Lipid nanoparticle targeted delivery system as well as preparation method and application thereof

Abstract

The invention discloses a lipid nanoparticle targeted delivery system as well as a preparation method and application thereof. The lipid nanoparticle targeted delivery system contains lipid nanoparticles, and nano enzyme particles are coated inside the lipid nanoparticles; the surfaces of the lipid nanoparticles are modified with targeted polypeptides. According to the invention, an ionizable cationic lipid material is creatively combined with nano-enzyme particles with enzyme-like catalytic activity, so that a nano-enzyme functionalized ionizable lipid nano-particle delivery system is constructed. The design breaks through the function limitation of traditional LNP single delivery of nucleic acid, and realizes targeted delivery of nano-enzyme. Experimental results show that the prepared nano-enzyme lipid nanoparticle targeting delivery system shows excellent enzyme-like activity in vitro, shows good tumor targeting toxicity on the cellular level, shows a remarkable treatment effect in a B-cell lymphoma animal model, and has good application prospects. And a new strategy is provided for expanding a biomedical scene of collaborative application of nano-enzyme catalytic treatment and a lipid nanotechnology.

Description

Technical Field

[0001] This invention relates to a lipid nanoparticle targeted delivery system, its preparation method, and its application, belonging to the field of nanomedicine technology. Background Technology

[0002] Lymphoma is a highly heterogeneous group of malignant tumors, encompassing a variety of subtypes. Diffuse large B-cell lymphoma (DLBCL), as one of the most common subtypes, has consistently been a focus of medical attention due to its treatment advancements. In recent years, significant breakthroughs have been achieved in DLBCL treatment thanks to the emergence of new drugs and continuous optimization of treatment strategies. From antibody-drug conjugates (ADCs) to bispecific antibodies and small molecule targeted therapies, a series of innovative therapies have brought new hope to some DLBCL patients, enabling them to achieve a cure through first-line immunochemotherapy (such as R-CHOP). However, some patients still experience disease progression (i.e., primary refractory) early in treatment or at the end of treatment, or relapse after initial remission. For these patients, existing treatment options are extremely limited, necessitating the development of new therapeutic drugs to drive the continuous diversification and personalization of lymphoma treatment.

[0003] Nanozymes are a class of novel nanomaterials that combine nanoscale (1-100 nm) characteristics with enzyme catalytic function. They integrate the advantages of natural enzymes and artificial catalysts, maintaining the high catalytic efficiency and substrate specificity of natural enzymes while possessing the excellent chemical stability, tunable surface properties, and feasibility for large-scale production of artificial nanomaterials. Due to their unique dual-function properties, nanozymes have attracted widespread attention in the biomedical field, especially in tumor therapy. As novel catalytic materials, nanozymes can continuously generate cytotoxic reactive oxygen species (ROS) through enzyme-like catalytic reactions, directly inducing tumor cell apoptosis. However, despite the significant potential of nanozymes in tumor therapy, their practical application in the complex tumor microenvironment still faces multiple challenges. For example, key performance indicators such as catalytic efficiency, targeting specificity, and biosafety of nanozyme systems have not yet met the requirements for clinical translation. Strategies such as surface functionalization modification, biomimetic structural design, and intelligent responsive development can effectively improve their adaptability to the tumor microenvironment and their therapeutic specificity.

[0004] Lipid nanoparticles (LNPs), as gene delivery carriers, have become one of the most promising technology platforms in the biomedical field due to their excellent biosafety, good in vivo tolerability, and modular design characteristics. Their core structure consists of ionizable cationic lipids, which self-assemble with negatively charged mRNA molecules through electrostatic interactions to form stable complexes. This unique encapsulation mechanism not only achieves efficient loading of nucleic acid molecules but also constructs a physical barrier against nuclease degradation.

[0005] The interaction mechanism between chemokine receptor 4 (CXCR4) and its homologous ligand CXCL12 (stromal cell-derived factor-1α, SDF-1α) has become a significant breakthrough in tumor metastasis research. This ligand-receptor system plays a crucial role in the progression of various malignant tumors by regulating the chemotactic migration, invasion, and metastasis cascade of tumor cells. Notably, in the molecular pathological mechanism studies of diffuse large B-cell lymphoma (DLBCL), abnormal activation of the CXCR4-CXCL12 signaling axis has been confirmed to be closely related to disease development and progression, making it a highly promising therapeutic target. Current research reports indicate that the E5 peptide can effectively block the CXCR4 / CXCL12 signaling pathway and has demonstrated good therapeutic effects in mouse models of acute myeloid leukemia (AML) and mouse models of breast cancer.

[0006] Given the potential value of nanozymes in lymphoma treatment and the unique advantages of LNPs, the limitations of traditional LNPs in delivering nucleic acids alone can be overcome. Summary of the Invention

[0007] Purpose of the invention: The technical problem to be solved by the present invention is to provide a lipid nanoparticle targeted delivery system, its preparation method and application, which can realize precise targeted treatment of diseases related to the CXCR4-CXCL12 biological axis.

[0008] Technical Solution: To solve the above-mentioned technical problems, the present invention provides a lipid nanoparticle targeted delivery system, which comprises lipid nanoparticles with a surface modified with a targeting peptide coating a nanoenzyme particle with a negatively charged surface; the lipid nanoparticles contain cationic lipids and lipids containing maleimide groups; the targeting peptide has a thiol group at its end, thereby achieving efficient loading of negatively charged nanoenzymes and covalent modification of tumor-targeting peptides on the particle surface.

[0009] The present invention also provides a method for preparing the lipid nanoparticle targeted delivery system, comprising the following steps: (1) Preparation of negatively charged nanoenzyme particles: A polymer was selected as the surface stabilizer for the nanoenzyme particles, and negatively charged nanoenzyme particles were prepared by chemical precipitation. Specifically, the polymer was mixed with the nanoenzyme precursor, and a reducing agent was added dropwise under mechanical stirring to react. Then, the excess reducing agent was removed by ultrafiltration to obtain the negatively charged nanoenzyme particle solution. (2) Preparation of nanoparticles loaded with nanoenzyme lipids: The aqueous phase of the nanoenzyme obtained in step (1) was adjusted to a weak acid and then mixed with the ethanol phase containing cationic lipids, auxiliary lipids, PEG lipids and cholesterol in a microfluidic chip. The organic solvent was then removed by ultrafiltration to obtain a solution of lipid nanoparticles loaded with nanoenzymes. (3) Preparation of nanoenzyme lipid nanoparticle targeted delivery system: The lipid nanoparticle solution containing nanoenzyme obtained in step (2) is adjusted to neutral, mixed with a targeted polypeptide with thiol group at the end, and mechanically stirred to react. Then, the unreacted polypeptide is removed by ultrafiltration to obtain the lipid nanoparticle targeted delivery system.

[0010] In step (1), the polymer is mercaptomethoxy polyethylene glycol (SH-mPEG); the nanozyme precursor is chloroplatinic acid hexahydrate; and the reducing agent is sodium borohydride.

[0011] The mass concentrations of the chloroplatinic acid hexahydrate solution were 9-11 mg / mL, the mercaptomethoxy polyethylene glycol solution was 2-4 mg / mL, and the sodium borohydride solution was 9-11 mg / mL.

[0012] The volume ratio of the chloroplatinic acid hexahydrate solution, the SH-mPEG aqueous solution, and the sodium borohydride solution is 1-3:15-17:1-3.

[0013] In step (1), the mechanical stirring speed for the synthesis of nanoenzyme particles is 900-1100 rpm, the reaction temperature is 20-30 ℃, and the reaction time is 20-40 min.

[0014] In step (1), the centrifuge tube used for ultrafiltration has a molecular weight of 10 kDa, a centrifugation speed of 2000-4000 rpm, a centrifugation time of 5-15 min, and a centrifugation frequency of 3-5 times.

[0015] In step (2), the pH adjuster is acetic acid with a mass concentration of 1-3% and a pH adjustment range of 4-5.

[0016] In step (2), the cationic lipid is SM-102, the auxiliary lipid is DSPC, and the PEG lipids are DSPE-PEG-Mal and mPEG. 2000 -DMG.

[0017] The SM-102 solution had a mass concentration of 2.83-2.85 mg / mL, the DSPC solution had a mass concentration of 0.631-0.633 mg / mL, and mPEG... 2000 The mass concentration of the DMG solution is 0.2-0.4 mg / mL, and the mass concentration of the DSPE-PEG-Mal is 0.25-1 mg / mL.

[0018] The cholesterol concentration in step (2) is 1.18-1.20 mg / mL.

[0019] In step (2), the ratio of the aqueous phase of the nanoenzyme to the ethanol phase containing cationic lipids, auxiliary lipids, PEG-lipids and cholesterol in the microfluidic chip is 2-4:0.5-2.

[0020] The mass ratio of cationic lipids, auxiliary lipids, PEG-lipids and cholesterol was 4.49:1:1.27:1.88.

[0021] In step (2), the centrifuge tube used for ultrafiltration has a molecular weight of 30 kDa, a centrifugation speed of 2000-4000 rpm, a centrifugation time of 5-15 min, and a centrifugation frequency of 5-10 times.

[0022] In step (3), the pH adjuster is ammonium bicarbonate with a concentration of 1-3 M, the pH value is adjusted to 6.5-7.5, the stirring reaction speed is 500 rpm / min, and the reaction time is 4-12 h.

[0023] In step (3), the centrifuge tube used for ultrafiltration has a molecular weight of 30 kDa, a centrifugation speed of 2000-4000 rpm, a centrifugation time of 5-15 min, and a centrifugation frequency of 3-5 times.

[0024] The present invention also provides the use of the lipid nanoparticle targeted delivery system in the preparation of drugs for the treatment and / or prevention of tumors associated with the CXCR4-CXCL12 biological axis.

[0025] The tumors associated with the CXCR4-CXCL12 biological axis include acute myeloid leukemia, breast cancer, or lymphoma.

[0026] The principle of this invention is as follows: Negatively charged nanozymes interact electrostatically with cationic lipids, resulting in efficient encapsulation within lipid nanoparticles. Simultaneously, a targeting peptide with terminal thiol groups covalently links to maleimide in the lipid component via specific thioether bonds, thus stably modifying the surface of the lipid nanoparticles. By constructing an LNP-nanozyme hybrid system and modifying its surface with an E5 peptide capable of targeting lymphoma cells, this strategy is expected to synergistically enhance the in vivo targeting delivery efficiency and circulating half-life of the nanozyme, aiming to overcome the technical bottleneck of traditional nanomedicines in targeted and precise delivery within the tumor microenvironment. Specifically, the E5 peptide primarily targets tumor cells that highly express CXCR4, while normal cells have low CXCR4 expression levels, resulting in less cellular uptake and therefore lower cytotoxicity to normal cells.

[0027] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: 1. This invention breaks through the functional limitation of traditional LNPs in single nucleic acid delivery, realizing the targeted delivery of nanozymes. 2. The nanozyme lipid nanoparticle targeted delivery system prepared by this invention has good stability, water solubility, and enzyme-like activity, enabling targeted therapy in the tumor microenvironment, effectively reducing toxic side effects on normal cells, and exhibiting good biocompatibility. 3. The lipid nanoparticle targeted delivery system containing nanozymes prepared by this invention can achieve the "Fenton reaction" through platinum nanozymes, disrupting the intracellular redox balance and further inducing immunogenic cell death; after being injected into B-cell lymphoma mice via tail vein, it exhibits significant anti-tumor effects and has potential clinical application value. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the synthesis of Pt-LNP@E5 prepared in this invention.

[0029] Figure 2 Hydrodynamic dimensions and potential statistics of Pt@mPEG, Pt-LNP and Pt-LNP@E5 prepared for this invention.

[0030] Figure 3 Transmission electron microscope image of Pt-LNP@E5 prepared for this invention and elemental distribution diagram of P, Pt and S.

[0031] Figure 4 The X-ray photoelectron spectrum of Pt-LNP@E5 prepared in this invention is shown.

[0032] Figure 5 The diagram shows the oxidase-like activity analysis of Pt@mPEG, Pt-LNP, and Pt-LNP@E5 prepared in this invention.

[0033] Figure 6 The diagram shows the peroxidase-like activity analysis of Pt@mPEG, Pt-LNP, and Pt-LNP@E5 prepared in this invention.

[0034] Figure 7 Electron paramagnetic resonance spectra of Pt@mPEG, Pt-LNP, and Pt-LNP@E5 prepared in this invention.

[0035] Figure 8 Cell viability graphs of Pt@mPEG, Pt-LNP, and Pt-LNP@E5 prepared in this invention after co-incubation with A20 cells for 24 h.

[0036] Figure 9 This is a confocal analysis diagram of Pt@mPEG, Pt-LNP, and Pt-LNP@E5 prepared in this invention after co-incubation with A20 cells for 6 h, showing the uptake of materials by the cells.

[0037] Figure 10 This is an analytical diagram showing how Pt@mPEG, Pt-LNP, and Pt-LNP@E5 prepared in this invention blocked the migration of A20 cells to MS-5 cells after co-incubation.

[0038] Figure 11 Flow cytometry analysis of intracellular ROS levels after co-incubation of Pt@mPEG, Pt-LNP, and Pt-LNP@E5 prepared in this invention with A20 cells for 6 h.

[0039] Figure 12 Flow cytometry analysis of apoptosis in A20 cells after co-incubation of Pt@mPEG, Pt-LNP, and Pt-LNP@E5 prepared in this invention with A20 cells for 24 h.

[0040] Figure 13 This is a confocal analysis image of the expression of CRT on the cell surface of A20 cells after co-incubation of Pt@mPEG, Pt-LNP and Pt-LNP@E5 prepared in this invention with A20 cells for 24 h.

[0041] Figure 14 The graph shows the ATP secretion level in the cell supernatant of A20 cells after co-incubation of Pt@mPEG, Pt-LNP and Pt-LNP@E5 prepared in this invention with A20 cells for 24 h.

[0042] Figure 15 Flow cytometry analysis of the maturation level of dendritic cells (DCs) in bone marrow after co-incubation of Pt@mPEG, Pt-LNP, and Pt-LNP prepared in this invention with A20 cells for 24 h.

[0043] Figure 16 Mice with B-cell lymphoma were treated by tail vein injection of PBS, Pt@mPEG, Pt-LNP and Pt-LNP. The treatment effect of mice on day 15 was recorded as (a) and the bioluminescence intensity was recorded as (b). Detailed Implementation

[0044] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0045] Unless otherwise specified, all chemical reagents are commercially available and can be used directly without further purification.

[0046] Chloroplatinic acid hexahydrate (H₂PtCl₆·6H₂O) and 3,3',5,5'-tetramethylbenzidine (TMB) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. SM-102 (CAS no.: 2089251-47-6), DSPC (CAS no.: 816-94-4), and mPEG were also mentioned. 2000DMG (CAS no.: 160743-62-4), DSPE-PEG-Mal (molecular weight 2000), and cholesterol were purchased from Xiamen Sinobond Biotechnology Co., Ltd. E5 peptide (Cys-Gly-Gly-Arg-Ser-Phe-Phe-Leu-Leu-Arg-Arg-Ile-Gln-Gly-Cys-Arg-Phe-Arg-Asn-Thr-Val-Asp-Asp) was purchased from Anhui Guoping Pharmaceutical Co., Ltd. Sodium borohydride (NaBH4), acetic acid, ammonium bicarbonate, and H2O2 were purchased from Sinopharm Chemical Reagent Co., Ltd. A20 cells (mouse B-cell lymphoma cell line) were purchased from Guangzhou Saiku Biotechnology Co., Ltd. RPMI-1640 medium was purchased from Jiangsu Kaiji Biotechnology Co., Ltd. Fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific. Cell Counting Kit-8 (CCK8) and 4',6-diamidinyl-2-phenylindole (DAPI) were purchased from Jiangsu Kaiji Biotechnology Co., Ltd. ROS detection kit and cell membrane far-infrared fluorescent probe (DID) were purchased from Shanghai Beyotime Biotechnology Co., Ltd. (Shanghai, China). Apoptosis detection kit (Annexin V-FITC / PI) was purchased from Jiangsu Kaiji Biotechnology Co., Ltd. ATP kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd. (Shanghai, China). Recombinant Alexa Fluor® 488 anti-CRT antibody was purchased from Abcam (ab196158). FITC anti-mouse CD11c Antibody (catalog number: 117305), PE anti-mouse CD80 Antibody (catalog number: 104707), and APC anti-mouse CD86 Antibody (catalog number: 105011) antibodies were purchased from Biolegend. BALB / c mice were purchased from Jiangsu Qinglongshan Experimental Animal Center. All water with a resistivity higher than 18.2 MΩ·cm used in all experiments was purified using a laboratory water purification system (AW-2002-H, ICP). Mercaptomethoxy polyethylene glycol (mPEG-SH, catalog number: MEPG-0236 (molecular weight 5000), Chongqing Yusi Pharmaceutical Technology Co., Ltd.) was also used.

[0047] Example 1: Preparation of Pt-LNP@E5 according to Figure 1The steps are as follows: First, weigh 20 mg of H₂PtCl₆·6H₂O and dissolve it in 2 mL of pure water. Next, weigh 40 mg of SH-mPEG and dissolve it in 16 mL of pure water. Then, mix the two solutions thoroughly and pour them into a reactor. Under mechanical stirring at 1000 rpm, add 2 mL of NaBH₄ solution (10 mg / mL) dropwise to the reactor. Maintain the reaction temperature at 25 °C throughout the reaction process, and allow the liquid-phase chemical precipitation reaction to continue for 30 min. After the reaction is complete, centrifuge at 3000 rpm for 10 min using a 10 kDa ultrafiltration tube. Repeat this operation three times to purify and obtain an aqueous solution of Pt@mPEG nanozyme.

[0048] Take the above Pt@mPEG nanoenzyme aqueous solution and adjust the pH to 4 with 2% acetic acid aqueous solution. Then weigh out 2.84 mg SM-102, 0.632 mg DSPC, and 0.3 mg mPEG sequentially. 2000 DMG, 1.19 mg cholesterol, and 0.5 mg DSPE-PEG-Mal were dissolved together in 1 mL of anhydrous ethanol to obtain a mixed lipid ethanol solution. Then, using microfluidics, the pH-adjusted Pt@mPEG nanoenzyme aqueous solution and the mixed lipid ethanol solution were respectively connected to a standard PDMS chip (ZX-LS-31, Suzhou Zhongxin Qiheng Scientific Instruments Co., Ltd.). The flow rates were set to 150 μL / min (Pt@mPEG solution) and 50 μL / min (mixed lipid ethanol solution), respectively, to allow them to mix within the chip. Finally, the solution was purified by centrifugation at 3000 rpm for 10 min using a 30 kDa ultrafiltration tube, repeated 8 times, to obtain a Pt-LNP aqueous solution.

[0049] The above-mentioned Pt-LNP aqueous solution was mixed with 0.5 mg of E5 peptide, and then the pH of the solution was adjusted to 7.5 using ammonium bicarbonate solution (1M). The mixture was stirred continuously at 500 rpm for 6 h at room temperature. After the reaction was completed, the reaction solution was transferred to an ultrafiltration tube with a molecular weight cutoff of 30 kDa and centrifuged at 3000 rpm for 10 min. This operation was repeated 3 times to finally purify the Pt-LNP@E5 aqueous solution.

[0050] Example 2 Morphology and particle size characterization of Pt-LNP@E5 Aqueous solutions of Pt@mPEG, Pt-LNP, and Pt-LNP@E5 synthesized in Example 1 (each with a concentration of 100 μg / mL, calculated as Pt, 1 mL) were taken, and their surface potential and hydrodynamic size were measured, respectively. The results are as follows: Figure 2As shown, after coating with lipid nanoparticles (LNPs), the particle size of Pt@mPEG increased from 6.92 nm to 31.34 nm, and when further modified with E5 peptide, the particle size continued to increase to 50.04 nm. Simultaneously, the potential of Pt@mPEG was -16.17 mV, which changed to 14.07 mV after lipid nanoparticle coating, and further increased to 27.97 mV after subsequent E5 peptide modification. These changes in potential and particle size confirm that Pt@mPEG was successfully coated with lipid nanoparticles and that the E5 peptide was also successfully modified.

[0051] The Pt-LNP@E5 aqueous solution (50 μg / mL, based on Pt) synthesized in Example 1 was dropped onto the surface of a copper mesh (Zhongjing Scientific Instruments Technology Co., Ltd.) covered with a carbon film, followed by the addition of phosphotungstic acid solution (2%, 20 μL). The mesh was then allowed to air dry at room temperature for transmission electron microscopy (TEM) imaging. The results are as follows: Figure 3 As shown in the TEM image, Pt@mPEG is clearly observed to be encapsulated within LNPs. Furthermore, elemental analysis reveals a uniform distribution of Pt, S, and P elements in Pt-LNP@E5. This result strongly supports the successful preparation of Pt-LNP@E5.

[0052] Example 3: X-ray photoelectron spectroscopy test The Pt-LNP@E5 aqueous solution synthesized in Example 1 was freeze-dried into powder, and then subjected to X-ray photoelectron spectroscopy (XPS) analysis. Analysis of the high-resolution Pt 4f spectrum revealed binding energy levels of 75.64 eV and 74.25 eV, which are consistent with Pt 4f... 5 / 2 The presence of electrons corresponds to this. Simultaneously, Pt 4f 7 / 2 The binding energies of electrons are 72.44 eV and 71.05 eV, respectively (e.g., ...). Figure 4 (As shown).

[0053] Example 4: Determination of Oxidase (OXD) Activity The aqueous solutions of Pt@mPEG, Pt-LNP and Pt-LNP@E5 synthesized in Example 1 were used for OXD activity testing. Using TMB as the chromogenic substrate, the oxidase activities of Pt@mPEG, Pt-LNP, and Pt-LNP@E5 were measured under different pH conditions. The test sample solutions contained 150 μL of buffer solutions at different pH values ​​[pH 3.92 (acetic acid / sodium acetate buffer), 5.03 (acetic acid / sodium acetate buffer), 5.60 (acetic acid / sodium acetate buffer), 6.54 (citric acid / sodium citrate buffer), 7.40 (PBS phosphate buffer), and 8.00 (MES buffer)], 25 μL of aqueous solutions of different nanozymes (Pt@mPEG, Pt-LNP, or Pt-LNP@E5 aqueous solutions were all at a concentration of 200 μg / mL, calculated as Pt), and 25 μL of TMB (10 mg / mL) solution. The absorbance at 650 nm was measured after the reaction solutions were placed in the wells for 2 h. The results are as follows: Figure 5 As shown, the absorbance gradually decreases with increasing pH value, indicating that the material exhibits good OXD activity under acidic conditions. Furthermore, the OXD activity of Pt@mPEG significantly decreases after being coated with LNP. This phenomenon is because the direct contact area between the nanoparticles and the chromogenic substrate TMB is reduced after the nanoparticles are coated inside the LNP, resulting in a decrease in color development.

[0054] Example 5: Assay of Peroxidase (POD) Activity The POD activity of the Pt@mPEG, Pt-LNP, and Pt-LNP@E5 aqueous solutions synthesized in Example 1 was tested. Using TMB as the chromogenic substrate, the POD activity of the Pt@mPEG, Pt-LNP, and Pt-LNP@E5 aqueous solutions under different pH conditions was investigated in the presence of H2O2. The test sample solution contained 170 μL of buffer solutions with different pH values ​​[pH 3.92 (acetic acid / sodium acetate buffer), 5.03 (acetic acid / sodium acetate buffer), 5.60 (acetic acid / sodium acetate buffer), 6.54 (citric acid / sodium citrate buffer), 7.40 (PBS phosphate buffer), and 8.00 (MES buffer)], 10 μL of different nanozyme aqueous solutions (Pt@mPEG, Pt-LNP, or Pt-LNP@E5 aqueous solutions, all at a concentration of 200 μg / mL, calculated as Pt), 10 μL of H2O2 solution (3.75%), and 10 μL of LTMB (10 mg / mL) solution. After reacting in the wells for 5 min, the absorbance at 650 nm was measured. The results are as follows: Figure 6As shown, the absorbance of Pt@mPEG, Pt-LNP, and Pt-LNP@E5 gradually decreased with increasing pH, indicating that Pt@mPEG, Pt-LNP, and Pt-LNP@E5 exhibit good POD activity under acidic conditions. Furthermore, the POD activity of Pt@mPEG also significantly decreased after being coated with LNP. This phenomenon is also due to the reduced direct contact area between the nanoparticles and H2O2 and the chromogenic substrate TMB after the nanoparticles are coated inside the LNP, resulting in a decrease in the degree of color development.

[0055] Example 6: Electron paramagnetic resonance (ESR) detection of ·OH The formation of ·OH from H₂O₂ catalyzed by Pt@mPEG, Pt-LNP, and Pt-LNP@E5 was detected using an A300-10 / 12 Bruker ESR spectrometer. Under H₂O₂ conditions, 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO) was used as a trapping agent to capture ·OH and form DMPO / ·OH adducts. First, 500 μL of pH 5.03 buffer (acetic acid / sodium acetate buffer) was mixed with 100 μL of 3.75% H₂O₂ and 200 μg / mL of the material (100 μL each of Pt@mPEG, Pt-LNP, or Pt-LNP@E5, concentration expressed as Pt). After reacting for 5 min, 100 μL of the reaction mixture was mixed with 100 μL of DMPO solution (100 mM) and placed in a glass capillary tube and sealed. The spectral characteristic lines were then recorded. H2O2, Pt@mPEG, Pt-LNP, or Pt-LNP@E5 were reacted with the trapping agent DMPO as control groups, respectively. Results are as follows: Figure 7 As shown, when Pt@mPEG, Pt-LNP, or Pt-LNP@E5 are present alone, or when H2O2 is present alone, no characteristic peak signal of the DMPO / ·OH adduct (1:2:2:1) is observed. In contrast, the ESR characteristic signal of the DMPO / ·OH adduct is significantly enhanced after the simultaneous addition of Pt@mPEG, Pt-LNP, or Pt-LNP@E5 and H2O2. This indicates that Pt@mPEG, Pt-LNP, and Pt-LNP@E5 can catalyze the production of ·OH from H2O2. In the three systems of Pt@mPEG, Pt-LNP, and Pt-LNP@E5, the core activity for catalyzing the production of ·OH from H2O2 originates from Pt@mPEG. Subsequent LNP encapsulation and E5 peptide functionalization modification of Pt@mPEG did not significantly improve the ·OH effect, but it maintained the original catalytic activity.

[0056] Example 7: Determination of relative cell viability using the CCK8 assay The cytotoxicity of Pt@mPEG, Pt-LNP, and Pt-LNP@E5 prepared in Example 1 was tested using A20 cells as a model. A20 cells in logarithmic growth phase were collected and cultured at 5 × 10⁻⁶ cells per cell line. 4 Cells were seeded at a density of 100 cells / well in 96-well plates, and then different concentrations of Pt@mPEG, Pt-LNP, and Pt-LNP@E5 solutions (final Pt concentrations of 0, 5, 10, 20, 40, 60, 80, and 100 μg / mL, respectively) were added. The plates were incubated at 37 °C for 24 h at 5% CO2. The culture medium used was RPMI-1640 complete medium supplemented with 100 U / mL streptomycin and 100 U / mL streptomycin, and 10% FBS. Afterward, 10% (v / v) CCK-8 solution was added, and the plates were cultured for another 2 h. Finally, the absorbance of each well was measured at 450 nm using a multi-mode microplate reader. PBS-treated A20 cells served as the control group, with cell viability recorded as 100%. PBS-treated medium served as blank control 1. Medium containing only Pt@mPEG, Pt-LNP, or Pt-LNP@E5 served as blank control 2. Results are shown below. Figure 8 As shown in the figure, within the experimentally defined concentration range, cytotoxicity gradually increased with increasing Pt concentration. This result indicates that Pt@mPEG nanozymes have good anti-tumor effects. However, under the same Pt ​​concentration conditions, the cytotoxicity was more significant after modification with lipid nanoparticles (LNP) and E5 peptides. This suggests that E5 peptide modification increases the uptake of nanozymes by cells, leading to greater toxicity. The cell survival percentage (%) was calculated as: [(Experimental group absorbance - Blank control 2) / (Control group absorbance - Blank control 1 absorbance)] * 100%.

[0057] Example 8 Cell uptake detection 10 mL of 200 μg / mL Pt-LNP and Pt-LNP@E5 (concentrations expressed as Pt) were added to 10 μL of FAM solution (6-carboxyfluorescein, 10 mg / mL), and the mixture was shaken for 4 h to obtain FAM-modified Pt-LNP and Pt-LNP@E5. A20 cells were then cultured at a specific density (5*10⁻⁶ cells / mL). 6 Cells (number per well) were seeded into 6-well plates, and then incubated with FAM-modified Pt-LNP or Pt-LNP@E5 for 6 h. After incubation, the cells were collected, washed, and fixed with 4% paraformaldehyde. The nuclei were stained with DAPI, and the cell membranes were stained with DID. Finally, imaging analysis was performed using laser confocal microscopy. The results are as follows: Figure 9As shown, compared with Pt-LNP, Pt-LNP functionalized with E5 was taken up by cells by a significantly greater amount when co-incubated with A20 cells for 6 h.

[0058] Example 9: Testing of Tumor Cell Migration Ability First, MS-5 cells were inoculated at a concentration of 5 × 10⁻⁶. 5 Cells were seeded at a density of cells / well in the lower chamber of a Transwell-24 well plate and incubated overnight. Subsequently, A20 cells were seeded at the same density (5 × 10⁶ cells / well). 5 A20 cells (number per well) were seeded into the upper chamber of a Transwell-24 microarray. These A20 cells had been pretreated for 12 h by co-incubation with Pt@mPEG, Pt-LNP, or Pt-LNP@E5 aqueous solutions (10 μg / mL, concentration expressed as Pt). Afterward, the A20 cells in the upper chamber were co-incubated with MS-5 cells in the lower chamber for 24 h. Following incubation, the number of A20 cells migrating to the lower chamber was counted using a cell counter, and the relative migration rate of A20 cells in different treatment groups (Pt@mPEG, Pt-LNP, or Pt-LNP@E5) was calculated. A PBS-treated group served as a control group. The experimental results are as follows: Figure 10 As shown, there were significant differences in the migration degree of A20 cells among different treatment groups. In particular, the migration degree of A20 cells to MS-5 cells was significantly inhibited under Pt-LNP@E5 treatment, which was significantly lower than that in the Pt-LNP treatment group. This result indicates that Pt-LNP@E5 can effectively block the CXCR4-CXCL12 biological axis, thereby facilitating its subsequent therapeutic effect.

[0059] Wherein, A20 cell migration rate = [(CA) / (BA)] * 100%, A = The number of A20 cells that leak out naturally without any treatment when there are no MS-5 cells in the lower chamber; B = The number of A20 cells that migrate downwards naturally without any treatment when there are MS-5 cells in the lower chamber; C = The number of A20 cells that migrate downward after treatment with PBS, Pt@mPEG, Pt-LNP, or Pt-LNP@E5 when MS-5 cells are present in the lower chamber.

[0060] Example 10 Detection of reactive oxygen species levels in cells To assess the effect of the material on intracellular reactive oxygen species (ROS) levels, A20 cells were cultured at 5 × 10⁻⁶ cells / year. 5Cells were seeded at a density of cells / well in 6-well plates, and then Pt@mPEG, Pt-LNP, or Pt-LNP@E5 (final concentration 20 μg / mL) were added to each well, with a PBS-treated group serving as a control. Cells were incubated for 6 h in a 37 °C incubator containing 5% CO2 using RPMI-1640 complete medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin, and 10% FBS. After incubation, cells were collected from each well, centrifuged to remove the original medium, washed with PBS, and then co-incubated with the DCFH-DA probe diluted in serum-free medium for 30 min. The DCFH-DA probe can be oxidized by ROS within the cells, generating DCF with green fluorescence. After incubation, cells were washed three times with PBS and then analyzed by flow cytometry. Results are as follows: Figure 11 As shown, compared with the Pt@mPEG or Pt-LNP groups, cells treated with Pt-LNP@E5 exhibited a higher percentage of ROS (36.7%), indicating that Pt-LNP@E5 was taken up by A20 cells in greater quantities and was able to induce more ROS production within the cells.

[0061] Example 11 Detection of Tumor Cell Apoptosis Using A20 cells as a model, the killing effects of Pt@mPEG, Pt-LNP, and Pt-LNP@E5 prepared in Example 1 on cancer cells were evaluated. A20 cells in the logarithmic growth phase were collected and cultured at a concentration of 5 × 10⁻⁶ cells / cells. 5 Cells were seeded at a density of 10 cells / well into 6-well plates. Subsequently, Pt@mPEG, Pt-LNP, or Pt-LNP@E5 aqueous solutions (final concentration 10 μg / mL) were added to each well, and the plates were incubated at 37 °C for 24 h in a 5% CO2 incubator. The culture medium used was RPMI-1640 complete medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin, and 10% FBS. After incubation, cells were collected from each well, centrifuged, washed three times with PBS, and then co-incubated for 10 min with Annexin V-FITC (a cell apoptosis detection reagent) and PI (Jiangsu Kaiji Biotechnology Co., Ltd.) diluted 100-fold with PBS. Finally, cell apoptosis was detected by flow cytometry. The PBS-treated group served as a control group. Results are as follows: Figure 12 As shown, no significant apoptosis was observed in the PBS group. Compared with the Pt@mPEG and Pt-LNP groups, the Pt-LNP@E5 treatment group had the highest proportion of late apoptosis, reaching 11.46%. This indicates that A20 cells took up more Pt-LNP@E5, which induced the cells to produce more ROS, thereby promoting apoptosis.

[0062] Example 12 Detection of Immunogenic Death and Dendritic Cell Maturation in Tumor Cells To verify the immunogenic cell death (ICD) effect induced by Pt@mPEG, Pt-LNP, and Pt-LNP@E5 prepared in Example 1 after tumor treatment, this invention uses A20 cells as a model to evaluate the expression levels of ICD markers—calreticulin (CRT) and adenosine triphosphate (ATP) after treatment with these materials, and evaluates the ICD-induced dendritic cell (DC) maturation level in bone marrow using Transwell assays.

[0063] Collect A20 cells in the logarithmic growth phase at a concentration of 5 × 10⁻⁶ cells / cells. 5 Cells were seeded at a density of 10 cells / well in 6-well plates. Pt@mPEG, Pt-LNP, or Pt-LNP@E5 (final concentration 30 μg / mL) were then added to each well, with a PBS-treated group serving as the control. The cells were incubated for 24 h in a 37°C incubator containing 5% CO2. The culture medium used was RPMI-1640 complete medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin, and 10% FBS. After incubation, the culture medium was collected, and the relative ATP content in the cell culture medium was detected using an ATP assay kit (Shanghai Beyotime Biotechnology Co., Ltd.). Cells were incubated with CRT-Alexa Fluor488 antibody (Abcam, ab196158) at a 1:100 volume ratio for 30 min, then fixed with paraformaldehyde for 15 min, and finally stained with DAPI to the cell nuclei. CRT expression on the cell surface was detected by laser confocal microscopy. Results are as follows: Figure 13 and Figure 14 As shown, the ATP release and CRT surface expression levels were significantly increased in the Pt-LNP@E5 group. Compared with the PBS group, although the CRT expression and ATP secretion levels in the Pt-LNP group were somewhat increased, they were still much lower than those in the Pt-LNP@E5 group. This result indicates that the chemokinetic treatment induced by Pt-LNP@E5 used in this invention can promote CRT expression and ATP secretion to varying degrees, inducing immunogenic cell death.

[0064] To evaluate the level of DC maturation induced by the materials prepared in Example 1 on treated cancer cells, this example uses A20 cells and DC cells extracted from bone marrow as models, and verifies the maturation of DC cells through a Transwell assay. The specific procedure is as follows: A20 cells in the logarithmic growth phase are collected and processed at a rate of 5 × 10⁻⁶ cells / year. 5Cells were seeded at a density of 10 cells / well into the upper chamber of a 12-well Transwell plate, followed by the addition of Pt@mPEG, Pt-LNP, or Pt-LNP@E5 (final concentration 30 μg / mL) to each well, and incubated for 24 h at 37°C with 5% CO2. The culture medium used was RPMI-1640 complete medium supplemented with 100 U / mL penicillin and 100 U / mL streptomycin, and 10% FBS. Simultaneously, dendritic cells (DCs) were extracted from the bone marrow of BALB / c mice and cultured at 5 × 10⁻⁶ cells / well. 5 Cells were seeded at a density of [number] cells / well in the lower chamber of a 12-well Transwell plate, using the same culture medium as for A20 cells, and cultured in a 37°C incubator containing 5% CO2. After 24 h of co-incubation with the materials (Pt@mPEG, Pt-LNP, or Pt-LNP@E5), the original culture medium from the upper chamber was collected. This collected original culture medium was then replaced with the culture medium for the DC cells in the lower chamber, and co-cultured for another 24 h. Subsequently, the DC cells from the lower chamber were collected and incubated with antibodies against DC cell maturation markers CD11c-FITC, CD80-PE, and CD86-APC at room temperature in the dark for 30 min. The cells were then washed three times with PBS, resuspended in PBS, and finally, the maturation status of the DC cells was detected by flow cytometry. The results are as follows: Figure 15 As shown, after treatment with Pt-LNP@E5, the expression levels of CD80 and CD86 proteins significantly increased (21.60%), indicating that the Pt-LNP@E5-based material can induce strong immunogenic cell death, thereby further inducing DC cell maturation. Compared with Pt-LNP@E5, the expression level of Pt-LNP was slightly lower, only 15.30%, indicating that cells have a stronger recognition and phagocytic ability for E5 peptide-modified Pt-LNP, which can enhance the immunogenic cell death effect induced by nanomedicine, thus giving Pt-LNP@E5 a strong ability to induce DC cell maturation.

[0065] Example 13 Evaluation of the therapeutic effect in mice with B-cell lymphoma All animal experiments were conducted strictly in accordance with animal protection association standards. Six- to seven-week-old female BALB / c mice used in the experiments were purchased from Jiangsu Qinglongshan Experimental Animal Center. A B-cell lymphoma model was established in the mice, and 1×10⁶ mice were used... 8A20-Luc cells were injected into mice via the tail vein and the mice were fed normally for one week. One week later, mice were injected intraperitoneally with D-fluorescein potassium solution (15 mg / mL, 150 mg / kg). Small animal in vivo imaging was used to detect disease progression; the presence of bioluminescent signals indicated successful modeling. The successfully modeled mice were divided into four groups (n = 5 mice per group): control group (PBS, 200 μL); Pt@mPEG group; Pt-LNP group; and Pt-LNP@E5 group (Pt dose 10 mg / kg, 200 μL, respectively), administered via tail vein injection. The day treatment began was designated as day 1, with treatment every 3 days for a total of 5 treatments. Bioluminescence imaging was performed on the mice on day 15 to detect disease progression. The results are as follows: Figure 16 As shown in Figure a, the fluorescence signals in different treatment groups were all lower than those in PBS, indicating that the material has varying degrees of inhibitory effect on tumor progression. Compared with Pt@mPEG and Pt-LNP, the morbidity of mice treated with Pt-LNP@E5 was effectively alleviated, as shown in the specific quantitative fluorescence intensity figures. Figure 16 As shown in b, Pt-LNP@E5 can effectively alleviate tumors and improve treatment efficacy in vivo.

Claims

1. A lipid nanoparticle targeted delivery system, characterized in that, It consists of lipid nanoparticles with a surface modified with a targeting peptide coating a nanoenzyme particle with a negatively charged surface; the lipid nanoparticles contain cationic lipids and lipids containing maleimide groups; the targeting peptide has a thiol group at its end.

2. A method for preparing the lipid nanoparticle targeted delivery system of claim 1, characterized in that, Includes the following steps: (1) Preparation of nanoenzyme particles with negative surface charge: The polymer is mixed with the nanoenzyme precursor, and a reducing agent is added dropwise under stirring to react. Then, the excess reducing agent is removed by ultrafiltration to obtain the solution of the negatively charged nanoenzyme particles. (2) Preparation of lipid nanoparticles loaded with nanozymes: The aqueous phase of the nanozymes obtained in step (1) was adjusted to a weak acid and then mixed with the ethanol phase containing cationic lipids, auxiliary lipids, PEG lipids and cholesterol in a microfluidic chip. The organic solvent was then removed by ultrafiltration to obtain a solution of lipid nanoparticles loaded with nanozymes. (3) Preparation of nanoenzyme lipid nanoparticle targeted delivery system: The lipid nanoparticle solution containing nanoenzyme obtained in step (2) is adjusted to neutral, mixed with a targeted polypeptide with thiol group at the end and stirred to react, and then the unreacted polypeptide is removed by ultrafiltration to obtain the lipid nanoparticle targeted delivery system.

3. The method according to claim 2, characterized in that, The polymer in step (1) is mercaptomethoxy polyethylene glycol; the nanozyme precursor is chloroplatinic acid hexahydrate; and the reducing agent is sodium borohydride.

4. The method according to claim 3, characterized in that, The mass concentration of the chloroplatinic acid hexahydrate solution is 9-11 mg / mL, the mass concentration of the mercaptomethoxy polyethylene glycol solution is 2-4 mg / mL, and the mass concentration of the sodium borohydride solution is 9-11 mg / mL.

5. The method according to claim 4, characterized in that, The volume ratio of chloroplatinic acid hexahydrate solution, mercaptomethoxy polyethylene glycol aqueous solution, and sodium borohydride solution is 1-3:15-17:1-3.

6. The method according to claim 2, characterized in that, The cationic lipid mentioned in step (2) is SM-102, the auxiliary lipid is DSPC, and the PEG lipids are DSPE-PEG-Mal and mPEG. 2000 -DMG.

7. The method according to claim 6, characterized in that, The mass concentration of SM-102 solution was 2.83-2.85 mg / mL, the mass concentration of DSPC solution was 0.631-0.633 mg / mL, and mPEG... 2000 The mass concentration of the DMG solution is 0.2-0.4 mg / mL, and the mass concentration of the DSPE-PEG-Mal is 0.25-1 mg / mL.

8. The method according to claim 2, characterized in that, The cholesterol concentration mentioned in step (2) is 1.18-1.20 mg / mL.

9. The method according to claim 2, characterized in that, In step (2), the ratio of the aqueous phase of the nanozyme to the ethanol phase containing cationic lipids, auxiliary lipids, PEG-lipids and cholesterol is 2-4:0.5-2 in the microfluidic chip.

10. The use of the lipid nanoparticle targeted delivery system of claim 1 in the preparation of drugs for the treatment and / or prevention of tumors associated with the CXCR4-CXCL12 biological axis.

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