Gene delivery nanoparticle as well as preparation method and application thereof

Abstract

The invention provides a gene delivery nanoparticle and a preparation method and application thereof, and relates to the technical field of biological medicines.The preparation method comprises the steps that 2-((t-butyloxycarboryl) amino) ethyl methacrylate serves as a monomer, mPEG5k-RAFT serves as a macromolecular chain transfer agent, azodiisobutyronitrile serves as an initiator, a reaction is conducted in a dimethylformamide solvent, and the gene delivery nanoparticle is obtained. Carrying out reversible addition-fragmentation chain transfer polymerization reaction, dialyzing and freeze-drying to obtain an intermediate; dissolving the intermediate in dichloromethane, adding trifluoroacetic acid, and reacting to obtain a polymer polyethylene glycol-polymethylacrylic acid amino ethyl ester; the preparation method comprises the following steps: dissolving sparfloxacin in a hydrochloric acid solution, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, dissolving polyethylene glycol-poly (aminoethyl methacrylate) in dimethyl sulfoxide, adding the solution into the aqueous solution, dialyzing and freeze-drying to obtain the final product polyethylene glycol-polysparfloxacin. The preparation method comprises the following steps: putting polyethylene glycol-polysparfloxacin into an aqueous solution, and carrying out self-assembly to form nano particles.

Description

Technical Field

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

[0002] Immunotherapy is an efficient tumor treatment model following surgery, radiotherapy, chemotherapy and targeted therapy. It has shown a breakthrough effect in the treatment of recurrent and refractory tumors and has become an important part of comprehensive tumor treatment. Among them, dendritic cells (DCs) are important antigen presenting cells in the immune system. They can take up, process and present antigens to T cells, initiate immune responses, and enhance anti-tumor immune responses.

[0003] DCs take up antigens released by tumor cells through receptors on their surfaces. Antigens include proteins, peptides or other molecules on the surface of tumor cells. DCs process the taken-up antigens, break them down into small peptide fragments, and bind to major histocompatibility complex (MHC) molecules to form antigen-MHC complexes. The processed antigen-MHC complexes are carried by DCs and migrate to lymphoid organs, such as lymph nodes. In lymphoid organs, DCs interact with T cells and interact with receptors on the surface of T cells through co-stimulatory molecules on their surfaces, providing signals required for T cell activation. At the same time, the antigen-MHC complex is presented to T cells, allowing them to recognize and bind to specific antigens. Activated effector T cells have the ability to kill tumor cells or regulate immune responses. Some activated T cells will form immune memory cells, which can survive for a long time and quickly initiate immune responses when encountering the same antigen again, providing long-lasting anti-tumor immune protection. Through the above process, DCs can effectively take up, process and present tumor antigens, activate T cell-mediated immune responses, and thus guide the occurrence 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 to develop more effective anti-tumor immunotherapy methods. Gene delivery activation of 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 by 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, etc., to enhance their immune activation ability.

[0005] For example, genes encoding immunostimulatory molecules (cytokines, co-stimulatory molecules, etc.) are introduced into DCs to make them express these molecules on the cell surface, thereby enhancing the interaction between DCs and T cells and promoting the activation and proliferation of T cells; tumor antigen genes are introduced into DCs to make them express tumor-associated antigens, and then these DCs are returned to patients to induce specific anti-tumor immune responses. Gene delivery technology can also be used to regulate the functions of DCs, such as enhancing their antigen uptake ability, promoting their maturation and migration, etc., 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, survival and functional maintenance of DCs, etc. 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 a preparation method and application thereof to solve the problem of

[0008] In order 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 a macromolecular chain transfer agent, azobisisobutyronitrile is an initiator, and a reversible addition-fragmentation chain transfer polymerization reaction is carried out in a dimethylformamide solvent, and an intermediate is obtained by dialysis and freeze drying; the intermediate is dissolved in dichloromethane, and trifluoroacetic acid is added to react to obtain a 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, and then polyethylene glycol-polyaminoethyl methacrylate is dissolved in dimethyl sulfoxide and added to the above aqueous solution, and the final product polyethylene glycol-polysparfloxacin is obtained by dialysis and freeze drying; 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 polyethylene glycol-polysparfloxacin to the immune adjuvant CpG is at least greater than 3:1, the binding mass ratio of polyethylene glycol-polysparfloxacin to PD-L1 small interfering RNA is at least greater than 1:1, and the binding mass ratio of polyethylene glycol-polysparfloxacin to OVA messenger RNA is at least greater than 1:1.

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

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

[0018] According to a gene delivery nanoparticle and its preparation method and application provided by the present invention, the preparation method of the novel gene delivery nanoparticle for activating DCs of the present invention is simple and easy to operate, and is formed by self-assembly through the interaction between the hydrophilic and hydrophobic segments of the copolymer, does not involve cumbersome 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 delivery efficiency of genes in DCs, prepares a sparfloxacin prodrug with long circulation in the body through RAFT polymerization, and efficiently loads bisoxalate through self-assembly technology to obtain nanoparticles loaded with bisoxalate, and an appropriate concentration of polyethylene glycol-polysparfloxacin can increase the level of reactive oxygen ROS in DCs, and the bisoxalate in the nanoparticles can break and release energy, activate the aggregated sparfloxacin to release higher ROS, promote the escape of the delivered CpG, siPD-L1 and OVA-mRNA genes through the endosome, thereby effectively activating and promoting the maturation of DCs cells, and inducing 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 drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0020] Figure 1 The synthesis reaction diagram of 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 diagram of PESPA and PESPA@CPPO nanoparticles obtained by light scattering and the transmission electron microscope (TEM) image using an electron beam as the light source in Example 2;

[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 The effect of PESPA and PESPA@CPPO nanoparticles of 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] Fig. 9 The effect of PESPA@CpG and PESPA@CPPO@CpG nanoparticles of Example 5 on BMDC cell maturation;

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

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

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

[0032] Fig.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 specific embodiments of the present invention are described in detail below 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 used 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), 2-((tert-butoxycarbonyl)amino)ethyl methacrylate (1.14565 g, 5 mmol) and initiator azobisisobutyronitrile (AIBN) (1.3421 mg, 0.01 mmol) were added, and the mixture was evacuated and purged with argon three times. The mixture was subjected to reversible addition-fragmentation chain transfer polymerization at 70 °C for 24 hours, and the intermediate was obtained by dialyzing and freeze-drying.

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

[0038] Sparfloxacin SPA (2.3544 g, 6 mmol) was dissolved in 10 mL of hydrochloric acid solution, 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, 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 is 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 is the NMR spectrum of PESPA copolymer. Figure 2 The characteristic resonance peaks of PEG and SPA can be clearly observed, indicating that the synthesis of PESPA copolymer is successful and its 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 in 1 mL of trifluoroethanol, or dissolve 20 mg of PESPA polymer and 10 mg of CPPO in 1 mL of trifluoroethanol, and then add dropwise to 10 mL of Milli-Q ultrapure water at a drop rate of 100 μL / min, stirring while dropping. Then continue to stir the solution for 48-72 hours until the trifluoroethanol evaporates completely. 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, and 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, and the staining solution was absorbed with filter paper. 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 detected by 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. Each test was balanced for 2 min before each test. Each sample was tested three times and the average value was taken.

[0046] The nanoparticles were diluted with Milli-Q ultrapure water to a 0.1 mg / mL solution, and their absorption peaks were detected by UV-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. Figure 4 It can be seen that both nanoparticles are positively charged, about 22.52 mV and 20.74 mV respectively.

[0049] Figure 5 Figure 2 is the light scattering particle size distribution diagram of PESPA NPs and PESPA@CPPO NPs in this embodiment, where the average particle sizes are about 60nm and 80nm 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 8×10 4 / well were seeded in a 12-well plate and divided into Control group, PESPA NPs group, and PESPA@CPPO NPs group. The dosage concentration of PESPA NPs and PESPA@CPPO NPs nanoparticles was 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 level 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 and incubated 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 no matter whether the nanoparticles were loaded with CPPO or not, there was no development at the incubation ratio of 1:3-1:5, while the fluorescence of the CpG positive control 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 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 2×10 5 / well were seeded 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. After 24 hours of administration, the cells were incubated with CD11c, CD80, and CD86 antibodies for 1 hour and detected by flow cytometer.

[0058] Fig. 9 This is the detection of the ability of the PESPA@CPPO@CpG nanoparticles of this example to promote the maturation of BMDC cells. Fig. 9 The results showed that the nanoparticles in the PESPA@CpG group and the PESPA@CPPO@CpG group could significantly increase the 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] In order 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 detected by 1% agarose gel electrophoresis at a constant voltage of 110 V for 20 minutes.

[0061] Fig.10 The results of gel UV imaging showed that regardless of whether the nanoparticles were loaded with CPPO, there was no development 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 2×10 5 / well were seeded in a 12-well plate 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. After 24 hours of administration, the cells were incubated with CD40, CD86, and SIINFEKL-H-2Kb antibodies for 1 hour and detected by flow cytometer.

[0064] Fig.11 This is a test of the ability of the PESPA@CPPO@OVA mRNA nanoparticles in Example 5 to promote the cross-presentation of antigens by BMDCs cells. Fig.11 The results showed that the nanoparticles in the PESPA@OVA mRNA group and the PESPA@CPPO@OVA mRNA group could significantly increase the fluorescence intensity of CD40 and CD86 of 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 with 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] Fig.12 The results of gel UV imaging 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 8×10 4The cells were inoculated at a density of 1.5 μg / mL in the Plasmid group and 1.5 μg / mL in the nanoparticle group. The cells were seeded at a density of 1.5 μg / mL and 1.0 μg / mL in the nanoparticle group. The cells were then divided into Control group, Plasmid group, lipo2000+Plasmid group, PESPA@Plasmid group and PESPA@CPPO@Plasmid group. The concentration of the Plasmid group was 1.5 μg / mL, and the concentration of the nanoparticles was 10 μg / mL. Pictures were taken with an inverted fluorescence microscope 24 hours after administration.

[0070] Fig.13 The fluorescence photography results of the plasmid transfection effect on DC2.4 cells showed that the plasmid transfection effect of lipo2000 reagent on DC2.4 cells was not obvious, and the positive cells had little green fluorescence, while the fluorescence of PESPA NPs and PESPA@CPPO NPs 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 protection scope of the present invention shall be subject to the scope defined by the claims.

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

[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 enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be 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 will not be limited to the embodiments shown herein, but rather 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: With 2-((tert-butyloxycarbonyl)amino)ethyl methacrylate as monomer, mPEG 5k -RAFT is a macromolecular chain transfer agent, azobisisobutyronitrile is an initiator, and 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 a polymer polyethylene glycol-polyaminoethyl methacrylate; Dissolving sparfloxacin in a hydrochloric acid solution, and adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, then dissolving polyethylene glycol-polyaminoethyl methacrylate in dimethyl sulfoxide and adding the mixture to the aqueous solution, dialyzing and freeze-drying to obtain the final product polyethylene glycol-polysparfloxacin; Polyethylene glycol-polysparfloxacin is placed in an aqueous solution and self-assembled to form nanoparticles.

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

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

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

5. A novel gene delivery nanoparticle, characterized in that: The gene delivery nanoparticles are prepared by the preparation method of claims 1-4, comprising nanoparticles formed by self-assembly of a polymer polyethylene glycol-polysparfloxacin, wherein the structural formula of the polyethylene glycol-polysparfloxacin is 6. The gene delivery nanoparticle according to claim 5, characterized in that The particle size of the nanoparticles is 50-250nm.

7. The gene delivery nanoparticle according to claim 5, characterized in that The nanoparticles can bind to the immune adjuvant CpG, PD-L1 small interfering RNA and 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 be used as carriers to transfect plasmids into dendritic cells.

10. Application of a novel gene delivery nanoparticle in tumor treatment, characterized in that: The nanoparticle is the gene delivery nanoparticle according to any one of claims 5-9.

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