Traditional Chinese medicine multi-component nano drug delivery system as well as preparation method and application thereof

Abstract

The invention relates to the technical field of anti-cancer drugs, and provides a traditional Chinese medicine multi-component nano drug delivery system as well as a preparation method and application thereof. According to the invention, honokiol (HN) is entrapped by adopting lipid nanoparticles formed by a berberine (BR)-disulfide bond-caprylic capric triglyceride compound, the surface of the lipid nanoparticles is coated with a gelatin layer, and quercetin (QE) is entrapped in the gelatin layer, so that simultaneous delivery of HN, BR and QE and programmed release outside and inside cells are realized. Furthermore, the surface of the gelatin layer is modified with PEG, so that the long circulation of the drug delivery system in blood is improved; furthermore, cRGD is connected to PEG, and the synergistic lung cancer resisting effect of HN, BR and QE is greatly improved through the targeting effect of cRGD. In conclusion, the drug delivery system provided by the invention can give full play to the effect of synergistically treating lung cancer by HN, QE and BR, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of anticancer drug technology, and in particular to a multi-component nano-drug delivery system of traditional Chinese medicine, its preparation method and application. Background Technology

[0002] Lung cancer is a leading cause of cancer death worldwide, posing a serious threat to human life and health. It is mostly discovered at an advanced stage, making it more difficult to treat and highly susceptible to drug resistance. Therefore, preventing its progression and designing more effective treatments are crucial. Currently, lung cancer treatments primarily rely on hormone therapy, surgical intervention, chemotherapy, photodynamic therapy, immunotherapy, radiotherapy, and gene therapy. However, these methods suffer from low response rates and significant damage to normal tissues, resulting in unsatisfactory treatment outcomes. Traditional Chinese medicine (TCM) treatment, combining syndrome differentiation and treatment, a holistic approach, and the concept of preventative medicine, complements Western medicine's shortcomings. It can enhance efficacy and reduce toxicity, improve clinical symptoms, prolong survival time with the tumor, improve quality of life, inhibit tumor growth and metastasis, and improve various functions in patients.

[0003] Traditional Chinese medicine (TCM), often used as an adjuvant in clinical chemotherapy, is characterized by its multiple mechanisms of action and low toxicity. Its monomers and extracts have become a hot topic in cancer treatment research. However, in-depth research into TCM's anti-cancer properties has revealed problems such as poor stability and targeting, low water solubility, and degradation by digestive enzymes in the acidic environment of the gastrointestinal tract, leading to low absorption and utilization rates by the human body. This significantly restricts the development and application of TCM. Nanoparticle drug delivery systems can encapsulate poorly soluble drugs, improving solubility and targeting, thereby enhancing drug stability and bioavailability, and playing a synergistic and toxicity-reducing role. However, current nanoparticle drug delivery systems are mostly based on single-component chemotherapy drugs, and there are no reports of multi-component TCM nanoparticle formulations used clinically.

[0004] Recent studies have shown that the effective components in the ancient formula for treating lung cancer, which focuses on "strengthening the spleen and replenishing qi, and eliminating toxins and pathogens," along with magnolol (HN), berberine (BR), and quercetin (QE), possess different anti-cancer mechanisms and can inhibit the proliferation of lung cancer cells, exhibiting significant anti-lung cancer effects. However, the poor water solubility of HN and QE, and the poor membrane permeability of BR, limit their application in the field of anti-lung cancer treatment. Summary of the Invention

[0005] In view of this, the present invention provides a multi-component nano-drug delivery system for traditional Chinese medicine, its preparation method, and its application. The drug delivery system provided by the present invention can simultaneously deliver HN, BR, and QE, solving the problems of poor water solubility of HN and QE and poor membrane permeability of BR.

[0006] This invention provides a multi-component nanoparticle drug delivery system for traditional Chinese medicine, comprising BR lipid nanoparticles loaded with HN, a gelatin layer covering the surface of the HN-loaded BR lipid nanoparticles, and QE encapsulated in the gelatin layer; the HN-loaded BR lipid nanoparticles include BR lipid nanoparticles and HN encapsulated within them; the BR lipid nanoparticles are formed from a BR-disulfide bond-caprylic / capric triglyceride compound (BR-SS-GTCC); the gelatin in the gelatin layer is type A gelatin. This invention utilizes BR-SS-GTCC-formed lipid nanoparticles to encapsulate HN, and simultaneously loads QE onto the surface of the lipid nanoparticles with a gelatin layer, thereby achieving simultaneous delivery of HN, QE, and BR, solving the problems of poor water solubility of HN and QE and poor membrane permeability of BR. The results of the examples show that in the multi-component nano-delivery system of traditional Chinese medicine of the present invention, gelatin can be degraded by MMP-2 (matrix metalloproteinase 2) to release QE and B-LNP@HN (BR lipid nanoparticles loaded with HN). BR-SS-GTCC in B-LNP@HN undergoes a reduction reaction with GSH (glutathione) to release BR and HN, thereby achieving the simultaneous delivery of three important active ingredients.

[0007] Furthermore, this invention modifies the gelatin layer surface with polyethylene glycol (PEG). By modifying PEG, this invention can prolong the in vivo retention time, achieving a long-circulation effect in vivo, reducing the dosage, and improving the therapeutic effect of the drug delivery system. The results of the examples show that the polyethylene glycol-modified multi-component traditional Chinese medicine nano-drug delivery system (PGB-LNP@HN, QE) provided by this invention can respond to MMP-2 and GSH, achieving programmed extracellular and intracellular release of QE, HN, and BR.

[0008] Furthermore, this invention also links cRGD to polyethylene glycol. The targeting effect of cRGD, combined with the tumor microenvironment-regulating effect of QE, achieves tumor penetration, enabling the drug delivery system to target deep into the tumor. Combined with the mitochondrial targeting function of BR and the mitochondrial-dependent anti-lung cancer effect of HN, the synergistic anti-lung cancer effect of HN, BR, and QE is significantly enhanced. This formulation simultaneously responds to the release of MMP-2 highly expressed in tumor tissue and GSH highly expressed in tumor cells, specifically binding to αvβ3 highly expressed in tumor tissue to achieve programmed drug release, fully leveraging the synergistic anti-lung cancer effect of each active ingredient. The nano-drug delivery system provided by this invention can target tumor sites in vivo and accumulate in tumor tissue through long-term in vivo circulation. Gelatin responds to the degradation of MMP-2 in tumor tissue, releasing QE to act on lung cancer tumor tissue, exerting the QE's effect of regulating the tumor microenvironment. B-LNP@HN responds to GSH, simultaneously releasing HN and BR to act on lung cancer tumor cells, while HN acts on the mitochondria of lung cancer tumor cells.

[0009] This invention evaluates the anti-lung cancer effects of PGB-LNP@HN,QR and cRGD-PGB-LNP@HN,QE through in vitro and in vivo experiments. PGB-LNP@HN,QE achieved long-term circulation in vivo and was capable of programmed release, exhibiting anti-tumor activity both in vitro and in vivo. cRGD-PGB-LNP@HN,QE, in addition to the effects of PGB-LNP@HN,QE, also achieved penetration to deep tumor sites. HN, BR, and QE have a synergistic anti-lung cancer effect, increasing the drug's targeting to A549 cells. PEG modification achieved long-term circulation in vivo, and the combination of cRGD modification and QE enabled penetration into deep tissues. The nanoparticles could target mitochondria, inducing mitochondrial membrane potential transitions and increasing ROS generation. Both PGB-LNP@HN,QE and cRGD-PGB-LNP@HN,QE achieved long-term circulation in vivo and could accumulate at lung cancer sites, demonstrating good in vivo anti-lung cancer activity.

[0010] This invention also provides a method for preparing the multi-component nano-drug delivery system of traditional Chinese medicine described above. The operation method provided by this invention is simple, and the resulting drug delivery system has a small particle size and high encapsulation efficiency of HN and QE. Furthermore, this invention uses single-factor investigation and star point design-response surface methodology to investigate surfactant, solvent type, stirring temperature, gelatin concentration, gelatin strength, gelatin pH, volume ratio of B-LNP@HN to gelatin, and QE dosage, and uses particle size and encapsulation efficiency of HN and QE as evaluation indicators to screen the optimal formulation process. Attached Figure Description

[0011] Figure 1 For the synthesis of berberine; Figure 2 The mass spectrum of berberine; Figure 3 The above shows the 1H NMR spectrum and 1C NMR spectrum of berberine. Figure 4 Synthesis of GTCC-SS-COOH; Figure 5 Synthesis of BR-SS-GTCC; Figure 6 This is a BR-SS-GTCC mass spectrum; Figure 7 The images show the BR-SS-GTCC 1H NMR spectrum (top) and 1C NMR spectrum (bottom). Figure 8 For PG validation results, where: A: Iodine powder colorimetric plate (from left to right: gelatin solution, PG solution, PEG solution), B: Free aminoninhydrin colorimetric plate; Figure 9FTIR spectra for AG, MPEG, MPEG+AG, PGB-LNP@HN,QE; Figure 10 The appearance of the PGB-LNP@HN,QE nanoparticle dispersion; Figure 11 The morphology of PGB-LNP@HN,QE (×530000) under transmission electron microscopy; Figure 12 The particle size (A) and potential (B) distributions of PGB-LNP@HN are shown. Figure 13 The results of stability tests for PGB-LNP@HN,QE are as follows: A: Changes in absorbance of PGB-LNP@HN,QE in FBS and serum; B: Changes in encapsulation efficiency of HN, QE, and BR of PGB-LNP@HN,QE; CE: Changes in PDI, particle size, and Zeta potential of PGB-LNP@HN,QE. Figure 14 The results of blood compatibility tests for B-LNP@HN and PGB-LNP@HN, QE are shown in Figure A, where A represents the appearance and B represents the hemolysis rate. Figure 15 Particle size (A) and potential (B) distribution of PGB-LNP@HN,QE after incubation with MMP 2 for 24 h; Figure 16 Particle size (A) and potential (B) distribution of PGB-LNP@HN,QE after incubation with MMP 2 for 48 h; Figure 17 Particle size (A) and potential (B) distribution of PGB-LNP@HN,QE after 48h incubation with BSA; Figure 18 Transmission electron microscopy (TEM) images of PGB-LNP@HN,QE (×530000) incubated with BSA / MMP-2; where: A: PGB-LNP@HN,QE after 24 h of incubation with MMP-2; B: PGB-LNP@HN,QE after 48 h of incubation with MMP-2; C: PGB-LNP@HN,QE after 48 h of incubation with BSA; Figure 19 The cumulative release curves of BR (left), HN (middle), and QE (right) in GB-LNP@HN,QE and PGB-LNP@HN,QE under different conditions; Figure 20CLSM images of DiO&DiL, SLN@DiO,DiL, B-LNP@DiO,DiL, PG-DiO,DiL-B-LNP and PG-DiO,DiL-B-LNP+MMP-2 after 2 hours of incubation with A549 cells. Figure 21 Two hours after the uptake of DiO&DiL, SLN@DiO,DiL, B-LNP@DiO,DiL, PG-DiO,DiL-B-LNP and PG-DiO,DiL-B-LNP+MMP-2, the culture dishes were replaced with fresh culture medium, and the results were monitored at 1 h, 3 h and 12 h (green: DiO channel; red: DiL channel). Figure 22 CLSM images of B-LNP@DiO,DiL, PG-DiO,DiL-B-LNP, and PG-DiO,DiL-B-LNP+MMP-2 after incubation with A549 cells for different times (green: DiO channel; red: DiL channel). Figure 23 Characterization of cRGD-PGB-LNP@HN,QE: (A) Appearance of cRGD-PGB-LNP@HN,QE; (B) Transmission electron micrograph of cRGD-PGB-LNP@HN,QE; (C) and (E) Particle size and potential diagrams of cRGD-PGB-LNP@HN,QE, respectively; (D) Infrared spectral density of cRGD-PEG-AG, cRGD-PGB-LNP@HN,QE, and AG. Figure 24 Particle size (A), PDI (B), and potential (C) for storage stability of cRGD-PGB-LNP@HN,QE, and particle size (D) and PDI (E) for serum stability. Figure 25 The blood compatibility appearance of different concentrations (50%, 25%, 12.5%, 6.25%, 3.125%) of B-LNP@HN, PGB-LNP@HN,QE, and cRGD-PGB-LNP@HN,QE; Figure 26 The cumulative release rates of QE, HN, and BR in cRGD-PGB-LNP@HN,QE under different conditions; Figure 27Cell uptake of A549 cells after incubation with DiO / DiL, B-LNP@DiO / DiL, PGB-LNP@DiO / DiL, PGB-LNP@DiO / DiL, QE, cRGD-PGB-LNP@DiO / DiL, and cRGD-PGB-LNP@DiO / DiL, QE for 1, 2, and 4 h: (A) Laser confocal microscopy image; (B) Average fluorescence intensity of A549 cells after incubation with different formulations by flow cytometry; (C) Comparison of cell uptake of A549 cells after incubation with different formulations by flow cytometry for 4 h. (n=3) <0.05, <0.01, <0.001; Figure 28 Cell inhibition rates of HN, HN+BR+QE, B-LNP@HN, PGB-LNP@HN,QE, and cRGD-PGB-LNP@HN,QE after incubation with A549 cells for 24 h, 48 h, and 72 h, respectively; (n=3) <0.05, <0.01, <0.001; Figure 29 To observe the changes in the size and specific volume of 3D tumor spheres after 15 days of incubation in different drug administration groups (control group, HN, QE, BR, HN+BR, HN+BR+QE, B-LNP@HN, PGB-LNP@HN,QE, cRGD-PGB-LNP@HN,QE) under an inverted fluorescence microscope: LLC (A) and A549 (B) (n=3); Figure 30 After 12 h of co-incubation with DiO, DiL, B-LNP@DiO / DiL, PGB-LNP@DiO / DiL, PGB-LNP@DiO / DiL, QE, cRGD-PGB-LNP@DiO / DiL, and cRGD-PGB-LNP@DiO / DiL, QE, the penetration ability of different formulations was photographed using laser confocal microscopy (n=3). Figure 31 To detect the apoptosis of A549 cells by different formulation groups using flow cytometry (n=3); Figure 32A549 cells were scratched and incubated with PGB-LNP@HN,QE, PGB-LNP@HN, cRGD-PGB-LNP@HN,QE, and cRGD-PGB-LNP@HN groups at 5 μg / mL for 0, 12, and 24 h, respectively. Cell migration was observed under an inverted fluorescence microscope (A) and the scratch area was compared (B). (n=3) <0.01, <0.001; Figure 33 The migration status of A549 cells after 24 h of treatment with different groups (control group; B-LNP@HN; PGB-LNP@HN; PGB-LNP@HN,QE; cRGD-PGB-LNP@HN; cRGD-PGB-LNP@HN,QE) under an inverted fluorescence microscope (n=3). Figure 34 Mitochondrial targeting of B-LNP@COU6, PGB-LNP@COU6, PGB-LNP@COU6,QE, cRGD-PGB-LNP@COU6,QE and A549 were observed after 1 sh, 2 h and 4 h of laser confocal microscopy (n=3). Figure 35 The mitochondrial membrane potential changes in A549 cells 12 h after administration of B-LNP@HN, PGB-LNP@HN, PGB-LNP@HN,QE, cRGD-PGB-LNP@HN, and cRGD-PGB-LNP@HN,QE containing HN 2.5, 10, and 15 μg / mL, respectively: (A) Mitochondrial membrane potential changes under laser confocal microscopy; (B) Green / red ratio of mitochondrial membrane potential in different formulation groups; (n=3) <0.01, <0.001; Figure 36 The ROS generation of the Control group, HN+BR+QE group, B-LNP@HN group, PGB-LNP@HN,QE group, and cRGD-PGB-LNP@HN,QE group after incubation with A549 cells for 24 h was captured by laser confocal microscopy (A), and the fluorescence intensity of the different drug-treated groups after incubation with A549 cells was compared by flow cytometry (B) (×530000) (n=3). Figure 37To observe the distribution and targeting of B-LNP@DiR; PGB-LNP@DiR; PGB-LNP@DiR,QE; cRGD-PGB-LNP@DiR; cRGD-PGB-LNP@DiR,QE in vivo over 120 h using in vivo imaging: (A) In vivo distribution of the formulations over 120 h; (B) Drug accumulation in major organs and tumors after 120 h; (C) Fluorescence intensity bar chart of the formulations in vivo over 120 h; (n=3) ns: no significant difference. <0.05, <0.01; Figure 38 The changes in tumor size in mice in the Control, HN+BR+QE, PGB-LNP@HN,QE, and cRGF-PGB-LNP@HN,QE groups after treatment for 3, 5, 7, 9, 11, 13, and 15 days are shown in Figure (A). A fluorescence intensity statistical graph on day 15 is also shown (B), and a comparison of tumor size after treatment is shown (C). (n=6) ns: No significant difference. <0.01, <0.001.

[0012] Figure 39 This is a graph showing the changes in mouse body weight during treatment; (n=6) ns: no significant difference. <0.01, <0.001; Figure 40 The results of H&E staining of heart, liver, spleen, and kidney tissues and organs of C57BL / 6 mice in each experimental group; Figure 41 Tuneel staining images and analysis results of tumor tissue sections from mice after administration of Control, HN+BR+QE, PGB-LNP@HN,QE, and cRGF-PGB-LNP@HN,QE; (n=6) <0.05, <0.005, ns: no significant difference; Figure 42 The results of PD-L1 immunohistochemistry and statistically significant differences in tumor tissues were presented; (n=6) <0.05, <0.005, ns: no significant difference. Detailed Implementation

[0013] This invention provides a multi-component nano-delivery system for traditional Chinese medicine, comprising HN-loaded BR lipid nanoparticles, a gelatin layer covering the surface of the HN-loaded BR lipid nanoparticles, and QE encapsulated in the gelatin layer; the HN-loaded BR lipid nanoparticles comprise BR lipid nanoparticles and HN encapsulated in the BR lipid nanoparticles. The BR lipid nanoparticles are formed from a BR-disulfide bond-caprylic / capric triglyceride compound; the structural formula of the BR-disulfide bond-caprylic / capric triglyceride compound is shown in Formula I. Formula I; The gelatin in the gelatin layer is type A gelatin (AG).

[0014] In this invention, the gelatin layer is preferably further modified with polyethylene glycol; the molecular weight of the polyethylene glycol is 2 kDa; by modifying the gelatin layer with polyethylene glycol, this invention can delay the release of drugs and improve the sustained-release properties of the drug delivery system.

[0015] In this invention, the polyethylene glycol is preferably further connected with cRGD; the targeting effect of cRGD combined with the tumor microenvironment regulation effect of QE enhances tumor penetration, enabling the drug delivery system to target deep into the tumor, and combined with the mitochondrial targeting function of BR to exert the mitochondrial-dependent anti-lung cancer effect of HN, significantly improving the synergistic anti-lung cancer effect of HN, BR and QE.

[0016] In a specific embodiment of the present invention, the unmodified polyethylene glycol multi-component traditional Chinese medicine nano-delivery system is denoted as G-BRB-LNP@HN,QE; the polyethylene glycol-modified polyethylene glycol multi-component traditional Chinese medicine nano-delivery system is denoted as PGB-LNP@HN,QE; and the polyethylene glycol multi-component traditional Chinese medicine nano-delivery system grafted with cGRD is denoted as cRGD-PGB-LNP@HN,QE.

[0017] The present invention also provides a method for preparing the multi-component nano-drug delivery system of traditional Chinese medicine described above.

[0018] First, the preparation method of the BR-disulfide bond-caprylic / capric triglyceride compound will be explained.

[0019] In this invention, the preferred method for preparing the BR-disulfide-caprylic / capric triglyceride compound (BR-SS-GTCC) includes: mixing 3,3'-dithiodipropionic acid (DTPA), oxalyl chloride, and an organic solvent to carry out a first reaction to obtain a first intermediate; mixing the first intermediate, caprylic / capric triglyceride (GTCC), triethylamine, and an organic solvent to carry out a second reaction to obtain a second intermediate; and mixing the second intermediate, an organic solvent, a catalyst, and BR to carry out a third reaction to obtain the BR-disulfide-caprylic / capric triglyceride compound.

[0020] In this invention, the molar ratio of 3,3'-dithiodipropionic acid and oxaloyl chloride is preferably 1:1~2, more preferably 1:1.5; the organic solvent is preferably anhydrous tetrahydrofuran; the temperature of the first reaction is preferably room temperature, and the time is preferably 3~5h, specifically 4h; in a specific embodiment of this invention, it is preferable to first dissolve 3,3'-dithiodipropionic acid in anhydrous tetrahydrofuran, then add oxaloyl chloride dropwise, and after the addition is complete, carry out the first reaction at room temperature; after the reaction is completed, remove the solvent under reduced pressure to obtain a white powdery product, which is the first intermediate.

[0021] In this invention, the molar ratio of 3,3'-dithiodipropionic acid and caprylic / capric glyceride is preferably 2-2.5:1, more preferably 2:1; the organic solvent used in the second reaction is preferably dichloromethane; the temperature of the second reaction is preferably 40-50°C, specifically 45°C, and the second reaction is preferably carried out under reflux conditions; the time of the second reaction is preferably 4-6 hours, specifically 5 hours; in a specific embodiment of this invention, the first intermediate is preferably placed in a reaction vessel, dichloromethane and triethylamine are added, and the mixture is sonicated until the first intermediate dissolves, and then caprylic / capric glyceride is added to carry out the second reaction; after the second reaction is completed, the resulting reaction solution is preferably separated and purified, and the solvent is removed under reduced pressure to obtain the second intermediate; this invention preferably uses column chromatography to separate and purify the reaction solution, and the column chromatography developing solvent system used for separation and purification is a dichloromethane-methanol system, wherein the volume ratio of dichloromethane to methanol in the dichloromethane-methanol system is preferably 50:1. In this invention, the second intermediate is denoted as GTCC-SS-COOH.

[0022] In this invention, the preferred method for preparing BR includes: activating BR by heating to obtain crude BR product, and purifying the crude BR product by column chromatography to obtain BR; the preferred temperature for the heating activation is 190°C, and the preferred time is 30 min; the preferred column chromatography solvent system for the column chromatography purification is a dichloromethane-methanol system, and the preferred volume ratio of dichloromethane to methanol in the dichloromethane-methanol system is 25:1.

[0023] In this invention, the molar ratio of the second intermediate to BR is preferably 5-6:1, more preferably 5:1; the catalyst is preferably DMAP (4-dimethylaminopyridine) and EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide); the molar ratio of BR, DMAP, and EDC is preferably 1:0.1:1.5; the organic solvent used in the third reaction is preferably anhydrous N,N-dimethylformamide; the temperature of the third reaction is preferably 40-50°C, specifically 45°C; the time of the third reaction is preferably 45-50 h, specifically 48 h; in a specific embodiment of this invention, it is preferable to first dissolve the second intermediate in an organic solvent, then add a catalyst to activate it for 1 h, and then add BR to carry out the third reaction. After the third reaction is completed, the reaction system is preferably extracted with dichloromethane and distilled water to separate the organic phase. The obtained organic phase is then extracted again with saturated brine and dehydrated with anhydrous sodium sulfate. The organic phase is then evaporated to dryness to obtain a crude product. The crude product is purified by column chromatography to obtain the BR-disulfide bond-caprylic / capric glyceride compound. The separation method of the column chromatography purification is preferably gradient separation. The gradient separation is preferably carried out by sequentially using a column-flushing and developing solvent system with a volume ratio of dichloromethane to methanol of 25:1, 25:2, and 25:4.

[0024] The preparation method of G-BRB-LNP@HN,QE is described below.

[0025] The preparation method of the G-BRB-LNP@HN,QE includes the following steps: HN, BR-caprylic / capric triglyceride compound and dimethyl sulfoxide were mixed to obtain an organic phase; The organic phase was added to the aqueous phase and heated and stirred, then cooled to obtain a dispersion of HN-loaded BR lipid nanoparticles; the aqueous phase was a poloxamer aqueous solution. QE was dispersed in an aqueous solution of type A gelatin to obtain a QE-gelatin solution; The HN-loaded BR lipid nanoparticle dispersion was added to the QE-gelatin solution and then heated and stirred to solidify, thereby obtaining the G-BRB-LNP@HN,QE.

[0026] In this invention, the preferred mass ratio of HN to BR-disulfide-caprylic / capric triglyceride compound is 1:4 to 6, specifically 1:5; the preferred amount of HN and dimethyl sulfoxide is 1 mg:1 to 3 mL, specifically 1 mg:1.5 mL; preferably, HN and BR-caprylic / capric triglyceride compound are added to dimethyl sulfoxide and heated to dissolve at 60°C to obtain an organic phase.

[0027] In this invention, the poloxamer is preferably poloxamer 188; the concentration of the poloxamer aqueous solution is preferably 2-4 wt%, more preferably 3 wt%; the volume ratio of the organic phase to the aqueous phase is preferably 1:5-10, more preferably 1:8; the temperature at which the organic phase is added to the aqueous phase for heating and stirring is preferably 60-70°C, and the time is preferably 15-30 min; in this invention, the organic phase is preferably rapidly injected into the aqueous phase under stirring, and then stirred at the above temperature for solvent dispersion; after the solvent dispersion is completed, the resulting liquid is preferably naturally cooled to room temperature, and then filtered through a 0.22 μm microporous membrane to obtain a dispersion of HN-loaded BR lipid nanoparticles (B-LNP@HN).

[0028] In this invention, the preferred method for preparing the type A gelatin aqueous solution includes: swelling type A gelatin in water, followed by water bath heating to obtain the type A gelatin aqueous solution; the swelling time is preferably 2 hours, and the water bath heating temperature is preferably 60°C. The concentration of the type A gelatin aqueous solution is preferably 0.5~1.5% w / v, more preferably 1% w / v; the mass ratio of QE to HN is preferably 1~4:1, specifically 1:1, 2:1, 3:1 or 4:1, most preferably 3:1. The preferred amounts of QE and type A gelatin aqueous solution are 3 mg: 20 mL.

[0029] In this invention, the volume ratio of the HN-loaded BR lipid nanoparticle dispersion to the type A gelatin aqueous solution is preferably 1:0.5~1.5, more preferably 1:1; the HN-loaded BR lipid nanoparticle dispersion is preferably added to the QE-gelatin solution by dropwise addition, preferably under stirring conditions, and the dropwise addition temperature is preferably 60~70℃; after the dropwise addition is completed, the resulting solution is preferably stirred at 800 rpm for 30 min, and then cured; the curing is preferably carried out under ice bath conditions, and the curing time is preferably 30 min.

[0030] The preparation method of the PEG-modified multi-component nano-delivery system for traditional Chinese medicine (PGB-LNP@HN, QE) is described below.

[0031] In this invention, the preparation method of PGB-LNP@HN,QE includes the following steps: mixing mPEG-COOH, buffer solution, and crosslinking agent to react and obtain a reaction solution; mixing the reaction solution with an aqueous solution of type A gelatin and incubating to obtain an aqueous solution of type A gelatin (PG) modified with polyethylene glycol; replacing the aqueous solution of type A gelatin modified with polyethylene glycol with the aqueous solution of type A gelatin, and preparing according to the preparation method of G-BRB-LNP@HN,QE to obtain a multi-component nano-delivery system of traditional Chinese medicine modified with polyethylene glycol on a gelatin layer. In this invention, the molecular weight of the mPEG-COOH is preferably 2 kDa; the buffer solution is preferably MES buffer, and the pH value of the MES buffer solution is preferably 5-6; the volume ratio of the mPEG-COOH to the buffer solution is preferably 3 mg: 1-5 mL, specifically 3 mg: 4 mL; the crosslinking agent is preferably EDC and NHS (N-hydroxysuccinimide), and the mass ratio of the mPEG-COOH, EDC and NHS is preferably 1-3:1-3:1-3, more preferably 1:1:1; the reaction time is preferably 3-5 h, more preferably 4 h, and the reaction can be carried out at room temperature.

[0032] In this invention, the method for preparing the concentration of the type A gelatin aqueous solution is the same as described above, and will not be repeated here. The preferred ratio of mPEG-COOH to type A gelatin aqueous solution is 3 mg: 1-4 mL, specifically 3 mg: 4 mL. The preferred incubation time is 15-20 h, more preferably 16 h. The incubation can be carried out at room temperature. After incubation, this invention preferably dialyzes the obtained incubation solution at room temperature to remove unreacted crosslinking agents. The preferred molecular weight cutoff for the dialyzed solution is 2 kDa.

[0033] After obtaining the PG aqueous solution, replace the type A gelatin aqueous solution with the PG aqueous solution and prepare according to the preparation method of G-BRB-LNP@HN,QE, which will not be elaborated here.

[0034] The preparation method of cRGD-PGB-LNP@HN,QE is described below: In this invention, the preparation method of cRGD-PGB-LNP@HN,QE includes the following steps: cRGD-PEG-COOH, buffer solution, and crosslinking agent were mixed and reacted to obtain a reaction solution. The reaction solution was then mixed with an aqueous solution of type A gelatin and incubated to obtain an aqueous solution of cRGD-polyethylene glycol modified gelatin (cRGD-PEG-AG). The aqueous solution of type A gelatin was replaced with the aqueous solution of cRGD-PEG-AG and prepared according to the preparation method of G-BRB-LNP@HN,QE to obtain cRGD-PGB-LNP@HN,QE.

[0035] In this invention, the preparation method of the cRGD-polyethylene glycol modified gelatin aqueous solution is the same as that of the polyethylene glycol modified gelatin aqueous solution, except that mPEG is replaced with cRGD-PEG-COOH.

[0036] The present invention also provides the application of the multi-component nano-delivery system of traditional Chinese medicine described in the above-described scheme or the multi-component nano-delivery system of traditional Chinese medicine prepared by the preparation method described in the above-described scheme in the preparation of anti-lung cancer drugs.

[0037] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0038] Example 1: Synthesis and Characterization of BR-SS-GTCC 1. Synthesis of berberine In a vacuum environment, BR was heated to 190°C and activated for 30 min to obtain berberine ( Figure 1 The crude product was then purified by column chromatography, with a column eluent system of Vdichloromethane:Vmethanol = 25:1. After purification, the product was analyzed by mass spectrometry (MS / MS). Figure 2 ) and NMR ( Figure 3 Its structure was confirmed.

[0039] 2. Synthesis of BR-SS-GTCC 3,3'-dithiodipropionic acid (DTPA, 2 mmol) was dissolved in anhydrous tetrahydrofuran solution at 0 °C and stirred until dissolved. Oxaloyl chloride (3 mmol) was then added dropwise at a uniform rate, and the mixture was immediately covered with a balloon after the addition was complete. The system was allowed to react at room temperature for 4 h until DTPA was completely reacted. After the reaction was complete, the solvent was removed by rotary evaporation under reduced pressure to obtain a white powdery intermediate. The prepared intermediate was placed in a round-bottom flask, and 200 mL of dichloromethane and 5 mL of triethylamine were added. The mixture was dissolved by sonication, and glyceryl caprylate (GTCC, 1 mmol) was added. The mixture was refluxed at 45 °C for five hours. Figure 4 After the reaction was completed, GTCC-SS-COOH was separated and purified by column chromatography. At room temperature, GTCC-SS-COOH (5 mmol) was dissolved in anhydrous N,N-dimethylformamide, and DMAP (0.1 mmol) and EDC (1.5 mmol) were added as catalysts. The mixture was activated for 1 h, and then berberine (1 mmol) was added. The reaction system was heated to 45 °C and refluxed for 48 h until the berberine was completely reacted. Figure 5 After the reaction was complete, the reaction system was quenched with equal volumes of dichloromethane and distilled water to separate the organic phase, which was then dehydrated sequentially with saturated brine and anhydrous sodium sulfate. The organic phase was evaporated to dryness, and the crude product was purified by column chromatography using gradient separation. The purified product was then analyzed by mass spectrometry (MS / MS). Figure 6 Its structure was confirmed by NMR. Figure 7 ).

[0040] Example 2: Preparation and Characterization of P-GB-LNP@HN,QE 1. Preparation and characterization of PGB-LNP@HN,QE 1.1 Synthesis of PG 3 mg of mPEG-COOH was weighed and placed in a vial. 3 mg of EDC and 3 mg of NHS were added, and the mixture was dissolved in 4 mL of MES buffer to activate the carboxyl groups in mPEG-COOH. After 4 h, 4 mL of 1% (w / w) type A gelatin aqueous solution was added, and the reaction was allowed to proceed for 16 h. The resulting solution was then placed in a 2500 Da dialysis bag and dialyzed for 2 days to obtain PG. The thin-layer chromatography results in Figure [Figure number missing] show the successful synthesis of PG (…). Figure 8 The grafting rate was calculated to be 70.19% ± 1.5% by ninhydrin colorimetric method.

[0041] 1.2 Validation of the PGB-LNP@HN,QE structure Free AG, mPEG, AG+mPEG, and PGB-LNP@HN,QE were mixed with KBer particles and pressed into a physical mixture, then subjected to compression at 4000~400 cm⁻¹. -1 Infrared scanning is performed within the specified range. For example... Figure 9 As shown: Compared with mPEG, PGB-LNP@HN, QE is at 1924 cm⁻¹ -1 and 1654cm -1 There are additional absorption bands, which are due to the -CH2- stretching vibration of gelatin and the amide bond, which usually produce a characteristic peak at 1650-1750 cm⁻¹. -1 The wavelength range indicates a successful gelatin coating. Compared to AG, PGB-LNP@HN, QE at 2886 cm⁻¹... -1 1539cm -1 and 952cm -1 There is an additional absorption zone at 1539 cm. -1 The tensile vibration of PG's -NH- shows the characteristic peak of PG at 1343 cm⁻¹. -1 The vibrational peak of COC at this point indicates the characteristic peak of PEG, signifying successful MPEG coating. Although the -CH2- peak of mPEG is 3328 cm⁻¹, the peak of mPEG+AG is also present. -1 With PGB-LNP@HN,QE 3328 cm -1 While the peak values ​​overlapped, the -CH2- peak in the FTIR of PGB-LNP@HN,QE was significantly larger than that of mPEG and mPEG+gelatin. Nevertheless, the presence of the characteristic -COC- peak was sufficient to confirm the successful coupling of PEG and gelatin, resulting in the successful preparation of PEG-modified gelatin nanoparticles.

[0042] 1.3 Preparation of PGB-LNP@HN,QE Based on single-factor investigation and star-point design-response surface methodology experiments, the optimal formulation process is determined to be: B-LNP@HN was prepared by solvent diffusion: 1 mg HN and 5 mg BR-SS-GTCC were accurately weighed and dissolved in 3 mL DMSO. The solution was heated in a 70℃ constant temperature water bath until fully dissolved, forming the organic phase. 24 mL of 3.3wt% P188 solution was used as the aqueous phase. The organic phase was rapidly injected into the aqueous phase under high-speed stirring, and the temperature was maintained at (70±2)℃. The mixture was stirred for 5 min, allowed to cool naturally to room temperature, and then filtered through a 0.22 μm microporous membrane to obtain B-LNP@HN.

[0043] 3.0 mg of QE was uniformly dispersed in 20 mL of 1% PG aqueous solution, heated and stirred in a water bath at 60 °C, and then B-LNP@HN solution was added dropwise to the above solution every 3 seconds. The mixture was stirred at 800 rpm for 30 min and then solidified in an ice bath for 30 min to obtain PGB-LNP@HN,QE.

[0044] 1.4 Characterization of PGB-LNP@HN,QE The appearance, morphology, particle size, potential, PDI, encapsulation efficiency, and stability of PGB-LNP@HN,QE were characterized, and the results are as follows: Figures 10~13 As shown. Test results show that PGB-LNP@HN,QE is roughly circular and relatively evenly distributed (e.g., Figure 11 The particle size was 151.2 ± 20.4 nm, the PDI was 0.231 ± 0.037, and the Zeta potential was 9.21 ± 2.8 mV (e.g. Figure 12 The encapsulation efficiencies of HN, BR, and QE in PGB-LNP@HN, prepared according to the optimal formulation process were 82.88±0.25%, 84.70±0.43%, and 84.33±0.45%, respectively.

[0045] Stability: The absorbance of PGB-LNP@HN,QE after incubation with FBS for 48 h showed no significant change. In terms of storage stability, the encapsulation efficiency, potential, particle size, and PDI of HN, BR, and QE showed no significant changes, indicating that PGB-LNP@HN,QE has good serum stability and storage stability (e.g., ...). Figure 13 ).

[0046] 1.5 PGB-LNP@HN,QE hemolysis test Blood was collected from the marginal ear vein of healthy rabbits to prepare a 2% erythrocyte suspension. 0.2 mL of this suspension was incubated at 37 °C for 2 h with purified water, PBS, and a series of different concentrations of reagents. The suspension was then collected, centrifuged at 2000 r / min for 6 min, and photographed. Figure 14 As shown, the hemolysis rates of B-LNP@HN, GB-LNP@HN,QE (preparation method and PGB-LNP@HN,QE, only the PG aqueous solution was replaced with type A gelatin aqueous solution) and PGB-LNP@HN,QE were all less than 5% in the concentration range of 50% to 1.56% (v / v), indicating that B-LNP@HN, GB-LNP@HN,QE and PGB-LNP@HN,QE all have good blood compatibility.

[0047] 1.6 Characterization of PGB-LNP@HN,QE in vitro MMP-2 response A 5 ng / mL aqueous solution of MMP-2 was prepared and mixed with PGB-LNP@HN,QE solution at a volume ratio of 1:1. After incubation for 24 h and 48 h, particle size and potential were measured, and the morphology of the formulation was analyzed by transmission electron microscopy. BSA was used as a control group instead of MMP-2. Gelatin in PGB-LNP@HN,QE underwent a specific reaction with MMP-2. From 24 h to 48 h, the particle size, potential, and PDI changed from 292.9±173.5 nm, 7.34±2.3 mV, and 0.695±0.037 to 587.0±520.8 nm, 8.87±1.4 mV, and 1.130±0.045, respectively. The results are as follows: Figure 15 , 16 The gelatin on PGB-LNP@HN,QE did not respond to BSA degradation, as shown in the results. Figure 17 After 48 h, the particle size was 207.1 ± 10.2 nm, PDI was 0.265 ± 0.031, and Zeta potential was 8.19 ± 1.7 mV. Transmission electron microscopy showed that the morphology of PGB-LNP@HN,QE and MMP-2 changed after incubation. Figure 18 As shown in Figures A and B, the morphology did not change after incubation with BSA for 48 hours, as shown in Figures A and B. Figure 18 As shown in C.

[0048] 1.7 Evaluation of in vitro release of PGB-LNP@HN,QE PGB-LNP@HN and QE were simultaneously added to PBS containing / without 2.50 μg / mL MMP-2 + 10.00 mM GSH; BR, QE, and HN were placed in PBS as control groups, respectively. The mixture was incubated at 100 r / min for 72 h in a constant temperature shaker at 37℃, and the cumulative release rates of HN, QE, and BR were tested. Results are as follows...Figure 19 As shown, the release rates of HN, BR, and QE in the release medium without MMP-2+GSH were 21.63±0.31%, 23.13±0.25%, and 23.83±0.25%, respectively; while in the release medium containing MMP-2+GSH, the release rates of HN, BR, and QE were 59.4±0.62%, 65.1±0.3%, and 74.1±0.36%, respectively, indicating that the nano-drug delivery system responds to the release of MMP-2 and GSH.

[0049] Example 3: Evaluation of the program release characteristics of P-GB-LNP@HN,QE This embodiment studies the responsiveness of PGB-LNP@HN,QE to MMP-2 and evaluates the programmed release characteristics of this formulation in response to MMP-2 and GSH in the extracellular and intracellular environments, respectively.

[0050] 1 Evaluation of the intracellular and extracellular programmed release characteristics of PGB-LNP@HN,QE A549 cells were selected as the research object, and solid lipid nanoparticles SLN@DiO,DiL, B-LNP@DiO,DiL, B-LNP, and PG-DiO,DiL-B-LNP were prepared by referring to the preparation method of nanodrug delivery system. Specifically, HN was replaced with 0.5 mg DiO and 0.5 mg DiI, and BR-SS-GTCC was replaced with GTCC. The remaining conditions were the same as those for B-LNP@HN, yielding SLN@DiO,DiI. HN was replaced with 0.5 mg DiO and 0.5 mg DiI. The remaining conditions were the same as those for B-LNP@HN, yielding B-LNP@DiO,DiL. Without DiO and DiL, the same procedure was performed to obtain BRB-LNP. 3.0 mg QE was replaced with 1.5 mg DiO and 1.5 mg DiI. The remaining conditions were the same as those for PGB-LNP@HN,QE, yielding PG-DiO,DiL-B-LNP.

[0051] (1) Evaluation of intracellular and extracellular programmed release characteristics Intracellular release characteristics: A549 cells were incubated for 2 h with media containing DiL&DiO (30 μg / mL), SLN@DiO,DiL, B-LNP@DiO,DiL, PG-DiO,DiL-B-LNP, and PG-DiO,DiL-B-LNP + MMP-2 (2.5 μg / mL), respectively. The culture medium was then replaced with normal medium and cultured for 1, 3, and 12 h, and the cells were examined under a laser confocal microscope. FRET (DiO&DiL) was used to observe the conditions under which the drug delivery systems SLN, B-LNP, and PGB-LNP released drugs into the cells. Figure 20 As shown, after incubating A549 cells with each group for 2 hours, green fluorescence of DiO and red fluorescence of DiL were observed within the cells. This indicates that most DiO and DiL are internalized and remain tightly encapsulated within LNP and SLN. After 2 hours of incubation, the intracellular fluorescence intensity was PG-DiO,DiL-B-LNP > PG-DiO,DiL-B-LNP + MMP-2. This suggests a specific reaction between extracellular MMP-2 and the gelatin layer, releasing fluorescent pairs. Only a small portion of PG-DiO,DiL-B-LNP is taken up by the cells, resulting in significantly stronger fluorescence in cells without MMP-2 compared to those containing MMP-2. Figure 21 As shown, for SLN@DiO,DiL, the fluorescence intensity remained strong in A549 cells for 12 hours, indicating that most DiO & DiL were not significantly released. The fluorescence intensity of B-LNP@DiO,DiL was: 1h > 3h > 12h, indicating that BR-SS-GTCC in B-LNP@DiO,DiL undergoes a reduction reaction with GSH in tumor cells, leading to disulfide bond breakage and rapid release of the fluorescent pair and BR in tumor cells, resulting in decreased fluorescence. These results demonstrate the specific reaction between the gelatin layer and MMP-2 on PGB-LNP@HN,QE outside tumor cells, releasing the drug into the tumor cell stroma. Simultaneously, it demonstrates that BR-SS-GTCC in B-LNP@HN is GSH-reduc sensitive in tumor cells, causing disulfide bond breakage and releasing HN. This indicates that PGB-LNP@HN,QE can achieve programmed release.

[0052] (2) Extracellular release characteristics: The incubation groups were the same as those for intracellular incubation. Incubation was performed for 0.5, 1, 2, 3, and 12 hours, followed by laser confocal microscopy measurements. The release of extracellular PGB-LNP@HN,QE was observed using DiO & DiL. A549 cells were incubated with DiO & DiL, PG-DiO,DiL-B-LNP, and PG-DiO,DiL-B-LNP+MMP-2 for different times (0.5, 1, 2, 3, 12 hours). Figure 22As shown, the fluorescence of each group increased with increasing incubation time. At each time point, the fluorescence of the formulation without MMP-2 was greater than that of the formulation containing MMP-2, indicating that most of PG-DiO, DiL-B-LNP was taken up by cells and not significantly released. However, when MMP-2 was present extracellularly, the gelatin on PG-DiO, DiL-B-LNP reacted specifically with MMP-2, causing some of the gelatin layer of PG-DiO, DiL-B-LNP to rupture and release the fluorescent pair and B-LNP extracellularly, thus reducing the amount of PG-DiO, DiL-B-LNP taken up by cells. This demonstrates the specific reaction between the gelatin layer on PGB-LNP@HN,QE and MMP-2, enabling the release of the drug extracellularly, indicating that PGB-LNP@HN,QE can achieve QE-programmed release extracellularly.

[0053] Example 4: Preparation and characterization of cRGD-PGB-LNP@HN,QE 1. The preparation process is the same as that of PGB-LNP@HN,QE in Example 2, except that the PG aqueous solution is replaced with cRGD-PEG-AG.

[0054] 2. Confirmation of the cRGD-PGB-LNP@HN,QE structure AG, cRGD-PEG-COOH, cRGD-PEG-AG, and cRGD-PGB-LNP@HN,QE were determined using Fourier transform spectroscopy. Figure 23 From D, it can be seen that cRGD-PEG-COOH is at 2890 cm⁻¹ -1 A -CH2- peak appears at 1110 cm⁻¹. -1 Absorption occurs at these two locations, and AG also shows absorption at these two locations. In the cRGD-PGB-LNP@HN,QE group, absorption at 2890 cm⁻¹ is observed. -1 1110 cm -1 The significantly increased absorption at this point indicates that cRGD-PGB-LNP@HN,QE contains the cRGD-PEG-COOH and AG components; while the absorption at 1680 cm⁻¹ in cRGD-PGB-LNP@HN,QE is significantly increased. -1 The peak at 1550 cm⁻¹ represents the C=O stretching vibration of the amide bond. -1 The NH bending vibration peak in the amide bond confirms the presence of an amide bond in the formulation, further demonstrating that cRGD-PEG-COOH is linked to AG via an amide bond.

[0055] 3. Characterization of cRGD-PGB-LNP@HN,QE Similar to the characterization of PGB-LNP@HN,QE, the appearance, morphology, grafting rate, potential, particle size, PDI, encapsulation efficiency, stability, and blood compatibility of cRGD-PGB-LNP@HN,QE were characterized.

[0056] The results showed that cRGD-PGB-LNP@HN,QE appeared as a pale yellow, clear solution. Figure 23 (A), appears as a nearly circular shape under a transmission electron microscope. Figure 23 (Type B), evenly distributed. The grafting rate of cRGD-PEG-COOH with 1% type A gelatin was 62.44%. Figure 23 The particles C and E have a size of 194.43±3.32 nm, a PDI of 0.117±0.034, and a zeta potential of 2.88±0.16 mV. Encapsulation efficiency measurements were as follows: HN 85.89±0.083%, BR 83.68±0.30%, and QE 81.44±0.20%. In stability tests, cRGD-PGB-LNP@HN and QE were stable during co-incubation with serum and storage, with particle sizes between 190 and 210 nm, PDI between 0.05 and 0.15, and zeta potential between 2.5 and 3.5 mV. Figure 24 In the blood compatibility test, no hemolysis occurred in any of the formulations, and the results were as follows. Figure 25 .

[0057] 4. In vitro release study The release medium was the same as in the in vitro release experiment and PGB-LNP@HN,QE, but the release time was extended to 120 h.

[0058] Free QE, HN, and BR were rapidly released under PBS conditions, with release rates of 92.47±3.93%, 97.73±0.28%, and 95.56±0.87% respectively at 36 h. In PBS, cRGD-PGB-LNP@HN,QE released 46.58±4.87%, 32.88±2.37%, and 26.98±0.46% of QE, HN, and BR, respectively. In a release medium containing MMP-2, the release of QE, HN, and BR from cRGD-PGB-LNP@HN,QE increased, reaching 78.05±3.71%, 50.33±1.65%, and 50.74±1.42%, respectively. In the release medium containing GSH, the cumulative release amounts of HN and BR were 37.27±1.65% and 40.67±1.84%, respectively. However, in the release medium containing MMP-2+GSH, the cumulative release amounts of HN and BR were 76.43±2.11% and 67.79±2.29%, respectively. These cumulative release amounts were significantly higher than those in the PBS release medium and the PBS release medium containing GSH. (See attached figures). Figure 26 The results showed that cRGD-PGB-LNP@HN,QE could respond to the release of MMP-2 and GSH, and significantly delayed the drug release time.

[0059] 1.5 Evaluation of the in vitro anti-lung cancer effect of cRGD-PGB-LNP@HN,QE 1.5.1 Cellular uptake experiment (1) Qualitative experiment on cell uptake Using DiO / DiL as a fluorescent probe, the uptake of different nanodelivery systems (DiO / DiL, B-LNP@DiO / DiL, PGB-LNP@DiO / DiL, PGB-LNP@DiO / DiL, QE, cRGD-PGB-LNP@DiO / DiL, cRGD-PGB-LNP@DiO / DiL, QE, prepared using the same method as before, except that HN was replaced with DiO / DiL) by A549 cells at different time points (1, 2, and 4 h). Figure 27As shown in Figure A, the fluorescence intensity of B-LNP@DiO / DiL was significantly stronger than that of PGB-LNP@DiO / DiL and cRGD-PGB-LNP@DiO / DiL. Among them, the fluorescence intensity of PGB-LNP@DiO / DiL,QE and cRGD-PGB-LNP@DiO / DiL,QE was significantly stronger than that of PGB-LNP@DiO / DiL and cRGD-PGB-LNP@DiO / DiL without QE coating, and was close to that of B-LNP@DiO / DiL. This indicates that incubation with PGB-LNP@DiO / DiL,QE and cRGD-PGB-LNP@DiO / DiL,QE significantly increased the uptake of the drug by A549 cells.

[0060] (2) Quantitative experiment on cell uptake Flow cytometry results were consistent with those obtained using laser confocal microscopy. B-LNP@HN exhibited the strongest fluorescence intensity, with average fluorescence intensities of DiO and DiL of 2081.00±4.36 and 2103.67±21.22 after 4 h of drug uptake. PGB-LNP@DiO / DiL,QE and cRGD-PGB-LNP@DiO / DiL,QE showed weaker average fluorescence intensities than B-LNP@HN. After 4 h of drug uptake, the average fluorescence intensities of DiO were 1900.33±22.72 and 1940.67±23.07, respectively, and those of DiL were 1910.33±23.46 and 1941.00±7.55, respectively. The fluorescence intensities of PGB-LNP@DiO / DiL and cRGD-PGB-LNP@DiO / DiL were significantly weaker than B-LNP@HN, indicating a lower fluorescence intensity after 4 h of drug uptake. The mean fluorescence intensities of DiO after h were 178.33±1.53 and 581.67±13.61, respectively, while those of DiL were 269.67±6.43 and 627.67±1.15, respectively. With the modification of QE with PEG and cRGD, the fluorescence intensity decreased significantly, further indicating that the formulation containing QE significantly increased drug uptake by A549 cells. The results are as follows... Figure 27 B and C in the text.

[0061] 1.5.2 Cytotoxicity assay (1) Determination of drug combination index Based on the ratio of HN, BR, and QE in the formulation, the measured OD values ​​were analyzed using CompuSyn software. The CI values ​​for the two-drug combination (HN:BR = 1:5) and the three-drug combination (HN:BR:QE = 1:5:3.6) were 0.5714 and 0.7930, respectively, at an HN concentration of 15 μg / mL. When CI < 1, the drugs exhibit synergistic effects; when CI = 1, an additive effect; and when CI > 1, an antagonistic effect. This demonstrates that at an HN concentration of 15 μg / mL, both the two-drug and three-drug combinations exhibit synergistic effects.

[0062] (2) Cytotoxicity assay The cytotoxicity of A549 was determined using the CCK-8 assay. The concentration of HN was fixed (15 μg / mL), and the groups were set as HN, HN+BR+QE, B-LNP@HN, PGB-LNP@HN,QE, and cRGD-PGB-LNP@HN,QE. At 48 h and 72 h, the cell inhibition rates of the formulation groups increased significantly. At 72 h, the cell inhibition rates of the free drugs HN and HN+BR+QE were 33.33±1.10% and 47.27±0.28%, respectively. The cell inhibition rates of B-LNP@HN, PGB-LNP@HN,QE, and cRGD-PGB-LNP@HN,QE in the formulation groups were 58.73±1.04%, 63.33±1.00%, and 79.38±0.036%, respectively, indicating that the formulations have certain anti-tumor effects. Furthermore, cRGD-PGB-LNP@HN,QE exhibited the strongest cell inhibition effect. Figure 28 As shown.

[0063] 1.5.3 Study on the inhibition of 3D tumor cell spheroid growth in vitro A549 cell tumor spheroids and LLC cell tumor spheroids were prepared using the suspension method. Drug administration was initiated when the diameter of both A549 and LLC cell tumor spheroids reached approximately 400 μm. Excluding the blank control group, groups were established with an HN concentration of 20 μg / mL: HN, HN+BR, HN+BR+QE, B-LNP@HN, PGB-LNP@HN,QE, and cRGD-PGB-LNP@HN,QE. The drug was administered via fluid exchange every other day, and the morphology and volume changes of the tumor spheroids were observed and photographed using an inverted fluorescence microscope, and the volume was calculated.

[0064] Inhibition of A549 cell 3D tumor spheroid growth: In the control group, after 15 days of growth, the tumor spheroid volume was 147.65±6.618% of the original size. In the HN alone group, the volume after 15 days was 53.77±6.086% of the volume on day one, demonstrating some anti-tumor activity. In the groups containing two drug components: the HN+BR group ultimately had a tumor volume of 74.24±2.821% of the original size; the B-LNP@HN group showed a decreasing cell inhibition rate over time, failing to inhibit tumor spheroid growth after day 11, with a final volume of 129.13±15.9%. 2%; The three drug components group: the free drug HN+BR+QE group showed a certain inhibitory effect on tumor spheroids, with the tumor spheroid volume after 15 days being 42.58±9.335% of that on day 1, and the inhibitory effect was significantly stronger than that of the HN+BR group; while the PGB-LNP@HN,QE group and the cRGD-PGB-LNP@HN,QE group showed the strongest inhibitory effect on A549 cell tumor spheroids, with the volumes after 15 days being 0.5542±0.04487% and 1.565±0.2705% of those on day 1, respectively, showing the strongest inhibitory effect on the growth of A549 cell 3D tumor spheroids. The results are as follows. Figure 29 A in the middle.

[0065] Inhibition of LLC cell 3D tumor spheroid growth: In the blank control group, the tumor spheroid volume after 15 days was 232.28±17.37% of the original volume; In the single free drug groups: the final tumor spheroid volumes in the HN, BR, and QE groups were 165.59±9.825%, 104.97±0.8136%, and 77.65±0.2151% of the original volume on day 1, respectively, indicating that HN and BR had a weak inhibitory effect on LLC cell tumor spheroid growth, while QE had a certain inhibitory effect; In the drug administration groups containing two drug components, the final volume of HN+BR was 116.72±1.565% of the original volume, and B-LNP@HN... The final tumor sphere volume was 82.46±0.5005% of the original volume, showing a stronger inhibitory effect than the HN+BR group. Among the groups containing the three drug components, the final tumor sphere volumes of HN+BR+QE, PGB-LNP@HN,QE, and cRGD-PGB-LNP@HN,QE were 114.69±0.1784%, 60.64±1.137%, and 51.57±0.04550% of the original volume, respectively. PGB-LNP@HN,QE and cRGD-PGB-LNP@HN,QE showed significantly stronger inhibitory effects on the growth of LLC cell 3D tumor spheres than the other drug groups. The results are as follows... Figure 29 B in the middle.

[0066] The above results of the inhibition experiments on the growth of 3D tumor spheres in A549 cells and LLC cells all indicate that PGB-LNP@HN,QE and cRGD-PGB-LNP@HN,QE have strong in vitro antitumor activity.

[0067] 1.5.4 In vitro tumor cell spheroid penetration study of cRGD-PGB-LNP@HN,QE When the A549 cell tumor spheres and LLC cell tumor spheres reached a diameter of approximately 400 μm, the culture medium in the wells was aspirated, and complete culture medium containing free DiO / DiL, B-LNP@DiO / DiL, PGB-LNP@DiO / DiL, PGB-LNP@DiO / DiL, QE, cRGD-PGB-LNP@DiO / DiL, and cRGD-PGB-LNP@DiO / DiL, QE was added and incubated for 12 h. The penetration of the tumor spheres was then observed using laser confocal microscopy.

[0068] Penetration of A549 tumor spheres: PGB-LNP@HN,QE and cRGD-PGB-LNP@HN,QE showed the highest fluorescence and brightness inside the tumor spheres. Comparison revealed that cRGD-PGB-LNP@HN,QE began to show fluorescence at a tumor sphere thickness of 30 μm and remained bright at 150 μm. PGB-LNP@HN,QE exhibited strong fluorescence at thicknesses of 50-110 μm, but its brightness gradually decreased beyond 110 μm. The fluorescence intensity of N and cRGD-PGB-LNP@HN was significantly weaker than that of the QE-encapsulated formulation, but they still exhibited some penetration ability into tumor spheres. The fluorescence intensity of the B-LNP@HN combination was stronger than that of PGB-LNP@HN and cRGD-PGB-LNP@HN, but its penetration ability was weak, with strong fluorescence intensity between 50-90 μm and very weak fluorescence intensity at other thicknesses. DiO and DiL were consistent with B-LNP@HN, only able to penetrate 30-70 μm, with very weak fluorescence at deeper tumor spheres. Figure 30 The left image in the image.

[0069] Penetration of LLC tumor spheres: The B-LNP@DiO / DiL group exhibited fluorescence at 130-220 μm, with fluorescence intensity gradually decreasing as the thickness of the tumor sphere increased. The penetration ability of the PGB-LNP@DiO / DiL group was consistent with that of the B-LNP@DiO / DiL group, but the cRGD-PGB-LNP@DiO / DiL group showed enhanced penetration ability, exhibiting significant fluorescence at 130 μm and maintaining fluorescence at a thickness of 280 μm without weakening, indicating that cRGD modification increased the drug's penetration ability through tumor spheres. In the formulations encapsulated with QE, fluorescence intensity increased significantly, indicating that QE encapsulation increased the formulation's penetration performance through tumor spheres. The PGB-LNP@DiO / DiL and QE groups both exhibited significant fluorescence at 190-280 μm, while the cRGD-PGB-LNP@DiO / DiL and QE groups showed stronger penetration ability, exhibiting stronger fluorescence intensity at 130-280 μm than all other groups. The results are as follows. Figure 30 The right image in the text.

[0070] 1.5.5 Apoptosis Experiment Flow cytometry was used to determine the apoptosis of cells in the following groups after incubation for 24 h: blank control group, PI group, FITC group, FITC / PI group, HN, BR, QE, HN+BR, HN+BR+QE, B-LNP@HN, PGB-LNP@HN, PGB-LNP@HN,QE, cRGD-PGB-LNP@HN, cRGD-PGB-LNP@HN,QE, and A549 cells. The results are as follows: Figure 31In the blank control group, no apoptosis was observed. In the free drug groups, the early apoptosis rates in the QE, BR, and HN groups were 0.41%, 1.16%, and 1.71%, respectively, and the late apoptosis rates were 2.11%, 3.11%, and 3.79%, respectively. In the HN+BR and HN+BR+QE groups, the early apoptosis rates were 3.44% and 4.18%, respectively, and the late apoptosis rates were 12.73% and 19.48%, respectively, indicating that the drug combination can increase cell apoptosis. In the formulation groups, the early apoptosis and late apoptosis rates in the B-LNP@HN group were 4.31% and 20.51%, respectively; the early apoptosis rates in the PGB-LNP@HN and cRGD-PGB-LNP@HN groups were 18.90% and 33.62%, respectively, and the late apoptosis rates were 5.05% and 10.24%, respectively, indicating that the addition of QE increased the apoptosis effect, and the apoptosis effect was enhanced compared with B-LNP@HN. The early apoptosis and late apoptosis rates in the PGB-LNP@HN,QE group and the cRGD-PGB-LNP@HN,QE group were 35.35% and 35.51%, and 11.99% and 13.05%, respectively. The apoptosis effect after cRGD-PEG modification was slightly stronger than that of the PEG-modified formulation, and the apoptosis effect was stronger compared with the PGB-LNP@HN and cRGD-PGB-LNP@HN groups, indicating that QE can increase the apoptosis effect.

[0071] 1.5.6 Cell Scratch Assay The effects of different drug-treated groups (groups were set according to HN concentration of 5 μg / mL: PGB-LNP@HN, QE group, PGB-LNP@HN group, cRGD-PGB-LNP@HN, QE group, and cRGD-PGB-LNP@HN group) on A549 cells after scratching and incubation for 0, 12, and 24 h were observed using an inverted fluorescence microscope. The changes in cell area were analyzed using ImageJ software. The results are as follows: Figure 32 In the control group, the scratch area had shrunk to 62.82±10.78% of the original area after 12 h, and to 49.84±18.29% after 24 h. In the PGB-LNP@HN, PGB-LNP@HN,QE, cRGD-PGB-LNP@HN, and cRGD-PGB-LNP@HN,QE groups, the scratch areas after 24 h of incubation with cells were 98.43±2.07%, 94.89±5.90%, 96.82±6.71%, and 97.07±3.30% of the original area, respectively, indicating that all the formulations could significantly inhibit the migration of A549 cells.

[0072] 1.5.7 Transwell cell migration experiment The ability of cells to migrate in a chamber after incubation with A549 for 24 h was observed using an inverted fluorescence microscope. The treatment groups (excluding the control group, the other groups were set up with an HN concentration of 15 μg / mL as B-LNP@HN, PGB-LNP@HN, QE, PGB-LNP@HN, cRGD-PGB-LNP@HN, QE, and cRGD-PGB-LNP@HN) were selected as the control group. The results are as follows: Figure 33 In the blank control group, large areas of dark purple A549 cells were clearly visible, indicating strong cell viability of A549 cells in the blank control group. In the B-LNP@HN group, the number of cells in each chamber gradually decreased, indicating that this preparation had a certain impact on cell viability. The PGB-LNP@HN group had slightly fewer cells than the B-LNP@HN group, but more cells than the PGB-LNP@HN,QE group, indicating that PEG-modified gelatin coating weakened some cell viability, and the addition of QE reduced cell viability. This is also evident in the comparison between the cRGD-PGB-LNP@HN and cRGD-PGB-LNP@HN,QE groups. Furthermore, the cRGD-PGB-LNP@HN and cRGD-PGB-LNP@HN,QE groups had the fewest cells in each chamber, indicating that cRGD modification could further reduce the viability of A549 cells.

[0073] 1.5.8 Evaluation of mitochondrial targeting Using COU6 as a fluorescent probe, A549 cells in the logarithmic growth phase were incubated with the same COU6 concentration in four groups: B-LNP@COU6, PGB-LNP@COU6,QE, PGB-LNP@COU6, cRGD-PGB-LNP@COU6,QE, and cRGD-PGB-LNP@COU6, for 1, 2, and 4 hours, respectively. Laser confocal microscopy was used to observe whether the formulation targeted mitochondria. The results are as follows: Figure 34 The Pearson coefficients of all treatment groups increased with time. The B-LNP@COU6 group had a Pearson coefficient of 0.51±0.061 at 4 h, indicating less drug accumulation in the mitochondria. The drug accumulation in the mitochondria of PGB-LNP@COU6 at 4 h was similar to that of cRGD-PGB-LNP@COU6,QE, with Pearson coefficients of 0.52±0.040 and 0.50±0.029, respectively. The mitochondrial accumulation of PGB-LNP@COU6,QE and cRGD-PGB-LNP@COU6,QE was significantly higher than other formulations, with Pearson coefficients of 0.55±0.017 and 0.75±0.023, respectively, which were higher than the formulations without QE, indicating that QE encapsulation can increase drug accumulation in the mitochondria.

[0074] 1.5.9 Mitochondrial membrane potential detection A549 cells and different drug-treated groups: Excluding the negative control group, positive control group, and different HN concentrations (2.5, 10, 15 μg / mL), the following groups were incubated for 12 h: B-LNP@HN, PGB-LNP@HN,QE, PGB-LNP@HN, cRGD-PGB-LNP@HN,QE, and cRGD-PGB-LNP@HN. Mitochondrial membrane potential transitions were observed, and the results are as follows: Figure 35 In the B-LNP@HN group, the green / red fluorescence ratio at an HN concentration of 15 μg / mL was 1.40±0.34, and significant green fluorescence was also observed according to laser confocal microscopy. In the PGB-LNP@HN group and the PGB-LNP@HN,QE group, the green / red fluorescence ratios at an HN concentration of 15 μg / mL were 0.69±0.20 and 0.60±0.28, respectively. According to laser confocal microscopy, the mitochondrial membrane potential of both groups gradually increased with increasing drug concentration, while the green fluorescence of the cRGD-PGB-LNP@HN group and the cRGD-PGB-LNP@HN,QE group was significant, with green / red fluorescence values ​​of 2.22±0.37 and 2.55±0.32 at an HN concentration of 15 μg / mL, respectively. Furthermore, the red fluorescence gradually disappeared with increasing HN concentration, as observed under laser confocal microscopy, indicating that this formulation significantly reduces mitochondrial membrane potential with increasing concentration.

[0075] 1.5.10 ROS Level Detection The generation of ROS in A549 cells after 12 h of incubation with different drug-treated groups was observed using laser confocal microscopy. The control group showed very weak fluorescence, while the fluorescence of the HN+BR+QE group in the free drug group was stronger than that of the HN group, indicating that the drug combination could increase ROS generation in A549 cells. Under laser confocal microscopy, the B-LNP@HN group showed higher green fluorescence intensity than the free drug group; the PGB-LNP@HN, cRGD-PGB-LNP@HN, PGB-LNP@HN,QE, and cRGD-PGB-LNP@HN,QE groups showed significant fluorescence, indicating that A549 cells produced the most ROS. The results are as follows: Figure 36 As shown in the figure. Flow cytometry was used to determine the ROS production of different drug-treated groups after incubation with A549 cells for 12 h. Free drug (HN+BR+QE) produced a certain amount of reactive oxygen species after incubation with A549 cells for 12 h. The amount of ROS produced in the drug-treated groups was greater than that in the free drug-treated groups. Flow cytometry showed that the peaks of the prepared B-LNP@HN, PGB-LNP@HN,QE, and cRGD-PGB-LNP@HN,QE cells shifted to the right, indicating that the drug-treated groups increased ROS production. The results are shown in the figure.Figure 36 As shown.

[0076] 1.6 Evaluation of in vivo anti-lung cancer effect 1.6.1 Establishment of an in situ lung cancer model A mouse orthotopic tumor model was constructed. The orthotopic tumors were used for experimental research 4 days after being seeded in LLC-LuC, and the tumor size was detected using D-fluorescein potassium salt.

[0077] 1.6.2 Biodistribution within the body Using DiR (500 μg / kg) as a probe, DiR, B-LNP@DiR, PGB-LNP@DiR,QE, and cRGD-PGB-LNP@DiR,QE (n=3) were prepared. Following tail vein administration, images were taken at 0, 2, 4, 6, 8, 12, 24, 36, 48, 72, 96, and 120 h. After imaging, mice were dissected, tumors and major organs were removed, rinsed with PBS, dried, and then photographed using a small animal in vivo imaging system. Data were analyzed using Living Image software after imaging.

[0078] The results are as follows Figure 37 The bioluminescence generated at 0 h by the combination of D-luciferin potassium salt and LLC-LuC facilitates tumor localization. The free DiR group did not accumulate at the tumor site from 0 to 120 h, but was distributed in the liver and spleen in vivo. As time increased, the fluorescence intensity at 120 h was 8209.56±226.77. At this time, there was no accumulation in the lungs, and the fluorescence intensity of this group was used as the background. The B-LNP@DiR group showed slight fluorescence in the lungs during mouse in vivo imaging. The fluorescence intensity at 120 h was 9558.94±2187.87, which was slightly stronger than that of the DiR group. Figure 37 The C-value in the figure shows that the fluorescence intensity of the PGB-LNP@DiR,QE group and the cRGD-PGB-LNP@DiR,QE group was significantly stronger than that of the DiR group and the B-LNP@DiR group in mice from 2 to 120 h. Observation using lung tumor localization and anatomical images showed that both groups accumulated at the tumor site for up to 120 h. The fluorescence intensity at 120 h was 22380.72±11346.78 and 37201.26±11416.12, respectively, indicating that the PEG-modified formulations all have a long-term circulating effect in vivo. Figure 37 The C-value shows that the fluorescence intensity accumulated at the tumor site in the cRGD-PGB-LNP@DiR,QE group is stronger than that in the PGB-LNP@DiR,QE group, indicating that the cRGD-PEG-COOH modified formulation has both targeting and long-term circulation in vivo, and the drug accumulates more at the tumor site to exert its effect.

[0079] 1.6.3 In vivo anti-lung cancer efficacy Four days after tumor implantation in mice, the in vivo anti-lung cancer efficacy was evaluated. Mice were randomly divided into four groups: saline group (control group), HN+BR+QE group, PGB-LNP@HN,QE group, and cRGD-PGB-LNP@HN,QE group (n=6). Drug administration was performed every other day. D-fluorescein potassium salt was injected intraperitoneally on days 3, 5, 7, 9, 11, 13, and 15. Tumor size was observed using a small animal in vivo imaging system, and mouse weight was recorded. Lung cancer tissue was harvested from the mice 15 days later. Results are as follows: Figure 38 As shown, in the control group, the tumors in mice grew larger over time, and some mice died. After 15 days, the fluorescence intensity of the tumors in the mice was 828.27±232.75. The HN+BR+QE group was able to inhibit tumor growth in the early stage, but the inhibitory effect gradually weakened over time. After 15 days of treatment, the fluorescence intensity of the tumors in the mice was 510.54±173.66, which was stronger than that of the control group. After 15 days of treatment, the fluorescence intensities of the tumors in the cRGD-PGB-LNP@HN,QE group and the PGB-LNP@HN,QE group were 357.43±169.42 and 441.91±136.07, respectively, which were significantly lower than those in the control group. This indicates that both preparations have anti-tumor effects in vivo, with the cRGD-PGB-LNP@HN,QE group showing the strongest anti-tumor effect, followed by the PGB-LNP@HN,QE group.

[0080] 1.7 Safety Evaluation 1.7.1 Changes in mouse body weight The mice were observed for changes in body weight 15 days after administration of the drug, and the results were as follows. Figure 39 As shown in the figure. Some mice in the control group died, so the weight records are incomplete. During the treatment, the weight of mice in the control group decreased significantly. The weight of mice in the free drug group (HN+BR+QE group) also decreased slightly. Compared to the free drug group, no mice died in the formulation groups, and their weights did not show a significant decreasing trend. This indicates that the prepared PGB-LNP@HN,QE and cRGD-PGB-LNP@HN,QE groups had a relatively small impact on mouse weight.

[0081] 1.7.2 HE staining After 15 days of treatment, H&E staining was performed on the heart, liver, spleen, and kidneys of C57BL / 6 mice. As shown in Figure 40, compared with the control group and the healthy group, no significant damage was observed in any organ tissue in either the nano-drug-loaded group or the free drug group. This indicates that cRGD-PGB-LNP@HN,QE and PGB-LNP@HN,QE did not cause significant damage to important metabolic organs when targeting tumor tissue, and the formulation has a certain degree of biosafety.

[0082] Example 5 1. Antitumor mechanism of cRGD-PGB-LNP@HN,QE After the in vivo antitumor activity experiment of cRGD-PGB-LNP@HN,QE was completed, the main organs of mice were removed and the tumors were examined to verify the antitumor activity mechanism.

[0083] 1.1 TUNEL staining Tumor tissues from each experimental group were analyzed by TUNEL staining. Figure 41 The effects of different formulations on cell apoptosis were evaluated. In the control group, the nuclei of tumor cells were stained blue, indicating virtually no tumor cell apoptosis. However, after treatment with different drug formulations, apoptosis was observed in the HN+BR+QE, B-LNP@HN, and PGB-LNP@HN,QE groups. The cRGD-PGB-LNP@HN,QE group showed a larger green coverage area and a corresponding increase in apoptotic tumor cells. The apoptosis signal intensity of the tumor cells was used to evaluate the effects of different formulations on cell apoptosis. Figure 41 Analysis showed that, compared with the control group, the cRGD-PGB-LNP@HN,QE (p<0.005) group was more effective in promoting cell apoptosis than other groups. Comparison of fluorescence intensity revealed no significant difference between HN+BR+QE (29.96±6.40) and the control group (2.67±2.19), while B-LNP@HN (37.08±19.73, p<0.05) and PGB-LNP@HN,QE (40.39±10.73, p<0.05) showed significant differences. The cRGD-PGB-LNP@HN,QE (86.77±22.64, p<0.005) group showed the greatest difference, indicating that the targeting of the nanodelivery system to tumor sites was enhanced by cRGD modification.

[0084] 1.2 Immunohistochemistry Immunohistochemistry of PD-L1 was performed on tumor tissues from each group, and significant differences were identified. Figure 42Analysis was performed to evaluate the effects of different formulations on PD-L1 expression at tumor sites. In the control group, the brown color was more pronounced, indicating a corresponding increase in PD-L1 protein. The B-LNP@HN,QE (p<0.05) and PGB-LNP@HN,QE (p<0.05) groups showed a slight pale blue tinge, indicating that BR (Breast Radical Injection) exhibited a certain inhibitory effect on PD-L1 expression. Compared to the control group, cRGD-PGB-LNP@HN,QE (p<0.005) showed a significantly more visible blue-purple color. Fluorescence intensity analysis (…) Figure 42 The fluorescence intensities were as follows: control group (2.67±2.19), HN+BR+QE (29.96±6.40), B-LNP@HN,QE (37.08±19.73), PGB-LNP@HN,QE (40.39±10.73), and cRGD-PGB-LNP@HN,QE (86.77±22.64). Except for the free drug, all others showed significant differences from the control group, and the fluorescence intensity of cRGD-PGB-LNP@HN,QE was significantly higher than that of the control group. The results suggest that BR, in the cRGD-PGB-LNP@HN,QE nanoformulation modified with PEG and cRGD, exhibits active targeting and long-circulation effects, resulting in a more significant effect on blocking PD-L1 protein expression.

[0085] As demonstrated by the above embodiments, the present invention successfully prepared PGB-LNP@HN,QE and cRGD-PGB-LNP@HN,QE, exhibiting good stability and hemolytic activity, and responding to the release of MMP-2 and GSH. They also showed good inhibitory effects on A549 cells. In vitro, they exhibited effects such as targeting mitochondria, increasing mitochondrial reactive oxygen species production, and altering mitochondrial membrane potential. In vivo, they achieved long-term circulation, targeting tumor tissue, penetrating deep into tumor tissue, inhibiting tumor growth, and reducing the toxicity of HN, providing a reference for multi-component traditional Chinese medicine therapy for tumors.

[0086] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A multi-component nano-drug delivery system for traditional Chinese medicine, characterized in that, The BR lipid nanoparticles include HN-loaded BR lipid nanoparticles, a gelatin layer covering the surface of the HN-loaded BR lipid nanoparticles, and QE loaded in the gelatin layer; the HN-loaded BR lipid nanoparticles include BR lipid nanoparticles and HN encapsulated in the BR lipid nanoparticles. The BR lipid nanoparticles are formed from a BR-disulfide bond-caprylic / capric triglyceride compound; the structural formula of the BR-disulfide bond-caprylic / capric triglyceride compound is shown in Formula I. The gelatin in the gelatin layer is type A gelatin.

2. The multi-component nano-drug delivery system for traditional Chinese medicine according to claim 1, characterized in that, The gelatin layer is also modified with polyethylene glycol.

3. The multi-component nano-drug delivery system for traditional Chinese medicine according to claim 2, characterized in that, The polyethylene glycol is also attached with cRGD.

4. The preparation method of the multi-component nano-drug delivery system of traditional Chinese medicine according to claim 1, characterized in that, Includes the following steps: HN, BR-disulfide bond-caprylic / capric triglyceride compound and dimethyl sulfoxide were mixed to obtain an organic phase; The organic phase was added to the aqueous phase and heated and stirred, then cooled to obtain a dispersion of HN-loaded BR lipid nanoparticles; the aqueous phase was a poloxamer aqueous solution. QE was dispersed in an aqueous solution of type A gelatin to obtain a QE-gelatin solution; The HN-loaded BR lipid nanoparticle dispersion was added to the QE-gelatin solution, heated and stirred, and then solidified to obtain the multi-component nano-drug delivery system of traditional Chinese medicine.

5. The multi-component nano-drug delivery system for traditional Chinese medicine according to claim 4, characterized in that, The preparation method of the type A gelatin aqueous solution includes: placing type A gelatin in water to swell, and then heating it in a water bath to obtain a type A gelatin aqueous solution.

6. The preparation method according to claim 5, characterized in that, The mass ratio of HN and BR-disulfide bond-caprylic / capric triglyceride compound is 1:4 to 6; The poloxamer is poloxamer 188; the concentration of the poloxamer aqueous solution is 2-4 wt%; the volume ratio of the organic phase to the aqueous phase is 1:5-10; The organic phase is added to the aqueous phase and heated and stirred at a temperature of 60–70°C for 15–30 min. The concentration of the type A gelatin aqueous solution is 0.5–1.5% w / v; The volume ratio of the HN-loaded BR lipid nanoparticle dispersion to the type A gelatin aqueous solution is 1:0.5 to 1.5; the mass ratio of QE to HN is 1 to 4:1; the HN-loaded BR lipid nanoparticle dispersion is added to the QE-gelatin solution by dropwise addition, which is carried out under stirring conditions, and the temperature of the dropwise addition is 60 to 70°C.

7. The preparation method of the multi-component nano-drug delivery system of traditional Chinese medicine according to claim 2, characterized in that, Includes the following steps: mPEG-COOH, buffer solution, and crosslinking agent are mixed and reacted to obtain a reaction solution; the reaction solution is then mixed with an aqueous solution of type A gelatin and incubated to obtain an aqueous solution of type A gelatin modified with polyethylene glycol. The aqueous solution of type A gelatin modified with polyethylene glycol was used to replace the aqueous solution of type A gelatin, and the preparation was carried out according to the preparation method of any one of claims 3 to 6 to obtain a multi-component nano-drug delivery system of traditional Chinese medicine with polyethylene glycol modified on the gelatin layer.

8. The preparation method according to claim 7, characterized in that, The crosslinking agents are EDC and NHS, and the mass ratio of mP EG-COOH, EDC and NHS is 1-3:1-3:1-3; the reaction time is 4-6 hours; and the incubation time is 15-20 hours.

9. The preparation method of the multi-component nano-drug delivery system of traditional Chinese medicine according to claim 3, characterized in that, Includes the following steps: cRGD-PEG-COOH, buffer solution and crosslinking agent are mixed and reacted to obtain a reaction solution. The reaction solution is then mixed with an aqueous solution of type A gelatin and incubated to obtain an aqueous solution of cRGD-polyethylene glycol modified gelatin. A cRGD-polyethylene glycol-modified gelatin aqueous solution was used to replace the type A gelatin aqueous solution, and the preparation was carried out according to any one of claims 3 to 6 to obtain a multi-component traditional Chinese medicine nano-delivery system with cRGD-polyethylene glycol modification on the gelatin layer.

10. The application of the multi-component nano-delivery system of traditional Chinese medicine according to any one of claims 1 to 3 or the multi-component nano-delivery system of traditional Chinese medicine prepared by the preparation method according to any one of claims 4 to 9 in the preparation of anti-lung cancer drugs.

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