.

[0040] The PA@pDNA complex, HPSOC, HPSOC+HAase product, and HPSOC+H2O2 product were analyzed: the particle size distribution, polydispersity index (PDI), and zeta potential of the PA@pDNA complex, HPSOC, HPSOC+HAase product, and HPSOC+H2O2 product were detected by dynamic light scattering (DLS).

[0041] pDNA and PA were incubated together at room temperature for 30 minutes at mass ratios of 1:2, 1:5, 1:10, 1:15, and 1:20. Then, HPSO, pDNA, and PA were incubated together for another 30 minutes at mass ratios of 20 μg, 2 μg, and 10 μg, respectively. The incubated nanoparticles were then added to the wells of an agarose gel and separated by electrophoresis at 120 V for 30 minutes.

[0042] 2. Experimental Results This invention measured the particle size and zeta potential of the PA@pDNA complex and HPSOC. The particle sizes of PA@pDNA and HPSOC were 92.5 ± 1.1 nm and 195.6 ± 0.5 nm, respectively. Figure 3 a). The zeta potentials of PA@pDNA and HPSOC were +19.7±1.6 mV and -30.5±2.0 mV, respectively. Figure 3 b).

[0043] To investigate the enzyme's responsiveness and degradability, HPSOC was co-incubated with hydrogen peroxide or hyaluronidase for 24 hours, respectively. After incubation, the particle size of HPSOC decreased from 195.6 ± 0.5 nm to 164.7 ± 2.5 nm and 106.2 ± 1.4 nm, respectively. Figure 3 a). Simultaneously, the Zeta potential of HPSOC changed from -30.5 ± 2.0 mV to -28.3 ± 2.1 mV and +15.4 ± 1.4 mV, respectively. Figure 3 b). Agarose gel electrophoresis showed that HPSOC could efficiently compress the loaded plasmid ( Figure 3 c).

[0044] Oxaliplatin in HPSO is a hydrophobic drug. This invention links it to HA via a chemical reaction, making it water-soluble. It can also form water-soluble nanoparticles without reducing transfection efficiency. Furthermore, it can activate the immune system.

[0045] The above experimental results demonstrate that the HPSOC prepared in this invention is responsive to hyaluronidase and H2O2.

[0046] Example 3: Verification of the transfection efficiency of HPSO, the non-viral outer shell material of the present invention. 1. Experimental Methods PEI 25K and PA were used as gene vectors to inoculate EGFP plasmids, resulting in PEI 25K@EGFP and PA@EGFP, respectively. PA was prepared according to the method described in Example 2. The mass ratio of PEI 25K to EGFP plasmid was 2.66 μg:2 μg, and after incubation for 20 minutes, PEI 25K@EGFP was obtained; the mass ratio of PA to EGFP plasmid was 20 μg:2 μg, and after incubation for 20 minutes, PA@EGFP was obtained. HPSO prepared in Example 1 of this invention (mass ratio of HPSO, PA, and EGFP plasmid was 40 μg:20 μg:2 μg) was added to PA@EGFP, and after incubation for 20 minutes, HPSO@EGFP was obtained. The prepared PEI 25K@EGFP, PA@EGFP, and HPSO@EGFP were added to LLC cells for transfection and co-incubation. After incubation for 6 hours, the medium was replaced with normal 1640 double-sided medium, and cultured for 48 hours. Fluorescence photography and flow cytometry were then performed.

[0047] 2. Experimental Results This invention verifies the tumor cell transfection capability of HPSOC, such as... Figure 4 As shown in 4a and 4b, the results indicate that, compared to the control group PEI 25K (transfection efficiency of approximately 32%), HPSOC exhibits a significantly higher cell transfection efficiency for lung cancer cells (transfection efficiency of approximately 90%), similar to that of cationic vectors. The experimental results demonstrate that adding the shell material HPSO of this invention does not reduce the transfection efficiency of the cationic gene vector.

[0048] Test Example 4: Toxicity Verification of HPSO, the Non-Virus Coating Material of the Present Invention 1. Experimental Methods The cytotoxicity of HPSO prepared in Example 1 was assessed using the MTT assay. LLC cells in logarithmic growth phase were seeded in 96-well plates (5000 cells / well) and cultured overnight to allow complete cell adhesion. Cells were treated with different concentrations (0, 2.5, 5, 10, 20, 40, 80, 100, 160, and 320 μg / mL) of HPSO for 48 hours. Then, 20 μL of MTT (5 mg / mL) was added to each well, and the cells were cultured for another 4 hours. Finally, the culture medium was discarded, and 150 μL of DMSO was added to each well to dissolve the formazan precipitate. The absorbance was measured at 570 nm to calculate cell viability. Each experiment was performed in triplicate.

[0049] 2. Experimental Results MTT assay results showed that the shell material HPSO prepared in this invention had no significant cytotoxicity and did not affect the expression of plasmid DNA delivered by the HPSOC system.

[0050] Experimental Example 5: Verification of the immune activation of HPSO, the non-viral shell material of the present invention. 1. Experimental Methods HPSOC was prepared according to the method described in Example 2. To investigate the in vitro activation and activating effects of HPSOC on DC cells and T cells, this invention set up an experiment involving HA and PEG. 2000 Oxa and HPSO were co-incubated with PA@pDNA (prepared according to the method described in Example 2) using the same method as in Example 2, except that HPSO was replaced. HPSO (which can be replaced with HA or PEG) was added to each well (6-well plate). 2000 The mass ratios of HAC, PEG, and Oxa, PA, and plasmid were 20 μg: 5 μg: 1 μg, and after 20 minutes, HAC and PEG were obtained, respectively. 2000 C, OxaC, and HPSOC. HAC and PEG. 2000 C and OxaC were mixed at a mass ratio of 1:1:1 (HPO group) and then added to LLC cells for transfection and co-incubation. HPSOC was added to LLC cells for transfection and co-incubation. After 6 hours, the medium was replaced with normal 1640 double-sided medium and cultured for 48 hours. Subsequently, the transfected tumor cells were co-incubated with DC cells for 72 hours. A portion of the DC cells were used to detect DC cell maturation and activation by flow cytometry (CD40, CD80, CD86, CD83) and ELISA (IFN-γ, TNF-α). Another portion of the DC cells were co-incubated with T cells, and after 72 hours, IFN-γ and TNF-α cytokines were detected by ELISA to characterize T cell activation.

[0051] 2. Experimental Results Experimental results show that ( Figure 5 In contrast, incubating HA, PEG, Oxa, and HPSO individually with the PA@pDNA complex and then co-incubating them with tumor cells (HPO group) did not induce the maturation and activation of dendritic cells (DCs) and T cells. This is mainly because the individual component combinations alone do not promote efficient plasmid transfection and immune activation. However, the HPSOC group effectively promoted DC maturation and activation, as well as T cell activation, indicating that HPSOC can effectively transfect tumor cells, enhance the synergistic tumor-killing ability of functional plasmids and drugs, and increase the release of effective antigens, thereby inducing immune activation. Therefore, this demonstrates that HPSOC is not simply a matter of component and functional superposition.

[0052] The above experimental results demonstrate that this invention successfully prepared a multifunctional shell material, HPSO, which carries a certain negative charge and can bind to cationic carriers, shielding them from positive charges. This overcomes the problem of existing cationic carriers binding to negatively charged proteins in blood, causing hemolysis and other highly toxic side effects. Furthermore, the shell material prepared from the specific raw materials of this invention exhibits a synergistic immune activation effect, and the transfection efficiency remains unchanged, showing excellent prospects for clinical application.

[0053] In summary, this invention provides a multifunctional shell material, HPSO. When combined with a gene-carrying cationic polymer gene vector, HPSO shields its positive charge, giving the gene vector a negative charge. This negatively charged gene vector can be administered intravenously for in vivo therapy, overcoming the problem of hemolysis and other severe toxic side effects caused by the binding of existing cationic polymer gene vectors to negatively charged proteins in the blood. Furthermore, the transfection efficiency of the cationic polymer gene vector is not reduced after combining with this shell material. In addition, the non-viral shell material HPSO of this invention can enhance immune activation. The HPSO shell material of this invention shows promising application prospects in the preparation of gene vectors for in vivo therapy.

Claims

1. A multifunctional shell material HPSO, characterized in that: The structure of the multifunctional shell material HPSO is shown in Formula I: Formula I Where x is 57 and y is 58.

2. The multifunctional shell material HPSO according to claim 1, characterized in that: It is made from the following raw materials in the following weight ratio: 10-20 parts of oxaliplatin oxide, 1-10 parts of linker molecule, and 50-100 parts of HA-PEG; The structure of the connecting molecule is as follows: ; The structure of the HA-PEG is as follows: x is 57, y is 58; The structure of the oxaliplatin oxide is as follows: .

3. The multifunctional shell material HPSO according to claim 2, characterized in that: It is made from the following raw materials in the following weight ratio: 10 parts of oxaliplatin oxide, 4-5 parts of linker molecules, and 50 parts of HA-PEG.

4. The multifunctional shell material HPSO according to claim 2 or 3, characterized in that: The preparation method of the oxaliplatin oxide includes the following steps: (a) Dissolve oxaliplatin in a solvent, add hydrogen peroxide to react, and obtain a reaction solution; (b) Dissolve and recrystallize the precipitate in the reaction solution to obtain oxaliplatin oxide; Preferably, In step (a), the solvent is water; And / or, in step (a), the concentration of hydrogen peroxide is 3% to 10%; And / or, in step (a), the mass-to-volume ratio of oxaliplatin to hydrogen peroxide is 1 g: (1~10) mL; And / or, in step (a), the reaction temperature is 20~40°C and the reaction time is 20~40h; And / or, in step (b), the solvent used for dissolution is anhydrous methanol; And / or, in step (b), the solvent for recrystallization is diethyl ether.

5. The multifunctional shell material HPSO according to claim 2 or 3, characterized in that: The preparation method of the HA-PEG includes the following steps: (A) Dissolve hyaluronic acid in a solvent, and then add EDCI and NHS for activation; (B) Add PEG 2000 -NH2 reacts; (C) After the reaction, dialyze and freeze-dry to obtain HA-PEG; Preferably, In step (A), the solvent is MES buffer; And / or, in step (A), the mass ratio of hyaluronic acid, EDCI and NHS is 100:1~10:1~10; And / or, in step (B), the PEG 2000 The mass ratio of -NH2 to hyaluronic acid is 1:1~5; And / or, in step (B), the reaction temperature is 20~40℃ and the reaction time is 30~60h.

6. The method for preparing the multifunctional shell material HPSO according to any one of claims 1 to 5, characterized in that: It includes the following steps: (1) The linker molecule is dissolved in a solvent, and oxaliplatin oxide, DMAP and DCC are added, and the reaction proceeds. (2) Add DMAP, HA-PEG and DCC to the reaction system obtained in step (1) and react; (3) Dialyze and freeze-dry the reaction product obtained in step (2) to obtain the final product.

7. The preparation method according to claim 6, characterized in that: In step (1), the solvent is formamide; And / or, in step (1), the mass ratio of oxaliplatin oxide, DMAP and DCC is 10:1~5:1~5; And / or, in step (1), the reaction is first carried out at 0~4℃ for 10~60 minutes, and then at 20~40℃ for 30~60 hours; And / or, in step (2), the mass ratio of HA-PEG, DMAP and DCC is 50:1~5:1~5; And / or, in step (2), the reaction is first carried out at 0~4℃ for 10~60 minutes, and then at 20~40℃ for 30~60 hours.

8. The use of the multifunctional shell material HPSO according to any one of claims 1 to 5 in the preparation of gene vectors.

9. The use according to claim 8, characterized in that: The multifunctional shell material HPSO is used as the shell material for gene vectors. Preferably, the gene vector is a cationic polymer nonviral gene vector.

10. A gene vector, characterized in that: It includes the multifunctional shell material HPSO as described in any one of claims 1 to 5.

Images