Nano-drug preparation based on collagenase infiltration strategy and application of nano-drug in deep tumor chemotherapy-immune synergistic treatment

Abstract

The invention relates to a nano-drug based on a collagenase infiltration strategy and application of the nano-drug in deep tumor chemotherapy-immune collaborative therapy. Aiming at the problems of insufficient permeation, low accumulation rate and large toxic and side effects of traditional chemotherapeutic drugs caused by a shielding effect of a tumor tissue microenvironment on the nano-drugs, bovine serum albumin is used as a carrier, collagenase and low-dose docetaxel are co-entrapped through a self-assembly method, and a nano-particle drug (COL / DTX-coated BSA NPs) is constructed. The nano-drug has the characteristics of uniform spherical morphology, high encapsulation efficiency and drug loading capacity, good collagenase activity maintenance, excellent biocompatibility and the like, and collagenase-mediated tumor penetration enhancement, a low-dose chemotherapy-immune synergistic mechanism and safety guarantee can be realized. Tumor targeted enrichment and deep penetration are realized in a breast cancer model, chemotherapy tumor inhibition effect and anti-tumor immunity are synergistically enhanced, system toxicity is remarkably reduced, and a new strategy is provided for high-efficiency and low-toxicity treatment of solid tumors.

Description

Technical field

[0001] The present invention belongs to the field of targeted nanomedicine materials, and specifically relates to the preparation of a nanomedicine based on collagenase penetration and low-dose chemotherapy-induced tumor immunotherapy, as well as its application in tumor chemotherapy-immunotherapy synergy. [Background Technology]

[0002] Tumors' shielding effect on nanomedicines can lead to problems such as insufficient penetration during tumor treatment. The complex tumor tissue microenvironment also reduces the efficacy of nanomedicines, leading to minimal accumulation of nanomedicines in solid tumors, thus reducing the effectiveness of drug therapy. Existing clinical chemotherapy drugs, such as docetaxel and cisplatin, have potential efficacy in treating breast, ovarian, lung, prostate, and head and neck cancers, for example, by improving drug penetration barriers in breast cancer. However, they also have significant toxic side effects. Previous scientific research has demonstrated that lower doses of docetaxel can help enhance immune function. Encapsulating collagenase and lower doses of docetaxel in bovine serum albumin and preparing them into nanoparticles can play a crucial role in tumor destruction and drug penetration, reducing the tumor's shielding effect on nanomedicines and improving their intratumoral penetration. Therefore, low-dose docetaxel chemotherapy can achieve anti-tumor effects while reducing its toxic side effects and enhancing immune function.

[0003] While nano-drug delivery systems have made significant progress in recent decades, various nano-drugs are subject to differences in their penetration and retention in tumor cells. This results in low accumulation of nano-drugs within tumor tissues, and a significant discrepancy between therapeutic effects and expectations, which remains a major challenge for current nano-drug delivery systems. Therefore, designing a nano-drug that improves tumor tissue penetration and retention, enhances the immune system, and ultimately achieves better tumor treatment outcomes is a key research topic for this patent.

[0004] Functionalized nanomaterials have been extensively studied for their anti-tumor applications. The complex tumor microenvironment (TME) contains multiple therapeutic barriers, such as a dense extracellular matrix (ECM) and abnormal vascular system, which greatly hinder the penetration of nanomaterials deep into solid tumors, making it difficult to achieve optimal therapeutic effects in deep-seated tumors.

[0005] This study aimed to develop a nanomedicine based on collagenase-mediated infiltration for chemotherapy-induced immunotherapy of deep-seated tumors. Collagenase (COL) and docetaxel (DTX) were co-encapsulated in bovine serum albumin (BSA) to construct COL / DTX@BSA nanoparticles (NPs). These nanoparticles were enriched in tumors by enhanced permeation and retention. The released collagenase not only acts as a "molecular scissor" to sever collagen fibers in the tumor's ECM but also facilitates deep penetration of DTX into the tumor, thereby exerting its chemotherapeutic effects. Furthermore, chemotherapeutic drugs can induce immunogenic cell death: low-dose DTX not only exhibits chemotherapy-related cytotoxicity but also induces immunogenic cell death, inducing tumor immunotherapy and triggering the release of damage-associated molecular patterns (DAMPs), such as calreticulin, adenosine triphosphate, and high-mobility group box 1 protein. DAMPs activate the immune system, triggering an immune response, ultimately leading to immunogenic cell death (ICD), thereby achieving a synergistic effect between chemotherapy and immunotherapy. This study provides a new reference for deep tumor nanodrug delivery strategies based on collagenase infiltration and expands the application of chemotherapy drug-induced tumor ICD in nanomedicine. [Summary of the invention]

[0006] In view of the shortcomings of the existing technology, the present invention intends to provide a method for preparing and applying nanomedicines for tumor immunotherapy induced by collagenase penetration and low-dose chemotherapy.

[0007] To achieve the above effects, the first object of the present invention is to provide a method for preparing nanomedicines based on collagenase penetration and low-dose chemotherapy-induced tumor immunotherapy. The nanomedicine is formed by self-assembly, including bovine serum albumin as a carrier of the nanomedicine and encapsulating collagenase and a low-dose chemotherapy drug docetaxel.

[0008] Furthermore, the bovine serum albumin in the present invention has good stability and solubility, and can simulate a complex biological environment while also having a certain protective effect on the encapsulated collagenase; collagenase can inhibit the migration and invasion ability of tumor cells and change the physical properties and biochemical signals in the tumor microenvironment; lower doses of docetaxel help to exert a suitable chemotherapy effect, activate the body's immune response through multiple mechanisms, and thus enhance the immune clearance ability of tumors.

[0009] The nanocarrier drug of the present invention is spherical in shape and has a uniform particle size distribution. The average particle size of the nanocarrier drug is 50 to 200 nm, preferably 50 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, or 200 nm.

[0010] The drug loading amount of docetaxel in the nano drug is 1.3-3.3%, preferably 1.3%, 1.7%, 2.1%, 2.5%, 2.9%, and 3.36%.

[0011] Preferably, the activity of collagenase in the nanomedicine is 0.06-0.07 Units / ml, more preferably 0.06 Units / ml, 0.062 Units / ml, 0.064 Units / ml, 0.066 Units / ml, 0.068 Units / ml, or 0.07 Units / ml.

[0012] The second object of the present invention is to provide a method for preparing the aforementioned nanomedicine, the main steps of which are: mixing bovine serum albumin, collagenase, and docetaxel uniformly, stirring for 12 hours, and then granulating, ultrafiltrating, and filtering to obtain the nanomedicine.

[0013] Furthermore, in the present invention, the nanomedicine assembly method comprises the following steps:

[0014] (1) Dissolve bovine serum albumin in PBS (pH 7.4), dissolve collagenase in PBS, and quickly mix the two solutions.

[0015] (2) dissolving docetaxel in acetone, then mixing the above three solutions, adding glutaraldehyde, and stirring overnight;

[0016] (3) Ultrafiltration is performed three times in a centrifuge using an ultrafiltration tube and filtration is performed to obtain the nanomedicine.

[0017] Preferably, when preparing the nanomedicine, the mass ratio of bovine serum albumin, collagenase, and docetaxel is 200:1:10;

[0018] Preferably, glutaraldehyde is added during preparation to form stable nanoparticles;

[0019] Preferably, the prepared nanomedicine is a clear and transparent milky white solution without white precipitation after standing.

[0020] Preferably, the pH of the solvent PBS is 7.2 to 7.6, and more preferably 7.2, 7.3, 7.5, 7.6, or 7.4;

[0021] Preferably, the stirring speed is 300-600 rpm, more preferably 450 rpm, and the stirring time is 12 h;

[0022] Preferably, the rotation speed during centrifugation is 4500-5500r, more preferably 4500r, 5000r, 5500r;

[0023] Preferably, the duration of each centrifugation is 2.5 to 3.5 minutes, more preferably 2.5 minutes, 3.0 minutes, or 3.5 minutes;

[0024] Preferably, the ultrafiltration centrifuge tube is 100kDa;

[0025] Preferably, the storage temperature of the nanomedicine is 4°C.

[0026] The third purpose of the present invention is to improve the tumor microenvironment, enhance the permeability and distribution of encapsulated drugs, and activate the body's immune response, thereby achieving efficient treatment of tumors.

[0027] Preferably, the tumor cells are human triple-negative breast cancer cell line 4T1 and mouse melanoma cell line B16F10.

[0028] Preferably, the normal tissue cells are mouse bone marrow-derived dendritic cell lines BMDCs and dendritic cells DCs.

[0029] In this nanomedicine, collagenase is used to hydrolyze collagen in the extracellular matrix, opening pathways deep into tumor tissue. By using a lower dose of docetaxel to reduce systemic toxicity, it inhibits deep-seated tumor cells and stimulates the body's immune system. This has important scientific and social implications for designing new strategies for tumor treatment and enhancing immunotherapy.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] This study uses bovine serum albumin (BSA) as a carrier to encapsulate collagenase and docetaxel via self-assembly to prepare the nanoparticle drug COL / DTX@BSANPs. The resulting nanoparticles are clear, uniform in size, and spherical in shape. The nanoparticles exhibit high drug content and encapsulation efficiency, are negatively charged, exhibit good stability, and maintain good COL activity. The protein secondary structure of BSA, used as a drug carrier, remains unchanged before and after synthesis. These nanoparticles demonstrate strong cytotoxicity against 4T1 breast cancer cells. In vitro 3D spheroid models demonstrate that these nanoparticles can effectively hydrolyze tumor collagen, demonstrating that collagenase can effectively hydrolyze tumor collagen and promote the maturation of bone marrow-derived dendritic cells. Western-Blot and immunofluorescence results showed that the nanoparticles could significantly induce apoptosis of 4T1 cells. At the same time, the quantification of ATP, calreticulin, immunofluorescence staining and immunogenic death marker products also showed a significant upregulation. The results of in vivo imaging of small animals showed that the nanoparticles could be well enriched at the tumor site and effectively penetrate into the tumor, while also showing a good retention effect. The upregulation of TNF-α, IFN-γ and CD3 / CD8 positive immune T cells proved that the immune capacity of mice was enhanced. The results of liver function, kidney function and blood biochemistry showed that the nanoparticles had good safety in vivo.

[0032] Nanoparticles were prepared by encapsulating COL with BSA and simultaneously loading it with a low dose of the chemotherapy drug DTX. COL is used to hydrolyze collagen in the extracellular matrix and open pathways to the deep layers of tumor tissue. Using a lower dose of DTX reduces systemic toxicity, inhibits deep-seated tumor cells, and stimulates the body's immunity. This invention significantly promotes the penetration and retention of nanomedicines into tumor cells and enhances immune capacity, which has important scientific significance and social value for designing new strategies for tumor treatment and enhancing immunotherapy.

Brief Description of the Drawings

[0033] Figure 1 Schematic diagram of collagenase-permeable nanomedicine for chemotherapy-induced tumor immunotherapy;

[0034] Figure 2 Preparation and characterization of bovine serum albumin-loaded collagenase / docetaxel nanoparticles (COL / DTX@BSANPs);

[0035] Figure 3 In vitro and in vivo permeability tests of COL / DTX@BSA NPs;

[0036] Figure 4 The in vitro antitumor effect of COL / DTX@BSA NPs;

[0037] Figure 5 To characterize the immunogenic cell death (ICD) and dendritic cell (DC) maturation induced by COL / DTX@BSA NPs;

[0038] Figure 6 The in vivo antitumor effect of COL / DTX@BSA NPs;

[0039] Figure 7 The inhibitory effect of COL / DTX@BSANPs on bilateral tumor-bearing;

[0040] Figure 8 This is the in vivo immune enhancement effect of COL / DTX@BSA NPs. [Specific implementation method]

[0041] The present invention will be described in detail with reference to specific embodiments and the accompanying drawings.

[0042] Example 1

[0043] In this embodiment, the nanomedicine is obtained by self-assembly, and the schematic diagram of the nanomedicine based on collagenase penetration for chemotherapy-induced tumor immunotherapy is shown in FIG. Figure 1 shown.

[0044] The nanomedicine of the present invention is prepared by the following method, which specifically comprises the following steps:

[0045] Dissolve 20 mg of bovine serum albumin in 2 mL of PBS (pH 7.4) to obtain a 10 mg / mL bovine serum albumin solution. Add this to a 10 mL flat glass vial with a lid. Dissolve 100 μg of collagenase in 0.5 mL of PBS to obtain a 0.2 mg / mL COL solution. Add 0.5 mL of collagenase solution to the bovine serum albumin vial over 10 seconds. Dissolve 1 mg of docetaxel in 10 μL–90 μL of acetone (the total volume of acetone should not exceed 100 μL), ensuring the solution is clear and free of particles. Slowly add this to the vial over 30 seconds. Add 10 μL of 50% glutaraldehyde to 990 μL of PBS, vortex for 10 seconds to mix, and then add 100 μL to the vial. Stir at room temperature for 12 hours.

[0046] The sample from the bottle was transferred to a 15ml 100kDa ultrafiltration tube and placed in a high-speed centrifuge (5000r / min) for ultrafiltration. After ultrafiltration of approximately 500μL, the inner tube of the ultrafiltration tube was filled with PBS to 2ml. Ultrafiltration was continued for 3 minutes at 5000r / min for a total of three centrifugations. Approximately 200μL of sample remained in the inner tube of the ultrafiltration tube. The sample in the inner tube of the ultrafiltration tube was transferred to an EP tube, and the inner tube of the centrifuge tube was rinsed with PBS. The rinse solution was combined with the sample. The sample was aspirated with a 1mL syringe and filtered through a disposable 0.22μm drainage filter to obtain the nanodrug COL / DTX@BSA NPs, which were stored at 4°C. COL@BSANPs and DTX@BSANPs were prepared using a similar method.

[0047] In this embodiment, a biological transmission electron microscope and a dynamic light scattering instrument are used to analyze the size and morphology of the nanomedicine. The transmission electron microscope sample preparation method is as follows: the prepared nanomedicine is diluted 10 times and dispersed by ultrasonication for 2 minutes. 7 μL of the diluted nanomedicine aqueous solution is dropped on the TEM test copper grid, and the solution is allowed to stand at room temperature. After the solvent is completely evaporated and dried, a 2% phosphotungstic acid negative stain solution is centrifuged at 12000 rpm for 5 minutes. 7 μL of the supernatant is gently dropped on the copper grid loaded with the nanomedicine and negatively stained for 1 minute. The negative stain solution is then absorbed with the side of the filter paper. After drying, the sample is observed using a biological transmission electron microscope. The results are shown as follows. Figure 2 As shown in B, the nanomedicine is spherical and well dispersed, with a size between 50 and 200 nm.

[0048] Dynamic light scattering results are as follows Figure 2 As shown in Figure C, the average hydrated particle size of the nanomedicine is about 50 nm, and the particle size distribution range is narrow.

[0049] Example 2

[0050] Characterization of COL / DTX@BSA NPs:

[0051] (1) Morphology and particle size characterization

[0052] Nanopharmaceuticals COL / DTX@BSA NPs were prepared by magnetic stirring self-assembly. The morphology and size of the nanopharmaceuticals were characterized. COL / DTX@BSA NPs were a clear, transparent milky white solution with no white precipitate after standing. The particle size was measured and the peak size was 70 nm. The PDI was 0.342. The prepared nanoparticles had a uniform particle size and a normal distribution ( Figure 2 B). The appearance of COL / DTX@BSA NPs was observed by electron microscopy (e.g. Figure 2 C), The appearance of COL / DTX@BSA NPs under TEM is uniform, with a particle size of about 50 nm and a regular spherical shape.

[0053] (2) Collagenase activity assay

[0054] COL@BSANPs were prepared and the enzyme activity of the prepared COL@BSANPs and COL standard was tested using a COL activity assay kit. The results showed that the average enzyme activity of the COL standard was 0.07666 Units / ml, while the average enzyme activities of the COL@BSA and COL / DTX@BSANPs prepared into nanomaterials were 0.05485 Units / ml and 0.06531 Units / ml, respectively. This indicates that after the nanoparticles were prepared, the COL used still retained good activity, indicating that it retained the ability to digest collagen fibers (e.g. Figure 2 D,E).

[0055] (3) Protein secondary structure determination

[0056] The protein secondary structure of COL / DTX@BSA NPs was determined in detail to evaluate the changes and retention of its structure during the preparation process. The protein secondary structures of natural BSA and COL / DTX@BSA NPs were compared and analyzed using circular dichroism spectrometer. The results showed that COL / DTX@BSA NPs maintained a high degree of consistency with natural BSA in terms of structural composition such as α-helix, β-sheet, β-turn and random coil (52.229% α-helix, 10.886% β-sheet, 14.486% β-turn and 24.328% random coil in natural BSA; 61.243% α-helix, 8.571% β-sheet, 12.871% β-turn and 19.129% random coil in COL / DTX@BSA NPs). This shows that the protein structure of BSA is well retained during the preparation of nanomedicines, which is of great significance for maintaining its biocompatibility and functionality (such as Figure 2 F,G).

[0057] Example 3

[0058] Evaluation of COL / DTX@BSANPs penetration and in vivo distribution

[0059] (1) Construction of tumor-bearing mouse model

[0060] 4T1 cells were cultured, digested with trypsin and the supernatant was removed, and washed twice with PBS; the cells were diluted with PBS and counted with a cell counting plate, and the final dilution concentration was 10 7 The outer side of the right leg of the mouse was shaved and then depilated with depilatory cream. The mouse was marked with an ear tag and each mouse was injected with 100 μl of cell suspension. The length and width of the subcutaneous tumor of the mouse were recorded every other day and the volume V = d 2 *D / 2, where D is the major diameter and d is the minor diameter, and the volume reaches 100mm 3 When performing the experiment, mice with the same growth condition and similar weight were selected for the experiment.

[0061] (2) In vivo fluorescence distribution and penetration evaluation

[0062] Preparation of drugs COL / DTX@BSA-cy7 NPs and DTX@BSA-cy7 NPs; labeling of 10 mice of similar body weight and size, with tumor volume reaching 100 mm 3 About 100 tumor-bearing mice were divided into two groups and administered with 10 mg / kg DTX via the tail vein, with 100 μl of COL / DTX@BSA-cy7NPs and DTX@BSA-cy7NPs injected respectively; they were anesthetized with chloral hydrate 2, 4, 6, 8, and 12 hours after administration, and photographed under a small animal fluorescence imager to observe the drug distribution in the body. The excitation wavelength was set to 700 nm, and the emission wavelength was 790 nm. The fluorescence intensity at the tumor was quantified to observe the changes in tumor drug accumulation; after the end, the mice were euthanized, and the heart, liver, spleen, lung, kidney and tumor were dissected and the fluorescence intensity was detected and recorded using the same method; after recording the fluorescence intensity, the tumor was placed in 4% paraformaldehyde and sent to the company for tumor collagen fiber, CD31, and DAPI immunofluorescence section staining, and the results were awaited.

[0063] (3) In vivo penetration and distribution evaluation

[0064] In the in vivo experiment, the distribution of Cy7-labeled COL / DTX@BSA NPs in tumor-bearing mice was observed by small animal near-infrared imaging technology ( Figure 3B). Image analysis results showed that the accumulation of COL / DTX@BSA NPs in the tumor area was significantly higher than that in the control group, indicating its excellent tumor penetration ability. In addition, quantitative analysis of fluorescence intensity at different time points further confirmed the continuous accumulation of nanomedicine in tumor tissue ( Figure 3 C). The results of the anatomical analysis showed that ( Figure 3 E), compared with Cy7-DTX@BSANPs, COL / DTX@BSA NPs were less distributed in major organs outside the tumor, which reduced potential toxic side effects and improved the biosafety of the drug. Finally, immunofluorescence staining ( Figure 3 -F), detailing the distribution of the nanomedicine in tumor tissue. Tumor sections revealed that COL / DTX@BSA NPs were able to deeply penetrate tumor tissue and co-localize with tumor-associated blood vessels and cell nuclei, further demonstrating their excellent permeability.

[0065] Example 4

[0066] In this example, the CCK-8 method was used to measure the effects of different concentrations of nanomedicines in different groups on the survival rate of triple-negative breast cancer cells 4T1 cells.

[0067] Collect 4T1 cells in the logarithmic phase and adjust the cell concentration to 10 4 Cells were inoculated into 96-well plates at 200 μL per well and incubated in a 37°C incubator with 5% CO2 for 24 h. COL / DTX@BSANPs containing different concentrations of DTX (DTX = 1.0, 5.0, 10.0, 25.0, 50.0, 100.0 μg / mL) diluted in culture medium were added at 200 μL per well. Six replicate wells were set for each concentration and incubated for 8 h. The culture medium was discarded, and CCK-8 diluted in culture medium was added at 200 μL per well. The culture was continued for 0.5 h. The absorbance of each well was measured at a wavelength of 450 nm using a microplate reader. A zero well and a control well were set to calculate the cell survival rate.

[0068] Cell viability (%) = (OD treatment / OD control) * 100%

[0069] Melanoma cell sphere models were constructed and divided into five groups: normal saline control group, COL@BSA group, Free DTX group, DTX@BSANPs group, and COL / DTX@BSANPs group. 150 μL of drug (DTX concentration was uniformly 100 M) was added to each well of a 96-well plate and incubated for 24 h. The scissors were sterilized and irradiated with UV for 30 min. A 200 μL pipette tip was cut off in a clean bench. The cell spheres were carefully removed and digested with EDTA-free protease. The cells were stained with the Annexin V-FITC / PI staining kit, and cell apoptosis was immediately analyzed by flow cytometry after staining.

[0070] In vitro tumor inhibition effect of COL / DTX@BSA NPs:

[0071] The experimental results showed that compared with free DTX and DTX@BSA NPs, COL / DTX@BSA NPs showed stronger tumor cell inhibition effect at the tested concentrations ( Figure 4 A). Cell viability assessed by the CCK-8 assay showed that the COL / DTX@BSA NPs-treated group had significantly lower cell viability than the control group, especially at higher concentrations. Furthermore, photographs of tumor spheroid growth in vitro and their volume change curves confirmed that COL / DTX@BSA NPs significantly inhibited tumor spheroid growth, with an IC50 value of 42.53 μg / mL, compared to 48.58 μg / mL for DTX@BSA NPs and 73.40 μg / mL for Free DTX. This finding was further supported by the statistical analysis of the number of tumor spheroids in vitro across the different treatment groups, demonstrating that COL / DTX@BSANPs effectively reduced the number of tumor spheroids.

[0072] Compared with free DTX and DTX@BSA NPs, COL / DTX@BSA NPs exhibited a stronger tumor cell inhibition effect at the tested concentrations. Cell viability assessed by CCK-8 assay showed that the cell viability in the COL / DTX@BSA NPs-treated group was significantly lower than that in the control group, especially at higher concentrations ( Figure 4 A). In addition, the growth photos of tumor cell spheres in vitro ( Figure 4 B) and its volume change curve ( Figure 4 C) It was also confirmed that COL / DTX@BSA NPs significantly inhibited the growth of tumor cell spheres, and its IC 50 The value was 42.53μg / ml, while DTX@BSA NPs and Free DTX were 48.58μg / ml and 73.40μg / ml respectively. Figure 4 D) Further supporting this finding, it is shown that COL / DTX@BSANPs effectively reduced the number of tumor cell spheres.

[0073] Further studies revealed the mechanism by which COL / DTX@BSA NPs inhibit tumor cells, including tumor cells and cells digested from tumor cell spheres. Flow cytometric analysis using an Annexin V-FITC / PI staining kit showed that the apoptosis rate in the COL / DTX@BSA NPs-treated group was significantly increased compared to the control group and other treatment groups. In particular, the main type of apoptosis observed was early apoptosis ( Figure 4E), indicating that COL / DTX@BSA NPs can effectively promote the early apoptosis of tumor cells.

[0074] Example 5

[0075] In this example, bone marrow-derived dendritic cells were extracted, separated and cultured in vivo to verify the ability of nanomedicine to promote the maturation of bone marrow-derived dendritic cells.

[0076] C57BL / 6J male mice were sacrificed and the skin and muscles were aseptically separated. The intact lower limb bones were removed, and large muscle tissue was roughly cut off and immersed in sterile PBS solution. The muscle tissue was removed and immersed in sterile RPMI-1640 culture medium. The bone marrow was flushed into a dry sterile culture dish with a syringe. Each long bone was repeatedly flushed until the bone marrow cavity was white and almost transparent. It was filtered through a sterile 200-mesh filter to remove impurities and tissue debris. The bone marrow cell suspension was collected and centrifuged. After discarding the supernatant, 5 mL of sterile red blood cell lysis buffer was added and the cells were mixed. The cells were allowed to stand at room temperature for 2 minutes. After the red blood cells were lysed, 6 mL of sterile saline was added to terminate the lysis. After centrifugation, the supernatant was discarded and the cells were washed twice. The cells were suspended in RPMI-1640 complete culture medium containing 10% fetal bovine serum, rm GM-CSF (10 ng / mL), and rm IL-4 (10 ng / mL). The cells were counted and the cell concentration was adjusted to 10 6 After culturing the cells at a concentration of 100 cells / ml, the culture medium was plated into 6-well cell culture plates, with 4 mL per well. After culturing the cells for 2 hours, observe cell morphology under an inverted fluorescence microscope, discard the supernatant, and retain only the adherent cells. Continue culturing under the same conditions until day 5-6, observe cell morphology under an inverted fluorescence microscope, and add 0.5 mL of culture medium to each well. When adherent cells reach approximately 90% or greater confluence, proceed to the next step of the experiment.

[0077] Five groups (control, COL@BSA, Free DTX, DTX@BSA NPs, and COL / DTX@BSANPs) were set up (DTX concentration was 100 M). Cells were treated with fresh culture medium for 24 hours. The culture medium of the tumor cells was collected, centrifuged, and the supernatant discarded. The cells were washed twice with saline, and the supernatant discarded. 330 μL of saline was added to resuspend the cells and counted. Saline was added to the volume to a total of 800 μL. 100 μL of cell suspension was added to each tube, blocked with blocking buffer, and incubated on ice for 10 minutes. Antibody labeling was added and incubated at room temperature in the dark for 20 minutes. After incubation, 3 ml of saline was added to each tube, and the supernatant discarded. The cells were resuspended in 500 μL of PBS per tube and stained for mature DCs, which were analyzed by flow cytometry (CD11C / CD80 / CD86).

[0078] ICD marker determination:

[0079] The experimental results clearly showed that compared with the control group, the tumor cells treated with COL / DTX@BSA NPs significantly increased the surface expression of ICD markers, including calreticulin (CRT) and high mobility group protein B1 (HMGB1), as well as the release of extracellular ATP. Figure 5 A) confirms this. The results of the ATP detection kit ( Figure 5 B) Further verification that COL / DTX@BSA NPs significantly promoted the release of ATP, another key hallmark of ICD.

[0080] For the maturation experiment of BMDCs, flow cytometry analysis revealed that COL / DTX@BSA NPs significantly promoted the expression of BMDC surface maturation markers CD80 and CD86 ( Figure 5 D). This result indicates that COL / DTX@BSA NPs can not only induce ICD of tumor cells but also activate the immune system and enhance the presentation of tumor antigens by promoting the maturation of DCs.

[0081] Example 6

[0082] The experimental results showed that compared with the control group, the COL / DTX@BSA NPs group showed a significant effect in inhibiting the growth of primary tumors and recurrent tumors ( Figure 6 C and D). In addition, compared with the other treatment groups, the body weight of mice in the COL / DTX@BSA NPs treatment group changed less, indicating its better biocompatibility and lower toxic side effects ( Figure 6 E). These findings confirm that COL / DTX@BSA NPs exhibit significant tumor inhibitory effects in vivo while maintaining a good safety profile.

[0083] Immunity enhancement mechanisms in vivo Figure 8 As shown in Figure A, nanomaterial treatment enhanced the immunogenicity of solid tumor cells, releasing damage-associated molecular patterns (DAMPs), recruiting dendritic cells (DCs) to the tumor site, enhancing DC function, and activating specific cytotoxic T lymphocytes (CTLs) to attack the tumor. At the end of treatment, tumor-draining lymph nodes (tdLNs), tumor tissue, spleen, and serum were obtained from the mice to prepare single-cell suspensions, and the proportions of different immune cells were analyzed by flow cytometry.

[0084] The proportion of mature dendritic cells in the tumor-draining lymph nodes (tdLNs) of mice treated with COL / DTX@BSA NPs increased to 35.97%, which was 2.67 times higher than that in the saline group, which was beneficial for antigen cross-presentation (e.g. Figure 8 B).

[0085] Subsequently, research focused on T cells infiltrating the tumor microenvironment (TME). Figure 8 As shown in Figures C and 8E, compared with the saline group, the proportion of CD8+T cells in CD3+T cells in the primary tumor and the proportion of CD8+T cells in CD3+T cells in the distal tumor increased by 23.64% in the COL / DTX@BSA NPs-treated group. In contrast, the proportion of CD4+T cells in the tissues on both sides of the tumor increased by nearly 21.5% ( Figure 8 D and 8E). A similar increase in CD8+ and CD4+ T cells was observed in the spleens of treated mice. This phenomenon is attributed to the permeability-enhancing effect of collagenase, which leads to higher accumulation of COL / DTX@BSANPs at the tumor site. Docetaxel (DTX) exerts its chemotherapeutic effects by inducing immunogenic cell death (ICD), enhancing immune activation, and effectively promoting CD8+ and CD4+ T cell infiltration.

[0086] ELISA was used to analyze the levels of tumor necrosis factor-α (TNF-α) in the serum of mice after different treatments. Figure 8 F), interferon-γ (IFN-γ) ( Figure 8 G) and interleukin-12 (IL-12) ( Figure 8 Quantitative analysis of the levels of IFN-γ, TNF-α, and IL-12 in solid tumors further confirmed that COL / DTX@BSA NPs can effectively stimulate immune responses, providing strong evidence for anti-tumor immunotherapy.

[0087] This patent successfully designed a novel nanodrug delivery system, COL / DTX@BSA NPs, to improve the efficiency of tumor treatment while reducing the side effects of chemotherapy drugs and enhancing the body's immunity. The anti-tumor effect and immune activation potential of COL / DTX@BSA NPs were comprehensively evaluated through physicochemical characterization and a series of in vitro and in vivo experiments. COL / DTX@BSA NPs were confirmed to have significant anti-tumor activity, effectively inhibiting tumor cell proliferation and promoting immunogenic cell death. Transmission electron microscopy and multiple biological assays confirmed the uniform morphology, stability, and efficient drug loading capacity of the NPs. Experimental results in an in vitro 3D cell spheroid model and an in vivo tumor model further validated the advantages of NPs in improving drug permeability and enhancing immune responses. Furthermore, COL / DTX@BSA NPs exhibited good biocompatibility and safety, providing a solid foundation for their future clinical application.

[0088] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the nanomedicine based on collagenase penetration and low-dose chemotherapy-induced tumor immunotherapy, its preparation method, and application, but the present invention is not limited to the detailed methods in the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary ingredients, selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims

1. A nanomedicine based on collagenase infiltration strategy, characterized in that: Bovine serum albumin (BSA) was used as a carrier to co-encapsulate collagenase (COL) and low-dose docetaxel (DTX) via self-assembly to form nanoparticles COL / DTX@BSANPs. Among them, the BSA carrier maintains the stability of the protein secondary structure and has a protective effect on COL; The COL is used to hydrolyze collagen in the extracellular matrix of tumor cells, thereby enhancing the deep tumor penetration ability of the nanoparticles; The dose of DTX is a low therapeutic dose, which is used to induce tumor immunogenic cell death and activate anti-tumor immune response.

2. The nanomedicine according to claim 1, characterized in that The average particle size of the nanoparticles is 50 to 200 nm.

3. The nanomedicine according to claim 1, characterized in that The drug loading of DTX was 1.3% to 3.3% (w / w).

4. The nanomedicine according to claim 1, characterized in that The activity of the COL is 0.06-0.07 Units / mg.

5. A method for preparing the nanomedicine according to any one of claims 1 to 4, characterized in that: The following steps are involved: (a) Dissolve BSA in PBS buffer (pH 7.2-7.6) to form a BSA solution; (b) dissolving COL in PBS buffer and mixing with the solution from step (a); (c) dissolving DTX in an organic solvent and adding the solution to the mixture of step (b); (d) adding glutaraldehyde as a cross-linking agent, and stirring at 450 ± 50 rpm for 12 hours; (e) The reaction solution was purified by ultrafiltration (ultrafiltration membrane molecular weight cutoff 100 kDa) and sterilized by filtration to obtain a nanoparticle suspension.

6. The preparation method according to claim 5, characterized in that: The organic solvent in step (c) is acetone; In step (d), the mass ratio of BSA, COL and DTX is 200:1:10; The ultrafiltration conditions in step (e) are: centrifugation at 4500-5500 rpm, 3 minutes each time, repeated 3 times.

7. The preparation method according to claim 5, characterized in that The obtained nanoparticles are a clear, transparent milky white solution with a stability of ≥7 days under storage conditions at 4°C.

8. Use of the nanomedicine according to any one of claims 1 to 4 in the preparation of a drug for treating solid tumors, characterized in that: The nanomedicine enhances deep penetration by hydrolyzing the tumor collagen barrier through COL and induces immunogenic cell death through low-dose DTX, synergistically activating anti-tumor immune responses.

9. The use according to claim 8, characterized in that The solid tumors include breast cancer and melanoma.

10. The use according to claim 8, characterized in that The anti-tumor immune response is manifested as at least one of the following: (i) HMGB1, ATP, and calreticulin expression are upregulated in the tumor microenvironment; (ii) increased secretion levels of TNF-α and IFN-γ; (iii) Increased CD8+ and CD4+ T cell infiltration.

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