Targeted nano adjuvant for tumor-associated fibroblasts as well as preparation method and application of targeted nano adjuvant

Abstract

The invention provides a tumor-associated fibroblast targeting nano adjuvant as well as a preparation method and application thereof, and belongs to the field of nano drugs. The nano adjuvant (named as PF9PEI and alpha PC NPs) provided by the invention is coupled with PEI25K modified by 3-perfluorobutyl-1, 2-epoxypropane by utilizing a PD-L2 targeting peptide nrirgenyryh, so that an efficient gene vector (PF9PEI) is formed, and is used for loading an alpha PD-L1-P2A-CD86 plasmid. According to the nano adjuvant, CAFs can be reprogrammed into engineered antigen presentation CAFs by expressing a costimulatory molecule CD86, so that T cell immunity of breast cancer specificity is activated, and T cell failure is retarded at the same time. Besides, the engineered CAFs generated by the nano adjuvant generate alphaPD-L1 locally, so that the immune escape mechanism of breast cancer cells is effectively blocked, and the immune surveillance function of T cells is recovered. After radiotherapy, by using the nano adjuvant for treatment, the generation of central memory T cells is promoted, the progress of the same solid tumor is inhibited, the tumor metastasis is eliminated, and the tumor recurrence is inhibited.

Description

Technical Field

[0001] The present invention belongs to the field of nanomedicines and relates to tumor-associated fibroblasts, and in particular to a tumor-associated fibroblast-targeted nanoadjuvant and a preparation method and application thereof. Background Art

[0002] Breast cancer is the most common malignancy in women. Breast-conserving surgery combined with radiotherapy (RT) is currently the standard treatment paradigm for breast cancer. RT successfully induces immunogenic cell death (ICD), enhancing tumor immunogenicity. However, the limited number of dendritic cells (DCs) in breast cancer tissue leads to insufficient antigen presentation and limited T cell activation. Extensive evidence indicates that RT triggers dynamic changes in the tumor stroma, activating immunosuppressive cancer-associated fibroblasts (CAFs). These CAFs competitively uptake limited tumor-specific antigens, further exacerbating immunosuppression. However, RT-activated CAFs, as non-professional antigen-presenting cells (APCs), lack the critical costimulatory signals required for successful T cell activation. Furthermore, under sustained antigen stimulation, CAFs upregulate immunosuppressive molecules such as programmed death ligand 2 (PD-L2), thereby inducing T cell dysfunction and even exhaustion. Furthermore, competitive antigen presentation by CAFs impairs the antigen uptake and presentation capacity of DCs, further exacerbating the immunosuppressive microenvironment of breast cancer. To address this challenge, current research focuses on directly depleting or inactivating CAFs in an effort to ameliorate CAF-induced immunosuppression.

[0003] In addition to CAF activation, the intrinsic immunosuppressive phenotype of breast cancer cells also hinders T cell recognition and activation. In particular, overexpression of PD-L1 in breast cancer cells inhibits T cell proliferation and function. Drug blockade of PD-L1 has demonstrated significant efficacy in various malignancies. However, intravenous administration of PD-L1 antibodies (αPD-L1) is limited by their short half-life, difficulty effectively accumulating within tumors, and potentially irreversible damage to normal tissues. While targeted delivery of αPD-L1 to tumors can enhance the efficacy of immune checkpoint blockade, the rigid tumor stroma hinders deep penetration of αPD-L1, limiting its efficacy. Genetically engineering CAFs to produce αPD-L1 in situ within the tumor microenvironment would not only improve αPD-L1's tumor penetration but also effectively reduce its side effects. Traditionally, viral vectors have been widely used for gene delivery due to their efficient transfection of large, negatively charged DNA. However, the potential for immune responses and biosafety issues associated with viral vectors have limited their therapeutic application. Therefore, the development of efficient and safe non-viral gene vectors is of vital importance to achieve CAFs reprogramming to express co-stimulatory signals and produce αPD-L1 in situ.

[0004] Prior art CN115844831B discloses a method for preparing an albumin-based CRISPR_Cas9 targeted delivery system. The main steps are: first, construct a CRISPR-Cas9 plasmid; then add substances such as human serum albumin aqueous solution to react; then add the inflammation-targeting peptide CBP to the solution and stir overnight at 4°C to allow CBP to fully couple with the albumin nanoparticles; finally, use an ultrafiltration tube to ultrafilter the nanoparticle solution to remove unbound albumin, glutaraldehyde, and ethanol residues to obtain TAP / pCas9-PAR2, which can transfect exogenous genes into cells. The disadvantages of this technology are that the components are complex, albumin needs to be added, and additional purification steps are required, resulting in low transfection efficiency.

[0005] A study has proposed a metabolic reprogramming strategy that targets and downregulates the key metabolic genes hexokinase 2 (HK2) and mitochondrial cytochrome c oxidase I (MTCO1) in CAFs through a specific CSF-targeted LNP nanoparticle LNP@D-siRNA-PD-1 delivery system. This dual inhibition leads to glucose overload and mitochondrial dysfunction in CAFs, thereby shifting CAFs from a tumor-promoting to an immune-supportive state. However, the drawback is the low transfection and delivery efficiency of this delivery system (Metabolic Reprogramming of Cancer-Associated Fibroblasts: Transforming Tumor Accomplices into Immunotherapeutic Allies, Adv. Funct. Mater. 2024). Summary of the Invention

[0006] In order to solve the problems of low transfection efficiency and delivery efficiency of existing delivery systems, the present invention provides a tumor-associated fibroblast-targeted nanoadjuvant and its preparation method and application; the nanoadjuvant (named PF9PEI@αPC NPs) is designed to alleviate radiotherapy-induced T cell exhaustion by reprogramming CAFs, thereby effectively preventing the recurrence of breast cancer. nrirgenyryh PEI modified with 3-perfluorobutyl-1,2-propylene oxide 25K Coupled to form a highly efficient gene carrier (PF9PEI) for loading αPD-L1-P2A-CD86 plasmid (αPC) to achieve targeted gene delivery (see schematic diagram Figure 1). Thanks to the modification with fluorinated PEI, the prepared PF9PEI@αPC NPs have excellent serum resistance and membrane permeability. At the same time, the PD-L2 targeting peptide modification promotes targeted gene delivery to CAFs, ensuring efficient gene transfection and CAFs reprogramming, aiming to prevent the recurrence of breast cancer after adjuvant radiotherapy. PF9PEI@αPC NPs use PD-L2 recognition to achieve precise targeting of CAFs. They can efficiently deliver genes and edit CAFs, transforming them into professional antigen-presenting cells, while also acting as a production factory for αPD-L1. Specifically, PF9PEI@αPC NPs can reprogram CAFs into engineered antigen-presenting CAFs by expressing the co-stimulatory molecule CD86, thereby activating breast cancer-specific T cell immunity while slowing down T cell exhaustion. In addition, the engineered CAFs generated by PF9PEI@αPC NPs produce αPD-L1 locally, effectively blocking the immune escape mechanism of breast cancer cells and restoring the immune surveillance function of T cells. Post-radiotherapy treatment with PF9PEI@αPC NPs promoted the generation of central memory T cells (Tcm), inhibited the progression of solid tumors of the same type, eliminated tumor metastasis, and suppressed tumor recurrence. This study proposes a strategy to overcome the immunosuppressive feedback associated with adjuvant radiotherapy, providing a reliable approach to address the challenge of cancer recurrence.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows: In one aspect, the present invention provides a method for preparing a tumor-associated fibroblast-targeting nanoadjuvant, the preparation method comprising the following steps: S1. preparing an expression plasmid comprising an αPD-L1 sequence and a CD86 sequence; S2. preparing a targeting peptide targeting PD-L2; S3, performing fluorination modification on polyethyleneimine to obtain fluorinated polyethyleneimine; modifying the targeting peptide onto the fluorinated polyethyleneimine to obtain a nanocarrier; S4. Mix the expression plasmid and the nanocarrier to prepare a nanoadjuvant.

[0008] Preferably, the αPD-L1 sequence in step S1 has the nucleic acid sequence shown in SEQ ID NO.2, and the CD86 sequence has the nucleic acid sequence shown in SEQ ID NO.4.

[0009] Preferably, the expression plasmid further comprises a CMV promoter, an Igκ leader sequence and a P2A sequence; the Igκ leader sequence has the nucleic acid sequence shown in SEQ ID NO.1, and the P2A sequence has the nucleic acid sequence shown in SEQ ID NO.3.

[0010] Preferably, the targeting peptide in step S2 has an amino acid sequence shown in SEQ ID NO.5.

[0011] Preferably, step S3 includes the following steps: (1) Dissolving polyethyleneimine and perfluorobutyl-1,2-epoxypropylene in methanol and reacting them at room temperature to obtain fluorinated polyethyleneimine; (2) Fluorinated polyethyleneimine and targeting peptide were dissolved in purified water, and N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide were added to react at room temperature. After purification and drying, the nanocarrier was obtained.

[0012] 6. The preparation method according to claim 5, characterized in that the polyethyleneimine in step (1) is PEI 25K ; The purification method in step (2) is dialysis, and the drying method is freeze-drying.

[0013] Preferably, the expression plasmid and nanocarrier in step S4 are dissolved in purified water and stirred at room temperature to react to obtain the nanoadjuvant.

[0014] On the other hand, the present invention provides a nanoadjuvant prepared by the above-mentioned preparation method.

[0015] On the other hand, the present invention provides the above-mentioned preparation method or use of the above-mentioned nanoadjuvant in nucleic acid delivery.

[0016] Preferably, the use includes use in preparing a product for enhancing transfection efficiency in serum.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. The nanoadjuvant provided by the present invention has high transfection efficiency and can achieve efficient gene delivery; 2. The nanoadjuvant provided by the present invention can reprogram CAFs into engineered antigen-presenting CAFs by expressing the co-stimulatory molecule CD86, thereby activating breast cancer-specific T cell immunity and slowing down T cell exhaustion; 3. The nanoadjuvant provided by this invention can generate engineered CAFs that locally produce αPD-L1, effectively blocking the immune escape mechanism of breast cancer cells and restoring the immune surveillance function of T cells. Treatment with this nanoadjuvant after radiotherapy promoted the generation of central memory T cells, inhibited the progression of solid tumors of the same type, eliminated tumor metastasis, and suppressed tumor recurrence. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a principle diagram.

[0019] Figure 2 This is a map of plasmid αPC, i.e., pCMV-Igκ-αPD-L1-P2A-CD86-Neo.

[0020] Figure 3 This is a map of plasmid αP, namely pCMV-Igκ-αPD-L1-Neo.

[0021] Figure 4 This is a map of plasmid C, pCMV-CD86-Neo.

[0022] Figure 5 This is a map of plasmid pEGFP, namely pcDNA3.1-EGFP.

[0023] Figure 6 Characterization diagram of the targeting peptide rirgenyryh; (A) HPLC spectrum and (B) ESI-MS spectrum.

[0024] Figure 7 Schematic diagram of the synthesis of PF9PEI.

[0025] Figure 8 Basic material characterization and gel retardation test results of PF9PEI@αPC NPs; (A) TEM image and hydrodynamic size distribution analysis of PF9PEI@αPC NPs, scale: 500 nm; (B) Stability evaluation of PF9PEI@αPC NPs under different conditions; (C) Zeta potential measurement of PEI@αPC, F9PEI@αPC, and PF9PEI@αPC NPs; (D) Gel retardation test of different ratios of PF9PEI and αPC NPs; (E) Protection of PF9PEI@αPC NPs against plasmid degradation by DNase.

[0026] Figure 9 Results of cell transfection ability test; (A) Flow cytometry analysis of transfection efficiency and verification by (B) fluorescence microscopy images, scale bar: 100 μm, transfected CAFs (green) were labeled with the reporter protein EGFP; (C) Serum adsorption characteristics at different serum concentrations.

[0027] Figure 10 The transfection efficiency of CAFs under different serum concentrations is shown in Figure 5. Scale bar: 200 μm.

[0028] Figure 11 Transfected CAFs were analyzed for flow cytometry.

[0029] Figure 12 Results of cell transfection ability test; (A) Trend analysis shows the efficiency at different serum concentrations, and (B) determination of surface amino group concentration.

[0030] Figure 13 Determination of PD-L2 targeting and blocking behavior; CLSM images show the distribution of NIH3T3, CAFs, and PD-L2-blocked CAFs after treatment with PF9PEI@DNACy5 NPs, scale bar: 20 μm.

[0031] Figure 14 Results of related gene and protein detection; (A) Analysis of changes in CD86 mRNA levels, (B) Flow cytometric evaluation of CD86-positive CAFs, (C) Evaluation of changes in αPD-L1 mRNA levels, scale bar: 50 μm; (D) Determination of secreted αPD-L1 protein concentration, (E) Flow cytometric analysis of PD-L1 expression after co-culture of transfected CAFs culture supernatant with 4T1 cells.

[0032] Figure 15 The following table shows the results of related gene and protein detection: (A) Immunofluorescence imaging of CD86 expression, scale bar: 50 μm; (B) Immunofluorescence evaluation of PD-L1 after co-culture of transfected CAFs culture supernatant with 4T1 cells. Scale bar: 50 μm.

[0033] Figure 16 Schematic diagram of the co-culture model.

[0034] Figure 17 Immunoexhaustion and immunotoxicity detection of CTLL-2 cells; (A) Proliferation level of T cells after treatment, (B) Evaluation of cytotoxicity of T cells against 4T1 after treatment and (C) quantification results, (D) IFN-γ concentration.

[0035] Figure 18 Immunodepletion and immunotoxicity detection of CTLL-2 cells; (A) Gra B concentration; (B) Images of CTLL-2 and 4T1 cells co-incubated, red arrows indicate CTLL-2 cells, green arrows indicate 4T1 cells, and blue arrows indicate apoptotic bodies. Scale bar: 50 μm. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described in detail below with reference to the examples. If the specific conditions are not specified in the examples, the experiments shall be carried out under conventional conditions or the conditions recommended by the manufacturer. All reagents or instruments without the manufacturer specified are conventional products that can be purchased commercially. In order to better illustrate the present invention, many specific details are given in the specific embodiments below. The specific embodiments described here are only used to explain the present invention and are not intended to constitute any limitation to the present invention. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present invention. Such structures and technologies are described in many publications, such as "Molecular Cloning Laboratory Manual (Fourth Edition)" (Cold Spring Harbor Laboratory Science Press), Ausubel, FM et al., Current Protocols in Molecular Biology , Greene Publishing Assoc., and Wiley-Interscience.

[0037] SPSS 23.0 software was used for data analysis. The t-test and one-way ANOVA test were used for significance analysis. P < 0.05 indicated a significant difference.

[0038] Preparation Example: Materials and Methods Concerning the Present Invention The main reagents involved in the present invention are shown in Table 1.

[0039] Table 1 Related reagent information.

[0040]

[0041] The main related instruments involved in the present invention are shown in Table 2.

[0042] Table 2 Related instrument information.

[0043]

[0044] The experimental materials involved in the present invention are described as follows: 4T1, DC2.4, and NIH3T3 cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). CT26 cell lines and bone marrow-derived dendritic cells (BMDCs) were cultured in Roswell Park Memorial Institute 1640 medium (RPMI 1640) supplemented with 10% FBS. CTLL-2 cell lines were cultured in RPMI 1640 supplemented with 10% FBS and 10 ng / mL IL-2. 4T1 cells were treated with IFN-γ to induce PD-L1 expression. NIH3T3 cells were treated with TGF-β (10 ng / mL) for 24 hours to induce CAFs.

[0045] BMDCs were isolated and cultured from mouse bone marrow. Femurs and tibias were extracted from 4-week-old female BALB / C mice (purchased from Zhuhai Baishitong Biotechnology Co., Ltd., weighing 14-16 g). The femoral and tibial cavities were repeatedly rinsed with cold PBS to collect the bone marrow. After centrifugation, the cells were resuspended in culture medium and cultured in a cell culture dish. After 24 hours, non-adherent cells were removed, and granulocyte-macrophage colony-stimulating factor (GM-CSF, 20 ng / mL) was added to the culture medium to induce stem cell differentiation into dendritic cells. After 7 days, immature BMDCs were harvested for subsequent experiments.

[0046] Example 1: Preparation and application of tumor-associated fibroblast-targeted nanoadjuvants (1) Radiotherapy Radiotherapy was performed by specialized personnel in accordance with the hospital's radiation oncology department's protocols. In in vitro experiments, a total radiation dose of 8 Gy was administered in a single fraction. In in vivo studies, BABL / C mice (purchased from Zhuhai Baishitong Biotechnology Co., Ltd., 6-8 weeks old, weighing 18-22 g) were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg / kg). Their solid tumors were then exposed to the radiotherapy area, while the rest of the body was shielded with lead blocks. A total radiation dose of 24 Gy was administered in three fractions.

[0047] (2) Plasmid construction The construction of all plasmids was completed by Wuhan Miaoling Company, and the extraction, purification and sequencing of the plasmids were performed by Guangzhou Ruibo Company. The αPD-L1-P2A-CD86 (αPC) plasmid consists of five main components: CMV promoter, Igκ leader sequence (SEQID NO.1), αPD-L1 (SEQ ID NO.2), P2A (SEQ ID NO.3) and CD86 (SEQ ID NO.4). The P2A sequence ensures that the αPD-L1 and CD86 genes are cleaved during transcription, allowing them to function independently. The CD86 sequence includes a transmembrane region that anchors CD86 to the cell membrane. In experimental controls, other partially functional plasmids were also synthesized, including αPD-L1 plasmid and CD86 plasmid. In order to evaluate transfection efficiency, the EGFP reporter gene sequence replaced the αPC sequence. The design of all plasmids used can be found in. Figure 2-Figure 5 .

[0048] (3) Synthesis and basic material characterization of PF9PEI@αPC NPs 3.1 Synthesis of PD-L2 Targeting Peptide PD-L2 targeting peptides nrirgenyryh (The amino acid sequence is SEQ ID NO.5: nrirgenyryh.) It was synthesized and purified by Hangzhou Peptide Biotechnology Co., Ltd. and characterized by ESI-MS and HPLC. nrigenyryh The peptides were synthesized by solid phase peptide synthesis (SPPS), characterized by electrospray ionization mass spectrometry (ESI-MS), and their purity was determined by high performance liquid chromatography (HPLC). Figure 6 A).

[0049] 3.2 Synthesis of PF9PEI PEI 25K It was dissolved in methanol with perfluorobutyl-1,2-epoxypropane and reacted at room temperature for 24 hours. After that, the product F9PEI was obtained through dialysis and freeze-drying. Next, F9PEI and PD-L2 targeting peptide were dissolved in ultrapure water, and appropriate amounts of N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were added and stirred at room temperature for 24 hours. After dialysis and freeze-drying again, the product PF9PEI was obtained. The coupling of the synthesized peptide with fluorinated PEI was confirmed by NMR spectroscopy ( Figure 6 B).

[0050] 3.3 Synthesis of PF9PEI@αPC NPs like Figure 7 As shown, PF9PEI and αPC were dissolved in ultrapure water and stirred for 20 minutes to prepare PF9PEI@αPC NPs. 25KThe control material PEI@αPC NPs was synthesized by dissolving it in ultrapure water and stirring it for 20 min.

[0051] 3.4 Basic material characterization of PF9PEI@αPC NPs PF9PEI@αPC NPs were added dropwise to a copper grid and air-dried before imaging by transmission electron microscopy (TEM). The hydrodynamic parameters and surface potential of PF9PEI@αPC NPs were characterized using a Malvern particle size analyzer. TEM showed that PF9PEI@αPC NPs were uniformly spherical in shape ( Figure 8 Furthermore, DLS measurements confirmed that the hydrodynamic diameter of the nanoparticles was approximately 203.62 nm, further indicating enhanced colloidal stability in aqueous environments, which is crucial for maintaining the integrity and functionality of the nanoparticles in systemic circulation ( Figure 8 In addition, the fluorination strategy of the present invention effectively reduces the positive charge of PEI@αPC NPs, reducing the surface potential to 0.18 times that of unmodified PEI@αPC NPs ( Figure 8 After peptide coupling, the surface potential of PF9PEI@αPC NPs was further reduced to approximately 4.2 mV. This modification is expected to significantly reduce serum protein adsorption and improve the feasibility of intravenous administration.

[0052] (4) Gel retardation experiment Weigh 1 g of agarose powder and add it to 100 mL of 1× TAE buffer. Mix thoroughly and heat until completely dissolved. Once the solution has cooled to approximately 50°C, add 5 μL of nucleic acid dye, mix thoroughly, and pour into an electrophoresis tank with a comb installed. Let it sit for 20-30 minutes until it solidifies. Place the solidified gel in the electrophoresis tank and add enough 1× TAE buffer to completely submerge the gel. Mix 5 μL of NPs with 1 μL of 6× DNA sample buffer and gently add it to the sample well of the gel. Also, add 5 μL of a DNA molecular weight standard to a separate sample well as a reference. Set the electrophoresis instrument voltage to 100 V and run the gel for approximately 30 minutes, until the dye front reaches 1 / 2 to 2 / 3 of the way up the gel. After electrophoresis, observe the gel using a UV imager and record the position and size of the target band.

[0053] To address different experimental objectives, the nanomaterials were pre-incubated with different solutions. αPC, PEI@αPC NPs, F9PEI@αPC NPs, and PF9PEI@αPC NPs were mixed with DNase I, and the protective effect of the carrier against DNase degradation was analyzed by gel electrophoresis. Furthermore, NPs formed from PF9PEI and αPC at varying mass ratios were analyzed by gel electrophoresis to compare the migration retardation of αPC in the gel at these different mass ratios.

[0054] Optimization of the gene delivery system showed that different mass ratios of PF9PEI to αPC had a significant effect on the encapsulation efficiency of PF9PEI. Gel retardation analysis showed that when the mass ratio of PF9PE:αPC exceeded 0.3, the plasmid DNA was completely encapsulated and the free plasmid band disappeared ( Figure 8 In addition, PF9PEI@αPC NPs have a good protective effect against DNase-mediated plasmid DNA degradation. Under simulated physiological conditions, the plasmid encapsulated by PF9PEI@αPC NPs has an intact structure, retaining clear DNA bands, and the naked plasmid DNA is completely degraded ( Figure 8 This result emphasizes the stability of PF9PEI@αPC NPs in maintaining the structural integrity and functionality of the plasmid during circulation.

[0055] (5) Cell transfection ability test NIH3T3 cells were treated with TGF-β (10 ng / mL) for 24 hours to induce CAFs. The culture medium was then replaced with basal medium and PF9PEI@pEGFR NPs (with a reporter plasmid (pEGFP) replacing αPC) were added. Transfection was terminated 24 hours later, and the transfection efficiency of CAFs was assessed by CLSM or flow cytometry. Transfected cells exhibited green fluorescence, which is the marker for EGFP.

[0056] To test the serum resistance of the vectors, PEI@αPC NPs, F9PEI@αPC NPs, and PF9PEI@αPC NPs were mixed with various concentrations of FBS, and changes in particle size were measured. Protein adsorption onto the different vectors was then analyzed by SDS-PAGE gel electrophoresis and Coomassie Brilliant Blue staining. Next, following a standard transfection procedure, the basal medium was replaced with medium containing varying concentrations of FBS to assess transfection efficiency in CAFs under these different FBS conditions.

[0057] To further evaluate transfection efficiency, a reporter plasmid (pEGFP) was used as a surrogate for αPC. Flow cytometry and fluorescence microscopy analysis revealed that transfection efficiency was less than 10% when the mass ratio of PF9PEI to αPC was low. However, when the weight ratio of PF9PEI to pEGFP reached 10:10, the transfection efficiency significantly increased to 23.6%, confirming this ratio as the optimal condition for subsequent experiments. Figure 9 A and B).

[0058] The stability of gene transfection nanosystems in serum is crucial for effective gene transfection, so the serum resistance of PF9PEI@αPC NPs was evaluated. Figure 9 As shown in Figure C, the particle size of PF9PEI@αPC NPs remained stable for nearly 12 h even in a 50% FBS environment. SDS-PAGE analysis showed that F9PEI@αPC NPs and PF9PEI@αPC NPs significantly reduced protein adsorption, while PEI@αPC NPs had limited resistance to protein binding. It is worth noting that only a faint protein band was detected at approximately 70 kDa, indicating that the amount of protein adsorption other than albumin was very small. These findings indicate that fluorination modification imparts hydrophobic and oleophobic properties to PF9PEI@αPC NPs, thereby enhancing their anti-serum ability. Figure 10 、 Figure 11 and Figure 12 As shown in Figure A, under serum-free conditions, PEI@pEGFP NPs, F9PEI@pEGFP NPs, and PF9PEI@pEGFP NPs showed comparable transfection efficiency. However, with increasing serum concentration, the transfection efficiency of PEI@pEGFP NPs decreased significantly. Notably, PF9PEI@pEGFP NPs still retained nearly half of their transfection capacity in serum-containing medium, reaching a transfection rate of 13.7%. After fluorination and peptide modification, the surface active amino groups were significantly reduced, which was manifested by a decrease in ninhydrin reactivity ( Figure 12 B). Specifically, the surface active amino groups of F9PEI@αPC NPs and PF9PEI@αPC NPs were reduced to 44.16% and 40.13%, respectively. This reduction is a key factor in the enhanced serum resistance of PF9PEI@αPC NPs. Taken together, these results demonstrate that the fluorination and amino-shielding strategy significantly improves the serum stability of the gene delivery system, demonstrating its potential as a candidate system for efficient in vivo gene transfection.

[0059] (6) PD-L2 targeting and blocking behavior assay NIH3T3, CAFs, and αPD-L2-treated CAFs were incubated with equal concentrations of PF9PEI@DNA. Cy5NPs (using Cy5-labeled DNA (DNA Cy5 ) was used to replace αPC and the cells were incubated together for 2 h. CLSM was used to observe the effects of different cell types on PF9PEI@DNA. Cy5 NPs uptake. Cy5 NPs and PF9PEI@DNA Cy5 NPs were incubated with CAFs for another 2 h, and CLSM and flow cytometry were used to assess the expression of PD-L2 protein on the surface of CAFs. The immunofluorescence assay steps for CLSM are as follows: 6.1 Fixation: After drug treatment, wash the confocal microplate with PBS and then fix with 4% paraformaldehyde for 15 minutes. After fixation, wash repeatedly with PBS.

[0060] 6.2. Blocking: Weigh 1.5 g of skim milk powder and dissolve it thoroughly in 30 mL of PBS. Add the milk powder supernatant to the culture dish and incubate at room temperature for 1 hour. After blocking, wash off the milk powder with PBS.

[0061] 6.3 Antibody Incubation: Dilute anti-PD-L2 antibody at a ratio of 1:500 in primary antibody diluent. Incubate cells with the primary antibody solution overnight. Wash with PBS after the primary antibody incubation. Then, incubate the cells with the fluorescent secondary antibody solution at room temperature for approximately 2 hours.

[0062] 6.4. DAPI Incubation: After the antibody incubation, incubate the cells with DAPI for 15 minutes to label the cell nuclei. Wash with PBS and then image using CLSM.

[0063] The gene delivery ability of PF9PEI@αPC NPs was then evaluated, focusing on the effect of PD-L2 peptide modification on CAFs-specific delivery. To track the gene delivery vector, Cy5-labeled DNA (DNA Cy5 ) instead of plasmid αPC, and assembled with PF9PEI to form NPs suitable for fluorescence observation. Figure 13 As shown in the figure, the internalization of the plasmid in NIH3T3 cells was almost negligible, while the internalization of PF9PEI@αPC NPs was clearly observed in CAFs. These results indicate that PF9PEI@αPC NPs exhibit specific targeting to CAFs, thereby reducing nonspecific gene transfection. In addition, pre-blocking with αPD-L2 significantly reduced the internalization of PF9PEI@DNA by CAFs. Cy5 The uptake of NPs further confirmed that the recognition and targeting of CAFs was mediated by PD-L2.

[0064] (7) Detection of related genes and proteins 7.1. PF9PEI@αPC NPs-induced CD86 expression To validate the efficacy of PF9PEI@αPC NPs in transforming CAFs into engineered antigen-presenting CAFs, transfected cells were analyzed. Standard transfection procedures were followed, and quantitative PCR (qPCR) was used to assess CD86 mRNA levels in engineered CAFs transfected with different plasmids. Expression of membrane-bound CD86 was assessed by CLSM and flow cytometry.

[0065] 7.2. PF9PEI@αPC NPs-induced αPD-L1 production Transfection was performed according to standard transfection procedures, and αPD-L1 mRNA levels in engineered CAFs transfected with different plasmids were assessed using qPCR. Supernatants from transfected cells were collected, and αPD-L1 concentrations were quantified by enzyme-linked immunosorbent assay (ELISA). Briefly, high-binding 96-well plates were coated with mouse recombinant PD-L1 protein (5 μg / mL) for 24 hours, blocked with nonfat dry milk for 1 hour, and then incubated with various concentrations of sample and 6x His-tagged PD-1 to mimic αPD-L1. After washing, anti-6x His-tag-HRP (1 μg / mL) was added and incubated for 1 hour. The reaction was stopped with sulfuric acid (5 mM), developed with tetramethylbenzidine (TMB), and absorbance was measured at 450 nm. To verify the ability of the secreted proteins from the transfected cells to block PD-L1 in 4T1 cells, the supernatant was concentrated to 200 μL and incubated with 4T1 cells overexpressing PD-L1 for 24 hours. Subsequently, CLSM and flow cytometry were used to detect the level of PD-L1 protein on the surface of 4T1 cells.

[0066] The actual antigen presentation ability of CAFs is limited due to the lack of co-stimulatory signals, resulting in poor stimulation of T cells. With the efficient gene delivery ability of PF9PEI@αPC NPs, the potential of genetically engineering CAFs into APCs and anti-PD-L1 production factories was evaluated. Figure 14 As shown in Figure A, PF9PEI@C NPs and PF9PEI@αPC NPs treatment significantly upregulated the mRNA level of CD86 in CAFs, and the proportion of CD86-positive CAFs increased to 45.3% and 41.6%, respectively. A significant enhancement of CD86 fluorescence was observed on the cell membrane ( Figure 14 B and Figure 15 These results indicate that PF9PEI@αPC NPs can efficiently upregulate the co-stimulatory molecule CD86, thereby transforming CAFs into engineered CAFs with antigen-presenting function and enhancing the antigen presentation ability to T cells.

[0067] In addition, the αPD-L1 mRNA level was also significantly increased in CAFs treated with PF9PEI@αP NPs or PF9PEI@αPC NPs ( Figure 14 ELISA results showed that the αPD-L1 protein concentration in the culture supernatant of CAFs treated with PF9PEI@αPC NPs reached approximately 2 ng / mL, which was approximately 22 times that of the PF9PEI@pEGFP NPs treatment ( Figure 14 Subsequently, the concentrated supernatant of these cells was co-incubated with 4T1 cells, resulting in a decrease in the PD-L1 level on tumor cells from 25.7% to 12.7% ( Figure 14 In addition, PD-L1 immunofluorescence on 4T1 cells was significantly reduced after treatment with cell supernatant ( Figure 15 These results indicate that PF9PEI@αPC NPs can induce CAFs to secrete αPD-L1, effectively blocking PD-L1 and reducing its levels on 4T1 tumor cells, thereby inhibiting immune escape.

[0068] (8) Immunoexhaustion and immunotoxicity detection of CTLL-2 cells After transfection of CAFs using a standard transfection procedure, radiotherapy was performed. Subsequently, the present invention used high-power ultrasound to treat 4T1 cells to extract their antigens. These engineered CAFs were co-incubated with the extracted antigens for 12 hours to transform them into engineered antigen-presenting CAFs. Next, the present invention used CFSE to label CTLL-2 cells and co-cultured them with engineered CAFs. At the same time, a control group was set up to co-incubate BMDCs with T cells. After 24 hours, the present invention detected the fluorescence intensity of CFSE on T cells to evaluate their proliferation. The schematic diagram of the above co-culture model is shown in Figure 16 . In addition, some of the co-cultured T cells were co-cultured with 4T1 or CT26 tumor cells again for 24 hours, and the culture medium was collected to measure the concentrations of gamma interferon (IFN-γ) and granzyme B (Gra B). These measurements help evaluate the immune effector function of T cells. Finally, the present invention collected tumor cells and detected and analyzed the apoptosis level of tumor cells by flow cytometry combined with Annexin V-APC / PI staining technology.

[0069] To further evaluate the role of engineered CAFs strategy in activating T cell immunity, CTLL-2 cells were co-incubated with untreated or treated CAFs or BMDCs to further detect the phenotypic changes of CTLL-2 cells and their killing effects on tumor cells. Figure 17As shown in Figure A, the proliferation of CTLL2 cells was monitored using CFSE labeling. The results showed that T cell proliferation was minimal when co-cultured with CAFs. However, after blocking the PD-L2 / PD-1 axis using PF9PEI@αPC NPs, T cell proliferation increased from 5.2% to 11.1%. BMDCs induced a strong proliferation response of 67.7%. Antigen-treated engineered CAFs also demonstrated a strong T cell proliferation induction ability, with a proliferation rate of 71.8%. These results further confirmed that the engineered CAFs strategy significantly alleviated the inhibitory effect of CAFs on T cell proliferation after radiotherapy, and further promoted T cell proliferation. Subsequently, the treated T cells were co-incubated with 4T1 tumor cells to assess the level of cell apoptosis. As shown Figure 17 As shown in Figures B and C, the survival rate of 4T1 cells in the RT-CAFs group was as high as 91.7%. In contrast, in the NPs-engineered CAFs group, the apoptosis and death rate of tumor cells increased to 11.02%, which was 1.36 times higher than that of the RT-CAFs group. Both engineered CAFs and BMDCs significantly activated T cells after antigen treatment, resulting in apoptosis and death rates of tumor cells of 18.81% and 14.87%, respectively. When T cells activated by antigen-treated engineered CAFs were co-incubated with tumor cells, the concentrations of IFN-γ and Gra B reached 57.51 pg / mL and 40.34 pg / mL, respectively, which were 4.73 times and 4.13 times that of the RT-CAFs group ( Figure 17 D and Figure 18 A). These results are consistent with the anti-tumor activity of activated T cells. In addition, when co-incubated with antigen-incubated engineered CAFs or RT-CAFs in a live cell workstation, the killing effect of T cells on 4T1 tumor cells was more obvious. Figure 18 As shown in Figure B, the real-time response of T cells to tumor cells was continuously observed in the same field of view. Green fluorescence indicates attached tumor cells, and red fluorescence indicates suspended T cells. T cells co-cultured with RT-CAFs failed to inhibit the rapid growth of tumor cells, even when they came into contact with them. In contrast, T cells co-cultured with engineered CAFs incubated with antigen immediately attacked tumor cells upon addition to the tumor culture dish. Tumor cell shrinkage was observed after 6 hours, apoptotic bodies appeared after 10 hours, and complete tumor cell disappearance occurred after 12 hours.

[0070] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a tumor-associated fibroblast-targeted nanoadjuvant, characterized in that: The preparation method comprises the following steps: S1. preparing an expression plasmid comprising an αPD-L1 sequence and a CD86 sequence; S2. preparing a targeting peptide targeting PD-L2; S3, performing fluorination modification on polyethyleneimine to obtain fluorinated polyethyleneimine; modifying the targeting peptide onto the fluorinated polyethyleneimine to obtain a nanocarrier; S4. Mix the expression plasmid and the nanocarrier to prepare a nanoadjuvant.

2. The preparation method according to claim 1, characterized in that The αPD-L1 sequence in step S1 has the nucleic acid sequence shown in SEQ ID NO.2, and the CD86 sequence has the nucleic acid sequence shown in SEQ ID NO.

4.

3. The preparation method according to claim 1 or 2, characterized in that The expression plasmid further comprises a CMV promoter, an Igκ leader sequence and a P2A sequence; the Igκ leader sequence has a nucleic acid sequence shown in SEQ ID NO.1, and the P2A sequence has a nucleic acid sequence shown in SEQ ID NO.

3.

4. The preparation method according to claim 1, characterized in that In step S2, the targeting peptide has the amino acid sequence shown in SEQ ID NO.

5.

5. The preparation method according to claim 1, characterized in that The step S3 comprises the following steps: (1) Dissolving polyethyleneimine and perfluorobutyl-1,2-epoxypropylene in methanol and reacting them at room temperature to obtain fluorinated polyethyleneimine; (2) Fluorinated polyethyleneimine and targeting peptide were dissolved in purified water, and N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide were added to react at room temperature. After purification and drying, the nanocarrier was obtained.

6. The preparation method according to claim 5, characterized in that The polyethyleneimine in step (1) is PEI 25K ; The purification method in step (2) is dialysis, and the drying method is freeze-drying.

7. The preparation method according to claim 1, characterized in that The expression plasmid and nanocarrier described in step S4 are dissolved in purified water and stirred for reaction at room temperature to obtain a nanoadjuvant.

8. The nanoadjuvant prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the preparation method according to any one of claims 1 to 7 or the nanoadjuvant according to claim 8 in nucleic acid delivery.

10. The use according to claim 9, characterized in that The application includes application in preparing a product for enhancing transfection efficiency in serum.

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