Multifunctional PEG lipid derivative and lipid nanoparticle co-delivery system

Abstract

The invention belongs to the technical field of drug delivery systems, and particularly relates to a multifunctional PEG lipid derivative and lipid nanoparticle co-delivery system. The multifunctional PEG (Polyethylene Glycol) lipid derivative is DSPE-PEG2000-R8-FRRG-IMI (Distearoyl Sulfonate Polymer). According to the lipid nanoparticle co-delivery system, lipid nanoparticles serve as a carrier, and siRNA and a cis-platinum prodrug are entrapped in the lipid nanoparticles. Wherein the lipid nanoparticles are prepared from an ionizable cationic lipid, DSPC (Distearoyl Pyrrolidone), cholesterol and a PEG (Polyethylene Glycol) lipid derivative, and the PEG lipid derivative is DSPE-PEG2000-R8-FRRG-IMI (Distearoyl Pyrrolidone Polyethylene Glycol 2000-R8-FRRG-IMI). The lipid nanoparticle co-delivery system not only can realize co-loading of a cis-platinum prodrug and siRNA and overcome cis-platinum drug resistance while killing tumors, but also has an active targeting property, stimulation responsiveness and an efficient intracellular escape function, and has a remarkable treatment effect on lung adenocarcinoma or breast cancer and the like.

Description

Technical Field

[0001] This invention belongs to the field of drug delivery system technology, specifically relating to a multifunctional co-delivery system of PEGylated lipid derivatives and lipid nanoparticles. Background Technology

[0002] Chemotherapy is one of the core methods for the clinical treatment of intermediate and advanced cancers. Cisplatin, as a first-line core chemotherapy drug, plays an irreplaceable role in clinical treatment by forming cross-links with tumor cell DNA, disrupting its structural integrity, thereby inhibiting tumor cell proliferation and inducing apoptosis. However, in long-term clinical use, tumor cells are prone to acquired resistance to cisplatin, which seriously limits the efficacy of cisplatin.

[0003] Studies have shown that the molecular mechanisms of cisplatin resistance mainly include the following three aspects: (1) Enhanced DNA damage repair capacity of tumor cells, with upregulation of repair-related genes such as BRCA1 and XRCC1, which can rapidly repair DNA damage caused by cisplatin, allowing tumor cells to escape apoptosis; (2) Overexpression of drug efflux proteins (such as P-glycoprotein, MRP1, etc.), thereby expelling cisplatin from the cell and causing the intracellular drug concentration to be lower than the effective killing threshold; (3) Abnormal activation of alternative survival signaling pathways such as PI3K / Akt and MAPK, enabling tumor cells to bypass DNA damage-dependent apoptosis pathways and maintain proliferative activity even if DNA is damaged, thus generating drug resistance. Current clinical practice often relies on increasing the dosage of cisplatin to try to overcome drug resistance; however, this not only has limited efficacy but also significantly increases toxic damage to normal tissues.

[0004] Lipid nanoparticles (LNPs) have made significant progress in cancer treatment in recent years as a core platform for delivering nucleic acid drugs (such as mRNA and siRNA). However, currently available LNP formulations generally rely on passive targeting to enter tumor tissues, resulting in a lack of drug specificity and limited delivery efficiency. Furthermore, during intracellular delivery, LNPs are often constrained by endosome / lysosome retention, leading to insufficient drug release and further weakening therapeutic effects. Although some studies have attempted to modify LNP surfaces with ligands or design responsive elements, a systematic solution that balances active targeting, stimulus responsiveness, and efficient endosome escape has yet to be developed.

[0005] Furthermore, the design of traditional LNPs focuses on the delivery efficiency of nucleic acid molecules and has not been optimized for the loading requirements of small molecule chemotherapy drugs, resulting in significant limitations in their application in cancer treatment: first, it is difficult to efficiently encapsulate both nucleic acid drugs and small molecule chemotherapy drugs simultaneously; second, there is a lack of prescription strategies to achieve synergistic release of the two. Summary of the Invention

[0006] To address the shortcomings of existing lipid nanoparticles in cisplatin resistance and cancer treatment, the present invention aims to provide a multifunctional PEGylated lipid derivative and lipid nanoparticle co-delivery system. This lipid nanoparticle co-delivery system not only enables the co-loading of cisplatin prodrugs and siRNA to overcome cisplatin resistance while killing tumors, but also possesses active targeting, stimulus responsiveness, and efficient intracellular escape capabilities, significantly improving the therapeutic effects of related cancers.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention first provides a multifunctional PEGylated lipid derivative, wherein the multifunctional PEGylated lipid derivative is DSPE-PEG. 2000 -R8-FRRG-IMI.

[0008] The synthesis method of the above-mentioned multifunctional PEGylated lipid derivatives is as follows: DSPE-PEG 2000 -MAL was dissolved in PBS buffer containing 8%~12% DMSO, and the pre-synthesized and purified polypeptide Cys-R8-FRRG-GGG-IMI was added at a molar ratio of 1:1. The reaction system was placed under nitrogen protection, protected from light, and magnetically stirred at room temperature for 10~14 hours. The product was then obtained by dialysis, freezing, and drying.

[0009] In the polypeptide Cys-R8-FRRG-GGG-IMI, "-RRRRRRRR-" represents an octaarginine peptide segment, where all arginine amino acids are D-type amino acids; "-FRRG-" represents a cathepsin B-responsive enzyme-sensitive peptide segment; and "IMI" represents a safflower toxin peptide segment, which consists of 12 amino acids (GCCSDPRCAWRC) and contains two disulfide bonds (linked to Cys). 2 –Cys 8 and Cys 3 –Cys 12 (between), its C-terminus is modified by amidation.

[0010] This invention also provides the application of the above-mentioned multifunctional PEGylated lipid derivatives in the preparation of lipid nanoparticles.

[0011] The present invention also provides a lipid nanoparticle co-delivery system, the lipid nanoparticle co-delivery system comprising: a lipid nanoparticle carrier and siRNA and cisplatin prodrug encapsulated within the lipid nanoparticles; The lipid nanoparticles are composed of ionizable cationic lipids, DSPC, cholesterol, and PEGylated lipid derivatives, wherein the PEGylated lipid derivatives are DSPE-PEG. 2000 -R8-FRRG-IMI.

[0012] Preferably, the molar ratio of the ionizable cationic lipid to DSPC is 0.75~5:1, and the PEGylated lipid derivative modification density is 1.5 mol%; The N / P ratio of the lipid nanoparticle co-delivery system is 10, and the molar ratio of the cisplatin prodrug to the lipid component is 0.2~5:1.

[0013] More preferably, the molar ratio of the ionizable cationic lipid to DSPC is 1:1; and the molar ratio of the cisplatin prodrug to the lipid component is 5:1.

[0014] Preferably, the ionizable cationic lipid is DLin-MC3-DMA.

[0015] The present invention also provides a pharmaceutical composition comprising the above-described lipid nanoparticle co-delivery system and pharmaceutically acceptable excipients.

[0016] The present invention also provides the application of the above-mentioned lipid nanoparticle co-delivery system in the preparation of antitumor drugs.

[0017] Preferably, the tumor is a lung adenocarcinoma cell or a breast cancer cell.

[0018] This invention provides a lipid nanoparticle co-delivery system. The system uses lipid nanoparticles as carriers, with siRNA and cisplatin prodrug encapsulated within them. The cisplatin prodrug, as a low-toxicity modified form of cisplatin, needs to be specifically activated at the tumor site to exert its killing effect, thus reducing systemic toxicity. siRNA can reverse the drug resistance phenotype of tumor cells at the molecular level by sequence-specifically silencing cisplatin resistance-related genes. This invention utilizes the synergistic effect of siRNA and cisplatin prodrug to effectively overcome the molecular mechanisms of cisplatin resistance while achieving tumor killing, thereby improving the efficacy of cancer chemotherapy.

[0019] To improve the encapsulation efficiency of siRNA and cisplatin prodrug, this invention first screened and optimized the formulation of the lipid particle nanodelivery system, effectively balancing the encapsulation efficiency of siRNA and cisplatin prodrug in the same delivery system while meeting the dosage requirements of both. This simplifies the issue of the ratio of the two active ingredients in combination therapy and reduces the types and amounts of membrane materials used in the delivery system.

[0020] Furthermore, this invention constructs a novel PEG lipid-derived material through surface modification design. Using this material as the PEG-lipid component in the aforementioned lipid nanoparticle delivery system endows it with active targeting, stimulus responsiveness, and enhanced endosome escape and cytoplasmic release properties, while simultaneously improving the encapsulation efficiency of siRNA and cisplatin prodrugs in the delivery system, thereby significantly enhancing cancer treatment efficacy. Attached Figure Description

[0021] Figure 1 Synthesis (A) and structural confirmation (B) of cisplatin prodrug.

[0022] Figure 2 For DSPE-PEG 2000 Synthetic route of -R8-FRRG-IMI.

[0023] Figure 3 Figure 1 shows a schematic diagram of LNP formulation preparation (A) and the physicochemical characterization of different LNP formulations. Figures BC and C show the particle size, PDI (B), and zeta potential (C) of different LNP formulations after storage at 4°C for 7 days. Figures DE and E show the particle size and PDI measurement results (D) and AGE determination results (E) of different LNP formulations after incubation with FBS. Figure F shows the hemolysis experiment results of different LNP formulations after incubation with 2% rat erythrocytes (scale bar: 20 μm) for 3 h. Figure G shows transmission electron microscopy images of different LNP formulations, showing particle morphology and dispersion (scale bar: 200 nm).

[0024] Figure 4 The results of cell uptake and tumor spheroid penetration detection for different LNP preparations are shown in Figure (A). The flow cytometry analysis results of Cy5 fluorescence intensity after A549 was incubated with different LNP preparations containing Cy5-siRNA for 8 hours are shown. Figure (B) shows the flow cytometry results of Cy5 fluorescence intensity after A549 / DDP cells were incubated with different LNP formulations loaded with Cy5-siRNA for 8 h; Figure (C) shows the intracellular Pt level of A549 / DDP cells after treatment with different LNP formulations loaded with Pro-DDP; Figure (D) shows CLSM images of A549 and A549 / DDP cells after incubation with different LNP formulations loaded with Cy5-siRNA for 8 h; cell nuclei were stained with Hoechst 33342 (blue); siRNA was labeled with Cy5 (red); scale bar: 20 μm; Figure (E) shows the MCTS results of different LNP formulations penetrating A549 / DDP cells as shown by Z-stack confocal images.

[0025] Figure 5 The results show the receptor binding and endosome / lysosome escape properties of the functionalized LNP formulation; Figure (A) shows the real-time calcium levels of A549 / DDP cells after treatment with PBS, LNP@siGLI1 / DDP, FI-LNP@siGLI1 / DDP, or RFI-LNP@siGLI1 / DDP, respectively; scale bar: 200 μm; Figure (B) shows the Ca²⁺ levels of multi-cell ROIs. +Quantitative analysis results of fluorescence intensity; Figure (C) shows the intracellular transport and endosome / lysosome escape of different functionalized LNPs after cellular uptake; Figure (D) shows the CLSM images of endosome / lysosome escape at different time points; Scale bar: 20 μm.

[0026] Figure 6 To verify the in vitro cytotoxicity of functionalized LNP formulations on A549 / DDP cells and their regulatory effect on the GLI1-mediated cisplatin resistance pathway, Figure (A) shows the dose-response (IC50) curves of free cisplatin in A549 and A549 / DDP cells as determined by the CCK-8 assay; Figure (B) shows the cell viability of A549 / DDP cells after treatment with different empty LNPs; Figure (C) shows the cell viability of A549 / DDP cells after treatment with LNPs loaded with siGLI1 alone (LNP@siGLI1); Figures (DE) show the IC50 curves and IC50 values ​​of A549 / DDP cells after treatment with different LNP formulations (LNP@siNC / DDP, LNP@siGLI1 / DDP, FI-LNP@siGLI1 / DDP, and RFI-LNP@siGLI1 / DDP); Figure (F) shows the GLI1... The results of qRT-PCR analysis of mRNA expression; Figure (G) shows the results of flow cytometry analysis of GLI1 protein expression level; Figures (H-K) show the results of flow cytometry analysis of ABCB1, ABCG2, ERCC1 and XRCC1 expression levels, respectively.

[0027] Figure 7 The results show the biodistribution and tumor targeting efficiency of functionalized LNP formulations in A549 / DDP tumor-bearing mice. Figure (A) shows whole-body fluorescence images of tumor-bearing mice at different time points (0.5, 2, 4, 6, 8, 10, 12 and 24 hours) after tail vein injection of free Cy5-siRNA or Cy5-siRNA encapsulated with unmodified LNPs, FI-LNPs or RFI-LNPs. Figure (B) shows in vitro fluorescence images of tumors and major organs of tumor-bearing mice 24 hours after injection.

[0028] Figure 8The results show the antitumor efficacy and cisplatin resistance pathway analysis of the functionalized LNP formulation in A549 / DDP tumor-bearing mice. Figure (A) is a schematic diagram of the treatment regimen (n = 5); Figure (B) shows images of tumors resected at the study endpoint; Figure (C) shows the tumor growth curve during treatment; Figure (D) shows the tumor weight measured at the end of treatment; Figure (E) shows the quantitative analysis of platinum accumulation in tumor tissue by ICP-MS; Figure (F) shows the expression of GLI1 in tumors of each treatment group detected by Western blotting; GAPDH was used as an internal control; Figure (G) shows the expression patterns of GLI1, ABCB1, ABCG2, ERCC1, and XRCC1 analyzed by immunohistochemistry; scale bar: 100 μm.

[0029] Figure 9 Figure 1 shows the in vivo safety evaluation results of the functionalized LNP formulations. Figure 2 shows the weight changes of A549 / DDP tumor-bearing mice during treatment. Figures 3 (B and C) show the hematological (B) and serum biochemical (C) parameters after treatment with different LNP formulations. Figure 3 (D) shows the H&E staining of the major organs (heart, liver, spleen, lung, and kidney) of the mice. Scale bar: 100 μm. Detailed Implementation Plan

[0030] Example 1: Formulation screening and optimization of LNPs co-loaded with Pro-DDP and siRNA 1. Preparation of LNPs co-loaded with Pro-DDP and siRNA The LNP preparation in this embodiment uses microfluidic technology and includes the following steps: Step (1) Organic phase preparation: Weigh out DLin-MC3-DMA, DSPC, Chol (cholesterol), and DSPE-PEG. 2000 Prepare an organic phase by dissolving it in ethanol in a specific ratio; Step (2) Aqueous phase preparation: Dissolve Pro-DDP and siRNA in citrate buffer at pH 4.0 to prepare the aqueous phase; Step (3) Microfluidic mixing: The organic phase and the aqueous phase are loaded into two syringes at a volume ratio of 1:3, and then placed in a microfluidic device. The organic phase is pushed into the microfluidic chip at a rate of 1 mL / min and the aqueous phase is pushed at a rate of 3 mL / min. The LNP is collected at the end of the chip into the ultrafiltration centrifuge tube of MW10000. Step (4) Purification: Replace the outer aqueous phase of LNP with an ultrafiltration centrifuge tube, centrifuge at 6000 rpm for 30 min, discard the lower layer of liquid in the centrifuge tube; add 1 mL of PBS with pH=7.4 to the upper layer, continue centrifuging for 30 min, repeat once, and you will get LNP with PBS co-loaded with Pro-DDP and siRNA in the outer aqueous phase.

[0031] In step (2), the synthesis of Pro-DDP is carried out according to the literature ( Wang, W., et al., 2024. Adv. Mater. 36(1): e2308762. The method reported is as follows, and the specific synthetic route is as follows: Figure 1 As shown in (A), the steps include: (1) Synthesis of intermediate c,c,t-[Pt(NH3)2Cl2(OH)2] Cisplatin (100 mg) was dissolved in an appropriate amount of deionized water, and 6.0 mL of 30% (w / v) hydrogen peroxide (H2O2) was slowly added dropwise. The mixture was stirred at 75°C in the dark for 8 hours. After the reaction was completed, the reaction solution was placed at -20°C overnight to promote crystallization. The obtained crystals were filtered and washed thoroughly with ice water, cold ethanol and anhydrous diethyl ether in sequence. Finally, the crystals were dried under vacuum to obtain the target intermediate c,c,t-[Pt(NH3)2Cl2(OH)2].

[0032] (2) Synthesis of cisplatin prodrug The prepared c,c,t-[Pt(NH3)2Cl2(OH)2] (50 mg, 0.15 mmol) was dissolved in 2 mL of anhydrous N,N-dimethylformamide (DMF), and succinic anhydride (60 mg, 0.6 mmol) was added. The reaction system was magnetically stirred at 70 °C for 24 hours under nitrogen protection in the dark until the solution became clear. After the reaction was completed, DMF was removed by rotary evaporation, and 5 mL of cold acetone was slowly added to the residue, followed by recrystallization at -20 °C. The resulting precipitate was then dissolved in 0.5 mL of methanol after rotary evaporation, and added dropwise to 10 mL of cold diethyl ether with rapid stirring to obtain a pale yellow solid. The final product was collected by suction filtration and dried under vacuum. The structure of the obtained final product, cisplatin prodrug, was confirmed by 1H NMR spectroscopy. Figure 1 As shown in (B), the ¹H-NMR spectrum shows a characteristic methylene peak (–CH2–) in the range of δ 2.38–2.50 ppm, which is a hallmark signal of succinic anhydride modification. A proton signal of the coordinating amino group (–NH3) is visible at δ 6.49 ppm. The absence of obvious impurity peaks in the spectrum indicates that the product has high purity and the structure is as expected.

[0033] 2. Screening and optimization of LNP formulations co-loaded with Pro-DDP and siRNA (1) Optimization of the molar percentage of ionizable cationic lipids to DSPC This embodiment uses DLin-MC3-DMA:DSPC:Chol:DSPE-PEG2000 (n / n) = 50: 10: 38.5:1.5, N / P = 10 as the basic formulation, with the lipid ratio of the fixed drug (Pro-DDP) at 1:5, and the DLin-MC3-DMA:DSPC adjusted to 5:1, 1:1, and 3:4 respectively. When the ratio of DLin-MC3-DMA to DSPC is 5:1: the siRNA encapsulation efficiency is 82.4% and the Pro-DDP encapsulation efficiency is 0.30%; when the ratio of DLin-MC3-DMA to DSPC is 1:1: the siRNA encapsulation efficiency is 80.1% and the Pro-DDP encapsulation efficiency is 0.46%. When the ratio of DLin-MC3-DMA to DSPC is 3:4, the siRNA encapsulation efficiency is 56.5%, and the Pro-DDP encapsulation efficiency is 0.09%.

[0034] (2) Optimization of drug (Pro-DDP) lipid ratio The fixed DLin-MC3-DMA:DSPC ratio was 1:1, and the lipid ratios of the adjusted drug (Pro-DDP) were 1 / 5, 3 / 1, and 5 / 1, respectively. When the drug (Pro-DDP) lipid ratio is 1 / 5: siRNA encapsulation efficiency is 82.9%, and Pro-DDP encapsulation efficiency is 0.46%; When the drug (Pro-DDP) lipid ratio is 3 / 1: siRNA encapsulation efficiency is 85.6%, and Pro-DDP encapsulation efficiency is 0.51%; When the drug (Pro-DDP) lipid ratio is 5 / 1: the ssiRNA encapsulation efficiency is 82.8% and the Pro-DDP encapsulation efficiency is 1.13%.

[0035] (3) N / P ratio optimization Fixed DLin-MC3-DMA: DSPC was 1:1, drug-lipid ratio was 5:1, and N / P ratio was adjusted to 10 and 20 respectively; When the N / P ratio is 20: siRNA encapsulation efficiency is 80.3%, and Pro-DDP encapsulation efficiency is 0.16%; When the N / P ratio is 10: siRNA encapsulation efficiency is 82.5%, and Pro-DDP encapsulation efficiency is 1.07%.

[0036] (4) PEG modification density optimization With a fixed DLin-MC3-DMA:DSPC ratio of 1:1 and a drug-to-lipid ratio of 5:1, the PEG modification densities were adjusted to 1.5% and 3%, respectively. When the PEG modification density is 3%, the siRNA encapsulation efficiency is 71.3%, and the Pro-DDP encapsulation efficiency is 0.32%. When the PEG modification density is 1.5%, the siRNA encapsulation efficiency is 85.2% and the Pro-DDP encapsulation efficiency is 1.09%.

[0037] (5) Screening of different preparation methods The LNPs were prepared using a fixed DLin-MC3-DMA:DSPC ratio of 1:1, a drug (Pro-DDP) lipid ratio of 5:1, an N / P ratio of 10, and a PEG modification density of 1.5 mol%. The methods employed included microfluidic one-step method, microfluidic two-step method (two microfluidic steps after lipid separation), microfluidic + post-insertion method, modified ethanol injection method, modified ethanol injection method + handheld extruder, and thin-film dispersion method. Comparison revealed that the microfluidic one-step method yielded the best encapsulation efficiency for both siRNA and Pro-DDP (both exhibited high encapsulation efficiency, meeting drug delivery requirements).

[0038] Microfluidic method (one-step method, as above): siRNA encapsulation efficiency 82.8%, Pro-DDP encapsulation efficiency 1.13%; Microfluidic method (two-step method): Dlin-MC3-DMA is dissolved in anhydrous ethanol as the organic phase, and siNC and Pro-DDP are dissolved in pH 4.0 citrate buffer as the aqueous phase. The two are first prepared into an LNP core by microfluidic control. Then, the core is used as the aqueous phase, and the remaining resin material is dissolved in anhydrous ethanol as the organic phase, and the LNP is prepared by microfluidic control.

[0039] The encapsulation efficiency of the obtained LNP siRNA was 30.1%, and the encapsulation efficiency of Pro-DDP was 0.05%.

[0040] Modified ethanol injection method + probe sonication method: Dissolve the lipid material in 200 μL of anhydrous ethanol according to the ratio, concentrate the ethanol to about 50 μL by stirring in a 65℃ water bath, inject it into 2 mL of pH 4.0 citrate buffer containing siNC and Pro-DDP at the same temperature, stir for 20 min, perform probe sonication in an ice water bath, replace the external aqueous phase in the ultrafiltration tube with PBS at pH 7.4, and the remaining steps are the same as above.

[0041] The encapsulation efficiency of the obtained LNP siRNA was 26.2%, and the encapsulation efficiency of Pro-DDP was 0.09%. Modified ethanol injection method + handheld extrusion method: The modified ethanol injection method is the same as above, except that the ultrasonic treatment of the ice water bath probe is replaced by a handheld extruder with 200 nm and 100 nm extrusion membranes placed in the middle respectively. Each membrane is extruded 10 times to control the particle size. Then, the membranes are transferred to an ultrafiltration tube to replace the external aqueous phase with PBS at pH 7.4. The remaining steps are the same as above.

[0042] The encapsulation efficiency of the obtained LNP siRNA was 31.3%, and the encapsulation efficiency of Pro-DDP was 0.02%. Comparative analysis revealed that the optimal combination of siRNA and Pro-DDP encapsulation efficiency of the obtained LNP was achieved using the microfluidic method (one-step method), with a DLin-MC3-DMA:DSPC ratio of 1:1, a drug (Pro-DDP) lipid ratio of 5 / 1, an N / P ratio of 10, and a PEG modification density of 1.5%, thus meeting the drug administration requirements.

[0043] Example 2 Synthesis of multifunctional PEGylated lipid derivatives This embodiment provides a method for synthesizing a multifunctional PEGylated lipid derivative.

[0044] The multifunctional PEGylated lipid derivative is DSPE-PEG. 2000 -R8-FRRG-IMI. It is in DSPE-PEG. 2000 -MAL is formed by coupling a cleavable peptide segment using a Michael addition reaction. The amino acid sequence of the cleavable peptide segment is C(RRRRRRRR)-FRRG-GGG-IMI, where the N-terminal cysteine ​​contains a thiol group, which can undergo a Michael addition reaction with maleimide to link the PEG lipid and the polypeptide; -RRRRRRRR- is an octaarginine peptide segment, where all arginines are D-type amino acids; -FRRG- is a cathepsin B-responsive enzyme-sensitive peptide segment, which will be cleaved under the action of cathepsin B; IMI is a safflower toxin peptide that can specifically bind to α7-nAChR.

[0045] In addition, this embodiment also designed another cleavable polypeptide without R8: C-FRRG-GGG-IMI, which was also combined with DSPE-PEG. 2000 -MAL linkage forms the PEGylated lipid derivative DSPE-PEG 2000 -FRRG-IMI.

[0046] Specifically, the DSPE-PEG 2000 The synthetic route of -R8-FRRG-IMI is as follows: Figure 2 As shown, the method is as follows: DSPE-PEG 2000-MAL was dissolved in PBS buffer containing 10% DMSO, and pre-synthesized and purified C-terminal thiol-modified peptide Cys-R8-FRRG-GGG-IMI was added at a molar ratio of 1:1. The reaction system was placed under nitrogen protection, protected from light, and magnetically stirred at room temperature for 12 hours to complete the Michael addition coupling reaction between the thiol and maleimide groups. After the reaction, the solution was dialyzed in deionized water for 48 hours using a MWCO 3.5 kDa dialysis bag to remove unreacted free peptides, small molecule impurities, and organic solvents, with the dialysate changed every 8 hours. The resulting solution was lyophilized to obtain DSPE-PEG. 2000 -R8-FRRG-IMI. Its structure was confirmed by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS).

[0047] For comparison, the same reaction conditions were used, but Cys-R8-FRRG-GGG-IMI was substituted for Cys-R8-FRRG-GGG-IMI, along with DSPE-PEG. 2000 -MAL is used for coupling to obtain DSPE-PEG 2000 -FRRG-IMI. Its purification and structure verification procedures are the same as those described above.

[0048] Structural confirmation confirmed that DSPE-PEG 2000 The mass spectrum of -MAL shows a molecular weight range of 2100-3500, DSPE-PEG 2000 The mass spectrum of -FRRG-IMI showed a major molecular ion peak (4947, 458), with the molecular weight highly consistent with the theoretical calculation. No obvious impurity peaks were observed, indicating that the product structure was correct and the purity was good. Similarly, DSPE-PEG... 2000 -R8-FRRG-IMI also showed the expected molecular weight peak (6199.198), confirming the successful synthesis of the control material.

[0049] Example 3 Preparation of LNP formulation In this example, the model siRNA is GLI1-siRNA (siGLI1), and the cisplatin prodrug is Pro-DDP synthesized in Example 1. For comparison, this example established the following three different multifunctional LNP formulations: (1) LNP@siGLI1 / DDP: DLin-MC3-DMA, DSPC, Chol, DSPE-PEG 2000 The LNP synthesized as a component (i.e., the unmodified LNP, denoted as LNP) was co-loaded with siGLI1 and Pro-DDP; (2) FI-LNP@siGLI1 / DDP: DLin-MC3-DMA, DSPC, Chol, DSPE-PEG 2000 -FRRG-IMI is a component-synthesized LNP (i.e., a target ligand-modified LNP, denoted as FI-LNP), co-loaded with siGLI1 and Pro-DDP; (3) RFI-LNP@siGLI1 / DDP: DLin-MC3-DMA, DSPC, Chol, DSPE-PEG 2000 -R8-FRRG-IMI is a component-synthesized LNP (an LNP modified with a targeting ligand and a membrane-penetrating peptide, denoted as RFI-LNP), co-loaded with siGLI1 and Pro-DDP.

[0050] Synthetic routes of different LNP formulations, such as Figure 3 As shown in (A). The specific steps are as follows: DLin-MC3-DMA, cholesterol (Chol), DSPC, and PEG-lipids (such as DSPE-PEG) 2000 or DSPE-PEG 2000 -FRRG-IMI or DSPE-PEG 2000 LNPs (-R8-FRRG-IMI) were dissolved in ethanol at a molar ratio of 30:30:38.5:1.5 to prepare the organic phase. siRNA (0.42 nmol / mL) and cisplatin prodrug (1.71 μmol / mL) were dissolved in citrate-sodium citrate buffer at pH 4.0 to prepare the aqueous phase. The N / P ratio was maintained at 10 throughout the preparation process. The aqueous and organic phases were loaded into two separate syringes and injected into the channels on either side of the chip via a microfluidic device, achieving rapid mixing in the central mixing area of ​​the chip. The flow rates of the organic and aqueous phases were set to 1 mL / min and 3 mL / min, respectively, with a volume ratio of 1:3. The LNPs suspension at pH 4 was transferred to an ultrafiltration centrifuge tube and centrifuged to allow small molecules in the outer aqueous phase to pass through the filter membrane into the lower layer. After discarding the filtrate, the LNPs were resuspended in PBS (pH 7.4). This process of centrifugation, filtrate discarding, PBS addition, and resuspension was repeated twice to ensure complete buffer replacement. Subsequently, functional lipid materials containing coupled peptides were introduced into the LNPs via post-insertion. Specifically, the functional materials were first prepared into micelles (1 mg / mL) using a thin-film dispersion method. These micelles were then added to the LNPs system at a ratio of 0.5% of the total lipid molar ratio and incubated at 37°C for 4 hours. Finally, excess functional materials not inserted into the LNPs were removed by dialysis.

[0051] Particle size, polymerization index (PDI), and zeta potential of different LNP formulations were determined using a Malvern laser particle size analyzer. To assess the stability of LNPs, they were stored at 4°C for 7 days, with particle size, PDI, and potential monitored daily. Simultaneously, equal volumes of fetal bovine serum (FBS) were mixed with different LNP formulations and incubated at 37°C for 24 hours to simulate the in vivo environment. The sensitivity of siRNA-LNPs to serum degradation was assessed using agarose gel electrophoresis (AGE) and particle size analysis. To assess the blood compatibility of each formulation, they were co-incubated with isolated rat erythrocytes (RBCs) for 3 hours to evaluate the degree of erythrocyte hemolysis.

[0052] Depend on Figure 3 (BC) As can be seen, the particle size of different LNP formulations did not change significantly within 7 days, indicating good storage stability. The average particle size of LNP@siGLI1 / DDP was 148.87 ± 0.42 nm, the polymerization index (PDI) was 0.13 ± 0.02, and the zeta potential was -8.56 ± 1.04 mV. After peptide modification with PEG lipid derivatives, the particle size and PDI of the resulting LNP formulations FI-LNP@siGLI1 / DDP and RFI-LNP@siGLI1 / DDP increased slightly, but remained within the nanoscale range. In addition, enzyme-linked immunosorbent assay (ELISA) showed that the encapsulation efficiency of RFI-LNP@siGLI1 / DDP for siRNA reached 91.5% ± 2.4%. ICP-MS analysis showed that the amount of cisplatin prodrug encapsulated per mg of total lipids was 11.12 ± 0.15 μg, indicating that the nanosystem has good dual-drug loading capacity. Figure 3 (DG) indicates that the LNP preparation has good serum stability and blood compatibility within 48 hours, with no hemolysis, and the morphology under transmission electron microscopy is spherical and uniform.

[0053] The above results indicate that the PEG lipid derivative, after peptide modification, does not significantly affect the particle size, potential, siRNA and chemotherapeutic drug encapsulation efficiency and stability of the LNP formulation, indicating that the LNP formulation was successfully constructed and has good stability.

[0054] Example 4: Efficacy Verification Experiment of LNP Formulation In this embodiment, non-drug-resistant (A549) and drug-resistant phenotype (A549 / DDP) of the human lung adenocarcinoma sensitive cell line A549 were used as cell models.

[0055] 1. Detection of cellular uptake and tumor spheroid permeability of LNP formulations 1.1 Flow cytometry analysis of cell uptake efficiency Experimental Methods: To evaluate the intracellular uptake efficiency of different LNP formulations, this study detected the concentrations of siRNA and cisplatin (Pt) in cells. First, in 6-well plates, at a concentration of 2 × 10⁶ cells per well... 5 A549 and A549 / DDP cells were seeded at a density of [number] cells / year. After 24 hours of adherent culture, three different LNP formulations loaded with Cy5-labeled siRNA (LNP@Cy5-siRNA, FI-LNP@Cy5-siRNA, and RFI-LNP@Cy5-siRNA) were added, along with free Cy5-labeled siRNA and a blank control. Cells were incubated for 8 hours. After treatment, cells were washed with PBS, trypsinized, centrifuged, and resuspended. Cells were then analyzed by flow cytometry at an excitation wavelength of 633 nm and an emission wavelength of 670 nm. (1×10⁶ cells / year) 4 The Cy5 fluorescence intensity of individual cells was analyzed, and the mean fluorescence intensity (MFI) was analyzed using FlowJo software.

[0056] like Figure 4 As shown in (AB), compared with the unmodified LNP formulation (LNP@Cy5-siRNA), the surface-modified R8-FRRG-IMI and FRRG-IMI peptide-targeted LNP formulations (RFI-LNP@Cy5-siRNA and FI-LNP@Cy5-siRNA) exhibited significantly enhanced cellular uptake capacity in A549 cells, especially RFI-LNP@Cy5-siRNA, which showed a more significant increase in average fluorescence intensity. Furthermore, its trend in A549 / DDP cells was consistent with that in A549 cells. This indicates that the cellular uptake efficiency of the targeted LNP formulations is not affected by the cisplatin resistance phenotype and can effectively overcome the uptake barrier of resistant cells.

[0057] 1.2 Detection of intracellular Pt accumulation by ICP-MS Experimental methods: To evaluate drug delivery efficiency, 5 × 10⁵ cells per well were used in a 6-well plate. 5 A549 / DDP cells were seeded at a density of [number] cells / mL and cultured adherently for 24 hours. Then, different LNP formulations (LNP@siNC / DDP, LNP@siGLII / DDP, FI-LNP@siGLII / DDP, RFI-LNP@siGLII / DDP) loaded with cisplatin prodrug (corresponding to a final cisplatin concentration of 3 μg / mL) were added and incubated for 8 hours. After treatment, the cells were washed with PBS, digested with trypsin, centrifuged, and counted. The cells were then digested, and the Pt concentration in the solution was determined by ICP-MS. The Pt concentration was calculated per 10-1 cells based on the cell count. 6 The amount of Pt accumulated in a single cell.

[0058] like Figure 4As shown in (C), compared with the control group, the intracellular Pt concentration of A549 / DDP was significantly increased after treatment with FI-LNP@siGLII / DDP and RFI-LNP@siGLII / DDP (P<0.05). In particular, the RFI-LNP@siGLII / DDP treatment group indicates that RFI-LNPs can encapsulate siGLII and Pro-DDP, which specifically recognize and efficiently enter lung cancer cells.

[0059] 1.3 CLSM observation of intracellular distribution Experimental Methods: To further observe the uptake of different LNP preparations by cancer cells using confocal microscopy, A549 and A549 / DDP cells were respectively injected with 1×10⁻⁶ LNPs. 5 Seeds were inoculated at a density of cells / well in glass-bottomed culture dishes. After adhesion, three different LNP formulations loaded with Cy5-labeled siRNA (LNP@Cy5-siRNA, FI-LNP@Cy5-siRNA, and RFI-LNP@Cy5-siRNA) were added and incubated. After 8 hours, the culture medium was discarded, the samples were washed three times with PBS, and incubated with Hoechst 33342 nuclear staining solution for 30 minutes. After staining, the samples were washed again with PBS. Confocal images were acquired using a laser scanning confocal microscope.

[0060] like Figure 4 (D) CLSM images (scale bar 20 μm) of A549 and A549 / DDP cells after 8 h of incubation with Cy5-siRNA-LNPs. It is evident that the nuclei of both cell groups were stained blue by Hoechst with clear outlines; a distinct red fluorescent signal (Cy5-siRNA) was visible in the cytoplasm, with uniform and widespread fluorescence intensity and no obvious aggregation on the cell membrane surface, indicating that the LNPs had been internalized and that Cy5-siRNA had been successfully released from the LNPs into the cytoplasm (a key site for siRNA gene silencing). There was no significant difference in red fluorescence intensity between A549 and A549 / DDP cells, further verifying that the intracellular delivery efficiency of LNPs is not affected by drug resistance phenotype, laying the foundation for siRNA silencing of drug resistance-related genes.

[0061] 1.4 Z-stack confocal imaging for detecting tumor spheroid penetration Experimental Methods: The penetration ability of different LNP formulations in three-dimensional tumor tissue was evaluated using a three-dimensional multicellular tumor spheroid (MCTS) model derived from A549 / DDP cells. To construct the in vitro MCTS model, A549 / DDP cells were seeded at a density of 1000 cells per well in ultra-low adhesion 96-well circular plates and cultured for 72 hours until the cells spontaneously aggregated to form dense spheroid structures. The average diameter of the formed tumor spheroids was approximately 200 μm. Three different LNP formulations loaded with Cy5-labeled siRNA (LNP@Cy5-siRNA, FI-LNP@Cy5-siRNA, and RFI-LNP@Cy5-siRNA) were added to the MCTS culture system and incubated for another 8 hours.

[0062] like Figure 4 As shown in (E), the RFI-LNP@Cy5-siRNA treatment group showed stronger penetration ability, with the red fluorescence signal penetrating deep into the central region of the sphere and the overall distribution being more uniform, suggesting that it has better tumor tissue penetration performance.

[0063] The above results indicate that targeted ligand combined with membrane-penetrating peptide modification helps improve the internalization efficiency of LNPs in lung cancer cells, as well as their gene knockout and cell-killing capabilities.

[0064] 2. Detection of receptor binding and endosome / lysosome escape properties of functionalized LNPs To verify the specific binding of IMI and α7-nAChR, the Fura-2 AM calcium ion probe was used to monitor intracellular Ca²⁺ in A549 / DDP cells in real time. + The effect of IMI peptide modification on calcium signaling in tumor cells was assessed by varying its concentration. Cells were seeded in glass-bottomed culture dishes and cultured adherently for 24 hours. After incubation with 1.0 μM Fura-2 AM for 35 minutes, free dye was removed by washing with HEPES buffered saline (pH 7.2) containing calcium and magnesium. Different functionalized LNP formulations (LNP@siGLI1 / DDP, FI-LNP@siGLI1 / DDP, RFI-LNP@siGLI1 / DDP) were added, and fluorescence images were continuously acquired using laser confocal microscopy to record dynamic changes in intracellular calcium ions. The receptor binding mediated by IMI peptide and its blocking effect on calcium influx were analyzed.

[0065] like Figure 5 As shown in (AB), compared with the PBS control group and the unmodified LNPs group, the intracellular Ca²⁺ levels in the IMI-modified FI-LNP@siGLI1 / DDP and RFI-LNP@siGLI1 / DDP treatment groups were significantly higher. + The fluorescence intensity decreased significantly.

[0066] To evaluate the endosome / lysosome escape ability of different LNP formulations in lung adenocarcinoma cells, this study used confocal laser scanning microscopy to observe the fluorescence colocalization of different LNP formulations with the lysosomal marker LysoTracker Green, and quantitatively analyzed the degree of colocalization using Pearson correlation coefficient (Pearson's R). A549 and A549 / DDP cells were treated with various LNP formulations loaded with Cy5-siRNA for 4 hours and then divided into two groups: one group underwent immediate fluorescence staining and imaging, while the other group was incubated in complete culture medium for another 2 hours before staining and observation, in order to dynamically track the release process of nanoparticles from endosomes / lysosomes.

[0067] Figure 5 (C) is a schematic diagram of intracellular transport and endosome / lysosome escape of different LNP formulations after cellular uptake.

[0068] like Figure 5 As shown in (D), the RFI-LNP@siGLI1 / DDP treatment group exhibited high colocalization at 4 hours (R = 0.56) and significantly decreased to 0.34 at 6 hours, indicating effective endosome escape. A similar trend was observed in A549 / DDP cells, further validating the superior escape efficiency of RFI-LNP@siGLI1 / DDP. This is because the FRRG peptide in RFI-LNP@siGLI1 / DDP is specifically cleaved by cathepsin B in lysosomes, exposing the transmembrane R8 peptide, which further promotes endosome / lysosomal escape and enhances the cytoplasmic release of the payload.

[0069] The above results indicate that, compared with LNP@siGLI1 / DDP and FI-LNP@siGLI1 / DDP, RFI-LNP@siGLI1 / DDP improves the intracellular delivery efficiency of siRNA and chemotherapeutic drugs.

[0070] 3. Cytotoxicity detection To investigate the in vitro cytotoxicity of different LNP formulations on A549 / DDP cells and their regulatory effects on the GLI1-mediated cisplatin resistance pathway, the cisplatin resistance phenotype of A549 / DDP cells, the biocompatibility of the LNP vector itself (no cytotoxicity), and the effect of siGLI1 alone on the proliferation of resistant cells were first verified.

[0071] The inhibition rate of proliferation of sensitive cells (A549) and drug-resistant cells (A549 / DDP) by different concentrations of free cisplatin was detected by CCK-8 assay. Dose-response curves were fitted and IC50 values ​​were calculated. Figure 6As shown in (A), the IC50 value of A549 / DDP cells is significantly higher than that of A549 (usually ≥3 times), proving that A549 / DDP cells do indeed have resistance to cis.

[0072] The viability of A549 / DDP cells was assessed after treatment with different empty LNPs (i.e., blank lipid nanoparticles LNP@siNC, FI-LNP@siNC, and RFI-LNP@siNC, which did not contain any drugs (siGLI1 or cisplatin prodrug)). Figure 6 As shown in (B), there was no significant difference in cell viability (viability ≥90%) among the different empty LNPs treatment groups.

[0073] like Figure 6 As shown in (C), the viability of A549 / DDP cells did not decrease significantly under different concentrations of LNP@siGLI1 treatment, indicating that siGLI1 itself has no obvious direct killing effect on A549 / DDP cells.

[0074] Next, the cytotoxicity of different functionalized LNP formulations was further tested.

[0075] Experimental Methods: A549 / DDP cells were seeded at a density of 5000 cells per well in 96-well plates and incubated overnight at 37°C and 5% CO2 to allow for adherence and growth. Then, free cisplatin (DDP, final concentration 0-80 μg / mL) and various LNP formulations containing equal doses of cisplatin prodrugs were added, and incubation continued for 48 hours. Untreated cells served as a negative control. After incubation, the original culture medium was discarded, and 100 μL of fresh F-12K medium containing 10% CCK-8 reagent was added to each well, with incubation continuing for 1–2 hours. The absorbance (OD) of each well was then measured using a microplate reader at 450 nm. 450 Cell viability was calculated by comparing the results with the control group, thereby assessing the cytotoxicity of each formulation.

[0076] like Figure 6As shown in (DE), the IC50 values ​​determined by the cytotoxicity assay indicated that the active-targeting LNP formulations (FI-LNP@siGLI1 / DDP and RFI-LNP@siGLI1 / DDP) significantly enhanced the cytotoxicity of DDP against A549 / DDP compared to their corresponding passive-targeting LNP formulations (LNP@siGLI1 / DDP). LNPs co-loaded with siGLI1 / DDP also significantly enhanced the cytotoxicity against A549 / DDP compared to their corresponding DDP-only LNPs. Comparing the simple targeted modified LNP formulation (FI-LNP@siGLI1 / DDP) with the targeted combined with transmembrane modified LNP formulation (RFI-LNP@siGLI1 / DDP), the latter showed significantly higher cytotoxicity against A549 / DDP than the former.

[0077] Gene knockout experiment A549 / DDP cells were seeded in 6-well plates and cultured adherently for 24 hours. The original medium was then replaced with different LNP formulations containing siGLI1 or siGLI1 / DDP, with a final siGLI1 concentration of 100 nM. After 24 hours of treatment, cells were collected, total RNA was extracted, reverse transcribed into cDNA, and analyzed by qRT-PCR to assess the relative expression level of GLI1. At the protein level, the LNP-mediated GLI1 knockdown effect was further analyzed by flow cytometry. After treating A549 / DDP cells with LNPs for 24 hours, the cells were washed three times with PBS and then incubated in fresh complete medium for another 24 hours. Subsequently, the cells were collected by trypsin digestion, fixed with 4% paraformaldehyde, and permeate with 0.1% Triton X-100. The cells were then incubated with the following primary antibodies: rabbit anti-human GLI1 antibody, ERCC1 antibody, and XRCC1 antibody; followed by fluorescent labeling with Alexa Fluor™ 647-labeled goat anti-rabbit IgG secondary antibody. Simultaneously, the cells were incubated with mouse anti-human ABCB1-FITC and ABCG2-PE antibodies, respectively, to detect the expression of the corresponding proteins. The staining process was carried out at 4°C in the dark for 30 minutes. After staining, the cells were washed three times with PBS and resuspended. Finally, the expression levels of the above proteins were detected by flow cytometry to analyze the expression changes among different treatment groups.

[0078] like Figure 6 (FK) results showed that all LNP formulations loaded with siGLI1 effectively knocked out GLI1-mRNA in A549 / DDP and downregulated the expression levels of GLI1 protein, as well as ABCB1, ABCG2, ERCC1, and XRCC1 signaling in A549 / DDP. Among them, RFI-LNP@siGLI1 / DDP showed the most significant effect.

[0079] The above results demonstrate that RFI-LNP can efficiently deliver siGLI1 to drug-resistant lung cancer cells and significantly inhibit GLI1 and its multiple drug resistance pathways, providing a clear molecular mechanism basis for targeted reversal of cisplatin resistance.

[0080] 5. Tumor targeting efficiency detection A549 / DDP cells were fed at a rate of 1×10 7 One dose per mouse was administered subcutaneously to the right scapular region of 4-6 week old female BALB / c nude mice (18-22g). Tumor growth was observed every two days after inoculation, and tumor volume was measured starting on day 14 post-inoculation. Tumors were monitored until they reached 200 mm in size. 3 Around 10:00 AM, tumor-bearing nude mice were randomly divided into four groups (n = 3). Each group received a tail vein injection of either free Cy5-labeled siRNA (Cy5-siRNA, dose 0.66 mg / kg) or different LNPs formulations containing an equivalent dose of Cy5-siRNA. In vivo imaging was performed at different time points after injection (0.5, 2, 4, 6, 8, 10, 12, and 24 h). Mice were anesthetized with 2% isoflurane during imaging, and were placed on a preheated IVIS Spectrum CT imaging system (PerkinElmer, USA) platform for whole-body fluorescence acquisition. After the final imaging, mice were sacrificed, and tumor tissue and major organs (heart, lung, liver, spleen, and kidney) were extracted for ex vivo fluorescence imaging.

[0081] like Figure 7 Small animal in vivo imaging results showed that, compared with free Cy5-siRNA, LNPs can effectively deliver their contents to tumor tissues, and actively targeted LNPs have better tumor targeting, indicating that targeting ligands can improve the tumor targeting efficiency of LNPs.

[0082] 6. Evaluation of in vivo anti-tumor effects A nude mouse A549 / DDP ectopic transplantation model was established, and the tumor was allowed to grow to 200 mm. 3 Drug treatment began around [time missing], with ultra-low doses of 1 mg / kg siGLI1 and DDP administered via tail vein at one-day intervals, for a total of 5 injections. Specific treatment protocol is as follows: Figure 8 As shown in (A). During treatment, the tumor proliferation trend was dynamically monitored. At the end of treatment, the tumor was removed and weighed. ICP-MS was used to quantify platinum accumulation in the tumor tissue. Western blotting was used to detect the expression of GLI1 in the tumor (GAPDH was used as an internal control). Immunohistochemistry (IHC) was used to analyze the expression patterns of GLI1, drug efflux proteins ABCB1 and ABCG2, and DNA repair proteins ERCC1 and XRCC1.

[0083] like Figure 8 (BG) As shown by the tumor growth curves and tumor tissue weighing results, compared with the control group and the free drug group, all LNP preparations significantly inhibited tumor growth. The tumor inhibition efficiency of each actively targeted LNP preparation group was higher than that of the passively targeted LNP preparation group, indicating that the targeted ligand improved the tumor targeting rate and thus improved the tumor inhibition efficiency. Among them, the RFI-LNP@siGLI1 / DDP treatment group significantly improved the tumor inhibition efficiency compared with other groups, indicating that the strategy of targeting combined with transmembrane modified LNPs to co-deliver siRNA and chemotherapeutic drugs can overcome chemotherapy resistance in NSCLC and effectively improve the tumor inhibition efficiency.

[0084] 7. Safety Evaluation Experimental Methods: During the pharmacodynamic studies described above, the in vivo safety of different LNP formulations was evaluated simultaneously. Forty-eight hours after the last administration, blood and major organs (heart, lung, liver, spleen, and kidney) were collected from mice for safety assessment. Blood samples were divided into two parts: one part was centrifuged at 5000 rpm for 15 minutes at 4°C to collect serum for serum biochemical analysis, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), and blood urea nitrogen (BUN); the other part was collected in EDTA anticoagulant tubes for hematological analysis, detecting white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), and platelet (PLT) parameters. For histological analysis, major organs and tumor tissues were fixed in 4% paraformaldehyde, routinely embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) to observe the effect of treatment on tissue morphology.

[0085] like Figure 9 As shown, the blood routine and blood biochemistry results of mice treated with different LNP formulations were generally within the normal range. The body weight of nude mice in each group remained relatively stable without significant decrease, and their food and water intake was normal during the pharmacodynamic study. After the pharmacodynamic study, no significant organic damage was found in the major organs of the mice in each group, indicating that each LNP formulation has high in vivo safety.

Claims

1. A multifunctional PEGylated lipid derivative, characterized in that, The multifunctional PEGylated lipid derivative is DSPE-PEG. 2000 -R8-FRRG-IMI.

2. The method for synthesizing the multifunctional PEGylated lipid derivative according to claim 1, characterized in that, DSPE-PEG 2000 -MAL was dissolved in PBS buffer containing 8%~12% DMSO, and the pre-synthesized and purified polypeptide Cys-R8-FRRG-GGG-IMI was added at a molar ratio of 1:

1. The reaction system was placed under nitrogen protection, protected from light, and magnetically stirred at room temperature for 10~14 hours. The product was then obtained by dialysis, freezing, and drying.

3. The application of the multifunctional PEGylated lipid derivative of claim 1 in the preparation of lipid nanoparticles.

4. A lipid nanoparticle co-delivery system, characterized in that, include: Lipid nanoparticle carrier and siRNA and cisplatin prodrug encapsulated within the lipid nanoparticles; The lipid nanoparticles are composed of ionizable cationic lipids, DSPC, cholesterol, and PEGylated lipid derivatives, wherein the PEGylated lipid derivatives are DSPE-PEG. 2000 -R8-FRRG-IMI.

5. The lipid nanoparticle co-delivery system according to claim 4, characterized in that, The molar ratio of the ionizable cationic lipid to DSPC is 0.75~5:1, and the PEGylated lipid derivative modification density is 1.5 mol%; The N / P ratio of the lipid nanoparticle co-delivery system is 10, and the molar ratio of the cisplatin prodrug to the lipid component is 0.2~5:

1.

6. The lipid nanoparticle co-delivery system according to claim 5, characterized in that, The molar ratio of the ionizable cationic lipid to DSPC is 1:1; the molar ratio of the cisplatin prodrug to the lipid component is 5:

1.

7. The lipid nanoparticle co-delivery system for cancer treatment according to claim 4, characterized in that, The ionizable cationic lipid is DLin-MC3-DMA.

8. A pharmaceutical composition, characterized in that, It comprises the lipid nanoparticle co-delivery system according to any one of claims 4-7, and pharmaceutically acceptable excipients.

9. The use of the lipid nanoparticle co-delivery system according to any one of claims 4-7 in the preparation of antitumor drugs.

10. The application according to claim 9, characterized in that, The tumor is either lung adenocarcinoma cells or breast cancer cells.

Images