Sulfonium co-polymeric peptide-based nucleic acid nanovesicles, methods of making and uses thereof

Abstract

The application discloses a kind of nucleic acid nano vesicles based on sulfonium copolymerization peptide and its preparation method and application.The vesicle is formed by the electrostatic self-assembly of a kind of sulfonium copolymerization peptide and polynucleotide, and is stable nano vesicle with hollow structure.The vesicle is different from conventional solid nanoparticles, and its unique hollow structure provides space for co-loading other therapeutic agents (such as enzymes, chemotherapeutic drugs).The application also provides a preparation method of the nano vesicle, and its application as a carrier for simultaneously loading nucleic acid drugs and other active ingredients for synergistic treatment.The nano vesicle has extraordinary long-term storage stability, and can deliver siRNA and catalase to tumor sites, block CD47 "don't eat me" signal through gene silencing, and enzymatically degrade immunosuppressive factor H2O2 in tumor microenvironment, thereby remodeling tumor immune microenvironment, efficiently activating macrophages, dendritic cells and T cells, and producing strong synergistic anti-tumor immune effect.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine delivery and tumor immunotherapy technology, specifically relating to nucleic acid nanovesicles based on thioonium copolymer peptides, their preparation methods, and applications. Background Technology

[0002] The success of tumor immunotherapy is often limited by the low immunogenicity of tumors and the immunosuppressive microenvironment. For example, the CD47 protein, highly expressed by tumor cells, sends a "don't eat me" signal to macrophages, helping them evade immune surveillance; simultaneously, high levels of metabolites such as hydrogen peroxide (H2O2) in the tumor microenvironment inhibit immune cell function, creating an immunosuppressive microenvironment. Therefore, developing combination therapies that can simultaneously silence immune checkpoint genes and modulate the immunosuppressive microenvironment is of great significance. However, efficiently co-delivering drugs with different properties (such as hydrophilic macromolecules and biological enzymes) to the same tumor cells and achieving synergistic effects remains a major challenge for current delivery technologies.

[0003] Traditional liposomes or polymer carriers, when combined with nucleic acids, often form solid nanoparticles, which limit the space and manner of drug delivery, making it difficult to achieve co-delivery and synergistic delivery of different types of drugs (Delivering more for less: nanosized, minimal-carrier and pharmacoactive drug delivery systems. Advanced Drug Delivery Reviews, 2021, 11(179), 113994; The RNA delivery dilemma—lipid versus polymer nanoparticle platforms. Drug Delivery and Translational Research, 2026.).

[0004] This invention discovers that thionium copolymer peptides with specific structures can self-assemble into hollow nanovesicles with uniform structure and controllable size through simple mixing. This unique vesicle structure provides an ideal platform for solving the aforementioned co-delivery challenges: its bilayer membrane structure originates from the electrostatic assembly of polymers and nucleic acids, endowing it with excellent nucleic acid binding and protection capabilities; while the hollow structure can effectively encapsulate a second type of therapeutic agent, achieving multi-drug co-delivery and synergistic therapy. Summary of the Invention

[0005] This invention aims to address the technical problem of the lack of nanocarriers in the prior art that can efficiently co-deliver drugs with different properties to achieve synergistic treatment, and provides a nucleic acid nanovesicle based on thioonium copolymer peptides, its preparation method and application.

[0006] The primary objective of this invention is to provide a nucleic acid nanovesicle based on thioonium copolymer peptides, which is formed by electrostatic self-assembly of thioonium copolymer peptide polymers and polynucleotides as shown in the figure below. This nucleic acid nanovesicle based on thioonium copolymer peptides has a hollow structure, which can be used to encapsulate a second active ingredient to achieve multi-drug co-delivery.

[0007]

[0008] In the structural formula, n is 45 or 113; m is any number from 2 to 11; x and y are any numbers from 3 to 200; q is any number from 1 to 3. R1 is selected from benzyl, PEG group or n-hexyl; R2 is selected from chlorine atom, oligoethylene glycol group or benzyloxy group.

[0009] Preferably, the thionium copolymer peptide polymer is SG. 37 OG 36 -O-Bn, where SG represents the sulfur-containing side chain in the copolymer peptide, OG represents the octyl side chain, and -O-Bn represents the benzyl group introduced via thioonylation. 37 OG 36 The numbers 37 and 36 in the text represent the number of the corresponding structural units in the polymer chain.

[0010] Preferably, the polynucleotide is small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotide (ASO), or sgRNA.

[0011] More preferably, the polynucleotide is a siRNA (siCD47) targeting CD47.

[0012] Preferably, the hollow structure of the nucleic acid nanovesicles based on thioonium copolymer peptides further encapsulates a second active ingredient, which is an enzyme, protein, chemotherapeutic drug, immune adjuvant, or photothermal / photodynamic therapy agent.

[0013] More preferably, the second active ingredient is catalase (CAT).

[0014] Preferably, the surface of the nucleic acid nanovesicles based on thioonium copolymer peptides is also coated with a targeting molecule for active targeted delivery, wherein the targeting molecule is hyaluronic acid (HA) or folic acid.

[0015] More preferably, the targeting molecule is hyaluronic acid.

[0016] The second objective of this invention is to provide a method for preparing the above-mentioned nucleic acid nanovesicles based on thionium copolymer peptides, which constructs nanovesicles in one step through a simple electrostatic self-assembly process, and can simultaneously achieve the encapsulation of the second active ingredient and the targeted modification of the surface.

[0017] Specifically, the preparation method includes the following steps: Thionium copolymer peptides were mixed with polynucleotides in a buffered aqueous solution and incubated at room temperature to form nucleic acid nanovesicles based on thioonium copolymer peptides through self-assembly.

[0018] Preferably, the buffer solution is a DEPC-treated sterile aqueous solution.

[0019] Preferably, the thioonium copolymer peptide polymer is mixed with polynucleotides at a charge ratio of 1:1 to 20:1.

[0020] More preferably, the charge ratio of the thioonium copolymer peptide polymer to the polynucleotide is 3:1-7:1.

[0021] Preferably, the incubation time is 10-30 minutes.

[0022] More preferably, the incubation time is 15-25 minutes, more preferably 20 minutes.

[0023] Preferably, a second active ingredient is added before or during the mixing of the thioonium copolymer peptides and polynucleotides. The second active ingredient is an enzyme, protein, chemotherapeutic drug, immune adjuvant, or photothermal / photodynamic therapy agent.

[0024] Preferably, a targeting molecule is added for incubation after vesicle formation; the targeting molecule is hyaluronic acid (HA) or folic acid.

[0025] A third objective of this invention is to provide a pharmaceutical composition comprising the above-described nucleic acid nanovesicles based on thioonium copolymer peptides and a pharmaceutically acceptable carrier.

[0026] A fourth objective of this invention is to provide the use of the above-mentioned nucleic acid nanovesicles based on thioonium copolymer peptides or the above-mentioned pharmaceutical compositions in the preparation of medicaments for treating tumors.

[0027] Preferably, the tumor is a solid tumor such as melanoma or colon cancer.

[0028] Preferably, the drug is used to simultaneously deliver siRNA and catalase, thereby reshaping the tumor immune microenvironment through the synergistic effect of CD47 gene silencing and H2O2 clearance, activating macrophages, dendritic cells and T cells, and producing an anti-tumor immune effect.

[0029] Compared with the prior art, the present invention can achieve the following beneficial effects: (1) Outstanding structural innovation: It is the first time that thioonium copolymer peptides and nucleic acids have been discovered to self-assemble into hollow nanovesicles, rather than conventional solid complexes. This unique vesicle structure provides an ideal platform for multi-drug co-delivery, and its cavity can efficiently encapsulate a second active ingredient, realizing the organic combination of gene therapy with enzyme therapy, chemotherapy, etc.

[0030] (2) The preparation process is simple and mild: a one-step self-assembly method is used, in which thioon copolymer peptides and polynucleotides are simply mixed in a buffer aqueous solution and incubated at room temperature for 10-30 minutes to form nanovesicles. If a second active ingredient needs to be loaded or surface modification is required, it can be done simultaneously with mixing or after vesicle formation, without the need for complicated emulsification, solvent evaporation or extrusion steps. The preparation conditions are mild, the reproducibility is good, and it is easy to scale up production.

[0031] (3) Excellent stability: The prepared nanovesicles exhibit exceptional long-term storage stability. After 14 months of storage at 4℃, PS siCD47 The transfection efficiency remained at 86.7%, with a decrease of less than 10% (Example 6), far exceeding the storage stability of conventional polymer nanoparticles such as PEI, laying a solid foundation for its clinical application and commercial development.

[0032] (4) Significant synergistic immunotherapy effect: Nanovesicles co-loaded with siCD47 and catalase (PSCH) siCD47 For example, in animal models, through the dual effects of CD47 silencing and H2O2 clearance, CD47 gene silencing blocks the "don't eat me" signal, promoting macrophage phagocytosis of tumor cells; catalase clears the immunosuppressive factor H2O2 from the tumor microenvironment, relieving functional inhibition of immune cells; and remodels the tumor immune microenvironment, significantly promoting M1 macrophage polarization, dendritic cell maturation, and cytotoxic T lymphocyte infiltration (Example 5). This gene-enzyme synergistic therapy strategy provides a novel approach to overcoming tumor immunosuppression.

[0033] (5) High biosafety: System safety evaluation (Example 5) showed that the weight of mice remained stable during treatment, indicating good in vivo biosafety. Attached Figure Description

[0034] Figure 1 For nucleic acid nanovesicles based on thionium copolymer peptides PS siRNA Morphology and characterization images: a) Negative staining TEM image; b) DLS particle size distribution image.

[0035] Figure 2 PSH, a nucleic acid nanovesicle based on thionium copolymer peptides siCD47 and PSCH siCD47 Characterization and in vitro functional testing: a) PSH siRNA negative staining TEM image; b) PSCH siRNA Negative staining TEM image.

[0036] Figure 3 PSH, a nucleic acid nanovesicle based on thionium copolymer peptides siCD47 and PSCHsiCD47 H2O2 degradation activity diagram.

[0037] Figure 4 For nucleic acid nanovesicles based on thionium copolymer peptides PS siCD47 PSH siCD47 and PSCH siCD47 Statistical graph of flow cytometry silencing efficiency in CT26 cells.

[0038] Figure 5 For nucleic acid nanovesicles based on thionium copolymer peptides PS Cy5-siRNA Uptake flow cytometry of B16-F10 cells at different time points.

[0039] Figure 6 For nucleic acid nanovesicles based on thionium copolymer peptides PS Cy5-siRNA Uptake rate of B16-F10 cells treated with different endocytosis pathway inhibitors.

[0040] Figure 7 The in vivo antitumor effect of nucleic acid nanovesicles based on thioonium copolymer peptides is shown in the figures: a) tumor photograph after treatment; b) tumor growth curve; G1: PBS control group, G2: PSH. siNC Group, G3: PS siCD47 Group, G4: PSH siCD47 Group, G5: PSCH siCD47 Group.

[0041] Figure 8 Figure 1 shows the effect of nucleic acid nanovesicles based on thioonium copolymer peptides on immune cells: a) Total macrophages in tumor tissue (F4 / 80) + CD11b + (a) Flow cytometry analysis of tumor tissue; (b) M1-like macrophages (F4 / 80) in tumor tissue. + CD11b + CD80 + (c) Flow cytometry analysis of tumor tissue; + CD8 + Flow cytometry analysis of T cells; d) Dendritic cells (CD11c) in lymph nodes + CD80 + CD86 + (e) Flow cytometry analysis of CD3 in the spleen; + CD8 + Flow cytometry analysis of T cells; G1: PBS control group, G2: PSH siNC Group, G3: PS siCD47 Group, G4: PSH siCD47 Group, G5: PSCH siCD47 Group.

[0042] Figure 9 This is a graph showing the change in body weight in mice during treatment with nucleic acid nanovesicles based on thioonium copolymer peptides; G1: PBS control group, G2: PSH control group. siNC Group, G3: PS siCD47 Group, G4: PSH siCD47 Group, G5: PSCH siCD47 Group.

[0043] Figure 10 For nucleic acid nanovesicles based on thionium copolymer peptides PS siRNA Long-term stability graphs: a) Transfection efficiency after 1 month of storage at 4℃ and 25℃; b) Particle size change after 1 month of storage at 4℃; c) Transfection efficiency after 14 months of storage at 4℃. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below with reference to embodiments. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0046] In the following specific embodiments and examples of the present invention, M represents mol / L, μM represents μmol / L, nM represents nmol / L, h represents hour, min represents minute, d represents day, rpm represents revolutions per minute, and eq represents molar equivalent. Flow cytometry data are expressed as mean ± standard deviation.

[0047] In the following specific embodiments and examples of the present invention, statistical analysis was performed using ANOVA. p <0.05 indicates a significant difference. p <0.01 indicates a highly significant difference. p <0.001 indicates an extremely significant difference. ns This indicates no significant difference.

[0048] B16-F10 mouse melanoma cells and CT26 colorectal cancer cells were routinely cultured in RPMI 1640 medium containing 10% (v / v) fetal bovine serum. Six- to eight-week-old BALB / c nude mice were housed in an SPF-grade environment.

[0049] The siRNA targeting CD47 (siCD47) sequence is as follows: siCD47-sense strand sequence: as shown in SEQ ID NO.1; siCD47-antisense strand sequence: as shown in SEQ ID NO.2. The non-targeting negative control siRNA (siNC) is a random sequence of the same length as the CD47-targeting siRNA but without homology, serving as the negative control group. Catalase (CAT), hyaluronic acid (HA), and all chemical reagents used were commercially available analytical grade products.

[0050] Example 1 This embodiment provides a thionium copolymer peptide SG. 37 OG 36 The preparation method of -O-Bn specifically includes the following steps: S1. Synthesis of N-(methylthio)propyl-N-carboxylic anhydride monomer: S11. Synthesis of N-(methylthio)propyl-substituted glycine hydrochloride: In a round-bottom flask, 20 g methionine, 40 g carvone, and 100 mL n-octanol were added, and the mixture was refluxed at 195 °C for 0.5 h. After cooling, 100 mL of 2 M hydrochloric acid was added, and the mixture was refluxed at 195 °C for 10 min. After cooling, 30 mL of glyoxylic acid aqueous solution (50 wt%) and 50 mL of 2 M hydrochloric acid were added, and the mixture was refluxed at 120 °C for 12 h. After the reaction was completed, the lower aqueous phase was collected, and the water was removed by rotary evaporation. The residue was dissolved in a small amount of methanol, precipitated in ice-cold diethyl ether, filtered, and dried under vacuum to give a white solid.

[0051] Synthesis of S12. N-tert-Butoxycarbonyl-N-(methylthio)propyl-substituted glycine: 15 g of the product obtained in S11 was dissolved in 300 mL of water, and 43.8 mL of di-tert-butyl dicarbonate and 52.1 mL of triethylamine were added. The reaction mixture was reacted at 37 °C for 72 h. The reaction solution was extracted with n-hexane (3 × 100 mL) to remove unreacted di-tert-butyl dicarbonate. The aqueous phase was adjusted to pH 2 with 2 M hydrochloric acid and then extracted with ethyl acetate (3 × 150 mL). The combined organic phases were washed with saturated brine and dried over anhydrous magnesium sulfate overnight. The mixture was filtered and rotary evaporated to obtain a yellow oil.

[0052] Synthesis of S13. N-(methylthio)propyl-N-carboxylic anhydride monomer: Under nitrogen protection, 15 g of the product obtained in S12 was dissolved in 150 mL of anhydrous dichloromethane, and phosphorus trichloride (1.5 eq) was added. The reaction was carried out at room temperature for 3 h. The solvent was removed by rotary evaporation, and the mixture was transferred to a glove box. It was precipitated three times with anhydrous tetrahydrofuran / n-hexane (1:10 (v / v)) mixed solvent. The solution was dried under vacuum to obtain a yellow transparent liquid, which was the target monomer.

[0053] S2. Synthesis of N-n-octyl-N-carboxylic anhydride monomer: S21. Synthesis of N-n-octyl-substituted glycine hydrochloride: 24.3 mL of n-octylamine and 30 mL of glyoxylic acid aqueous solution (50 wt%) were dissolved in 100 mL of 2 M hydrochloric acid and refluxed at 120 °C for 12 h. The water was removed by rotary evaporation, the residue was dissolved in methanol, precipitated in ice-cold diethyl ether, filtered, and dried under vacuum to give a white solid.

[0054] Synthesis of N-tert-butoxycarbonyl-N-octyl-substituted glycine (S22): 20 g of the product obtained in S21 was dissolved in 200 mL of water, and di-tert-butyl dicarbonate (2.5 eq) and triethylamine (5 eq) were added. The mixture was reacted at 37 °C for 72 h. Subsequent treatment was the same as in S12, yielding a colorless oil.

[0055] S23. Synthesis of N-n-octyl-N-carboxylic anhydride monomer: Under nitrogen protection, 15 g of the product obtained in S22 was dissolved in 100 mL of anhydrous dichloromethane, and phosphorus trichloride (1.5 eq) was added. The reaction was carried out at room temperature for 3 h. The post-treatment was the same as in S13, yielding a white solid, which was the target monomer.

[0056] S3. Polymerization reaction: Under nitrogen protection, the N-(methylthio)propyl-N-carboxylic anhydride monomers prepared in S1 and the N-n-octyl-N-carboxylic anhydride monomers prepared in S2 were dissolved separately in anhydrous tetrahydrofuran at a concentration of 100 mg / mL. 2.5% (w / w) benzylamine was used as the initiator in anhydrous tetrahydrofuran as the solvent. The two monomer solutions and the initiator were then mixed sequentially in a molar ratio of 40:40:1, and the mixture was transferred to a 55°C oil bath and stirred for 48 h. After the reaction was complete, the reaction solution was dropped into excess ice-cold diethyl ether to settle, centrifuged (9500 rpm, 5 min), and the supernatant was discarded. This process was repeated three times, and the mixture was dried under vacuum to obtain poly(N-(methylthio)propylglycine- r -N-octylglycine (SG) 37 OG 36 The degree of polymerization is x=37 and y=36.

[0057] S4. Thionyl modification: Take 200 mg of SG 37 OG 36 Dissolved in 10 mL of acetic acid (0.2 M), benzyl glycidyl ether (5 eq of the thioether side group) was added, and the reaction was carried out at 40 °C for 72 h. After the reaction was completed, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 1000 Da, and dialyzed against 0.1 M hydrochloric acid for 2 days, followed by dialyzed against deionized water for 2 days, changing the water 3 times a day. The thioonium copolymer peptide (SG) was obtained by freeze-drying. 37 OG 36 -O-Bn).

[0058] Example 2 This embodiment provides nucleic acid nanovesicles (PS) based on thioonium copolymer peptides. siRNAThe preparation and characterization of [the substance] specifically include the following steps: S1. Nucleic acid nanovesicles based on thioonium copolymer peptides (PS) siRNA Preparation: The SG prepared in Example 1 was used... 37 OG 36 -O-Bn polymer was dissolved in DEPC-treated sterile water (1 mg / mL), and mixed with 20 μL of siRNA at an N / P charge ratio of 4:1. The mixture was vortexed and incubated at room temperature for 20 minutes to obtain PS. siRNA The solution forms nucleic acid nanovesicles based on thionium copolymer peptides (PS). siRNA .

[0059] S2. Morphological characterization: Take 10 μL Ps siRNA The solution was dropped onto a copper grid, negatively stained with 2% (g / mL) phosphotungstic acid, and observed under a transmission electron microscope.

[0060] S3. Particle size and potential determination: Take PS siRNA After the solution was diluted, the hydrated particle size and polydispersity index were determined using a dynamic light scattering instrument.

[0061] Figure 1 Nucleic acid nanovesicles based on thionium copolymer peptides (PS) are shown. siRNA The morphology and characterization images are shown, where a) is a negative staining TEM image and b) is a DLS particle size distribution image.

[0062] Depend on Figure 1 From a), we can see that PS siRNA It exhibits a distinct hollow spherical structure, with a diameter of approximately 119 ± 11.5 nm; composed of Figure 1 From b), we can see that PS siRNA The hydrated particle size is 115.4 nm, and the polydispersity index is 0.196 ± 0.02, indicating that the particle size is uniform and the dispersibility is good.

[0063] Example 3 This embodiment provides nucleic acid nanovesicles based on thioon copolymer peptides (PSH). siCD47 and PSCH siCD47 The construction and characterization of [the data] specifically include the following steps: S1. Nucleic acid nanovesicles based on thioonium copolymer peptides with targeting function (PSH) siCD47 Construction: PS was prepared according to the method in S1 of Example 2. siCD47 After nanovesicle formation (with siCD47 as the siRNA), hyaluronic acid solution (1 mg / mL) was added, and the mixture was incubated at room temperature for 30 minutes. This allowed the hyaluronic acid to be electrostatically adsorbed onto the positively charged vesicle surface, resulting in PSH. siCD47 Solution formation of thiamonium copolymer peptide-based nucleic acid nanovesicles (PSH) with targeting function siCD47 .

[0064] S2. Enzyme-encapsulated, targeting-functional thioonium copolymer peptide-based nucleic acid nanovesicles PSCH siCD47 Construction of enzyme-loaded targeted nanovesicles: Following the method in S1 of Example 2, catalase (CAT, 2 mg / mL) was added for loading during the preparation of nanovesicles. Then, hyaluronic acid solution (1 mg / mL) was added and incubated at room temperature for 30 minutes, allowing it to be electrostatically adsorbed onto the positively charged vesicle surface, resulting in PSCH. siCD47 Solution, forming enzyme-encapsulated, targeted thioonium copolymer peptide-based nucleic acid nanovesicles PSCH siCD47 .

[0065] S3. Morphological characterization: Take PSH siCD47 and PSCH siCD47 The solution was subjected to TEM observation according to the method in S2 of Example 2. The results are as follows: Figure 2 As shown, Figure 2 The characterization and in vitro functionalization of functionalized nanovesicles are shown in the diagram, where a) is PSH. siRNA Negative staining TEM image, b) is PSCH siRNA Negative-stained TEM image. (By...) Figure 2 As shown in a) and b), both maintain a hollow vesicle structure. The PSH and PSCH are slightly larger than PS, and the PSCH is slightly larger than PSH.

[0066] S4. Enzyme activity assay: Add 90 μl of free CAT and PSH... siCD47 and PSCH siCD47 The mixture was co-incubated with 10 μl of 10 μM H2O2 solution (to bring the final H2O2 concentration to 100 μM), and the residual H2O2 concentration was determined using an H2O2 detection kit. The results are as follows: Figure 3 As shown, Figure 3 Nucleic acid nanovesicles based on thioonium copolymer peptides (PSH) were demonstrated. siCD47 and PSCH siCD47 The H2O2 degradation activity diagram shows that PSCH siCD47 It can effectively degrade H2O2, while PSH siCD47 It exhibits no significant degradation activity.

[0067] S5. Gene silencing efficiency assay: CT26 cells were seeded in 24-well plates (5 × 10⁻⁶ cells / well). 4 (cells / well), and PS siCD47 PSH siCD47 and PSCH siCD47(3 μl nanovesicles per well, dissolved in 497 μl of basal medium), incubated for 8 h, then replaced with fresh medium and cultured for another 40 h. Cells were collected, and the percentage of CD47-negative cells was determined by flow cytometry. PEI was also set up. siCD47 Control group (using the gold standard transfection PEI) 25k (N / P ratio = 3) forms a nucleic acid complex with siCD47, 5 μl PEI per well. siCD47 (dissolved in 495 μL of basal medium), free siCD47 group, and PBS control group. Results are as follows: Figure 4 As shown, Figure 4 Nucleic acid nanovesicles based on thionium copolymer peptides (PS) are shown. siCD47 PSH siCD47 and PSCH siCD47 The flow cytometry silencing efficiency statistics in CT26 cells show that PSH siCD47 and PSCH siCD47 The CD47 knockdown efficiencies were 84.3% and 82.4%, respectively, compared to PS. siCD47 The groups (86.0%) were comparable, both significantly higher than PEI. siCD47 57.3% (***) p <0.001), the concentrations of siRNA used for transfection were the optimal transfection concentrations for each group, indicating that neither HA surface coating nor CAT loading affected the gene silencing function of vesicles.

[0068] Example 4 This embodiment provides an in vitro cellular uptake and mechanism study of nucleic acid nanovesicles based on thioonium copolymer peptides, specifically including the following steps: S1. Preparation of fluorescently labeled thiamonium copolymer peptide-based nanovesicles: Cy5-labeled siRNA was assembled into PS according to the method in S1 of Example 2. Cy5-siRNA .

[0069] S2. Cell uptake kinetics: B16-F10 cells were seeded in 24-well plates (5 × 10⁶ cells / well). 4 (cells / well), and PS Cy5-siRNA (siRNA final concentration 30 nM) Incubated for 0.5, 1, 2, 4, 6, and 8 hours, and the percentage of Cy5-positive cells was detected by flow cytometry. Results are as follows: Figure 5 As shown, Figure 5 Nucleic acid nanovesicles based on thionium copolymer peptides (PS) are shown. Cy5-siRNA Flow cytometry plots of uptake in B16-F10 cells at different time points. The figure shows that PS... Cy5-siRNA It is rapidly absorbed within 30 minutes and reaches its peak value in 6 hours.

[0070] S3. Uptake Mechanism Study: B16-F10 cells were pre-incubated for 1 h with different inhibitors of endocytosis: amiloride, sodium methyl-β-cyclodextrin (M-β-CD), chlorpromazine (CPZ), and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). The culture medium was then replaced with fresh medium, and PS was added. Cy5-siRNA (siRNA final concentration 30 nM) Incubated for 4 h, uptake rate was detected by flow cytometry. Results are as follows. Figure 6 As shown, Figure 6 Nucleic acid nanovesicles based on thionium copolymer peptides (PS) are shown. Cy5-siRNA The uptake rate of B16-F10 cells treated with different endocytosis pathway inhibitors is shown in the figure. It can be seen from the figure that the thiol-mediated endocytosis inhibitor DTNB only caused a slight decrease in uptake rate, while the uptake rate of the chlorpromazine (clathrin inhibitor) treatment group was significantly reduced, indicating that clathrin-mediated endocytosis is its main uptake mechanism.

[0071] Example 5 This embodiment provides an in vivo antitumor effect evaluation of nucleic acid nanovesicles based on thioonium copolymer peptides, specifically including the following steps: S1. Animal model establishment: 6-8 week old BALB / c nude mice were subcutaneously inoculated with CT26 cells (1×10⁻⁶) on the right posterior back. 6 (each tumor), until the tumor volume reaches approximately 100 mm. 3 The experiment will begin at that time.

[0072] S2. Grouping and Administration: Tumor-bearing mice were randomly divided into 5 groups (n=5): G1: PBS control group, G2: PSH control group, G3: PBS control group, G4: PBS control group, G5: PBS control group, G6: PBS control group, G7: PBS control group, G8: PBS control group, G9: PBS control group, G1: PBS control group, G2 ... siNC Group (where the siRNA was selected as a non-targeted negative control siRNA, and the preparation method is as described in Example 2), G3: PS siCD47 Group, G4: PSH siCD47 Group, G5: PSCH siCD47 Group. Nucleic acid nanovesicles based on thioonium copolymer peptides were injected via tail vein (siRNA dose 0.5 OD / animal / dose) once every 2 days for a total of 5 doses.

[0073] S3. Tumor Growth Monitoring and Photography: The long and short diameters of the tumor were measured every two days, and the tumor volume was calculated. After treatment, the tumor tissue was dissected and photographed. Results are as follows: Figure 7 As shown, Figure 7 The in vivo antitumor effect of nucleic acid nanovesicles based on thioonium copolymer peptides is shown in the figure, where a) is a tumor photograph after treatment and b) is a tumor growth curve; G1: PBS control group, G2: PSH siNC Group, G3: PS siCD47 Group, G4: PSH siCD47 Group, G5: PSCHsiCD47 Group.

[0074] Depend on Figure 7 It can be seen that, compared with the PBS control group, all treatment groups significantly inhibited tumor growth. Figure 7 From a) and b), we can see that PSCH siCD47 The group had the smallest tumor volume, PSH siCD47 Group 2, PS siCD47 The results of this study demonstrate the good anti-tumor effect of nucleic acid nanovesicles based on thioonium copolymer peptides.

[0075] S4. Immune Microenvironment Analysis: After treatment, mice were sacrificed, and tumor tissue, spleen, and lymph nodes were collected. Single-cell suspensions were prepared, and flow cytometry was used to detect immune cell subsets. Results are as follows: Figure 8 As shown, Figure 8 The diagram shows the effect of nucleic acid nanovesicles based on thioonium copolymer peptides on immune cells, where a) represents the total number of macrophages (F4 / 80) in tumor tissue. + CD11b + (a) Flow cytometry analysis of tumor tissue, and (b) M1-like macrophages (F4 / 80) in tumor tissue. + CD11b + CD80 + The flow cytometry analysis diagram (c) shows CD3 in the tumor tissue. + CD8 + Flow cytometry analysis of T cells, d) shows dendritic cells (CD11c) in lymph nodes. + CD80 + CD86 + (e) Flow cytometry analysis of CD3 in the spleen. + CD8 + Flow cytometry analysis of T cells; G1: PBS control group, G2: PSH siNC Group, G3: PS siCD47 Group, G4: PSH siCD47 Group, G5: PSCH siCD47 Group.

[0076] Depend on Figure 8 It can be seen that PSCH siCD47 Group A showed significantly higher scores than other groups in all detection indicators, including PSH. siCD47 Group 2, PS siCD47 The second group was next. Specifically, compared with the control group, PSCH siCD47 Total macrophages in the tumor tissue (F4 / 80) + CD11b + ) and M1 macrophages (F4 / 80) + CD11b +CD80 + ) ratio, CD3 + CD8 + The number of T cell infiltrations and mature dendritic cells (CD11c) in lymph nodes + CD80 + CD86 + The proportion of CD3 in the spleen, and the proportion of CD3 in the spleen + CD8 + The proportion of T cells was significantly increased.

[0077] S5. Safety evaluation: Changes in mouse body weight were recorded during treatment, and the results are as follows: Figure 9 As shown, Figure 9 The figure shows the body weight change curve of mice during treatment with nucleic acid nanovesicles based on thioonium copolymer peptides; G1: PBS control group, G2: PSH siNC Group, G3: PS siCD47 Group, G4: PSH siCD47 Group, G5: PSCH siCD47 Groups. As shown in the figure, the body weight of mice in each group remained stable, demonstrating good biocompatibility.

[0078] Example 6 This embodiment provides a storage stability evaluation of nucleic acid nanovesicles based on thioonium copolymer peptides, specifically including the following steps: S1. Short-term stability: The PS prepared in Example 2 siCD47 The samples were stored at 4℃ and 25℃ for one month, and samples were taken at the following times: fresh preparation, one week of storage, and one month of storage. PEI was also set up. siCD47 Control group and PBS control group. Transfection efficiency was detected according to the method in S5 of Example 3 (B16-F10 cells were selected), and particle size was detected using a dynamic light scattering instrument.

[0079] S2. Long-term stability: The PS prepared in Example 2 siCD47 The samples were stored at 4°C for 14 months, and samples were taken at fresh preparation time, 8 months of storage time, and 14 months of storage time. PEI was also set up. siCD47 Control group and PBS control group. Transfection efficiency was assessed according to method S1.

[0080] S3. Result: Figure 10 Nucleic acid nanovesicles based on thionium copolymer peptides (PS) are shown. siRNA The long-term stability graphs are shown, where a) represents the transfection efficiency after 1 month of storage at 4℃ and 25℃, b) represents the particle size change after 1 month of storage at 4℃, and c) represents the transfection efficiency after 14 months of storage at 4℃.

[0081] Depend on Figure 10As shown in a), after storage at 4°C for one month, the siRNA delivery efficiency was 93.8%, and after storage at 25°C for one month, the siRNA delivery efficiency was 92.0%, comparable to the 94.9% efficiency of freshly prepared siRNA. Meanwhile, PEI... siCD47 After being stored at 4°C for one month, the efficiency of the group significantly decreased to 41.3%. Figure 10 From b), we can see that PS siCD47 The nanovesicles maintained stable particle size for one month at 4℃ and 25℃, showing no significant change. These results indicate that the nucleic acid nanovesicles based on thiamonium copolymer peptides (PS) exhibit stable particle size. siRNA It exhibits good short-term storage stability.

[0082] Depend on Figure 10 As shown in c), after 14 months of storage, PS siCD47 The transfection efficiency remained at 86.7%, a decrease of less than 10%, compared to the significant decrease in PEI. siCD47 The contrast between the two groups demonstrates their exceptional long-term storage stability.

[0083] In summary, the hollow nanovesicles constructed based on thioonium copolymer peptides in this invention have multiple advantages, including structural innovation, simple preparation, excellent stability, significant synergistic therapeutic effects, and high biosafety. They provide a novel and efficient nanodrug delivery platform for tumor immunotherapy and have important translational application value in the biomedical field.

[0084] Those skilled in the art will understand that, based on the mechanism demonstrated in the embodiments of the present invention—the self-assembly of siRNA and thionium copolymer peptides through electrostatic interactions to form hollow nanovesicles—other small nucleic acid molecules with similar charge densities, such as microRNA, antisense oligonucleotides, and sgRNA, can also be expected to form similar vesicle structures. sgRNA, as a key component of CRISPR gene editing, has similar length and negative charge characteristics to siRNA, and its efficient delivery via cationic carriers has been reported in the literature. Therefore, the nanovesicles of the present invention are also suitable for sgRNA delivery and can be used to construct gene editing delivery systems based on CRISPR technology.

[0085] It should be understood that the various forms of processes shown above can be used to reorder, add, or delete steps. For example, the steps described in this invention disclosure can be executed in parallel, sequentially, or in different orders, as long as the desired result of the technical solution disclosed in this invention can be achieved, and this is not limited herein.

[0086] The specific embodiments described above do not constitute a limitation on the scope of protection of this invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A nucleic acid nanovesicle based on thioonium copolymer peptides, characterized in that, These are stable, hollow nanovesicles formed by electrostatic self-assembly of thioon copolymer peptides with the structure shown in the figure below and polynucleotides. Where n is 45 or 113; m is any number from 2 to 11; x and y are any numbers from 3 to 200; q is any number from 1 to 3; R1 is selected from benzyl, PEG group or n-hexyl; R2 is selected from chlorine atom, oligoethylene glycol group or benzyloxy group.

2. The nucleic acid nanovesicles based on thioonium copolymer peptides according to claim 1, characterized in that, The polynucleotide is a small interfering RNA, microRNA, antisense oligonucleotide, or sgRNA.

3. The nucleic acid nanovesicles based on thioonium copolymer peptides according to claim 1, characterized in that, The hollow structure of the nucleic acid nanovesicles based on thioonium copolymer peptides also encapsulates a second active ingredient, which may be an enzyme, protein, chemotherapeutic drug, immune adjuvant, or photothermal / photodynamic therapy agent.

4. The nucleic acid nanovesicles based on thioonium copolymer peptides according to claim 1, characterized in that, The surface of the nucleic acid nanovesicles based on thioonium copolymer peptides is also coated with a targeting molecule, which is hyaluronic acid or folic acid.

5. A method for preparing nucleic acid nanovesicles based on thionium copolymer peptides as described in claim 1, characterized in that, Includes the following steps: Thionium copolymer peptides and polynucleotides were mixed in a buffered aqueous solution and incubated at room temperature to form nucleic acid nanovesicles based on thioonium copolymer peptides through self-assembly.

6. The preparation method according to claim 5, characterized in that, A second active ingredient is added before or during the mixing of thioonium copolymer peptides and polynucleotides. The second active ingredient is an enzyme, protein, chemotherapy drug, immune adjuvant, or photothermal / photodynamic therapy agent.

7. The preparation method according to claim 5, characterized in that, After vesicle formation, a targeting molecule is added for incubation. The targeting molecule is hyaluronic acid or folic acid.

8. The preparation method according to claim 5, characterized in that, The thioon copolymer peptides and polynucleotides are mixed at a charge ratio of 1:1 to 20:1; the incubation time is 10-30 minutes.

9. A pharmaceutical composition, characterized in that, It comprises the nucleic acid nanovesicles based on thioonium copolymer peptides as described in any one of claims 1-4, and a pharmaceutically acceptable carrier.

10. The use of the nucleic acid nanovesicles based on thioonium copolymer peptides according to any one of claims 1-4 or the pharmaceutical composition according to claim 9 in the preparation of a medicament for treating tumors.

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