A dual-targeting cascade delivery nanoparticle for remodeling pancreatic cancer microenvironment and preparation method and application thereof

Abstract

The application discloses a dual-targeting cascade delivery nanoparticle for remodeling pancreatic cancer microenvironment and a preparation method and application thereof. SPP1 The nanoparticle is a core-shell structure; the inner core of the core-shell structure is a cationized PLGA nanoparticle loaded with a CRISPR / Cas9 complex targeting SPP1 gene; the surface of the inner core is modified with a viral polypeptide capable of promoting lysosome escape as an interlayer; the outer shell of the core-shell structure is a hybrid cell membrane of macrophages and pancreatic cancer fibroblasts coated on the interlayer, and the surface of the membrane is modified with a tumor penetrating peptide. The application realizes the dual homologous targeting of CAFs and TAMs by using the hybrid membrane for the first time, and masks the toxicity of the viral polypeptide through a clever spatial structure. The nanoparticle not only can efficiently penetrate the dense stroma of pancreatic cancer, but also can trigger the conformational change of the viral polypeptide in the acidic lysosome, realize efficient endosome escape and SPP1 gene editing. The application realizes the reduction of type I collagen deposition and the inhibition of the polarization of TAMs to M2 type from the source, and has great clinical transformation value for the targeted gene therapy of pancreatic cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a dual-targeting cascade delivery nanoparticle for reshaping the pancreatic cancer microenvironment, its preparation method, and its application. Background Technology

[0002] Pancreatic ductal adenocarcinoma (PDAC) is known as the "king of cancers," characterized by its extremely dense connective tissue stroma (primarily composed of tumor-associated fibroblasts (CAFs) and their secreted type I collagen) and a highly immunosuppressive network (mainly mediated by tumor-associated macrophages (TAMs)). Existing antitumor nanomedicine delivery systems suffer from the following insurmountable technical limitations when targeting pancreatic cancer: Single-targeting limitations prevent a comprehensive reshaping of the tumor microenvironment: Existing targeted delivery systems typically target only a single cell (e.g., only cancer cells or a single stromal cell). However, the physical barriers (CAFs) and immune barriers (TAMs) of the PDAC microenvironment crosstalk each other, and intervention in a single cell alone cannot dismantle the protective network of the tumor microenvironment.

[0003] Large nucleic acid systems (CRISPR / Cas9) have extremely low delivery efficiency: Currently widely used FDA-approved polymeric materials (such as PLGA) have negatively charged surfaces, making it extremely difficult to efficiently load similarly negatively charged large CRISPR / Cas9 plasmids. Furthermore, nanomedicines, after being endocytosed by cells, are easily trapped in endosomes / lysosomes and degraded by acidic hydrolases (i.e., "endosome escape" is difficult), resulting in extremely low gene editing efficiency.

[0004] The systemic toxicity bias of traditional viral peptides: To facilitate lysosomal escape, current technologies attempt to modify the surface of nanoparticles with viral peptides such as Sendai virus fusion protein (F / HN). However, direct exposure of these viral peptides to the bloodstream can induce strong immunogenicity and non-specific cell fusion toxicity, leading to off-target effects and greatly limiting their in vivo application. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies, such as difficulty in effectively delivering macromolecular gene editing tools, poor microenvironment penetration, and inability to simultaneously intervene in multiple tumor stromal cells, and to provide a dual-targeting cascade delivery nanoparticle for reshaping the pancreatic cancer microenvironment.

[0006] A second objective of this invention is to provide a method for preparing the dual-targeted cascade delivery nanoparticles.

[0007] A third objective of this invention is to provide applications of the aforementioned dual-targeted cascade delivery nanoparticles.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution: A dual-targeting cascade delivery nanoparticle, wherein the nanoparticle has a core-shell structure; the inner core of the core-shell structure is loaded with a target SPP1 The CRISPR / Cas9 complex of the gene is a cationic polylactic acid-glycolic acid copolymer nanoparticle with an inner core surface modified with a viral polypeptide that promotes lysosomal escape as an interlayer; the outer shell of the core-shell structure is a hybrid cell membrane of macrophages and pancreatic cancer fibroblasts covering the interlayer, and the membrane surface is modified with tumor-penetrating peptides.

[0009] The dual-targeted cascade delivery nanoparticles of this invention comprise, from the inside out: a drug-loaded core layer, a polypeptide interlayer, and a hybrid biomimetic membrane shell layer; the material composition of the virus biomimetic nanoparticles having a three-layer spatial structure of "core-polypeptide interlayer-hybrid membrane shell"; (1) Drug-carrying core layer: encapsulated with target-secreting phosphoprotein 1 ( SPP1 Positively charged cationic polylactic-co-glycolic acid (PLGA) nanoparticles of the CRISPR / Cas9 complex of the gene; this invention uses SPP1 Using genes as pivotal targets, this invention simultaneously achieves a dual reshaping of both the tumor's physical barrier and its immunosuppressive barrier. SPP1 Genes, acting as a common pathological signaling hub connecting CAF activation and TAM immune polarization, promote collagen synthesis in CAFs by activating the TGF-β pathway, while in TAMs they drive polarization towards the immunosuppressive M2 phenotype. This invention utilizes hybrid membrane-mediated dual-target specificity to... SPP1 Gene knockout acts simultaneously on both cell types, inhibiting matrix formation in CAFs (e.g., reducing Collagen I) and blocking and reversing M2 polarization in TAMs. With an integrated design logic of "one vector, one target, two cells, and dual effects," it produces a synergistic therapeutic effect that is significantly superior to single-cell intervention strategies.

[0010] (2) Peptide interlayer: A viral peptide covalently linked or electrostatically adsorbed onto the surface of the drug-loaded core layer, which promotes lysosomal escape; the present invention designs the viral peptide as a structural interlayer hidden between the cationic PLGA core and the hybrid biomimetic membrane shell. During systemic circulation, the outer hybrid cell membrane completely isolates the viral peptide from blood components, eliminating its immunogenicity and non-specific membrane fusion toxicity. When the nanoparticles are endocytosed by the target cell into the acidic endosome / lysosome, the outer hybrid membrane degrades under hydrolytic enzyme and acidic conditions, and the viral peptide is spatiotemporally and specifically exposed. Through a cascade mechanism of "receptor recognition - conformational change - hydrophobic insertion - hexagonal bundle contraction", it actively pierces the endosome membrane and efficiently releases the CRISPR / Cas9 complex into the cytoplasm. The above mechanism strictly limits the activity of the viral peptide to the specific spatiotemporal node of the target cell endosome, completely avoiding off-target toxicity while overcoming the inherent limitations of the low efficiency of traditional passive escape strategies.

[0011] (3) Hybrid biomimetic membrane outer shell: A hybrid cell membrane formed by the fusion of macrophage membrane and tumor-associated fibroblast (CAF) membrane, which endows the nanoparticles with natural homology affinity for both TAMs and CAFs, two core matrix cells, enabling co-delivery of the two target cells in a single administration. Furthermore, the outer surface of the hybrid cell membrane is modified with tumor matrix-penetrating peptides or fibronectin-targeting peptides.

[0012] Furthermore, traditional cell membrane biomimetic nanoparticles rely on passive encapsulation through physical extrusion, resulting in weak interfacial bonding and easy cell membrane detachment. This invention modifies the PLGA core with cationization to make its surface positively charged, creating a well-defined electrostatic attraction gradient between the core and the naturally negatively charged outer hybrid cell membrane vesicles. This drives the hybrid membrane to spontaneously, tightly, and uniformly encapsulate the core surface. This strategy is significantly superior to simple physical extrusion methods, while ensuring stable anchoring of the peptide interlayer, effectively overcoming the problem of poor long-range storage stability of multi-component nanoparticles.

[0013] The nanoparticles of this invention possess a two-step cascade effect to overcome the dense matrix barrier of pancreatic cancer: First, tumor-penetrating peptides modified on the outer surface of the hybrid membrane actively open physical permeation channels for the nanoparticles within the tumor matrix by activating the integrin and neurociliary-1-dependent CendR pathway; second, the CRISPR / Cas9 system residing in CAFs... SPP1 Precise gene knockout continuously disrupts the ability of CAFs to synthesize and secrete type I collagen at the transcriptional level, leading to a sustained decrease in tumor stroma pressure and fundamentally improving the conditions for material transport within the tumor. This sequential cascade strategy of "physical penetration and biological disruption" not only enhances the delivery efficiency of the nanoparticles themselves but also creates favorable conditions for the subsequent penetration of combined chemotherapy drugs.

[0014] Further, the CRISPR / Cas9 complex is a CRISPR / Cas9 plasmid or a CRISPR / Cas9 ribonucleoprotein complex. The CRISPR / Cas9 ribonucleoprotein complex is a functional gene editing complex formed in vitro by the pre-assembly of purified Cas9 nuclease protein and chemically synthesized / in vitro transcribed single-guide RNA (sgRNA) through intermolecular forces.

[0015] Furthermore, the cationization is performed using polyethyleneimine. That is, the cationized polylactic acid-glycolic acid copolymer is a PLGA-PEI block copolymer. The hydrophobic PLGA segments form the inward core, and the cationic PEI segments form the outward stable positively charged shell. The PEI is preferably branched low molecular weight PEI, and the molecular weight of the PEI is preferably 10 kDa.

[0016] Furthermore, the viral polypeptide is the Sendai virus fusion polypeptide F / HN; specifically, it is a fusion protein composed of the fusion protein (F) on the surface of the Sendai virus (SeV) envelope and hemagglutinin-neuraminidase (HN).

[0017] Furthermore, the macrophages include, but are not limited to, mouse monocyte-macrophage RAW 264.7, human macrophage THP-1, bone marrow-derived macrophages (BMDM), peritoneal macrophages (PMs), peripheral blood mononuclear cells (PBMCs), and macrophages isolated from mouse pancreatic cancer tissue.

[0018] Furthermore, the tumor-penetrating peptide is iRGD, and the amino acid sequence of iRGD is c(CRGDKGPDC).

[0019] The present invention also provides a method for preparing any of the above-described nanoparticles, comprising the following steps: S1. Target Construction SPP1 The CRISPR / Cas9 complex of genes; S2. The target obtained in step S1 SPP1 The CRISPR / Cas9 complex of the gene was blended with cationic polylactic acid-glycolic acid copolymer to prepare the target gene encapsulated in step S1. SPP1 Cationic polylactic acid-glycolic acid copolymer nanoparticles of genes; S3. The surface modification of the nanoparticles described in step S2 can promote the escape of lysosomes by viral polypeptides; S4. Mix the cell membranes of macrophages and pancreatic cancer fibroblasts in equal proportions (referring to the ratio of membrane protein mass), then co-extrude them, and insert tumor-penetrating peptide-functionalized amphiphilic phospholipid material to obtain a hybrid cell membrane. S5. Blend the hybrid film from step S4 with the nanoparticles from step S3 to coat the surface of the nanoparticles from step S3 with the hybrid film.

[0020] Further, the targeting described in step S1 SPP1 Targeting genes in the CRISPR / Cas9 complex SPP1 The sgRNA sequence of the gene is sgRNA1: TGCTGTGTCCTCTGAAGAAA or sgRNA2: GCAGAATCTCCTTGCGCCAC or sgRNA3: AAGGTGAAAGTGACTGATTC.

[0021] Furthermore, the reaction described in step S2 includes, but is not limited to, solvent evaporation emulsion method, nanoprecipitation method or double emulsion method (W / O / W).

[0022] Furthermore, the modification method described in step S3 is covalent bonding or electrostatic adsorption.

[0023] Further, the amphiphilic phospholipid material functionalized with the tumor-penetrating peptide described in step S4 is DSPE-PEG-iRGD, namely distearylphosphatidylethanolamine-polyethylene glycol-iRGD peptide, which is an amphiphilic phospholipid material with targeting function. This compound consists of three parts: the phospholipid head DSPE (distearylphosphatidylethanolamine), the hydrophilic linker PEG (polyethylene glycol), and the targeting peptide iRGD. Its structural formula is as follows:

[0024] Structurally, DSPE-PEG-iRGD exhibits a typical triblock structure. The DSPE segment acts as a hydrophobic anchoring group, enabling stable embedding within the lipid bilayer; the PEG segment acts as a hydrophilic spacer arm, providing excellent water solubility and steric hindrance; and the iRGD peptide acts as a targeting ligand, specifically recognizing integrin receptors on the surface of tumor cells.

[0025] The nanoparticles of this invention utilize the hybrid membrane of macrophages and cancer cell fibroblasts (CAFs), combined with tumor-penetrating peptides, to achieve a cascade effect of dual "homological targeting" and "physical penetration" of pancreatic cancer CAFs and tumor-associated membranes (TAMs). This is achieved through delivery... SPP1 Knocking out plasmids inhibits collagen secretion at the source on a biological level, softens the tumor matrix, and reverses TAMs polarization, thus comprehensively reshaping the pancreatic cancer microenvironment and achieving the treatment of pancreatic cancer.

[0026] Therefore, the present invention also provides the use of any of the above-described nanoparticles in the preparation of pancreatic cancer therapeutic drugs.

[0027] The present invention also provides a pancreatic cancer treatment drug, wherein the drug contains any of the nanoparticles described above.

[0028] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0029] Compared with the prior art, the present invention has the following beneficial effects: This invention discloses a dual-targeting cascade delivery nanoparticle for reshaping the pancreatic cancer microenvironment. By constructing a spatiotemporally responsive "sandwich masking structure," it utilizes a biomimetic hybrid cell membrane shell to shield internal viral peptides from toxicity in the blood, and achieves specific exposure in lysosomes for efficient escape, thus solving the problem of low gene delivery efficiency. Utilizing a hybrid membrane of macrophages and CAFs, combined with tumor-penetrating peptides, a cascade effect of dual "homologous targeting" and "physical penetration" of pancreatic cancer CAFs and TAMs is achieved. By delivering an SPP1 knockout plasmid, collagen secretion is inhibited at the source at the biological level, softening the tumor matrix and TAMs, while simultaneously reversing TAM polarization, comprehensively reshaping the pancreatic cancer microenvironment, thereby achieving effective treatment of pancreatic cancer. This has significant clinical translational value for targeted gene therapy of pancreatic cancer. Attached Figure Description

[0030] Figure 1 This image shows the distribution characteristics and functions of SPP1 in the pancreatic cancer microenvironment. A shows immunohistochemical and immunofluorescence staining images of SPP1 expression in normal pancreatic tissue and in situ pancreatic cancer tissue; red represents SPP1, and blue represents cell nuclei. Scale bars: 1000 μm (first row), 100 μm (second row). B shows the Western blot results of ARG1, CD74, and PD-L1 protein expression in RAW 264.7 cells after treatment with recombinant SPP1 protein (rSPP1). C shows the analysis results of SPP1 expression levels in mouse and human pancreatic cancer fibroblast subsets. D shows the proportion of different immune cells in pancreatic cancer patients with high and low SPP1 expression.

[0031] Figure 2 This study validates the inhibition of SPP1 expression and activation phenotype in fibroblasts by the CRISPR / Cas9 system targeting SPP1. In Figure A, Western blot results show the expression of SPP1 and α-SMA proteins in SPP1-overexpressing fibroblasts transfected with empty vector EV or a CRISPR / Cas9 plasmid containing SPP1. Figure B shows immunofluorescence staining images of SPP1, α-SMA, and CollagenI in fibroblasts after the corresponding treatments. Red represents the target protein, and blue represents the cell nucleus. Scale bar: 50 μm.

[0032] Figure 3Results of fibroblast remodeling of macrophage secretory phenotype. After co-culturing SPP1 knockout fibroblasts with macrophages, the secretion levels of TNF-α, IL-6, IL-10, and TGF-β in the co-culture system were detected by ELISA.

[0033] Figure 4 These are cryo-transmission electron microscopy (CTEM) images of the nanoparticles. From left to right, these are CTEM images of the core nanoparticle (PNP), the hybrid membrane-coated nanoparticle (Hym@PNP), and the multi-level nanoparticles modified with the targeted penetrating peptide designed in this invention (RGD-Hym@PNP). Scale bar: 100 nm.

[0034] Figure 5 This is a laser confocal microscopy image of the hybrid macrophage membrane and CAF cell membrane on the surface of nanoparticles. In the image, the DiI channel (red fluorescence) represents the CAF cell membrane, the DiO channel (green fluorescence) represents the macrophage membrane, and the Merge plot is a combined image of the fluorescence channels of both. Scale bar: 50 μm.

[0035] Figure 6 Laser confocal microscopy images show the in vitro cellular uptake capacity of nanoparticles. Blue fluorescence represents DAPI-labeled cell nuclei, and red fluorescence represents RITC-labeled nanoparticles. Scale bar: 20 μm.

[0036] Figure 7 The figures show the in vivo antitumor efficacy and safety evaluation data curves for each group of nanoparticles. The left figure shows the tumor volume change of subcutaneous pancreatic cancer in mice over time, and the right figure shows the body weight change curves of mice in each treatment group during the treatment period. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0038] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0039] Example 1: Validation method for fibroblast-derived SPP1-induced immunosuppressive polarization in macrophages 1. Method Panc02 cells (1×10⁻⁶) were applied to C57BL / 6 mice. 6An orthotopic pancreatic cancer model was constructed. Tumor tissue and normal pancreatic tissue were obtained for immunohistochemistry and immunofluorescence staining to detect SPP1 protein expression. In in vitro experiments, RAW264.7 macrophages (Mφ) were treated with culture medium containing recombinant SPP1 protein (rSPP1, concentration 1.0 μg / mL). Macrophage phenotypic changes were detected by Western blotting and immunofluorescence (IF). Clinical samples were validated using bioinformatics analysis.

[0040] 2. Results The results are as follows Figure 1 As shown, SPP1 expression in mouse pancreatic cancer tissues was significantly higher than in normal tissues, and it was mainly distributed in the tumor stroma region enriched with fibroblasts. Figure 1 A). In vitro rSPP1 treatment significantly upregulated the expression of ARG1, CD74, and PD-L1 proteins in macrophages. Figure 1 B). Bioinformatics analysis showed that SPP1 levels in pancreatic cancer fibroblasts were higher than in normal fibroblasts ( Figure 1 C), and the proportion of immunosuppressive macrophages in tumor tissues of patients with high SPP1 expression was significantly higher than that of patients with low SPP1 expression (C). Figure 1 D). This demonstrates that SPP1 is a key factor driving immunosuppressive polarization in macrophages.

[0041] Example 2: A method for reshaping macrophage immune polarization using a CRISPR / Cas9 system targeting SPP1. 1. Method Construct a CRISPR / Cas9 plasmid vector (eSpCas9-2A-GFP) targeting SPP1 (sgRNA is sgRNA1: TGCTGTGTCCTCTGAAGAAA (used in this example) or sgRNA2: GCAGAATCTCCTTGCGCCAC or sgRNA3: AAGGTGAAAGTGACTGATTC), and transfect it into SPP1-overexpressing fibroblasts (SPP1). + (FIB) screening was used to obtain SPP1 knockout fibroblasts. An in vitro co-culture system was constructed to co-culture SPP1 knockout fibroblasts with normal macrophages. Macrophage phenotype was assessed by Western blotting and ELISA, and the secretion levels of TNF-α, IL-6, IL-10, and TGF-β in the co-culture system were detected.

[0042] 2. Results The results are as follows Figure 2 As shown, knocking out CAF-derived SPP1 using this gene editing method significantly reduced SPP1 expression in cells. Figure 2A), and at the same time, the expression of α-SMA, a marker of CAF cell activation, was significantly downregulated, and Collagen I synthesis was reduced ( Figure 2 (B) indicates that the activation of CAFs was significantly inhibited. Furthermore, as... Figure 3 As shown, the expression of PD-L1 and CD74 in macrophages in the co-culture system decreased significantly, and the cellular secretory profile reversed towards an anti-tumor direction (TNF-α and IL-6 were significantly increased). This demonstrates that targeting SPP1 using nucleic acid tools is an effective way to reverse TAM polarization. This indicates that... SPP1 Genes serve as a common pathological signaling hub connecting CAF activation and TAM immune polarization. In CAFs, they promote collagen synthesis by activating the TGF-β pathway, while in TAMs, they drive the polarization towards the immunosuppressive M2 phenotype.

[0043] Example 3: Preparation and Physicochemical Characterization of Multilayer Cascaded Delivery Nanoparticles 1. Method (1) A cationic PLGA core encapsulating SPP1-Cas9 plasmid was prepared using a two-emulsion method. 45 mg of PLGA, 4 mg of PLGA-PEG, and 1 mg of PLGA-PEG-Mal were weighed and added to 2 mL of dichloromethane. The mixture was vortexed or magnetically stirred until completely dissolved to obtain the polymer organic phase. The PLGA was preferably a polylactic-co-glycolic acid copolymer with a lactic acid / glycolic acid molar ratio of 50:50 or 75:25, and the weight-average molecular weight was preferably 20–50 kDa, more preferably 30–40 kDa. The PLGA-PEG was preferably an amphiphilic block copolymer with a PLGA segment molecular weight of 10–30 kDa and a PEG segment molecular weight of 2–5 kDa, such as PLGA20k-PEG5k (used in this embodiment). The PLGA-PEG-Mal was preferably a PLGA-PEG derivative with a maleimide terminal group, such as PLGA20k-PEG5k-Mal (used in this embodiment). The mass ratio of PLGA, PLGA-PEG and PLGA-PEG-Mal can be 80-95:3-15:0.5-5, preferably 90:8:2 (used in this embodiment).

[0044] In another example, 50 μg of the SPP1-Cas9 plasmid constructed in Example 2 was diluted to 200 μL with nuclease-free water. PEI solution (10 kDa) was added. The N / P ratio of PEI was 10:1 (nitrogen in PEI to phosphate in plasmid DNA). After mixing, the mixture was gently blown to homogenize and incubated at room temperature for 20 min to allow the SPP1-Cas9 plasmid and PEI to form a PEI / DNA complex through electrostatic interaction. The 200 μL PEI / SPP1-Cas9 complex was used as the inner aqueous phase and slowly added dropwise to 2 mL of dichloromethane organic phase containing PLGA, PLGA-PEG, and PLGA-PEG-Mal. The mixture was then sonicated under ice bath conditions using a probe at 80 W, with a 2 s working and 2 s rest interval, for a total sonication time of 60 s, forming a primary emulsion W1 / O.

[0045] The resulting colostrum was then rapidly added dropwise to 10 mL of a 2% (w / v) PVA aqueous solution as the external aqueous phase W2. The PVA was preferably polyvinyl alcohol with a molecular weight of 30–70 kDa and a degree of hydrolysis of 87%–89% or 98%–99%, more preferably PVA with a molecular weight of approximately 31–50 kDa and a degree of hydrolysis of 87%–89%. A second ultrasonic emulsification was performed under ice bath conditions at a power of 120 W, with a 2-second interval between cycles, for a total ultrasonic time of 120 s, yielding a W1 / O / W2 double emulsion. The double emulsion was magnetically stirred at room temperature for 4 h at a stirring speed of 500 rpm to allow the dichloromethane to fully evaporate and promote the solidification of the nanoparticles. The emulsion was centrifuged at 15000 g for 20 min, the supernatant was discarded, and the precipitate was collected. The precipitate was washed three times with PBS, centrifuged at 15000 g for 10 min each time, to remove free PVA, unencapsulated plasmids, and residual solvent. Finally, the precipitate was resuspended in 1 mL PBS to obtain the core nanoparticles encapsulating the SPP1-Cas9 plasmid, denoted as NP.

[0046] (2) Modification of Sendai virus F / HN peptide on the surface of core nanoparticles This step utilizes the maleimide groups exposed on the surface of the nanoparticles by the PLGA-PEG-Mal introduced in step (1) to undergo a thiol-maleimide Michael addition reaction with the thiol-containing Sendai virus F / HN polypeptide, thereby covalently linking the Sendai virus F / HN polypeptide to the surface of the PLGA nanocore.

[0047] Take the Mal-PNP / SPP1-Cas9 nanoparticle suspension obtained in step (1), wash and resuspend it with PBS or HEPES buffer, and adjust the nanoparticle concentration to 1-5 mg / mL, preferably 5 mg / mL (used in this example). The pH of the buffer solution is preferably 6.8-7.2 to ensure the reaction efficiency between maleimide groups and thiol groups, while reducing maleimide hydrolysis.

[0048] The Sendai virus F / HN polypeptide is preferably a terminal cysteine-modified F / HN functional polypeptide, i.e., F / HN-Cys or Cys-F / HN, which contains free thiol groups that can participate in the Michael addition reaction. Before use, the F / HN-Cys polypeptide is dissolved in HEPES buffer to prepare a polypeptide solution of 0.5–2 mg / mL. If necessary, a reducing agent TCEP can be added for a short-term treatment to keep the polypeptide thiol groups in a reduced state. Free TCEP is then removed by desalting column or ultrafiltration to avoid interference with subsequent reactions. The F / HN-Cys polypeptide is added to the Mal-PNP / SPP1-Cas9 nanoparticle suspension, such that the mass ratio of the F / HN-Cys polypeptide to the total PLGA mass in the nanoparticles is 1:10–1:50, preferably 1:20 (used in this embodiment). For example, 50 μg of F / HN-Cys polypeptide is added to every 1 mg of Mal-PNP / SPP1-Cas9 nanoparticles. After mixing, gently shake and react for 2–12 h at room temperature or 4°C, preferably overnight at 4°C, or 4 h at room temperature.

[0049] After the reaction was complete, the reaction solution was centrifuged at 15000 g for 20 min, the supernatant was discarded, and the precipitate was collected. The precipitate was washed three times with PBS, centrifuged at 15000 g for 10 min each time, to remove unreacted free F / HN-Cys peptides. Finally, the precipitate was resuspended in PBS to obtain a PLGA nanocore with surface covalently modified Sendai virus F / HN peptides, denoted as PNP.

[0050] (3) Extract the cell membranes of RAW 264.7 cells and CAFs cells to prepare iRGD-modified hybrid membranes. Cell membranes were extracted from mouse macrophages (RAW 264.7) and pancreatic cancer fibroblasts (CAFs) to prepare iRGD-modified hybrid membranes. Mouse macrophages (RAW 264.7) and pancreatic cancer-associated fibroblasts (CAFs) were cultured separately. When cell confluence reached 80%–90%, the culture medium was discarded, and the cells were washed three times with pre-cooled PBS. Cells were collected using a cell scraper, centrifuged at 800g for 5 min, and the supernatant was discarded; the cell pellet was collected.

[0051] Add pre-cooled hypotonic lysis buffer, comprising 10 mM Tris-HCl, 1 mM EDTA, and a protease inhibitor, to the cell pellet at pH 7.4. Per 1 × 10 7 Add 1 mL of hypotonic lysis buffer to each cell. Incubate on ice for 30 min, then homogenize 30 times to ensure complete cell lysis. Centrifuge the homogenate at 800 g for 10 min to remove nuclei and unly lysed cells. Collect the supernatant and centrifuge at 10000 g for 20 min to remove mitochondria and large organelles; then ultracentrifuge the supernatant at 100000 g for 60 min to collect the cell membrane pellet. Wash the pellet once with pre-chilled PBS, centrifuge again at 100000 g for 30 min, and finally resuspend in PBS to obtain RAW 264.7 cell membranes and CAF cell membranes, respectively. The membrane protein concentrations of the two cell membrane suspensions were determined using the BCA method. Mix RAW 264.7 cell membranes and CAF cell membranes at a 1:1 molecular weight ratio. For example, mix 1 mg of RAW 264.7 cell membrane protein with 1 mg of CAF cell membrane protein, and add PBS to bring the volume to 1 mL.

[0052] The above-mentioned mixed membrane suspension was sequentially extruded through 400 nm, 200 nm, and 100 nm polycarbonate membranes, with each pore size being extruded 10 times to ensure complete fusion of the two cell membranes, forming hybrid cell membrane vesicles, denoted as Hym. Subsequently, DSPE-PEG-iRGD was added to the Hym hybrid membrane suspension at a mass ratio of DSPE-PEG-iRGD to membrane protein of 1:20. For example, 50 μg of DSPE-PEG-iRGD was added for every 1 mg of membrane protein. After mixing, the mixture was incubated at 37 °C for 30 min to allow the DSPE lipid tails to insert into the hybrid membrane lipid bilayer. After incubation, the mixture was ultracentrifuged at 100,000 g for 30 min, the supernatant was discarded, and any uninserted free DSPE-PEG-iRGD was removed. The precipitate was resuspended in PBS to obtain the iRGD-modified hybrid cell membrane, denoted as RGD-Hym.

[0053] (4) The hybrid film is coated on the surface of the PLGA core by electrostatic self-assembly. Take the PNP nanocore suspension obtained in step (2) and adjust its concentration to 1 mg / mL. Take the RGD-Hym and Hym hybrid membrane suspensions obtained in step (3) and mix them at a PNP nanocore to membrane protein mass ratio of 1:1. For example, take 1 mg of nanocore and mix it with RGD-Hym or Hym containing 1 mg of membrane protein. When mixing, slowly add the RGD-Hym suspension to the PNP nanocore suspension while gently vortexing. After the addition is complete, incubate at room temperature for 30 min to allow the hybrid membrane to adsorb and coat the nanocore surface through electrostatic interaction and membrane fusion effect.

[0054] The mixture was then extruded sequentially through 400 nm, 200 nm, and 100 nm polycarbonate membranes, with each pore size repeated 10 times to promote uniform rearrangement of the hybrid membrane on the PNP core surface and the formation of a complete shell. After extrusion, the membrane was centrifuged at 12000 g for 15 min to remove uncoated free membrane vesicles and large particle aggregates. The precipitate was washed twice with PBS and resuspended to obtain the final iRGD-modified hybrid membrane-coated PNP nanoparticles, denoted as RGD-Hym@PNP. Following the same method, a control group of unmodified iRGD nanoparticles, denoted as Hym@PNP, was prepared by adding Hym suspension to the PNP nanocore suspension.

[0055] (5) TEM and DLS characterization of nanoparticles Take the nanoparticle suspensions from each group, dilute them with deionized water or PBS to 0.1-0.5 mg / mL, mix thoroughly, and add them to disposable particle size analysis dishes. Measure the hydrated particle size, polydispersity index (PDI), and zeta potential using a dynamic light scattering instrument. Each sample group was tested three times, and the average value was taken.

[0056] 3-5 μL of sample was dropped onto a carbon film copper grid treated with glow discharge and allowed to stand for 10-30 s for adsorption. Excess liquid was then absorbed using filter paper, allowing a thin liquid film to form on the copper grid surface. The copper grid was immediately and rapidly immersed in liquid ethane for vitrification, with the liquid ethane pre-cooled by liquid nitrogen. Observation was performed under a cryo-transmission electron microscope, with an accelerating voltage of 200 kV preferred, using low electron dose mode for image acquisition.

[0057] 2. Results Morphological verification: TEM images such as Figure 4 As shown, the uncoated core nanoparticles (PNP group) exhibit a smooth, dense, solid spherical morphology. After being coated with the hybrid membrane, both the Hym@PNP and RGD-Hym@PNP groups showed a distinct, uniformly thick, light-colored halo around their nanoparticles, exhibiting a typical and complete core-shell structure. This morphological feature directly confirms the successful coating of the hybrid cell membrane on the surface of the nanocore.

[0058] Particle size and surface potential analysis: As shown in Table 1, the hydrated particle size of PNP is 147.5 ± 0.9 nm, and the Zeta potential is -16.14 ± 0.52 mV, consistent with the basic characteristic of PLGA nanoparticles being negatively charged. After hybrid membrane coating, the particle size of Hym@PNP increased to 169.3 ± 3.7 nm, and the potential was approximately neutral at -2.82 ± 0.38 mV, mainly due to the outer cell membrane. Further insertion of a penetrating peptide into the outer membrane resulted in a slight increase in the particle size of RGD-Hym@PNP to 172.5 ± 4.0 nm, with a potential of -2.49 ± 0.41 mV. These significant changes in particle size and potential demonstrate the successful preparation of the multilayer cascaded biomimetic nanoparticles of this invention.

[0059] Table 1. Hydrated particle size (Size) and surface zeta potential of nanoparticles

[0060] Example 4: Fusion Verification of Hybrid Membranes 1. Method Macrophage membranes were labeled with DiO (green fluorescence) and CAF cell membranes with DiI (red fluorescence), respectively. These were used to prepare RGD-Hym@PNP nanoparticles, and the co-localization effect of fluorescence was observed by confocal microscopy to investigate the hybridization effect of cell membranes.

[0061] 2. Results Laser confocal microscope images as follows Figure 5 As shown, the CAF cell membrane (red fluorescence) and macrophage cell membrane (green fluorescence) coated on the surface of RGD-Hym@PNP nanoparticles exhibit a high degree of fluorescence overlap (a significant orange-yellow co-localization signal is observed in the Merge plot), indicating that the two cell membranes form a hybrid membrane on the nanoparticle surface. This result directly confirms that the two cell membranes from different sources successfully fused to form a bilayer structure after co-extrusion treatment, laying a structural foundation for subsequent homologous targeting using cell membranes.

[0062] Example 5: Verification of Cellular Uptake Capacity 1. Method The core nanoparticles were labeled with RITC (red fluorescence). The nanoparticles were incubated with target cells for 2 h, and the cell nuclei were stained with DAPI (blue fluorescence). The uptake efficiency of the nanoparticles in the cells was observed by confocal microscopy.

[0063] 2. Results Results of in vitro cell uptake experiments as follows Figure 6As shown, the cell entry efficiency of nanoparticles with different modifications differed significantly within target cells. The PNP group nanoparticles exhibited only extremely weak red fluorescence signals in the cytoplasm, indicating very low cellular uptake efficiency of the core nanoparticles. Hym@PNP nanoparticles showed significantly enhanced red fluorescence signals in cells compared to the PNP group, confirming that the homologous targeting of macrophages and CAF hybrid membranes promoted nanoparticle uptake within cells. The multi-layered biomimetic nanoparticles RGD-Hym@PNP constructed in this invention exhibited the strongest red fluorescence signal in cells, with a large number of nanoparticles accumulating in the region surrounding the cell nucleus. These results fully validate that modification with the iRGD tumor-penetrating peptide can effectively improve the cellular uptake and penetration capabilities of nanoparticles.

[0064] Example 6: Evaluation of in vivo antitumor effect 1. Method Panc02 cells in the logarithmic growth phase and in good condition were collected, washed with PBS, digested with trypsin, centrifuged, and resuspended in a mixture of sterile PBS and Matrigel to adjust the cell concentration to 5 × 10⁻⁶ cells / mL. 7 / mL. 100 μL of cell suspension was subcutaneously injected into the right axilla or right back of each C57BL / 6 mouse, i.e., 5 × 10⁹ cells per mouse. 6 One Panc02 cell was inoculated. Tumor formation in mice was observed periodically after inoculation, and the long and short diameters of the tumors were measured using calipers.

[0065] Mice were randomly divided into four groups of six: a PBS group, a PNP core nanoparticle group, a Hym@PNP cell membrane-coated nanoparticle group, and a RGD-Hym@PNP multilayer biomimetic nanoparticle group. Day 0 was the day the administration began. Mice in each group received the corresponding formulation via tail vein injection. Each nanoparticle formulation group was administered the same dose based on the amount of plasmid DNA it contained, preferably 1 mg / kg of plasmid DNA. The total dose of nanoparticles, calculated based on the plasmid drug loading, was preferably approximately 10 mg / kg. Each tail vein injection was administered in a volume of 100–200 μL per mouse, preferably 200 μL per mouse.

[0066] The preferred dosing cycle is once every 3 days, for a total of 5 doses, administered via tail vein injection on days 0, 3, 6, 9, and 12. The general condition of the mice is observed during the dosing process. From the first dosing day, the subcutaneous tumor volume is measured every 2 days, and changes in mouse body weight are recorded. Tumor volume is calculated using the formula V = 1 / 2 × L × W. 2The tumor diameter was calculated, where L represents the longest diameter of the tumor and W represents the shortest diameter perpendicular to the longest diameter. Tumor growth curves were plotted based on tumor volumes measured at each time point, and the in vivo safety of different formulations was evaluated based on changes in body weight. The experimental observation period typically lasted until day 21, or the experiment was terminated when the tumor volume in the control group approached the ethical endpoint. If, during the experiment, mouse body weight decreased by more than 20% or tumor volume exceeded 1500-2000 mm², the experiment was considered terminated. 3 Euthanasia may be performed in advance in cases of tumor rupture, mobility impairment, or significant pain.

[0067] 2. Results Tumor volume growth curve as shown Figure 7 As shown, during the 21-day observation period, tumors in both the PBS control group and the PNP group exhibited exponential rapid growth, with an average terminal volume of approximately 1200 mm. 3 Injection of Hym@PNP nanoparticles showed a certain antitumor effect in mice, with the tumor volume reaching approximately 600 mm² in the terminal stage. 3 In comparison, the RGD-Hym@PNP nanoparticles of this invention exhibited the best tumor-suppressive effect, with the average tumor volume at terminal stage controlled at 300 mm. 3 Within [a certain range]. Furthermore, the body weight of mice in each treatment group maintained a steady upward trend throughout the experimental period, demonstrating that the nanosystem has good biosafety and clinical translation potential.

Claims

1. A nanoparticle, characterized in that, The nanoparticles have a core-shell structure; the inner core of the core-shell structure is loaded with a target. SPP1 The CRISPR / Cas9 complex of the gene is a cationic polylactic acid-glycolic acid copolymer nanoparticle with an inner core surface modified with a viral polypeptide that promotes lysosomal escape as an interlayer; the outer shell of the core-shell structure is a hybrid cell membrane of macrophages and pancreatic cancer fibroblasts covering the interlayer, and the membrane surface is modified with tumor-penetrating peptides.

2. The nanoparticles according to claim 1, characterized in that, The CRISPR / Cas9 complex is a CRISPR / Cas9 plasmid or a CRISPR / Cas9 ribonucleoprotein complex.

3. The nanoparticles according to claim 1, characterized in that, The cationization is performed using polyethyleneimine.

4. The nanoparticles according to claim 1, characterized in that, The viral polypeptide is Sendai virus fusion polypeptide F / HN.

5. The nanoparticles according to claim 1, characterized in that, The tumor-penetrating peptide is iRGD.

6. The method for preparing the nanoparticles according to any one of claims 1 to 5, characterized in that, Includes the following steps: S1. Target Construction SPP1 The CRISPR / Cas9 complex of genes; S2. The target obtained in step S1 SPP1 The CRISPR / Cas9 complex of the gene was blended with cationic polylactic acid-glycolic acid copolymer to prepare the target gene encapsulated in step S1. SPP1 Cationic polylactic acid-glycolic acid copolymer nanoparticles of genes; S3. The surface modification of the nanoparticles described in step S2 can promote the escape of lysosomes by viral polypeptides; S4. Mix the cell membranes of macrophages and pancreatic cancer fibroblasts in equal proportions, then co-extrude them, and insert tumor-penetrating peptide-functionalized amphiphilic phospholipid material to obtain a hybrid cell membrane. S5. Blend the hybrid cell membrane from step S4 with the nanoparticles from step S3 to coat the surface of the nanoparticles from step S3 with the hybrid membrane.

7. The preparation method according to claim 6, characterized in that, The target described in step S1 SPP1 Targeting genes in the CRISPR / Cas9 complex SPP1 The sgRNA sequence of the gene is sgRNA1: TGCTGTGTCCTCTGAAGAAA or sgRNA2: GCAGAATCTCCTTGCGCCAC or sgRNA3: AAGGTGAAAGTGACTGATTC.

8. The preparation method according to claim 5, characterized in that, The amphiphilic phospholipid material for tumor-penetrating peptide functionalization in step S4 is DSPE-PEG-iRGD.

9. The use of the nanoparticles according to any one of claims 1 to 5 in the preparation of a pancreatic cancer treatment drug.

10. A pancreatic cancer treatment drug, characterized in that, It contains the nanoparticles described in any one of claims 1 to 5.

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