Gene delivery nanoparticles and their preparation method and application

Abstract

The present invention provides a gene delivery nanoparticle and its preparation method and application, which relates to the field of biomedicine. The preparation method comprises: using 2-((tert-butyloxycarbonyl)amino)ethyl methacrylate as a monomer, mPEG 5k The invention relates to a method for preparing a novel nanostructured polymer comprising: a polymer comprising polyethylene glycol-poly(aminoethyl methacrylate) ester, ...

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to dendritic cell gene delivery nanoparticles. Background Art

[0002] Immunotherapy is a highly effective cancer treatment modality following surgery, radiotherapy, chemotherapy, and targeted therapy. It has demonstrated breakthrough efficacy in the treatment of recurrent and refractory tumors and has become a crucial component of comprehensive cancer treatment. Dendritic cells (DCs) are key antigen-presenting cells in the immune system. They can take up, process, and present antigens to T cells, initiating immune responses and enhancing anti-tumor immunity.

[0003] DCs, through receptors on their surfaces, absorb antigens released by tumor cells. Antigens include proteins, peptides, or other molecules on the tumor cell surface. They then process the antigens, breaking them down into small peptide fragments that bind to major histocompatibility complex (MHC) molecules to form antigen-MHC complexes. These processed antigen-MHC complexes are carried by DCs and migrate to lymphoid organs, such as lymph nodes. Within these organs, DCs interact with T cells and, through co-stimulatory molecules on their surfaces, interact with receptors on T cell surfaces, providing the signals necessary for T cell activation. Simultaneously, the antigen-MHC complexes are presented to T cells, enabling them to recognize and bind to specific antigens. Activated effector T cells have the ability to kill tumor cells or modulate immune responses. Some activated T cells become memory cells, which can survive long-term and rapidly initiate immune responses upon re-encountering the same antigen, providing long-lasting anti-tumor immune protection. Through these processes, DCs are able to efficiently absorb, process, and present tumor antigens, activating T cell-mediated immune responses and thereby guiding the onset of anti-tumor immune responses. In addition, DCs can regulate the function of immune cells by secreting cytokines and chemokines, further enhancing the anti-tumor immune response.

[0004] In order to enhance the anti-tumor immune response of DCs, researchers are exploring various methods, such as optimizing the culture and activation conditions of DCs, using tumor-specific antigens or antigenic peptides for loading, and combining with other immunotherapy strategies. These studies aim to improve the immune activation ability of DCs in order to develop more effective anti-tumor immunotherapy methods. Gene delivery to activate DCs is an immunotherapy strategy that aims to enhance the antigen presentation ability and immunostimulatory activity of DCs by genetically modifying them, thereby inducing an effective anti-tumor immune response. Genes can be DNA, RNA or other biological molecules, which can be combined with nanoparticles through physical adsorption, chemical binding or electrostatic interaction. Through gene delivery technology, specific genes can be introduced into DCs to express immunostimulatory molecules, co-stimulatory molecules or tumor antigens to enhance their immune activation ability.

[0005] For example, genes encoding immunostimulatory molecules (cytokines, co-stimulatory molecules, etc.) can be introduced into DCs, causing them to express these molecules on their cell surface, thereby enhancing the interaction between DCs and T cells and promoting T cell activation and proliferation. Tumor antigen genes can also be introduced into DCs, causing them to express tumor-associated antigens. These DCs can then be infused back into the patient to induce a specific anti-tumor immune response. Gene delivery techniques can also be used to modulate the functions of DCs, such as enhancing their antigen uptake capacity, promoting their maturation and migration, thereby improving the immune activation effect of DCs. Research on gene delivery to activate DCs is of great significance in tumor immunotherapy.

[0006] However, this technology still faces some challenges, such as low gene transfection efficiency, poor stability of gene expression, and maintenance of DCs’ survival and function. So far, an ideal low-toxic and high-efficiency DCs gene delivery reagent has not been found for clinical practice. Summary of the Invention

[0007] The purpose of the present invention is to provide a gene delivery nanoparticle and its preparation method and application to solve

[0008] To solve the above problems, the present invention first provides a method for preparing gene delivery nanoparticles, comprising the following steps: using 2-((tert-butyloxycarbonyl)amino)ethyl methacrylate as a monomer, mPEG 5k-RAFT is used as a macromolecular chain transfer agent and azobisisobutyronitrile is used as an initiator. A reversible addition-fragmentation chain transfer polymerization reaction is carried out in a dimethylformamide solvent, and the intermediate is dialyzed and freeze-dried to obtain the intermediate; the intermediate is dissolved in dichloromethane, and trifluoroacetic acid is added to react to obtain the polymer polyethylene glycol-polyaminoethyl methacrylate; sparfloxacin is dissolved in a hydrochloric acid solution, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added. The polyethylene glycol-polyaminoethyl methacrylate is then dissolved in dimethyl sulfoxide and added to the above aqueous solution, and the final product polyethylene glycol-polysparfloxacin is obtained by dialyzing and freeze-drying; the polyethylene glycol-polysparfloxacin is placed in an aqueous solution and self-assembled to form nanoparticles.

[0009] Furthermore, the concentration of the polyethylene glycol-polysparfloxacin is 1-40 mg / mL.

[0010] Furthermore, the nanoparticles can be self-assembled to form empty nanoparticles and bisoxalate-loaded nanoparticles.

[0011] Furthermore, the concentration of the bisoxalate is 0.5-20 mg / mL.

[0012] The present invention also provides a novel gene delivery nanoparticle, which is prepared by the preparation method of the gene delivery nanoparticle described in the above technical solution, and comprises nanoparticles formed by self-assembly of the polymer polyethylene glycol-polysparfloxacin, wherein the structural formula of the polyethylene glycol-polysparfloxacin is

[0013] Furthermore, the particle size of the nanoparticles is 50-250 nm.

[0014] Furthermore, the nanoparticles can bind to the immune adjuvant CpG, PD-L1 small interfering RNA and OVA messenger RNA respectively through electrostatic adsorption.

[0015] Furthermore, the binding mass ratio of the polyethylene glycol-polysparfloxacin to the immune adjuvant CpG is at least greater than 3:1, the binding mass ratio of the polyethylene glycol-polysparfloxacin to the PD-L1 small interfering RNA is at least greater than 1:1, and the binding mass ratio of the polyethylene glycol-polysparfloxacin to the OVA messenger RNA is at least greater than 1:1.

[0016] Furthermore, the nanoparticles can serve as carriers to transfect plasmids into dendritic cells.

[0017] The present invention also provides a novel gene delivery nanoparticle for use in tumor treatment, characterized in that the nanoparticle is the gene delivery nanoparticle described in the above technical solution.

[0018] The present invention provides a gene delivery nanoparticle, its preparation method, and application. The preparation method of the novel DC-activating gene delivery nanoparticle of the present invention is simple and easy to operate. It is formed by self-assembly through the interaction between the hydrophilic and hydrophobic segments of the copolymer, does not involve tedious chemical reactions and toxic organic chemical reagents, and is conducive to production and clinical transformation. The present invention proposes a new strategy to improve the efficiency of gene delivery in DCs. Through RAFT polymerization, a long-circulating sparfloxacin prodrug in the body is prepared. The bisoxalate is efficiently loaded through self-assembly technology to obtain bisoxalate-loaded nanoparticles. Appropriate concentrations of polyethylene glycol-polysparfloxacin can increase the level of reactive oxygen species (ROS) in DCs. The bisoxalate in the nanoparticles can break and release energy, activating the aggregated sparfloxacin to release higher ROS, promoting the escape of the delivered CpG, siPD-L1, and OVA-mRNA genes through endosomal escape, thereby effectively activating and promoting DC cell maturation and inducing a systemic immune response. The present invention provides technical support for the early realization of the clinical transformation of nano-drug activated immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0020] Figure 1 The synthesis reaction diagram of the PESPA copolymer provided in Example 1;

[0021] Figure 2 is the nuclear magnetic resonance spectrum of the PESPA copolymer in Example 1;

[0022] Figure 3 UV spectra of SPA, PESPA, CPPO, and PESPA@CPPO nanoparticles of Example 2;

[0023] Figure 4 The potential diagram of PESPA and PESPA@CPPO nanoparticles in Example 2;

[0024] Figure 5 The particle size distribution of PESPA and PESPA@CPPO nanoparticles of Example 2 obtained by light scattering, and the transmission electron microscopy (TEM) image using an electron beam as the light source;

[0025] Figure 6The particle size diagram of PESPA and PESPA@CPPO nanoparticles in Example 2 obtained by light scattering in PBS and PBS containing 10% fetal bovine serum (FBS);

[0026] Figure 7 Effects of PESPA and PESPA@CPPO nanoparticles in Example 3 on ROS levels in DC2.4 cells;

[0027] Figure 8 The gel electrophoresis diagram of DNA bound to CpG in Example 4 and PESPA and PESPA@CPPO nanoparticles at different concentration ratios;

[0028] Figure 9 The effect of PESPA@CpG and PESPA@CPPO@CpG nanoparticles on BMDC cell maturation in Example 5;

[0029] Figure 10 The gel electrophoresis diagram of DNA bound to OVA-mRNA and PESPA and PESPA@CPPO nanoparticles at different concentration ratios in Example 6;

[0030] Figure 11 The effects of PESPA@OVA mRNA and PESPA@CPPO@OVA mRNA nanoparticles in Example 7 on BMDC cell maturation and antigen cross-presentation ability;

[0031] Figure 12 The DNA gel electrophoresis diagram of siPD-L1 bound to PESPA@CPPO nanoparticles at different concentration ratios in Example 8;

[0032] Figure 13 This is a fluorescence image of the transfection of the plasmid H128pLenti-EF1a-EGFP-3FLAG-PGK-Puro by PESPA and PESPA@CPPO nanoparticles in Example 9. DETAILED DESCRIPTION

[0033] In order to make the above-mentioned objects, features and advantages of the present invention more clearly understood, the following detailed description of the specific embodiments of the present invention is given in conjunction with the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0034] Example 1 Synthesis of Copolymer PESPA

[0035] The preparation process of the copolymer polyethylene glycol-polysparfloxacin is as follows, and it is named PESPA:

[0036] First, the macromolecular chain transfer agent mPEG 5k-RAFT (0.26735 g, 0.05 mmol) was dissolved in 4 mL of dimethylformamide (DMF), and 2-((tert-butoxycarbonyl)amino)ethyl methacrylate (1.14565 g, 5 mmol) and initiator azobisisobutyronitrile (AIBN) (1.3421 mg, 0.01 mmol) were added. The mixture was evacuated and purged with argon three times. Reversible addition-fragmentation chain transfer polymerization was carried out at 70 ° C for 24 hours, and the intermediate was obtained by dialyzed and freeze-dried.

[0037] The intermediate was dissolved in 5 mL of dichloromethane (DCM), and 5 mL of trifluoroacetic acid (TFA) was added. The mixture was stirred at room temperature for 6 hours to obtain a polymer, polyethylene glycol-polyaminoethyl methacrylate, which was named PEPA.

[0038] Sparfloxacin SPA (2.3544 g, 6 mmol) was dissolved in 10 mL of hydrochloric acid solution, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC (1.7253 g, 9 mmol) and N-hydroxysuccinimide NHS (2.07144 g, 18 mmol) were added. PEPA was dissolved in 5 mL of dimethyl sulfoxide (DMSO) and added to the above aqueous solution. The mixture was reacted at room temperature for 5 days, dialyzed and freeze-dried to obtain the final product PESPA.

[0039] Finally, the chemical composition of the polymer was analyzed by nuclear magnetic resonance, mass spectrometry and other techniques.

[0040] Figure 1 This is the synthesis reaction diagram of PESPA copolymer in this embodiment; Figure 2 The NMR spectrum of PESPA copolymer is shown in FIG. Figure 2 The characteristic resonance peaks of PEG and SPA can be clearly observed, indicating that the synthesis of PESPA copolymer is successful and the structure and composition are clear.

[0041] Example 2 Preparation of PESPA NPs and PESPA@CPPO NPs

[0042] The unloaded nanoparticles were named PESPA NPs, and the bisoxalate-loaded nanoparticles were named PESPA@CPPONPs.

[0043] Dissolve 20 mg of PESPA polymer or 20 mg of PESPA polymer and 10 mg of CPPO in 1 mL of trifluoroethanol. Add the solution dropwise to 10 mL of Milli-Q ultrapure water at a rate of 100 μL / min with stirring. Continue stirring for 48–72 hours until the trifluoroethanol evaporates. Finally, centrifuge at 3000 rpm for 5 minutes to obtain PESPA NPs and PESPA@CPPO NPs.

[0044] The nanoparticles were diluted to a 1 mg / mL solution with Milli-Q ultrapure water. 10-20 μL of the sample was added dropwise to a 230-mesh copper grid containing a carbon support film and dried at a constant temperature. 5 μL of 1-2% uranyl acetate was stained for 5 min, the staining solution was absorbed with filter paper, and after drying at room temperature, the sample morphology was observed using a transmission electron microscope (TEM, FEI, Tecnai G220S-TWIN, 200 kV, USA).

[0045] The nanoparticles were diluted to a 1 mg / mL solution with Milli-Q ultrapure water, and the particle size and potential size were measured using a laser particle size analyzer (DLS, Malvern, UK, Zetasizer NanoZS) at a temperature of 25°C, an angle of 90°, and a laser wavelength of 633 nm. The sample was balanced for 2 min before each measurement. Each sample was tested three times, and the average value was taken.

[0046] The nanoparticles were diluted to a 0.1 mg / mL solution with Milli-Q ultrapure water, and their absorption peaks were detected by ultraviolet-visible spectrophotometer (UV) at a temperature of 25 °C, an angle of 90°, and a laser wavelength of 200-800 nm. Each sample was tested three times and the average value was taken.

[0047] See the test charts for Figure 3-6 .in, Figure 3 The UV spectra of PESPA NPs and PESPA@CPPO NPs in this embodiment are shown in FIG. Figure 3 It can be seen that the absorption peaks of PESPA@CPPO NPs and CPPO overlap at around 400 nm, indicating the successful loading of CPPO.

[0048] Figure 4 The potential diagram of PESPA NPs and PESPA@CPPO NPs in this embodiment is shown in FIG. Figure 4 It can be seen that both nanoparticles are positively charged, about 22.52 mV and 20.74 mV, respectively.

[0049] Figure 5 The light scattering particle size distribution of PESPA NPs and PESPA@CPPO NPs in this embodiment is shown in Figure 2, where the average particle sizes are about 60 nm and 80 nm, respectively. Figure 5 It can also be seen from the TEM images that both PESPA NPs and PESPA@CPPO NPs nanoparticles have regular spherical structures, and the particle size is consistent with the DLS results.

[0050] Example 3 Effects of PESPA NPs and PESPA@CPPO NPs on ROS levels in DC2.4 cells

[0051] In order to detect the effects of PESPA NPs and PESPA@CPPO NPs on the ROS level of DC2.4 cells, DC2.4 cells were cultured at a density of 8 × 10 4 The cells were seeded at a density of 100 μg / well in a 12-well plate and divided into control group, PESPA NPs group, and PESPA@CPPO NPs group. The PESPA NPs and PESPA@CPPO NPs nanoparticles were administered at a concentration of 20 μg / mL. 24 hours after administration, the ROS level in DC2.4 cells was detected using the oxidized diacetate of 2',7'-dichlorodihydrofluorescein (DCFH-DA) fluorescent probe.

[0052] Figure 7 The flow cytometry results of ROS levels in DC2.4 cells showed that compared with the control group, the proportion of ROS-positive cells in the PESPA NPs and PESPA@CPPO NPs groups increased significantly, indicating that both PESPA NPs and PESPA@CPPO NPs nanoparticles can effectively increase the ROS levels in DC2.4 cells, and at the same concentration, PESPA@CPPO NPs can cause DC2.4 cells to produce higher levels of ROS.

[0053] Example 4 Determination of Binding Efficiency of PESPA NPs and PESPA@CPPO NPs to CpG

[0054] In order to detect the binding efficiency of PESPA NPs and PESPA@CPPO NPs with CpG, CpG (1 mg / mL) was mixed with PESPA NPs (1 mg / mL) and PESPA@CPPO NPs (1 mg / mL) at different concentration ratios. After incubation at room temperature for 1 hour, the binding efficiency with CpG was detected by 1% agarose gel electrophoresis at a constant voltage of 110 V for 20 minutes.

[0055] Figure 8 The results of gel UV imaging showed that regardless of whether the nanoparticles were loaded with CPPO, no fluorescence was observed at the incubation ratio of 1:3-1:5, while the CpG positive control fluorescence was significant, indicating that PESPA@CpG and PESPA@CPPO@CpG at all concentration gradients starting from 1:3 were successfully bound.

[0056] Example 5 Detection of the ability of PESPA@CPPO@CpG nanoparticles to promote the maturation of bone marrow-derived dendritic cells (BMDC)

[0057] Bone marrow stem cells were extracted from C57 mice and cultured in GM-CSF+IL-4+1640 medium+inactivated FBS for 5 days. BMDC cells were cultured at a density of 2×10 5The cells were seeded at a density of 1 / well in a 12-well plate and divided into control group, CpG group, PESPA@CpG group and PESPA@CPPO@CpG group. The CpG administration concentration was 2 μg / mL, and the nanoparticle administration concentration was 10 μg / mL. Twenty-four hours after administration, the cells were incubated with CD11c, CD80 and CD86 antibodies for 1 hour and detected by flow cytometry.

[0058] Figure 9 This is the detection of the ability of the PESPA@CPPO@CpG nanoparticles of this example to promote the maturation of BMDC cells. Figure 9 The results showed that the nanoparticles in the PESPA@CpG and PESPA@CPPO@CpG groups could significantly increase the expression of CD80 + CD86 + The ratio of BMDCs cells indicated that both nanoparticles could effectively promote the maturation of DCs cells.

[0059] Example 6 Determination of Binding Efficiency of PESPA NPs and PESPA@CPPO NPs to OVA mRNA

[0060] To detect the binding efficiency of PESPA NPs and PESPA@CPPO NPs with OVA mRNA, OVA mRNA (1 mg / mL) and NPs (1 mg / mL) were mixed at different concentration ratios and incubated at room temperature for 1 hour. The binding efficiency with OVA mRNA was then detected by 1% agarose gel electrophoresis at a constant voltage of 110 V for 20 minutes.

[0061] Figure 10 The gel UV imaging results showed that regardless of whether the nanoparticles were loaded with CPPO, no image was detected in the incubation ratio of 1:1-1:10, while the OVA mRNA positive control fluorescence was significant, indicating that PESPA@OVA mRNA and PESPA@CPPO@OVA mRNA at all concentration gradients starting from 1:1 were successfully bound.

[0062] Example 7 Detection of the cross-presentation ability of PESPA@CPPO@OVA mRNA nanoparticles on bone marrow-derived dendritic cells (BMDCs)

[0063] Bone marrow stem cells were extracted from C57 mice and cultured in GM-CSF+IL-4+1640 medium+inactivated FBS for 5 days. BMDCs were cultured at a density of 2×10 5The cells were seeded at a density of 10 μg / mL in 12-well plates and divided into control group, OVA mRNA group, PESPA@OVAmRNA group and PESPA@CPPO@OVA mRNA group. The OVA mRNA administration concentration was 2 μg / mL, and the nanoparticle administration concentration was 10 μg / mL. Twenty-four hours after administration, the cells were incubated with CD40, CD86, and SIINFEKL-H-2Kb antibodies for 1 hour and detected by flow cytometry.

[0064] Figure 11 This is a test of the ability of PESPA@CPPO@OVA mRNA nanoparticles in Example 5 to promote the cross-presentation of antigens by BMDCs cells. Figure 11 The results showed that the PESPA@OVA mRNA group and the PESPA@CPPO@OVA mRNA group nanoparticles could significantly increase the fluorescence intensity of CD40 and CD86 on BMDCs cells, indicating that both nanoparticles could effectively promote the maturation of DC cells. The high expression of SIINFEKL-H-2Kb antibody in the PESPA@OVA mRNA group and the PESPA@CPPO@OVA mRNA group represented that the two nanoparticles effectively promoted the presentation of OVA mRNA antigen by DC cells.

[0065] Example 8 Determination of Binding Efficiency of PESPA@CPPO NPs and siPD-L1

[0066] In order to detect the binding efficiency of PESPA@CPPO NPs to siPD-L1, siPD-L1 (1 mg / mL) and PESPA@CPPO NPs (1 mg / mL) were mixed at different concentration ratios and incubated at room temperature for 1 hour. The binding efficiency with siPD-L1 was detected by 1% agarose gel electrophoresis at a constant voltage of 110 V for 20 minutes.

[0067] Figure 12 The gel UV imaging results showed that there was no development at incubation ratios of 1:3-1:15, while the siPD-L1 positive control fluorescence was significant, indicating that PESPA@CPPO@siPD-L1 at all concentration gradients starting from 1:3 was successfully bound.

[0068] Example 9 Determination of Plasmid Transfection Efficiency of PESPA NPs and PESPA@CPPO NPs in DC2.4 Cells

[0069] In order to detect the plasmid transfection efficiency of PESPA NPs and PESPA@CPPO NPs on DC2.4 cells, DC2.4 cells were cultured at a density of 8×10 4The cells were seeded at a density of 10 μg / mL / well in a 12-well plate and divided into control group, Plasmid group, lipo2000+Plasmid group, PESPA@Plasmid group and PESPA@CPPO@Plasmid group. The Plasmid group was administered at a concentration of 1.5 μg / mL, and the nanoparticles were administered at a concentration of 10 μg / mL. Pictures were taken using an inverted fluorescence microscope 24 hours after administration.

[0070] Figure 13 The fluorescence photography results of the plasmid transfection effect of DC2.4 cells showed that the plasmid transfection effect of DC2.4 cells using lipo2000 reagent was not obvious, and the positive cells had little green fluorescence, while the fluorescence of the PESPA NPs and PESPA@CPPO NPs groups was significant, indicating that both PESPA NPs and PESPA@CPPO NPs nanoparticles can improve the plasmid transfection efficiency of DC2.4 cells.

[0071] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the scope defined by the claims.

[0072] Finally, it should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or device. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of additional identical elements in the process, method, article, or device comprising the element.

[0073] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0074] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing gene delivery nanoparticles, characterized in that: The following steps are involved: Using 2-((tert-butyloxycarbonyl)amino)ethyl methacrylate as monomer, mPEG 5k -RAFT is a macromolecular chain transfer agent and azobisisobutyronitrile is an initiator. In dimethylformamide solvent, a reversible addition-fragmentation chain transfer polymerization reaction is carried out, and the intermediate is obtained by dialysis and freeze-drying. The intermediate is dissolved in dichloromethane and trifluoroacetic acid is added to react to obtain the polymer polyethylene glycol-polyaminoethyl methacrylate; Sparfloxacin is dissolved in a hydrochloric acid solution, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added. Polyethylene glycol-polyaminoethyl methacrylate is dissolved in dimethyl sulfoxide and added to the above solution. The product is dialyzed and freeze-dried to obtain the final product, polyethylene glycol-polysparfloxacin. Polyethylene glycol-polysparfloxacin is placed in an aqueous solution and self-assembles to form nanoparticles.

2. The method for preparing gene delivery nanoparticles according to claim 1, wherein: The concentration of the polyethylene glycol-polysparfloxacin is 1-40 mg / mL.

3. The method for preparing gene delivery nanoparticles according to claim 1, wherein: It can self-assemble to form empty nanoparticles and bisoxalate-loaded nanoparticles.

4. The method for preparing gene delivery nanoparticles according to claim 3, wherein: The concentration of the bisoxalate is 0.5-20 mg / mL.

5. A gene delivery nanoparticle, characterized in that The gene delivery nanoparticles are prepared by the preparation method of any one of claims 1 to 4.

6. The gene delivery nanoparticle according to claim 5, characterized in that The particle size of the nanoparticles is 50-250 nm.

7. The gene delivery nanoparticle according to claim 5, characterized in that The nanoparticles can be bound to the immune adjuvant CpG, PD-L1 small interfering RNA or OVA messenger RNA respectively through electrostatic adsorption.

8. The gene delivery nanoparticle according to claim 7, characterized in that The binding mass ratio of the polyethylene glycol-polysparfloxacin to the immune adjuvant CpG is at least greater than 3:1, the binding mass ratio of the polyethylene glycol-polysparfloxacin to the PD-L1 small interfering RNA is at least greater than 1:1, and the binding mass ratio of the polyethylene glycol-polysparfloxacin to the OVA messenger RNA is at least greater than 1:

1.

9. The gene delivery nanoparticle according to claim 5, characterized in that The nanoparticles can serve as carriers to transfect plasmids into dendritic cells.

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