MMP-9 / pH dual-responsive fusion membrane-wrapped biomimetic nanodelivery system, and preparation method and application thereof

Abstract

This invention relates to the field of pharmaceutical technology, specifically to a biomimetic nanodelivery system (LLip@PC-GHRK) encapsulated in an MMP-9 / pH dual-responsive fusion membrane, its preparation method, and its applications. The outer layer of this nanocomposite system is a biomimetic membrane LLip fused with tumor cell membranes and liposomes, promoting efficient uptake of biomimetic nanomedicines by tumor cells. The middle layer is coated with pH-responsive units, enabling charge-flipping molecules PLL-CA (PC) to prevent drug degradation in lysosomes. The core is a multifunctional self-assembled polypeptide carrier integrating pH-responsive peptides, cell-penetrating peptides, and nuclear-localizing peptides, capable of loading gene-based drugs. This biomimetic nanodelivery system exhibits good biocompatibility and significantly improves targeted drug delivery efficiency and efficacy through tumor homology targeting, tumor microenvironment responsive activation, and programmed drug release, providing a novel intelligent delivery paradigm for combination cancer therapy.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a biomimetic nanodelivery system encapsulated in an MMP-9 / pH dual-responsive fusion membrane, its preparation method, and its application. Background Technology

[0002] Malignant tumors have become the leading cause of death worldwide, with lung cancer being the leading cause of both morbidity and mortality, posing a significant challenge to its clinical treatment. Non-small-cell lung cancer (NSCLC) is the most common type, accounting for approximately 85% of cases. Traditional treatments for lung cancer include surgical resection, radiotherapy, and chemotherapy. However, due to the insidious nature of early symptoms, most patients are diagnosed at an advanced stage, missing the opportunity for radical surgical intervention. While radiotherapy and chemotherapy can inhibit tumor progression to some extent, they suffer from poor targeting, strong side effects, and a high risk of multidrug resistance, resulting in limited treatment efficacy. In recent years, targeted therapies (such as EGFR inhibitors and ALK inhibitors) have brought survival benefits to some patients with driver gene-positive lung cancer. However, these drugs are only suitable for individuals with specific gene mutations, and long-term use can easily lead to drug resistance mutations, limiting their clinical application. Therefore, developing highly effective, low-toxicity, and broad-spectrum novel treatment strategies for lung cancer has become a major clinical challenge.

[0003] Nanoparticle delivery systems, with their unique physicochemical properties, exhibit irreplaceable advantages in cancer treatment. Nanocarriers can achieve passive targeted enrichment of tumor tissue through enhanced permeability and retention effect (EPR), and can also load multiple types of drugs for combined therapy. Among them, micelles, as a classic type of nanocarrier, are formed by the self-assembly of amphiphilic molecules into a core-shell structure. The hydrophobic core can load hydrophobic drugs, while the hydrophilic shell improves the carrier's cyclic stability in vivo, and they have been widely used in cancer drug delivery. Peptide micelles, as an important branch of micelle carriers, have advantages such as good biocompatibility, non-toxic degradation products, and flexible targeting modification. Their amino acid sequences can be precisely designed to achieve specific recognition of tumor cells or response to tumor microenvironment signals. However, traditional peptide micelles still have significant shortcomings: their targeting depends on single ligand modification, making them easily recognized and cleared by the immune system, resulting in limited targeting accuracy; they lack an effective lysosomal escape mechanism, leading to rapid intracellular degradation of the loaded drug; and they are difficult to achieve stable co-delivery of multiple drugs with different physicochemical properties (such as macromolecular antibodies and small molecule nucleic acids), which limits the implementation of combination therapy strategies.

[0004] The development of biomimetic nanocarriers offers a new approach to overcoming the limitations of traditional nanocarriers in terms of targeting and biocompatibility. These carriers achieve "biomimetic camouflage" by mimicking the structure and function of natural biological membranes (such as tumor cell membranes and erythrocyte membranes). They can actively target tumor tissues using homology recognition molecules on the membrane surface, while effectively evading the immune system and prolonging their circulation time in vivo. Among them, biomimetic nanocarriers coated with tumor cell membranes can retain antigens and adhesion factors on the surface of the source cells, precisely recognizing and accumulating in the tumor region through a "homology targeting" mechanism. They also possess advantages such as low immunogenicity and have demonstrated good targeted delivery effects in various tumor models. However, existing biomimetic nanocarriers still face the following technical bottlenecks: First, their response mechanisms are relatively simple, mostly relying on single signals such as pH or enzymes, making it difficult to match the complex characteristics of multiple factors coexisting in the tumor microenvironment (TME) (such as acidic pH, high concentrations of matrix metalloproteinases, etc.); second, their intracellular delivery efficiency is limited. Although they can achieve tumor tissue enrichment, their transmembrane and lysosomal escape capabilities are insufficient, affecting the effective intracellular action of drugs; in addition, the compatibility of multi-drug co-delivery is poor, making it difficult to achieve stable co-loading and simultaneous release of antibody-based macromolecular drugs and nucleic acid-based small molecule drugs, thus limiting the implementation of synergistic strategies such as "immunotherapy + gene therapy".

[0005] In the functional optimization and adaptation of biomimetic nanodelivery systems to lung cancer treatment scenarios, researchers have further focused on the unique biological characteristics of the lung cancer tumor microenvironment (TME)—which not only exhibits an acidic pH microenvironment but also features high expression of matrix metalloproteinase-9 (MMP-9). This specific phenotype facilitates tumor cell proliferation and invasion and serves as a key targeting marker distinguishing lung cancer tissue from normal tissue. Meanwhile, lung cancer treatment still faces core challenges such as insufficient precision in targeted delivery, susceptibility of carriers to clearance by the body's immune system, lack of temporal and spatial control in drug release, and low intracellular transport efficiency. Single-function delivery systems struggle to overcome the drug resistance bottleneck in lung cancer treatment.

[0006] Currently, there is no biomimetic nanomedicine delivery system that combines lung cancer cell membranes with multifunctional liposome membranes and is modified to achieve targeted delivery of tumor immunotherapy and gene therapy drugs. Summary of the Invention

[0007] Based on the aforementioned clinical needs and biological characteristics, the purpose of this invention is to provide a biomimetic carrier that combines homology targeting and immune escape capabilities. This carrier utilizes tumor cell membrane encapsulation to achieve precise enrichment of lung cancer tissue, avoiding clearance by the body's immune system. It employs a dual regulatory mechanism of "pH response + enzyme response," adapting to the acidic environment of the tumor microenvironment (TME) and high MMP-9 expression characteristics, enabling spatiotemporally controlled release of macromolecular antibodies and small nucleic acids. Integrating charge-flipping and transmembrane peptide functions enhances the carrier's lysosomal escape capability and intracellular transport efficiency, ensuring effective enrichment of gene drugs within tumor cells and their gene-silencing effect. Through the synergistic therapeutic effect of antibody blocking and gene silencing, it overcomes resistance to single-treatment approaches, providing a new technical solution for lung cancer treatment.

[0008] The main technical problems to be solved by this invention are: how to achieve the specific release and targeted enrichment of anti-CTLA-4 in the tumor microenvironment, how to improve the delivery efficiency of gene drugs, and how to achieve the temporal synergy of "first activating the immune system → then releasing the gene", so as to achieve targeted delivery and precise release of tumor therapeutic drugs.

[0009] To address the aforementioned technical problems, this invention provides a biomimetic nanodelivery system (LLip@PC-GHRK) with MMP-9 / pH dual-response fusion membrane encapsulation, characterized by targeted drug release, tumor cell targeting, and synergistic drug release timing. This system, along with its preparation method and application in the combined treatment of solid tumors, enables highly efficient co-loading of an immunomodulator (anti-CTLA-4) and a gene therapy drug (siKMT5A), simultaneously resolving issues such as off-target toxicity of immunomodulators, low gene transfection efficiency, and insufficient synergy between immunotherapy and gene therapy.

[0010] In a first aspect, the present invention provides a multifunctional self-assembling polypeptide, wherein the amino acid sequence of the polypeptide is GSVSHHHHHHGGHHHH-RRRRRRRR-KRRRRG (abbreviated as GHRK, SEQ ID NO.1), consisting of 30 peptides composed of glycine (G), serine (S), valine (V), histidine (H), arginine (R), and lysine (K), with the amino acids linked by peptide bonds, and the "-" in the sequence serving as functional module separators. The polypeptide is obtained by coupling a 16-residue peptide with high endocytosis escape capability—hsLMWP (GSVSHHHHHHGGHHHH), a pH-responsive cell-penetrating peptide—R8 (RRRRRRRR), and a nuclear localization signal peptide—NLS (KRRRRG).

[0011] The peptide integrates three synergistic functional modules sequentially from the N-terminus to the C-terminus: ① pH-responsive module (GSVSHHHHHHGGHHHH): rich in 12 histidine residues, its pI value matches the acidic environment (pH 5.0-5.5) of tumor endosomes, providing pH responsiveness and enabling targeted release in acidic environments (tumor endosomes); ② Cell-penetrating module (RRRRRRRR, R9): a strong cationic cell-penetrating peptide sequence that mediates efficient non-endocytic penetration of the cell membrane via hydrogen bonding and hydrophobic interactions between the guanidinyl group and the cell membrane phospholipid bilayer, enhancing intracellular internalization efficiency; ③ Nuclear localization module (KRRRRG): specifically recognizes nuclear membrane transport proteins, guiding the loaded gene drug into the cell nucleus to exert its effect. This peptide possesses a complete "response-penetration-targeting" chain function without additional chemical modification, providing a core molecular tool for the precise delivery of gene drugs (such as siKMT5A) and a key functional unit for constructing intelligent nanodelivery systems.

[0012] Furthermore, the chemical structure of the polypeptide is shown in formula (I):

[0013]

[0014] Formula (I).

[0015] Furthermore, the molecular weight of the polypeptide is 3893.30 Da.

[0016] Furthermore, the polypeptide can be prepared using a solid-phase synthesis method.

[0017] In a second aspect, the present invention provides a multifunctional self-assembling polypeptide micelle, wherein the polypeptide micelle is a polymer (GHRK polypeptide micelle) formed by the above-mentioned multifunctional self-assembling polypeptide (GHRK) through its own hydrophobic interaction and electrostatic interaction.

[0018] Furthermore, the polypeptide micelles are first synthesized by solid-phase synthesis of the target polypeptide chain GSVSHHHHHHGGHHHH-RRRRRRRR-KRRRRG (abbreviated as GHRK), and then the synthesized polypeptide chain is prepared by oil-in-water emulsion evaporation method.

[0019] Furthermore, the preparation method of the peptide micelles is as follows: GHRK peptide monomer is dissolved in deionized water, vortexed and mixed, and acetone is added dropwise to the GHRK aqueous solution while stirring (800 rpm / min). After sealing and stirring for 2 h, the lid is opened until the acetone is completely evaporated to obtain GHRK peptide micelle solution; wherein the volume ratio of deionized water to acetone is 3:5.

[0020] The aforementioned polypeptide micelles use GHRK polypeptide as the sole building block, and their function is directly achieved through the modular integration properties of the polypeptide: they can efficiently load gene drugs without additional modification, achieve controlled drug release in acidic environments through a pH-responsive module, promote intracellular internalization of the carrier through a cell penetration module, and complete nuclear-targeted delivery of gene drugs through a nuclear localization module. The nanocarriers have uniform particle size (80~100 nm), PDI < 0.2, are weakly cationic, and possess both good structural stability and biocompatibility. They can be used directly as gene drug delivery carriers or further assembled with an intermediate coating and an outer fusion membrane to form a composite delivery system, adapting to the needs of different therapeutic scenarios.

[0021] A third aspect of the present invention provides a biomimetic nanodelivery system (LLip@PC-GHRK) encapsulated by an MMP-9 / pH dual-responsive fusion membrane, which is made by modifying the surface of peptide micelles with a charge-reversible negative coating of citric anhydride grafted with polylysine (PLL-CA, PC), and then encapsulating it with a fusion membrane of tumor cell membrane and functionalized liposome membrane.

[0022] Further, the prepared fusion membrane of tumor cell membrane and functionalized liposome membrane (obtained by fusing tumor cell membrane and functionalized liposome membrane at a mass ratio of 1:1 through thin film hydration) is mixed with peptide micelles modified with negatively charged coating PC at a mass ratio of 1:2 and then sonicated in a water bath for 3 min to obtain the final product.

[0023] Furthermore, the tumor cell membrane in the fusion membrane is derived from non-small cell lung cancer.

[0024] Furthermore, the negatively charged coating modification is prepared by preparing a 1 mg / mL solution of citric anhydride-grafted polylysine (PLL-CA, PC) and incubating it at room temperature for 30 min at a molar ratio of GHRK amino to PC carboxyl groups of 1:3 in the polypeptide micelles.

[0025] Furthermore, the functionalized liposome is embedded in DSPE-PEG2000-MAP-MAL (DSPE-PEG-MAP), and its MAP polypeptide sequence is R9-PVGLIG-EGGEGGEGG.

[0026] Further, the peptide micelles are PC-GHRK / siKMT5A drug-loaded micelles. siKMT5A and GHRK peptide micelles are vortexed for 10 s and incubated at room temperature for 30 min to form GHRK / siKMT5A. 1 mg / mL PC solution is added at a GHRK amino to PC carboxyl molar ratio of 1:3, and the mixture is incubated at room temperature for 30 min to obtain PC-GHRK / siKMT5A drug-loaded micelles. The N / P ratio of the GHRK peptide micelles to siKMT5A is > 1.5.

[0027] The described biomimetic nanodelivery system (LLip@PC-GHRK) is an MMP-9 responsive and charge-reversible middle-outer layer composite modified structure. This structure is based on a charge-reversible coating and tumor membrane-liposome fusion membrane technology. Through targeted adaptation innovation and functional synergistic expansion, it achieves precise modification of the core (GHRK). Specifically, it includes a charge-reversible intermediate coating and a multifunctional liposome-homogeneous tumor cell membrane complex outer layer (LLip). These two components synergistically construct a dual-response mechanism and time-sequential drug release function.

[0028] (1) Charge-reversible intermediate coating: The negatively charged coating, citric anhydride grafted polylysine (PLL-CA, PC), has a potential of approximately -25 mV in a physiologically neutral environment (pH 7.4). It tightly coats the core surface through electrostatic interaction, forming a stable "core-intermediate coating" composite structure. After entering the acidic environment (pH 5.0-5.5) of tumor cell endosomes, the CA group in the PC molecule undergoes specific hydrolysis and detaches, exposing the positively charged amino group of PLL, thus achieving a charge reversal from "negative to positive".

[0029] This coating can achieve the following: ① The charge reversal effect disrupts the electrostatic interaction between the outer fusion membrane and the endosome membrane, inducing fusion membrane rupture and providing conditions for core exposure; ② The "proton sponge effect" of the positively charged PLL synergizes with the pH-responsive module of the core GHRK to accelerate endosome swelling and rupture, enabling endosome escape and preventing siKMT5A from being degraded by lysosomes; ③ The negatively charged shielding effect under neutral conditions can reduce carrier non-specific cell adhesion, reduce normal tissue uptake, and further reduce off-target risk.

[0030] (2) The outer layer of the multifunctional liposome-homogeneous tumor cell membrane complex (LLip): formed by the fusion of homologous tumor cell membranes (such as lung cancer LLC cells) and multifunctional liposomes (Lip), wherein the multifunctional liposomes are embedded in DSPE-PEG2000-MAP-MAL (DSPE-PEG-MAP), and its MAP polypeptide sequence is R9-PVGLIG-EGGEGGEGG (refer to Chen L, et al. ACSNano. 2020; 14 (6): The enzyme-responsive transmembrane peptide backbone reported in 6636-6648); unlike the general tumor membrane of the previous patent (application number: CN202111368883.8), this invention selects a homologous tumor cell membrane for target tumors with high MMP-9 expression (such as lung cancer). Its surface retains integrins (such as αvβ3 of LLC cells) and adhesion molecules (E-cadherin) specifically expressed by this type of tumor, and improves the targeted enrichment efficiency of tumor tissue through a "self-recognition" mechanism; ② Enzyme-responsive coupling of antibodies: the MAL group of DSPE-PEG-MAP is covalently linked to the thiol group of the antibody, so that the antibody is stably anchored on the surface of the fusion membrane, realizing the precise loading of immunomodulators;

[0031] The response and functional mechanisms of this outer membrane are as follows: ① MMP-9 response activation in the tumor microenvironment: MMP-9, which is highly expressed in the tumor site, enzymatically digests the PVGLIG-sensitive sequence in the MAP peptide, detaches the (EGG)3 negatively charged shielding layer, and exposes the R9 positively charged electroporative peptide, promoting the interaction and internalization between the carrier and the tumor cell membrane; ② Site-specific release of immunomodulators: MMP-9 enzymatic digestion simultaneously triggers the detachment of antibodies from the fusion membrane, allowing for precise release within the tumor microenvironment, avoiding non-specific activation of antibodies in normal tissues, and increasing drug concentration in the tumor microenvironment; ③ Biocompatibility optimization: The homologous tumor membrane retains natural cell membrane components, reducing carrier immunogenicity and minimizing clearance by the reticuloendothelial system.

[0032] A fourth aspect of the present invention provides the application of the MMP-9 / pH dual-response fusion membrane-encapsulated biomimetic nanodelivery system (LLip@PC-GHRK) as described above in the preparation of targeted delivery of antitumor drugs.

[0033] Furthermore, the MMP-9 / pH dual-response fusion membrane-encapsulated biomimetic nanodelivery system, in combination with immune checkpoint inhibitors or gene therapy drugs (siRNA or DNA gene therapy drugs), is used in the preparation of antitumor drug formulations.

[0034] Furthermore, the gene drug is loaded via polypeptide micelles, then modified with a negatively charged PC coating, and then encapsulated by a fusion membrane of tumor cell membrane and functionalized liposome membrane; the immune checkpoint inhibitor is linked via the DSPE-PEG-MAP group of the liposome.

[0035] Furthermore, the tumor targeted by the anti-tumor drug refers to a tumor of the same type as the cancer cell membrane in the fusion membrane. Even further, the tumor is non-small cell lung cancer.

[0036] This system leverages the core characteristics of "homogeneous targeting + MMP-9 / pH dual response + drug co-loading" to achieve precise delivery and efficient release of anti-tumor drugs. Details are as follows:

[0037] (1) Targeted tumor type: The targeted tumor is a tumor type that is homologous to the cancer cell membrane in the fusion membrane, including but not limited to various solid tumors such as lung cancer, breast cancer, and melanoma; relying on the homologous targeting function of the cancer cell membrane, cancer cell membranes from different types of tumors can specifically target their homologous tumors, adapting to the precision treatment needs of various tumors.

[0038] (2) Applicable drug types: Applicable anti-tumor drugs include antibodies and gene drugs, which can achieve the individual or co-delivery of the above two types of drugs, and are suitable for monotherapy or combination therapy.

[0039] (3) Application of core mechanisms: The cancer cell membrane components of the fusion membrane endow the system with biomimetic "camouflage" properties, which can evade the body's immune clearance system, reduce the uptake of the reticuloendothelial system, prolong the blood circulation half-life in the body, and lay the foundation for the enrichment of tumor sites; the cancer cell membrane in the fusion membrane specifically targets the same type of tumor cells through the homology recognition mechanism, realizes the precise enrichment of tumor sites, and improves the drug targeting selectivity; Dual response activation and drug release: ① Tumor microenvironment MMP-9 activation: MMP-9 highly expressed in the tumor site enzymatically digests the PVGLIG sensitive sequence of DSPE-PEG-MAP on the surface of the fusion membrane, exposes the R9 transmembrane peptide, and promotes the efficient internalization of the system into tumor cells to release antibodies; ② Intracellular pH-responsive drug release: After entering the endosome (pH 5.0-5.5), the intermediate coating PC achieves "negative charge → positive charge" flip, induces the fusion membrane to rupture, and, in conjunction with the pH-responsive characteristics of the core GHRK, triggers the precise release of gene drugs, ensuring the effective intracellular drug concentration.

[0040] (4) Core advantages of application: The system can efficiently achieve the whole chain of delivery of “immune escape → homologous targeting → tumor microenvironment activation → precise intracellular drug release”, which is suitable for the treatment of various homologous tumors. It can deliver hydrophobic drugs or gene drugs alone, or deliver both together, significantly improving the precision and synergy of anti-tumor treatment and reducing off-target toxicity.

[0041] In a fifth aspect, the present invention provides an MMP-9 / pH dual-response fusion membrane-encapsulated biomimetic nanoparticle drug delivery system, wherein an immune checkpoint inhibitor or gene therapy drug is loaded by the biomimetic nanoparticle delivery system described above; wherein the gene therapy drug is siRNA or DNA gene therapy drug.

[0042] Furthermore, the gene therapy drug is siKMT5A.

[0043] A sixth aspect of the present invention provides a method for preparing the MMP-9 / pH dual-response fusion membrane-encapsulated biomimetic nanoparticle drug delivery system (Anti-CTLA-4-LLip@PC-GHRK / siKMT5A) as described above, comprising the following steps:

[0044] (1) Synthesis and purification of multifunctional self-assembled peptide GHRK: GHRK peptide was prepared by solid-phase synthesis (C-terminal → N-terminal extension): ① Resin swelling: Fmoc-Gly-WangResin resin was soaked in DCM for 2 h to swell and remove Fmoc protecting groups; ② Amino acid coupling: Fmoc protected amino acids, PyBop, and DIEA were dissolved in DMF and a reaction system was formed at 0℃. The reaction system was injected into the reaction column and coupled at room temperature for 1 h. The reaction endpoint was confirmed by ninhydrin color development. The coupling-deprotection procedure was repeated until the target sequence was synthesized; ③ Pyrolysis purification: Resin and pyrolysis reagent were mixed at a ratio of 1 g: 10 mL and shaken in an ice bath for 2 h to remove side chain protecting groups. The crude peptide was filtered through a 0.45 μm filter membrane and purified by HPLC gradient elution. After concentration, it was freeze-dried to obtain high-quality GHRK peptide.

[0045] (2) Preparation of GHRK peptide micelles: The oil-in-water emulsion evaporation method was adopted. GHRK peptides were dissolved in deionized water, and acetone (aqueous phase: organic phase = 1:1~1:10, v / v) was added dropwise while stirring at 800 rpm. After sealing and stirring for 2 h, the lid was opened to evaporate the acetone and obtain a uniformly dispersed GHRK peptide micelle solution.

[0046] (3) PC polymer synthesis and preparation of drug-loaded micelles PC-GHRK / siKMT5A ① PC synthesis: PLL was dissolved in pH 7.4 PBS buffer, and CA was added dropwise under nitrogen protection (PLL to CA molar ratio 1:8.74). The system was adjusted to neutral by 0.1 M NaOH, and the reaction was stirred at 300 rpm for 12 h. The mixture was dialyzed with deionized water at 4℃ for 48 h in a dialysis bag (molecular weight cutoff 1 kDa). The PC polymer was obtained by freeze drying. ② Drug loading assembly: siKMT5A and GHRK peptide micelles were vortexed for 10 s and incubated at room temperature for 30 min to form GHRK / siKMT5A. 1 mg / mL PC solution was added at a molar ratio of GHRK amino to PC carboxyl of 1:3 and incubated at room temperature for 30 min to obtain PC-GHRK / siKMT5A drug-loaded micelles.

[0047] (4) Extraction of LLC tumor cell membrane: ① Cell culture: LLC cells were seeded in DMEM medium containing 10% fetal bovine serum and 1% penicillin antibody and cultured at 37℃ and 5% CO2 until the confluence reached 80%-90%; ② Cell membrane separation: The collected cells were washed twice with pre-cooled PBS, mixed with membrane protein extraction reagent containing 1 mM PMSF at a ratio of 1:5 (v / v), vortexed in an ice bath, and then cyclically frozen and thawed three times at -37℃ in liquid nitrogen. Cell debris was removed by centrifugation at 700×g at 4℃ for 10 min, and cell membrane precipitate was collected by centrifugation at 14000×g at 4℃ for 30 min. The precipitate was resuspended in PBS and the protein concentration was adjusted. The cells were stored at -80℃ for later use.

[0048] (5) Preparation of Anti-CTLA-4 modified liposomes (Anti-CTLA-4-Lip): ① Antibody thiolation: Anti-CTLA-4 antibody and Traut's reagent were dissolved in pH 8.0 carbonate buffer containing 4 mM EDTA at a molar ratio of 1:20, reacted at 4℃ in the dark for 1 h, and dialyzed with PBS for 30 min to obtain Anti-CTLA-4-SH; ② Coupling reaction: DSPE-PEG-MAP and Anti-CTLA-4-SH were reacted in pH 7.4 HBS buffer containing 4 mM EDTA at a molar ratio of maleimide group to antibody of 20:1, reacted at 4℃ in the dark overnight, and dialyzed with PBS for 12 h to purify DSPE-PEG-MAP-Anti-CTLA-4; ③ Liposome preparation: DSPC:cholesterol:DSPE-PEG-MAP-Anti-CTLA-4 was dissolved in dichloromethane at a molar ratio of 45:50:5, and rotary evaporated (100 A lipid membrane was formed at 50°C and 10% sucrose solution was added for hydration for 30 min. After sonication in an ice bath for 3 min, the membrane was squeezed three times through 0.2 μm and 0.1 μm polycarbonate membranes and purified by ultracentrifugation to obtain Anti-CTLA-4-Lip.

[0049] (6) Fusion membrane assembly and system formation: ① Fusion membrane preparation: At a liposome to cell membrane mass ratio of 1:1, LLC cell membrane suspension was added to a 10% sucrose hydration system of Anti-CTLA-4-Lip. After vortexing and mixing, the mixture was squeezed through a 100 nm polycarbonate membrane 3-5 times and centrifuged at 10000×g for 30 min to remove free membrane fragments, thus obtaining the Anti-CTLA-4-LLip fusion membrane; ② System assembly: Anti-CTLA-4-LLip was mixed with PC-GHRK / siKMT5A and sonicated for 3 min. A three-layer structure was formed through electrostatic interaction and hydrophobic interaction. After purification by ultracentrifugation, 5% mannitol was added, and the mixture was freeze-dried (pre-frozen at -40℃ for 4 h, sublimated at -20℃ for 12 h) and stored in a sealed container at -20℃.

[0050] The advantages of this invention are:

[0051] 1. The core of this invention employs a multifunctional self-assembling peptide GHRK (GSVSHHHHHHGGHHHH-RRRRRRRR-KRRRRG), integrating three functional modules: pH response (histidine-rich module), cell penetration (R9 sequence), and nuclear localization (KRRRRG sequence), achieving a precise delivery pathway of "extracellular stabilization → intracellular internalization → nuclear targeting." The nuclear localization sequence directly guides siKMT5A into the cell nucleus, overcoming the bottleneck of traditional gene vectors' inability to penetrate the nuclear membrane. Combined with the highly efficient membrane-penetrating ability of the R9 sequence, it enhances the nuclear delivery efficiency of siKMT5A, significantly improving the targeting and effectiveness of gene therapy.

[0052] 2. This invention constructs a dual regulatory mechanism of "MMP-9 + pH-responsive drug release." In the outer fusion membrane, DSPE-PEG-MAP, under the action of highly expressed MMP-9 in the tumor microenvironment, degrades the PVGLIG sequence, exposing the R9 positively electroporable peptide, achieving tumor-specific enrichment. The intermediate PC coating undergoes charge flipping in the acidic environment of the integrons, disrupting the membrane structure through the "proton sponge effect" and releasing nuclear-targeted gene drugs, preventing premature drug leakage. Biomimetic modification of the homologous tumor cell membrane reduces the clearance rate and immunogenicity of the reticuloendothelial system, ensuring that anti-CTLA-4 is released only in the tumor microenvironment, significantly reducing immune-related adverse reactions and achieving a balance between efficacy and safety.

[0053] 3. This invention's system simultaneously carries an immunomodulator (anti-CTLA-4) and a gene therapy (siKMT5A), forming a synergistic therapeutic network of "immune activation + gene inhibition": anti-CTLA-4 activates anti-tumor immunity, while siKMT5A inhibits tumor cell glycolysis and enhances immunogenicity. The synergistic effect of these two agents increases the tumor inhibition rate by more than 50% compared to single-treatment modalities. Furthermore, the modular design of the homologous tumor cell membrane (such as lung cancer LLC cells) is adaptable to various solid tumors, and the biodegradable properties of the functionalized liposome membrane and PC coating ensure that the carrier does not accumulate in vivo, exhibiting superior biocompatibility compared to traditional polymeric carriers.

[0054] Cancer drug therapy suffers from poor targeting and is easily inactivated by lysosomes after entering cells, thus failing to fully exert its anti-tumor efficacy. This invention constructs a nanocomposite system through a biomimetic delivery strategy, using a triple synergistic delivery mechanism to solve the above problems: The outer layer is a biomimetic membrane LLip fused with tumor cell membrane and liposomes. When the biomimetic nanodrug reaches the tumor target site, the MAP group in the liposome membrane responds to the high MMP-9 in the tumor microenvironment, triggering the release of antibodies at specific sites and exerting an immunomodulatory effect. Subsequently, the homologous targeting of the tumor cell membrane in LLiP and the membrane-penetrating peptide R9 sequence in the MAP group of the liposome membrane can promote the efficient uptake of biomimetic nanodrugs by tumor cells; The middle layer is coated with a pH-responsive unit—the charge-reversible molecule PLL-CA (PC), which is negatively charged at physiological pH to maintain the stability of the nanoparticles. After entering the acidic environment of tumor endosomes, the PC charge reverses, causing membrane rupture and enabling lysosome escape, preventing drug degradation by entering the lysosome; The core is a multifunctional self-assembled polypeptide carrier integrating pH-responsive peptides, cell-penetrating peptides, and nuclear-localizing peptides, which can load gene drugs. This carrier exhibits good biocompatibility and can significantly improve the efficiency and efficacy of targeted drug delivery through tumor homology targeting, tumor microenvironment response activation, and programmed drug release, providing a novel intelligent delivery paradigm for combination cancer therapy. Attached Figure Description

[0055] Figure 1HPLC purity determination of GHRK monomer;

[0056] Figure 2 HPLC purity determination of GHRK monomer;

[0057] Figure 3 Optimization of single-factor investigation of GHRK blank micelles;

[0058] Figure 4 The BCA protein reagent method was used to investigate the optimal coating ratio between lung cancer cell membranes and liposome membranes.

[0059] Figure 5 Evaluation of LLip@PC-GHRK / pEGFP transfection efficiency under different N / P conditions;

[0060] Figure 6 Investigation of the encapsulation capacity of GHRK peptide micelles for nucleic acids under different N / P conditions;

[0061] Figure 7 N / P represents the particle size (A) and potential diagram (B) of the fusion membrane biomimetic nanodelivery system at 20°C.

[0062] Figure 8 N / P is a transmission electron microscope image of the fusion membrane biomimetic nanodelivery system at 20°C.

[0063] Figure 9 The cytotoxicity of different concentrations of GHRK on LLC cells at 24h and 48h was investigated.

[0064] Figure 10 Hemolysis experiment investigation of the fusion membrane biomimetic nanodelivery system;

[0065] Figure 11 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (A) and protein imprinting (B) of the fusion membrane biomimetic nanodelivery system;

[0066] Figure 12 Release rates of Anti-CTLA-4 and siKMT5A under different release media conditions;

[0067] Figure 13 Figure 1. Flow cytometry results of FAM-siKMT5A uptake mediated by the fusion membrane biomimetic nanodelivery system in lung cancer cells;

[0068] Figure 14 Co-localization results of the fusion membrane biomimetic nanodelivery system in lung cancer cells; FAM (green fluorescent) labeling of the intracellular siKMT5A and DAPI (blue fluorescent) labeling of the cell nucleus;

[0069] Figure 15The study investigated lysosomal escape from a fusion membrane biomimetic nanodelivery system; FAM (green fluorescent) labeling was used to label the internally loaded siKMT5A and LysoTracker Red (red fluorescent) labeling of the lysosomes;

[0070] Figure 16 Figure 1 shows the results of a study on the anti-proliferation effect of a lung cancer cell membrane-functionalized liposome membrane fusion biomimetic nanodelivery system on lung cancer cells.

[0071] Figure 17 Figure 1 shows the anti-migration analysis results of a lung cancer cell membrane-functionalized liposome membrane fusion biomimetic nanodelivery system in lung cancer cells.

[0072] Figure 18 In vivo targeting experiments of the MMP-9 / pH dual-response fusion membrane biomimetic nanodelivery system. Detailed Implementation

[0073] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments.

[0074] Example 1: Synthesis of GHRK peptide

[0075] The target peptide GHRK was prepared using a solid-phase synthesis method. The specific steps are as follows:

[0076] 1) Weigh 3333.33 mg of Fmoc-Gly-WangResin resin (loading is 0.30 mmol / g) and place it in a solid-phase synthesis reactor. Soak and swell it in dichloromethane (DCM) for 2 h and then remove the Fmoc protecting group.

[0077] 2) Accurately weigh 1946.31 mg Fmoc-Arg(Pbf)-OH, 1560.12 mg benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (PyBop), and 1.21 mL N,N-diisopropylethylamine (DIEA). Dissolve them in 50 mL N,N-dimethylformamide (DMF) at low temperature (0℃) to form a homogeneous reaction system. After the reactants are completely dissolved, inject the mixture into the reaction column and carry out the coupling reaction at room temperature for 1 h. The reaction endpoint is confirmed by a negative colorimetric test using ninhydrin. After the cascade reaction was completed, the following purification procedure was performed sequentially: the reaction solution was removed by filtration, the resin was washed three times with 50 mL DMF, then 35 mL of a 20% hexahydropyridine DMF solution was injected and reacted for 5 min, the solution was filtered again and washed with DMF once, and finally the deprotection step of 20% hexahydropyridine DMF solution was repeated (50 mL of solution reacted for 10 min). After three DMF washes, two DCM washes and one final DMF rinse, the single-cycle synthesis was completed.

[0078] 3) Based on the C→N-terminal extension principle of the target polypeptide sequence, the coupling-deprotection procedure in step 2 is executed cyclically. The side-chain protecting groups for serine (Ser), histidine (His), arginine (Arg), and lysine (Lys) are tert-butyl, triphenylmethyl, 2,2,4,6,7-pentamethyldihydrobenzofuran-3-sulfonyl, and tert-butyloxycarbonyl, respectively; all α-amino acids are protected using Fmoc symmetry, ultimately completing the coupling of Fmoc-Gly-OH and the deprotection of Fmoc.

[0079] 4) After terminating the coupling reaction, remove the supernatant from the reaction system and perform three cycles of washing with 60 mL DMF (3 min each time). Then, perform three gradient elutions with analytical grade methanol, with each solvent volume being 5 mL. After solvent replacement, place the resin in a vacuum drying oven and dry it to constant weight at 40°C. Finally, determine the mass using a precision balance.

[0080] 5) The solid-phase synthesis product (resin-linear peptide complex) was mixed with a lysis reagent (trifluoroacetic acid:triisopropylsilane:water = 95:2.5:2.5, v / v) at a ratio of 10 mL lysis buffer / 1 g resin, and the mixture was shaken in an ice bath for 2 h. This yielded a peptide with all side-chain protecting groups removed.

[0081] 6) First, the crude peptide solution was pretreated using a 0.45 μm microporous membrane to effectively remove insoluble impurities. Then, gradient elution purification was performed by HPLC. The purified product was concentrated to near-dry state using a rotary evaporator in a 45℃ water bath. Following this, freeze-drying was carried out (pre-freezing stage: -40℃ for 2 h, sublimation drying stage: -20℃ for 12 h), finally yielding the refined peptide.

[0082] The results are as follows Figure 1 As shown, the polypeptide monomer exhibits a single symmetrical chromatographic peak with a retention time of 8.375 min and good symmetry. Apart from the solvent peak, no other obvious impurity peaks were observed, and the purity is >95%, which can be used for subsequent research.

[0083] Example 2: Determination of purity and molecular weight of GHRK peptide monomers

[0084] Its structure and purity were identified using mass spectrometry and HPLC, respectively.

[0085] 1) Purity determination: The obtained purified peptide was dissolved in water to prepare a 50 μg / mL solution, and its purity was determined using high-performance liquid chromatography (HPLC). The chromatographic conditions were as follows:

[0086] Chromatographic column: Agilent 5 HC-C18(2) 150×4.6 mm;

[0087] Mobile phase: A: water containing 0.1% trifluoroacetic acid; B: acetonitrile containing 0.1% trifluoroacetic acid;

[0088] Detection wavelength: 220 nm;

[0089] Flow rate: 1.0 mL / min;

[0090] 2) Molecular weight determination: Electrospray ionization mass spectrometry (ESI-MS) is suitable for the analysis of biomacromolecules (proteins, peptides) and complex matrix samples, offering high sensitivity, high resolution, and accuracy. Therefore, ESI-MS was used to determine the molecular weight of the synthesized peptide GHRK. The detection conditions are as follows:

[0091] Atomizing gas flow rate: 500 L / h;

[0092] Ion source: ESI;

[0093] Bending and desoldering voltage: +25 V;

[0094] Ion source voltage for positive ions: 3.5 kV;

[0095] Ion source voltage for negative ions: -0.2 KV;

[0096] The results are as follows Figure 2 As shown, the measured molecular weight of the GHRK polypeptide monomer is 3891.4. The theoretical molecular weight of the target sequence polypeptide GHRK is 3893.3, and the error between the molecular weight and the molecular weight measured by mass spectrometry is within two hydrogen ions, which confirms the successful synthesis of the target polypeptide.

[0097] Example 3: Construction of GHRK peptide micelles

[0098] Preparation of GHRK peptide micelles: GHRK peptide micelles were prepared using an oil-in-water emulsion evaporation method. 5 mg of GHRK peptide monomer was weighed and dissolved in 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL of deionized water, respectively. The solutions were vortexed and mixed thoroughly. While maintaining stirring (800 rpm / min), 5 mL of acetone was added dropwise to the GHRK aqueous solution. The mixture was sealed and stirred for 2 h. The solution was then opened until the acetone had completely evaporated, yielding the GHRK peptide micelle solution.

[0099] Preliminary experiments were conducted to determine the basic formulation of GHRK micelles. With the total mass of GHRK remaining constant, the volume ratio of deionized water to acetone was set at six levels: 1:10, 1:5, 2:5, 3:5, 4:5, and 1:1. Single-factor analysis was performed to investigate the particle size variation of GHRK micelles at different ratios. As shown in (A: size and PDI changes; B: zeta potential changes), the average particle size, zeta potential, and PDI of blank micelle GHRK at different ratios were measured using a nanoparticle size analyzer and a zeta potential analyzer. The results showed that a volume ratio of deionized water to acetone of 3:5 resulted in excellent nanoparticles. The size of these nanoparticles remained around 108.25 nm. The PDI was less than 0.3, and the zeta potential was approximately 24.53 mV; therefore, this concentration will be used in subsequent experiments.

[0100] Example 4: Construction and ratio optimization of MMP-9 / pH dual-response fusion membrane biomimetic nanocarrier

[0101] Preparation of drug-loaded micelles PC-GHRK: 50 mg of polylysine (PLL, 0.01 mmol) was dissolved in 2 mL of pH 7.4 phosphate buffer saline (PBS). Under nitrogen protection, 87.4 mg of citraconic anhydride (CA) was slowly added dropwise. After the reaction system was adjusted to neutral with 0.1 M NaOH solution, the reaction was continued for 12 h with a magnetic stirrer (300 rpm). The mixture was transferred to a dialysis bag with a molecular weight cutoff of 1 kDa and dialyzed in deionized water at 4 °C for 48 h (with the dialysate replaced every 8 h). The dialysate was collected and freeze-dried (pre-freezing temperature -40 °C for 4 h, sublimation drying stage -20 °C for 12 h) to obtain the powdered product PC. GHRK / siKMT5A was obtained by vortexing siKMT5A (Sense: 5'-CCAUUAGCUGGAAUCUACAGG-dTdT-3', SEQ ID NO.2; Antisense: 5'-UGUAGAUUCCAGCUAAUGGUU-dTdT-3', SEQ ID NO.3) with GHRK peptide micelles for 10 s and incubating at room temperature for 30 min. The compressibility of the GHRK peptide micelles with the gene drug siKMT5A was investigated using agarose gel electrophoresis (AGE). A suitable amount of PC was weighed to prepare a 1 mg / mL solution, and PC-GHRK / siKMT5A was obtained by incubating at room temperature for 30 min at a molar ratio of GHRK amino to PC carboxyl groups of 1:3.

[0102] Tumor cell culture and cell membrane extraction: Tumor cells frozen in liquid nitrogen were thawed by rapid shaking in a 37°C water bath for 1-2 minutes. The cell suspension was transferred to centrifuge tubes containing complete culture medium (DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin antibiotics), centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in complete culture medium and seeded into T25 cell culture flasks. The flasks were then incubated at 37°C in a 5% CO2 incubator. When the cell confluence reached 80%-90%, the cells were passaged and seeded into new culture flasks at a 1:2 ratio for further culture. Tumor cells were cultured using the above method until they reached the logarithmic growth phase (80%-90% confluence). The culture medium was removed, and the cells were washed twice with pre-chilled PBS (5 mL / wash). DMEM medium (pre-warmed to 37°C) was then added, and adherent cells were scraped off using a sterile cell scraper. The cell suspension was collected and counted. The cell suspension (2-5 × 10⁻⁶ cells / mL) was then collected into T25 cell culture flasks. 7 Cells / mL) were mixed with pre-cooled membrane protein extraction reagent A at a volume ratio of 1:5. Reagent A was pre-added to a final concentration of 1 mM phenylmethylsulfonylfluoride (PMSF). The mixture was vortexed three times in an ice bath (5 min intervals each time), followed by a liquid nitrogen-37°C-cycle freeze-thaw method (3 cycles) to disrupt the cell membrane structure. The disrupted product was centrifuged at 700×g for 10 min at 4°C. The supernatant was carefully collected and centrifuged at 14000×g for 30 min at 4°C. The supernatant was discarded to obtain the LLC cell membrane fraction. The protein concentration was determined using a BCA protein quantification kit (detection wavelength 562 nm). After aliquoting, the fraction was stored at -80°C for later use.

[0103] The DSPE-PEG2000-MAP-MAL (DSPE-PEG-MAP) peptide was synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. The MAP peptide sequence is: R9-PVGLIG-(EGG)3, where PVGLIG is the MMP-9 sensitive sequence, R9 is the positively charged electroporating peptide sequence, (EGG)3 is the negatively charged peptide sequence, and MAL is the maleimide group. Subsequently, 1.3 mg of dipalmitoylphosphatidylcholine, 0.3 mg of cholesterol, and 2.4 mg of DSPE-PEG-MAP were weighed and dissolved in 5 mL of dichloromethane, and thoroughly mixed to form a homogeneous lipid solution. The solution was transferred to a round-bottom flask, and the organic solvent was evaporated under reduced pressure using a rotary evaporator (100 rpm, 50°C) until a homogeneous lipid film formed on the inner wall of the flask. 2 mL of 10% sucrose solution was added to the flask for hydration for 30 min, followed by the addition of 3 mL of PBS, and gentle stirring was continued to promote liposome dispersion. The hydrated liposome suspension was sonicated in an ice bath for 3 min, then repeatedly squeezed through 0.2 μm and 0.1 μm polycarbonate membranes three times. Finally, the liposomes were purified by ultracentrifugation to remove unencapsulated molecules, yielding a TME-activated liposome membrane (Lip). During the Lip preparation process described above, 1.85 mL of 10% sucrose solution and 0.15 mL of tumor cell suspension (containing 2 mg of cell membrane protein, liposome to cell membrane mass ratio 1:1) were added during hydration. After vortexing and mixing, the mixture was transferred to a polycarbonate membrane extrusion device. The membrane was extruded 3-5 times (100 nm pore size) to form a homogeneous liposome-cell membrane fusion membrane (LLip). The membrane was then ultracentrifuged (10000×g, 30 min) to remove free cell membrane debris, and the purified LLip was collected. The LLip was then mixed with PC-GHRK and sonicated for 3 min to form LLip@PC-GHRK.

[0104] LLip and PC-GHRK NPs were incubated in 1 mL of phosphate buffer (pH 7.4) at a predetermined ratio (1:10 to 5:1, w / w) for 30 min. After homogenization by vortexing (300 rpm, 5 min), the mixture was subjected to a 2-min pulsed ultrasonic treatment (5 s on, 5 s off) using a 40 kHz ultrasonic processor. The mixture was then centrifuged at 11000 × g for 30 min at 4°C to effectively remove unbound free membrane components. The supernatant was filtered through a 0.22 μm filter, and the surface membrane protein content of LLip@PC-GHRK was determined using a BCA protein quantification kit to explore the optimal coating mass ratio for subsequent experiments.

[0105] See results Figure 4When the mass ratio of LLip to PC-GHRK NPs is less than 1:2, the protein content of the biomimetic nanoparticle surface membrane is significantly positively correlated with the mass ratio; when the mass ratio increases to above 1:2, the protein content of the surface membrane gradually reaches a plateau. This phenomenon indicates that at a mass ratio of 1:2, LLip has achieved complete coating of PC-GHRK NPs, and further increasing the amount of membrane material no longer significantly improves the coating efficiency. Based on this saturation characteristic, subsequent studies selected 1:2 as the optimal preparation parameter.

[0106] Example 5: Evaluation of in vitro transfection efficiency of LLip@PC-GHRK / pEGFP-N1

[0107] Mouse Lewis lung cancer cell line LLC cells (purchased from Wuhan Pronosai Life Science Technology Co., Ltd.) in good condition were seeded into 6-well plates at a rate of 50w / well. 2 mL of DMEM medium (Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco, USA) was added and the cells were cultured for 24 hours. After the cell confluence reached 60%-80%, the medium was replaced with serum-free medium.

[0108] LLip@PC-GHRK solution was prepared according to the method in Example 4. pEGFP-N1 was dissolved in water to prepare an aqueous solution. LLip@PC-GHRK / pEGFP-N1 solutions were prepared with N / P ratios of 5, 10, 20, 40, and 80. After vortexing for 10 seconds and allowing to stand for 30 minutes, the solutions were added to wells of a plate. Lipo2000 / pEGFP-N1 was used as a control. Three replicates were set for each group. Cells were incubated in a cell culture incubator for 4 hours, then replaced with 2 mL of fresh complete culture medium and cultured for another 24 hours. Cell transfection was observed and recorded using a fluorescence microscope. Figure 5 As shown.

[0109] As shown in Figure 5, when N / P=20, LLip@PC-GHRK exhibits transfection efficiency comparable to Lipo2000, so this ratio will be used in the future.

[0110] Example 6: Preparation and characterization of a biomimetic nanodelivery system (anti-CTLA-4-LLip@PC-GHRK / siKMT5A) with dual-response MMP-9 / pH fusion membrane

[0111] Take 1 mg of anti-CTLA-4 antibody solution and add PBS (pH 7.2) + dithiothreitol (DTT) to a final volume of 2 mL, making the final DTT concentration 5 mM. Incubate at 37°C for 30 minutes, then concentrate by centrifugation using a 30 kDa ultrafiltration centrifuge tube (4°C, 8000 rpm). Replace with PBS and repeat 3 times to completely remove residual DTT. Adjust the final antibody concentration to 1 mg / mL. Take DSPE-PEG-MAP powder and dissolve it in a small amount of sterile deionized water by sonication to prepare a 10 mg / mL stock solution. Under nitrogen protection, slowly add the DSPE-PEG-MAP stock solution dropwise to the pretreated anti-CTLA-4 antibody solution at a DSPE-PEG-MAP:Anti-CTLA-4 molar ratio of 5:1, and mix gently. Incubate at 4°C in the dark for 2 hours, gently mixing every 15 minutes during this period. After incubation, 5 mM mercaptoethanol was added, and the mixture was incubated at room temperature for 10 minutes to block unreacted maleimide groups, thus terminating the coupling reaction. A Sephadex G-50 column was used with PBS (pH 7.4) as the elution buffer. The first elution peak (DSPE-PEG-MAP-anti-CTLA-4, molecular weight >150 kDa) was collected, and subsequent smaller peaks were discarded, yielding DSPE-PEG-MAP-anti-CTLA-4. Subsequently, in the preparation of Lip liposome membranes in Example 4, DSPE-PEG-MAP was replaced with DSPE-PEG-MAP-anti-CTLA-4, while maintaining the other preparation steps, to obtain anti-CTLA-4-Lip liposomes with different antibody ratios. The above-mentioned anti-CTLA-4-Lip was hydrated, and 1.85 mL of 10% sucrose solution and 0.15 mL of tumor cell suspension (containing 2 mg of cell membrane protein, with a mass ratio of tumor cell membrane protein to liposome membrane of 1:1) were precisely added. After thorough mixing, membrane fusion was completed to obtain the anti-CTLA-4-LLip fusion membrane.

[0112] When preparing GHRK peptide micelles according to the method in Example 4, the GHRK peptide micelles and 1 μg siKMT5A were vortexed for 10 s with different N / P ratios (0, 0.5, 1, 1.5, 2, 4, 6, 8) and incubated at room temperature for 30 min to obtain the GHRK / siKMT5A solution. The encapsulation capacity of the GHRK peptide micelles for siKMT5A was investigated by AGE, and the results are as follows. Figure 6 When N / P > 1.5, GHRK peptide micelles can completely encapsulate the drug, preventing its migration on the electrophoresis plate.

[0113] Anti-CTLA-4-LLip@PC-GHRK / siKMT5A was prepared according to the above method. The encapsulation efficiency of siKMT5A in the nanoparticles was calculated to be (65.36 ± 5.20)%, and the anti-CTLA-4 linkage efficiency was (5.62 ± 3.51)%. siKMT5A was added at N / P = 20, and the particle size and potential were measured. The morphology of the nanocomposite was observed under a transmission electron microscope. Figure 7 and 8 .

[0114] Depend on Figure 7 (A: Particle size distribution diagram; B: zeta potential) It can be seen that the obtained nano-co-loaded micelles have a particle size of 193.91 ± 1.35 and a potential of -30.80 ± 1.39, which can ensure gene loading efficiency and good membrane penetration effect. Figure 8 Transmission electron microscopy results show that the nanocomposite has a regular morphology, is nearly spherical and uniformly dispersed, and can effectively avoid aggregation.

[0115] Example 7: Safety Study of Fusion Membrane Bionic Nanoscale Blank Carrier

[0116] CCK-8 assay: The cytotoxicity of GHRK blank micelles and LLip@PC-GHRK blank delivery vector was examined using the CCK-8 assay. The specific steps are as follows: LLC cells in logarithmic growth phase were collected and subjected to a cytotoxicity assay of 1×10⁻⁶ cells. 4 Cells were seeded at a density of 100 μL of DMEM complete medium in each well of a 96-well plate and incubated at 37°C in a 5% CO2 incubator for 24 h. After discarding the medium, the cells were washed twice with PBS, and equal volumes of different concentrations (12.5, 25, 50, 100, 150, 300, and 600 μg / mL) of GHRK blank micelles and LLip@PC-GHRK NPs were added to each well, with 6 replicates per group. LLC cells without the loading were used as a control group and incubated for another 24 h in a cell culture incubator. After culture, the culture medium in each well was discarded, and the cells were washed twice with PBS. 100 μL of DMEM medium containing 10 μL of CCK-8 solvent was added to each well. Wells containing 100 μL of cell-free DMEM medium and 10 μL of CCK-8 solvent served as a control group. The wells were incubated in the dark for 1 h. The 96-well plate was then removed, and the absorbance (OD value) of each well at 450 nm was measured using a microplate reader. Cell viability was calculated to assess the safety of the culture medium. Results are as follows: Figure 9 As shown.

[0117] Depend on Figure 9As shown in Figures (A: 24 h cell viability; B: 48 h cell viability), within a concentration range of 0–600 μg / mL, cell viability treated with both GHRK peptide micelles and LLip@PC-GHRK blank carrier remained above 80%. This indicates that both the GHRK peptide micelles and the LLip-modified PC-GHRK nanocarrier possess good biocompatibility, confirming the safety of this delivery system in drug delivery applications.

[0118] Hemolysis test: The blood compatibility of the LLip@PC-GHRK blank vector was evaluated by hemolysis test. The procedure is as follows: Fresh blood from healthy mice was collected in an anticoagulant tube, centrifuged at 4°C (1500 rpm, 5 min) to separate red blood cells, and then washed three times with gradient (PBS, pH 7.4) to remove plasma impurities. The washed red blood cells were resuspended in PBS to prepare a 5% red blood cell suspension. 0.5 mL of red blood cell suspension was mixed with 0.5 mL of LLip@PC-GHRKNPs solutions at different concentrations (25, 50, 100, 200, 300, 600, and 1200 μg / mL) to achieve final LLip@PC-GHRK NPs concentrations of 12.5, 25, 50, 100, 150, 300, and 600 μg / mL. A positive control group (0.5 mL red blood cell suspension + 0.5 mL ddH2O) and a negative control group (0.5 mL red blood cell suspension + 0.5 mL PBS) were also established. After standing at room temperature for 3 h, the mixture was centrifuged at 1500 rpm for 5 min. The color of the supernatant (pale red / colorless) was assessed, and the absorbance was measured at 540 nm using a microplate reader. The hemolysis rate was calculated to evaluate the blood compatibility of the nanoparticles.

[0119] The results are as follows Figure 10 As shown, when the red blood cell suspension was mixed with ddH2O and centrifuged, the supernatant turned red, indicating significant hemolysis in the positive control sample. Conversely, when different concentrations of LLip@PC-GHRK blank vector solution were added to the red blood cell suspension for co-incubation and centrifugation, the supernatant remained clear and transparent, indicating no significant hemolysis occurred. Using an ELISA reader to measure the absorbance of the supernatant in each treatment group, it was found that the hemolysis rate gradually increased with increasing LLip@PC-GHRK blank vector concentration, but within the experimental concentration range, the hemolysis rate remained below 4%, indicating that the LLip@PC-GHRK blank vector has good blood compatibility within the experimental concentration range.

[0120] Example 8: Investigation of Membrane Protein Retention

[0121] First, protein sample processing: Mix the protein extracts of each group with the loading buffer at a ratio of 4:1, denature them in a 100℃ metal bath for 5 min, then add the samples, followed by loading, electrophoresis, staining, and destaining until the bands are clear. Finally, observe and photograph them using a gel imaging system.

[0122] The prepared LLCM, LLip, LLip@PC-GHRK / siKMT5A, and anti-CTLA-4-LLip@PC-GHRK / siKMT5A samples were separated into proteins according to molecular weight using SDS-PAGE. After electrophoresis, the proteins on the gel were transferred to a polyvinylidene fluoride membrane and transferred at 120 V for 2 h. The membrane was then blocked with 5% skim milk blocking buffer at room temperature for 1 h. After blocking, residual liquid on the membrane surface was blotted off with filter paper, and the membrane was incubated overnight in primary antibody solution at 4 °C. The membrane was then washed three times with TBST for 10 min each time. Subsequently, the membrane was incubated with secondary antibody for 1 h, washed three times with TBST, and then placed on a clean culture dish. The target protein bands were visualized using chemiluminescence, and images were captured and analyzed using an imaging system.

[0123] The results are as follows Figure 11 (A: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of different components; B: Expression maps of LLC characteristic proteins of different components) As shown, compared with LLCM, LLip, LLip@PC-GHRK / siKMT5A NPs, and anti-CTLA-4-LLip@PC-GHRK / siKMT5A NPs preserved cell membrane proteins in LLCM better. Western blot results showed that the expression of the cell membrane marker pan-cadherin could be observed in LLC, LLCM, LLip, LLip@PC-GHRK / siKMT5A NPs, and anti-CTLA-4-LLip@PC-GHRK / siKMT5A NPs; however, the nuclear marker histone H3 and the mitochondrial marker protein COX IV were only present in LLC. These results demonstrate the successful preparation of LLip and the successful coating of LLip on the surface of the PC-GHRK delivery vector.

[0124] Example 9: In vitro drug release study of MMP-9 / pH dual-response fusion membrane biomimetic nanodelivery system

[0125] The dual-response release behavior of MMP-9 enzyme and pH in the anti-CTLA-4-LLip@PC-GHRK / siKMT5A nanosystem was evaluated using dialysis. The specific steps were as follows: For the anti-CTLA-4 release study, 2 mL of FITC-anti-CTLA-4-LLip@PC-GHRK / siKMT5A NPs (containing 150 μg siKMT5A and 100 μg anti-CTLA-4) was taken, and MMP-9 solution was added to achieve a concentration of 1 μg / mL. Separately, 2 mL of FITC-anti-CTLA-4-LLip@PC-GHRK / siKMT5A solution was taken without enzyme treatment. The NP suspension was placed in a 300 kDa dialysis bag, which was then placed in PBS solution at pH 7.4 and shaken at 37°C and 100 rpm / min. At 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 36 h, and 48 h, 1 mL of dialysis fluid was aspirated and replenished with the same volume of PBS. The FITC-Anti-CTLA-4 content in the dialysis medium was detected using a fluorescence spectrophotometer (Ex / Em = 495 nm / 519 nm), and release curves were plotted. For the release investigation of siKMT5A, sealed dialysis bags containing anti-CTLA-4-LLip@PC-GHRK / FAM-siKMT5A nanosuspension were placed in buffer media with different pH values ​​(7.4, 6.5, 5.5). The remaining steps were the same as for the FITC-anti-CTLA-4 release investigation. The in vitro release curves were plotted by detecting the fluorescence signal of FAM-siKMT5A in the dialysis fluid (Ex / Em = 490 nm / 520 nm). Figure 9 As shown.

[0126] Depend on Figure 12 (A) It is evident that antibody release was significantly stronger in the presence of MMP-9 enzyme than in the absence of MMP-9 treatment, with a cumulative release rate of approximately 82% after 48 hours. This indicates that MMP-9 enzyme triggers the cleavage of enzyme-sensitive peptides on the carrier surface, promoting the dissociation of the antibody from the liposome membrane. This difference confirms the carrier's specific response to the high expression of MMP-9 in the tumor microenvironment. Figure 11(B) It is known that the anti-CTLA-4-LLip@PC-GHRK / FAM-siKMT5A nanosystem has the lowest cumulative release rate of siKMT5A at pH 7.4, which simulates the physiological environment; the release rate increases as the pH decreases to the characteristic acidity of the tumor microenvironment (pH 6.5); in an acidic environment closer to that of endosomes (pH 5.5), the release rate can reach (68.26 ± 0.51)% after 48 h, which is attributed to the charge change of the carrier component PC under acidic conditions, which disrupts the membrane structure and accelerates the release of siRNA from the nanocore.

[0127] Example 10: Flow Cytometry Uptake of FAM-siKMT5A by a Fusion Membrane Biomimetic Nanopeptide Delivery System in Lung Cancer Cells

[0128] The uptake of FAM-siKMT5A by cells mediated by the LLip@PC-GHRK biomimetic carrier was investigated by flow cytometry. The specific steps were as follows: a suspension of LLC single cells in logarithmic growth phase was prepared, with a cell density of 3 × 10⁶ cells / cells. 5 Cells were seeded into 12-well plates, and 1 mL of DMEM complete medium was added to each well. The plates were incubated at 37°C and 5% CO2 for 24 h until the cells reached 80% confluence. The medium was aspirated from the wells, and the cells were gently washed with PBS. Then, PBS, free FAM-siKMT5A, anti-CTLA-4-LLip@PC-GHRK / FAM-siKMT5A NPs, and anti-CTLA-4-LLip@PC-GHRK / FAM-siKMT5A (MMP-9) (FAM-siKMT5: 1×10⁻⁶) were added to each well. 6 A nano-solution (M, N / P = 20:1) was prepared, with three replicates per group. Fresh DMEM medium (without FBS) was added to a volume of 1 mL, and the cells were incubated for 4 h. The medium was then discarded, and the cells were washed with PBS to remove unbound particles. Trypsin was added for 2 min of digestion. Once the cells detached from the cell wall, digestion was immediately terminated with DMEM medium containing 10% FBS. The cells were then transferred to centrifuge tubes and centrifuged at 1300 rpm for 5 min. The supernatant was discarded, and the precipitate was resuspended in 200 μL PBS (pH 6.0, sterile). The fluorescence intensity of the cells in each treatment group was detected by flow cytometry. 10,000 cells were collected for each sample, with untreated cells used as a negative control to calibrate background fluorescence. Results are as follows: Figure 13 As shown.

[0129] Depend on Figure 13(A: Flow cytometry uptake plots of LLC cells in different treatment groups; B: Statistical graphs of positive cell proportions and average fluorescence intensity in different treatment groups) It can be seen that in LLC cells, the positive cell ratio of anti-CTLA-4-LLip@PC-GHRK / FAM-siKMT5A (MMP-9) was significantly higher than that of the anti-CTLA-4-LLip@PC-GHRK / FAM-siKMT5A treatment group, and both were higher than the positive cell ratio of FAM-siKMT5A. This indicates that LLip@PC-GHRK can effectively promote the entry of gene drugs into cells.

[0130] Example 11: Intracellular localization and distribution of anti-CTLA-4-LLip@PC-GHRK / FAM-siKMT5A observed by laser confocal microscopy

[0131] LLC cells were cultured until they reached approximately 80% confluence as observed under a microscope. The culture medium was discarded, and the cells were gently washed once with PBS. 1 mL of trypsin was added, and the cells were incubated at 37°C for 2 min to digest. Once the cell edges became rounded, DMEM complete culture medium was added to terminate the digestion, and the cells were pipetted to prepare a single-cell suspension. 20 μL of the cell suspension was counted using a counting chamber at a cell density of 4 × 10⁶ cells / cells. 4 Cells were seeded into pre-equilibrated confocal culture dishes. 100 μL of DMEM complete medium was added to each well, and the dishes were incubated at 37°C with 5% CO2 for 1 h. After cell attachment, 900 μL of DMEM complete medium was added, and the dishes were cultured overnight. The old medium was discarded, and freshly prepared FAM-siKMT5A, anti-CTLA4-LLip@PC-GHRK / FAM-siKMT5A NPs, and anti-CTLA4-LLip@PC-GHRK / FAM-siKMT5A (MMP-9) NPs were added (FAM-siKMT5: 1×10⁻⁶). 6 Cells (M, N / P = 20:1) were incubated in an incubator for 4 h, after which the culture medium was removed, and the cells were washed three times with PBS to remove unbound particles. Cells were fixed with 4% paraformaldehyde solution for 30 min, and then washed three times with PBS to remove any remaining fixative. 5 μL of a fluorescence-quenching mounting medium containing 4′,6-diamino-2-phenyl indole (DAPI) was added to a culture dish, and observation was performed under laser confocal microscopy. The blue fluorescence of DAPI-labeled cell nuclei was detected by 405 nm excitation light, and the green fluorescence of FAM-siKMT5A was detected by 490 nm excitation light. Fluorescence signals from different channels were collected and superimposed to analyze the intracellular localization and distribution characteristics of the biomimetic nanosystem.

[0132] The results are as follows Figure 14(A: Intracellular distribution of siKMT5A in LLC cells under different treatment groups; B: Statistical graph of fluorescence intensity in different treatment groups) As shown, consistent with the results of flow cytometry, the green fluorescence intensity around the cell nucleus of the anti-CTLA-4-LLip@PC-GHRK / FAM-siKMT5A(MMP-9) NPs and anti-CTLA-4-LLip@PC-GHRK / FAM-siKMT5A NPs groups was higher than that of the FAM-siKMT5A group. This indicates that LLip@PC-GHRK mediates a greater uptake of siKMT5A by LLC cells.

[0133] Example 12: Laser confocal microscopy observation of lysosomal escape of anti-CTLA-4-LLip@PC-GHRK / FAM-siKMT5A

[0134] First, LLC cells in the logarithmic growth phase were arranged at a cell density of 4 × 10⁻⁶ cells / year. 4 Cells were seeded in pre-equilibrated confocal culture dishes, with 500 μL of DMEM complete medium added to each well. The dishes were incubated overnight at 37°C with 5% CO2 until cell adhesion was achieved. The medium was discarded, and the cells were washed with PBS. Then, a FAM-siKMT5A and anti-CTLA4-LLip@PC-GHRK / FAM-siKMT5A (MMP-9) nanosol (FAM-siKMT5: 1×10⁻⁶) solution was added. 6 M, N / P = 20:1). After incubation at 37℃ and 5% CO2 for 2 h and 4 h respectively, 50 nM LysoTracker Red dye was added, and the cells were incubated in the dark for 30 min to label live cell lysosomes. The staining solution was removed, the cells were washed three times with PBS, and fixed with 4% paraformaldehyde for 15 min. After fixation, the cells were washed three more times with PBS to remove residual fixative. Subsequently, 5 μL of anti-fluorescence quenching mounting medium containing DAPI was added to the culture dish, and images were captured and analyzed by laser confocal microscopy.

[0135] The results are as follows Figure 15As shown, compared with the FAM-siKMT5A group, the anti-CTLA-4-LLip@PC-GHRK / FAM-siKMT5A (MMP-9) group exhibited enhanced green fluorescence, suggesting that the camouflage modification of the LLip@PC-GHRK nanocarrier effectively improved the lysosomal escape efficiency of FAM-siKMT5A. In the early incubation period (2 h), FAM-siKMT5A and LysoTracker Red-labeled lysosomes showed strong co-localization, indicating that the carrier mainly entered the lysosomal compartment through endocytosis at this time. However, as the incubation time increased to 4 h, the co-localization signal between the nanosystem and lysosomes significantly weakened, and the green fluorescence gradually diffused from the punctate aggregated lysosomal region to the cytoplasm, exhibiting a uniform distribution. This dynamic process confirms that the LLip@PC-GHRK nanocarrier can mediate the effective lysosomal escape of FAM-siKMT5A, thereby reducing the risk of FAM-siKMT5A being degraded by lysosomal enzymes and providing a key guarantee for its gene silencing function.

[0136] Example 13: Investigation of the anti-LLC cell invasion ability of the fusion membrane biomimetic nanopeptide delivery system loaded with FAM-siKMT5A

[0137] LLC cells in the logarithmic growth phase were collected after being starved in serum-free medium for 24 h, and the cell density was adjusted to 1×10⁻⁶. 5 Cells / mL. Before the experiment, thaw the matrix gel overnight at 4°C, and pre-cool the pipette tips and centrifuge tubes. Place the Transwell chambers (8 μm pore size) into a 24-well plate containing 100 μL of BMEM complete medium and incubate at 37°C for 1 h to hydrate the basement membrane. Dilute the matrix gel with serum-free medium at a ratio of 1:8 on ice, mix thoroughly by pipetting, and then evenly spread 60 μL onto the upper chamber of the Transwell chamber. Incubate at 37°C for 3 h to cure the matrix gel, and carefully discard any uncured matrix gel solution. Add 600 μL of DMEM complete medium to the 24-well plate, and vertically place the Transwell chambers containing the cured matrix gel into the wells, ensuring the medium completely submerges the bottom of the chamber without air bubbles. 100 μL of cell suspension was added to the upper chamber of a Transwell chamber. The experimental groups were supplemented with LLip@PC-GHRK NPs, siKMT5A, GHRK / siKMT5A NPs, and LLip@PC-GHRK / siKMT5A NPs (FAM-siKMT5: 1×10⁻⁶). 6The cell culture medium (M, N / P = 20:1) was used, with a negative control RNA (siRNA Negative Control, siNC) as the control group. Each group had three replicates. Cells were incubated at 37°C with 5% CO2 for 24 h. The Transwell chambers were carefully removed, the culture medium was discarded, and the cells were washed twice with PBS. Cells were then fixed with 4% paraformaldehyde for 15 min. The fixative was discarded, and the cells were washed three times with PBS, followed by staining with 0.5% crystal violet solution for 15 min. Unmigrated cells on the upper surface of the chamber were gently wiped away with a moistened cotton swab. Six bright-field images were captured under an inverted microscope. Cells that migrated across the membrane to the lower surface of the chamber were counted using ImageJ software, and the average values ​​were used for inter-group comparisons.

[0138] The results are as follows Figure 16 As shown, there was no significant difference in the number of invasive cells among the siNC, LLip@PC-GHRK blank vector groups, and the free siKMT5A group. Compared with the free siKMT5A treatment group, the number of invasive cells in the LLip@PC-GHRK / siKMT5A NPs group was significantly reduced, decreasing by 29.56% (siKMT5A group: 9947 ± 473 cells / field; LLip@PC-GHRK / siKMT5A group: 7007 ± 256 cells / field). This indicates that the biomimetic nanosystem delivery of siKMT5A can significantly inhibit the invasion of LLC cells.

[0139] Example 14: Investigation of the anti-LLC cell migration ability of the fusion membrane biomimetic nanopeptide delivery system loaded with FAM-siKMT5A

[0140] LLC cells were fed at a rate of 3 × 10 5 Cells were seeded at a density of 1:10⁻⁶ wells in 6-well plates, and incubated at 37°C with 5% CO₂ until cell confluence reached 80% or higher. A sterile 200 μL pipette tip was used to make a straight incision on the bottom of each well. Cells were gently washed twice with PBS to remove cell debris and impurities from the incision area, and then replaced with fresh complete culture medium. The experimental groups were supplemented with siNC, siKMT5A, LLip@PC-GHRK NPs, GHRK / siKMT5A NPs, and LLip@PC-GHRK / siKMT5A NPs (FAM-siKMT5: 1×10⁻⁶). 6 (M, N / P = 20:1), with 3 parallel replicates per group. A fixed field of view was selected under an inverted microscope, and initial images of the scratches were acquired at 0 h. The culture plate was then returned to the incubator for further incubation, and scratch images were taken at the same location at 24 h and 48 h. ImageJ software was used to analyze changes in scratch width.

[0141] The results are shown in Figure 17: After 48 h of incubation, the wound closure rates of the LLip@PC-GHRK NPs, siKMT5A, GHRK / siKMT5A NPs, and LLip@PC-GHRK / siKMT5A NPs treatment groups were (60.33 ± 2.74)%, (63.25 ± 3.80)%, (46.94 ± 9.74)%, and (21.78 ± 3.86)%, respectively. LLip@PC-GHRK / siKMT5A NPs showed a stronger inhibitory effect on migration in LLC cells than GHRK / siKMT5A NPs. These results indicate that the LLip@PC-GHRK / siKMT5A nanodelivery system can inhibit the migration behavior of LLC cells by efficiently delivering siKMT5A, and its inhibitory effect is superior to that of free siKMT5A and non-biomimetic modified GHRK / siKMT5A nanocarriers.

[0142] Example 15: In vivo targeting performance of the MMP-9 / pH dual-response fusion membrane biomimetic nanodelivery system

[0143] LLC cells in logarithmic growth phase were digested with trypsin, centrifuged (1000 rpm, 5 min), and the supernatant was discarded. The cells were resuspended in serum-free DMEM medium and counted. The pre-thawed matrix gel (thawed overnight at 4°C) was mixed with the pre-chilled cell suspension at a 1:1 volume ratio, and the cell density was adjusted to 1 × 10⁶ cells / year by adding culture medium. 7 Cells / mL, temporarily stored at 4°C for later use.

[0144] Six-week-old male C57BL / 6 mice were selected, and the skin on the right upper limb shoulder and back was shaved 24 hours in advance. Before inoculation, the shaved area was disinfected with 75% ethanol cotton balls, and 100 μL of cell-Matrix gel mixture was slowly injected subcutaneously along the right shoulder and back of the mouse using a 1 mL sterile syringe. After inoculation, the mice were housed in an SPF-grade animal room, and their condition was observed and recorded. When the tumor volume reached 80 mm, the mice were monitored. 3 Once the LLC subcutaneous xenograft model is established, subsequent experiments can begin.

[0145] A mouse model of LLC subcutaneous tumor was constructed using the method described above, and the tumor volume was allowed to grow to 80 mm. 3Mice were randomly divided into two groups (n = 3). The two groups were administered different treatments via tail vein: Cy5-labeled siKMT5A solution alone, and anti-CTLA-4-LLip@PC-GHRK / Cy5-siKMT5A solution (100 μL for both). Fluorescence distribution in mice was observed and photographed using an in vivo imaging system (Ex / Em = 748 / 780 nm) at 2 h, 6 h, 12 h, and 18 h post-administration. Twenty-four h after administration, mice were euthanized by cervical dislocation, and major organs such as the heart, liver, spleen, lungs, and kidneys, as well as tumor tissue, were rapidly isolated and subjected to in vitro fluorescence imaging using the in vivo imaging system. All fluorescence signals were quantified using QuickView 3000 analysis software, and differences in fluorescence intensity at different time points and between tissues were compared to evaluate the in vivo targeting efficiency of siKMT5A mediated by the biomimetic carrier.

[0146] See results Figure 18 As shown in the figure, the free Cy5-siKMT5A group exhibited significant enrichment in the liver parenchyma. Compared with the free Cy5-siKMT5A group, the anti-CTLA-4-LLip@PC-GHRK / Cy5-siKMT5A NPs group demonstrated a specific tumor localization effect. In vitro tissue fluorescence also showed that the tumor tissue of the anti-CTLA-4-LLip@PC-GHRK / Cy5-siKMT5A NPs group had strong fluorescence, while the tumor site of the free Cy5-siKMT5A group showed almost no Cy5 fluorescence signal. In vitro quantitative analysis also indicated that the drug accumulation in the tumor site of the anti-CTLA-4-LLip@PC-GHRK / Cy5-siKMT5A NPs group was significantly better than that of the free Cy5-siKMT5A group (p < 0.0001).

[0147] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A multifunctional self-assembling polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. A multifunctional self-assembling polypeptide micelle, characterized in that, The polypeptide micelles are polymers formed by the multifunctional self-assembling polypeptide GHRK described in claim 1 through its own hydrophobic interactions and electrostatic interactions.

3. A biomimetic nanodelivery system encapsulated in an MMP-9 / pH dual-responsive fusion membrane, characterized in that, The peptide micelles described in claim 2 are modified with a charge-reversible negatively charged coating PC, and then coated with a fusion membrane of tumor cell membrane and functionalized liposome membrane.

4. The biomimetic nanodelivery system according to claim 3, characterized in that, The fusion membrane is prepared by mixing the fusion membrane with the peptide micelles modified with a negatively charged coating at a mass ratio of 1:2 and then sonicating in a water bath for 3 minutes. The fusion membrane is prepared by fusing tumor cell membranes and functionalized liposome membranes at a mass ratio of 1:1 using a thin-film hydration method. The negatively charged coating is prepared by preparing a 1 mg / mL PC solution and incubating at room temperature for 30 minutes with a molar ratio of GHRK amino to PC carboxyl groups of 1:3 in the peptide micelles.

5. The biomimetic nanodelivery system according to claim 3, characterized in that, The functionalized liposome is embedded in DSPE-PEG2000-MAP-MAL, and its MAP polypeptide sequence is R9-PVGLIG-EGGEGGEGG.

6. The application of the MMP-9 / pH dual-response fusion membrane-encapsulated biomimetic nanodelivery system as described in claim 3 in the preparation of targeted delivery antitumor drugs.

7. The application according to claim 6, characterized in that, The application of the MMP-9 / pH dual-response fusion membrane-encapsulated biomimetic nanodelivery system in combination with immune checkpoint inhibitors or gene therapy drugs in the preparation of antitumor drug formulations; wherein the gene therapy drug is siRNA or DNA gene therapy drug.

8. The application according to claim 7, characterized in that, The gene drug is loaded with polypeptide micelles, then modified with a negatively charged PC coating, and then encapsulated by a fusion membrane of tumor cell membrane and functionalized liposome membrane; the immune checkpoint inhibitor is linked by the DSPE-PEG-MAP group of the liposome.

9. The application according to claim 8, characterized in that, The tumor targeted by the anti-tumor drug refers to a tumor of the same type as the tumor cell membrane in the fusion membrane; the tumor is non-small cell lung cancer.

10. A biomimetic nanoparticle drug delivery system encapsulated in an MMP-9 / pH dual-response fusion membrane, characterized in that, The biomimetic nanodelivery system of claim 3 loads an immune checkpoint inhibitor or a gene therapy drug; the gene therapy drug is siRNA or DNA gene therapy drug.

Images