Lactic acid bacteria vesicle-based targeted tumor carrier and its preparation method and application

Abstract

The present invention belongs to the technical field of pharmaceutical carriers for targeting tumors, and specifically relates to a targeted tumor carrier based on lactic acid bacteria vesicles, and its preparation method and application. The targeted tumor carrier based on lactic acid bacteria vesicles of the present invention comprises lactic acid bacteria vesicles and DSPE-PEG2000-cRGD anchored on the surface of the lactic acid bacteria vesicles. The targeted tumor carrier of the present invention can effectively and actively deliver SR-18292 and doxorubicin into tumor cells, thereby effectively inhibiting tumor cell apoptosis, significantly inhibiting tumor cell migration and invasion, and thus exerting a significant anti-tumor therapeutic effect. In addition, the tumor drug delivery system of the present invention has good biosafety.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical carriers targeting tumors, and specifically relates to a tumor-targeting carrier based on lactic acid bacteria vesicles, and a preparation method and application thereof. Background Art

[0002] Ovarian cancer is the second most common gynecological malignancy. Due to its insidious nature and ineffective treatment, it has the highest mortality rate among gynecological malignancies. Most cases of ovarian cancer are diagnosed at an advanced stage, and current treatment options include surgery, radiotherapy, and chemotherapy. First-line chemotherapy agents for ovarian cancer include platinum-based chemotherapy agents and paclitaxel, but their efficacy is limited and recurrence and drug resistance are common. Chemoresistance to platinum-based chemotherapy is a major challenge in the clinical treatment of ovarian cancer. To overcome recurrence caused by drug resistance and prolong survival in patients with advanced ovarian cancer, it is necessary to explore novel treatment modalities to improve the clinical efficacy of ovarian cancer. Emerging treatments for ovarian cancer, including immunotherapy, gene therapy, and targeted therapy, have demonstrated positive results in the treatment of ovarian cancer due to their respective advantages. Combining them with chemotherapy may offer even better efficacy. With in-depth research into the mechanisms of chemoresistance in cancer cells, the active exploration of new targets to combat chemoresistance is also an effective approach to improve the quality of life for patients with advanced ovarian cancer. The recent development and maturity of nanotechnology has become a valuable alternative for improving drug delivery efficiency and reducing drug toxicity and side effects. Nanoparticles can precisely deliver drugs to cancer cells, leading to higher drug concentrations at the tumor site while reducing the toxic side effects of free drugs. Nanotechnology-based drug delivery systems have the potential to enhance the therapeutic efficacy of anti-tumor drugs, reduce chemotherapy drug resistance, and thus improve the prognosis of patients with advanced ovarian cancer.

[0003] Nanocarriers can enhance drug bioavailability at tumor sites through passive or active targeting. Passive targeting of nanoparticles relies on the enhanced permeability and retention effect (EPR) at tumor sites, also known as the EPR effect. Compared to normal tissue, solid tumors exhibit abnormally proliferating blood vessels, wide interstitial spaces, and poor lymphatic drainage, resulting in selective permeability and retention for certain macromolecules, leading to their increased tendency to accumulate in tumor tissue. Actively targeted nanocarriers further modify the nanoparticle surface with ligands or antibodies that specifically interact with tumor cells, enabling the nanoparticles to actively target tumor cells and promote selective drug accumulation at the tumor site. Nanodelivery vehicles have been a research hotspot in targeted tumor therapy, and some have achieved clinical translation. Nanomaterials are diverse, offering advantages such as adjustable shape and size, ease of synthesis and modification, and the ability to be designed and functionalized based on their inherent properties to enhance drug delivery efficiency. Currently, widely used lipid nanoparticles, inorganic nanoparticles, and polymer nanoparticles achieve effective targeted drug delivery, but they suffer from drawbacks such as high toxicity, poor stability, and poor metabolism. As more novel nanomaterials are developed for anti-tumor drug delivery, the toxicity and safety of carriers remain a challenge. Therefore, it is still necessary to find nanocarriers with good biocompatibility and high drug delivery efficiency to achieve better anti-tumor therapeutic effects. Summary of the Invention

[0004] One of the purposes of the present invention is to provide a tumor-targeting carrier based on lactic acid bacteria vesicles with good biocompatibility.

[0005] A second object of the present invention is to provide a method for preparing a tumor-targeting carrier based on lactic acid bacteria vesicles.

[0006] The third purpose of the present invention is to provide an application of a tumor-targeting carrier based on lactic acid bacteria vesicles.

[0007] One of the purposes of the present invention is achieved by the following technical solution:

[0008] A tumor-targeting carrier based on lactic acid bacteria vesicles comprises lactic acid bacteria vesicles and DSPE-PEG2000-cRGD anchored on the surface of the lactic acid bacteria vesicles.

[0009] Furthermore, the lactic acid bacteria vesicles are obtained by inoculating and culturing Lactobacillus rhamnosus and concentrating through differential centrifugation-ultrafiltration; the average diameter of the lactic acid bacteria vesicles is 80-100 nm, and the hydrated particle size is 85-105 nm.

[0010] Furthermore, the structural formula of the DSPE-PEG2000-cRGD is as follows:

[0011]

[0012] The second object of the present invention is achieved by adopting the following technical solution:

[0013] The preparation method of the above-mentioned tumor-targeting carrier based on lactic acid bacteria vesicles comprises the following steps:

[0014] The DSPE-PEG2000-cRGD solution was mixed with the lactic acid bacteria vesicles, incubated, and then centrifuged to obtain the lactic acid bacteria vesicle-based targeted tumor vector, which was designated as cRGD-CMVs.

[0015] Furthermore, the mass ratio of the DSPE-PEG2000-cRGD and the lactic acid bacteria vesicles is 1-2:1, and the concentration of the DSPE-PEG2000-cRGD solution is 0.5-1.5 mg / mL.

[0016] Furthermore, the incubation temperature is 35-40° C. and the incubation time is 3.5-4.5 hours.

[0017] Furthermore, the centrifugation process is: centrifugation at 3500×g-4500×g for 8-12 minutes at 3-5°C in an ultrafiltration centrifuge tube, washing with PBS, and then centrifuging at 130,000×g-150,000×g in a centrifuge for 2.5-3.5 hours.

[0018] Furthermore, the molecular weight cut-off of the ultrafiltration centrifuge tube is 100KDa.

[0019] The third object of the present invention is achieved by adopting the following technical solution:

[0020] The above-mentioned tumor-targeted carrier based on lactic acid bacteria vesicles is used to prepare a tumor-targeted drug delivery system that is simultaneously loaded with SR-18292 and chemotherapy drugs.

[0021] Furthermore, the tumor is ovarian cancer; the loading adopts electroporation technology; and the chemotherapy drug is doxorubicin.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention successfully constructed a targeted tumor carrier based on lactic acid bacteria vesicles. Experimental results showed that the targeted tumor drug delivery system obtained by loading cRGD-CMVs with SR-18292 and the chemotherapy drug doxorubicin can effectively and actively deliver SR-18292 and doxorubicin into tumor cells, thereby effectively inhibiting tumor cell apoptosis and significantly inhibiting tumor cell migration and invasion, thereby exerting a significant anti-tumor therapeutic effect. In vivo imaging results showed that cRGD-CMVs can concentrate at the tumor site and have good tumor targeting. In addition, based on the changes in mouse weight, blood routine and liver and kidney function indicators, as well as tissue sections, the targeted tumor drug delivery system obtained by loading cRGD-CMVs with SR-18292 and doxorubicin has good biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the process of CMVs extraction and cRGD-CMVs construction in Example 1;

[0025] Figure 2 This is a graph showing the yield of CMVs extracted in Example 1;

[0026] Figure 3 is the particle size and potential diagram of CMVs extracted in Example 1;

[0027] Figure 4 is a TEM morphology image of CMVs extracted in Example 1;

[0028] Figure 5 This is a diagram showing the connection of cRGD-CMVs prepared in Example 1;

[0029] Figure 6 is a graph showing the particle size and potential results of cRGD-CMVs prepared in Example 1;

[0030] Figure 7 is a TEM image of cRGD-CMVs prepared in Example 1;

[0031] Figure 8 This is the result diagram of the optimal mass ratio of cRGD-CMVs and SR-18292;

[0032] Figure 9 Figure 2 is the particle size and potential results of cRGD-CMVs@SR / DOX prepared in Example 2;

[0033] Figure 10 This is the TEM morphology of cRGD-CMVs@SR / DOX prepared in Example 2;

[0034] Figure 11 This is a diagram showing the effect of electroporation on CMVs-targeted vesicles;

[0035] Figure 12 : is a graph showing the toxicity test results of the cRGD-CMVs vector prepared in Example 1 and different preparations on ovarian cancer cells; Figure 12 A is a graph showing the toxicity test results of the cRGD-CMVs vector prepared in Example 1 on ovarian cancer cells. Figure 12 B is the toxicity test results of different preparations on ovarian cancer cells;

[0036] Figure 13 This is an analysis chart of the qualitative uptake results of different preparations on ovarian cancer cells;

[0037] Figure 14 This is a 4-hour qualitative uptake analysis of different preparations on ovarian cancer cells;

[0038] Figure 15 This is an analysis of the mitochondrial membrane potential results of different preparations on ovarian cancer cells;

[0039] Figure 16 This is an analysis of the effects of different preparations on ROS levels in ovarian cancer cells;

[0040] Figure 17 This is an analysis of the effects of different preparations on the apoptosis of ovarian cancer cells;

[0041] Figure 18 This is an analysis chart of the expression of intracellular apoptosis-related proteins by different preparations;

[0042] Figure 19 This is the analysis of the effects of different preparations on the tumor size of tumor-bearing mice;

[0043] Figure 20 The results of the analysis of the tumor inhibition rate of different preparations on tumor mice are shown in Figure A, where Figure A is the tumor volume result diagram and Figure B is the tumor inhibition rate diagram;

[0044] Figure 21 This is an analysis of the effects of different preparations on the apoptosis level of tumor tissue;

[0045] Figure 22 This is a graph analyzing the semi-quantitative results of TUNEL fluorescence of different preparations;

[0046] Figure 23 This is the result analysis of the expression levels of the anti-apoptotic protein BCL-2 and the pro-apoptotic protein BAX in different preparations;

[0047] Figure 24The in vivo safety evaluation results of different preparations are analyzed in Figure A, where Figure A shows weight changes (n=5), Figure B shows blood routine parameters (n=3), Figure C shows liver function indicators ALT and AST (n=3), and Figure D shows renal function indicators UREA and CREA (n=3);

[0048] Figure 25 These are H&E-stained sections of different preparations. DETAILED DESCRIPTION

[0049] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but this does not constitute a limitation to the protection scope of the present invention.

[0050] In the following examples, DSPE-PEG2000-cRGD was prepared by Nanjing Taopu Biotechnology Co., Ltd. according to the inventor's requirements; SR-18292 was purchased from CSN Pharm; doxorubicin hydrochloride (DOX) was purchased from Dalian Meilun Biotechnology Co., Ltd.; Lactobacillus rhamnosus (ATCC53103) was purchased from Hangzhou Baosai Biotechnology; DSPE-PEG2000-FITC was purchased from Shanghai Pengshuo Biotechnology Co., Ltd.; penicillin G sodium was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; BCA protein quantification kit was purchased from Shanghai Yazyme Biotechnology Co., Ltd.; and DID kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd. All other reagents and materials, unless otherwise specified, are commercially available.

[0051] Example 1

[0052] The targeted tumor carrier (cRGD-CMVs) based on lactic acid bacteria vesicles of this embodiment includes lactic acid bacteria vesicles and DSPE-PEG2000-cRGD anchored on the surface of the lactic acid bacteria vesicles.

[0053] The preparation process of the lactic acid bacteria vesicles (CMVs) is as follows:

[0054] Lactobacillus rhamnosus (LGG) strain, frozen at -80°C, was thawed in warm water at 37°C, inoculated onto plates using the streak method, and incubated at 30°C for 36 hours. Single colonies were selected and activated by static culture for 20 hours. The culture was then inoculated into blank MRS liquid medium at a volume ratio of 1:100, with blank medium serving as a control. The OD600 value of the bacterial culture was measured using a microplate reader. When the OD600 value reached 0.5, 0.5 μg / mL penicillin G sodium was added. When the OD600 value reached 1, the culture was centrifuged at 5000 × g for 30 minutes at 4°C to remove most of the cells. The supernatant was filtered through 0.45 μm and then 0.22 μm microporous membranes to remove residual cells. The supernatant was then concentrated using an ultrafiltration centrifuge tube (MWCO = 100 kDa) at 4000 × g for 10 minutes at 4°C. Impurities generated during concentration were removed by filtration through a 0.22 μm filter. Then, centrifuge in an ultracentrifuge at 150,000 × g for 3 hours at 4°C, discard the supernatant, resuspend in PBS, transfer to an ultrafiltration centrifuge tube, wash repeatedly with PBS, and concentrate to purify CMVs. Sterilize by filtering with a 0.22 μm filter. Finally, centrifuge at 150,000 × g for 3 hours at 4°C, and resuspend the pellet in PBS to obtain lactic acid bacterial vesicles for future use.

[0055] The tumor-targeting carrier of the lactic acid bacteria vesicles in this embodiment is a DSPE-PEG2000-cRGD-linked lactic acid bacteria vesicles by a biolinking method. After the linking, the lactic acid bacteria vesicles are fluorescently co-localized using laser confocal microscopy to determine whether the lactic acid bacteria vesicles and the cyclic peptide cRGD are successfully coupled.

[0056] Schematic diagram of lactic acid bacteria vesicle extraction, lactic acid bacteria vesicles and cyclic peptide cRGD bioconnection through DSPR-PEG2000 to prepare cRGD-CMVs Figure 1 The specific steps are as follows:

[0057] The chemically synthesized DSPE-PEG2000-cRGD (synthesized by a biotechnology company) was fully dissolved in PBS to obtain a DSPE-PEG2000-cRGD solution with a concentration of 1 mg / mL. The solution was evenly mixed with CMVs at a mass ratio of 1:1 and incubated at 37°C for 4 hours. The solution was then centrifuged at 4000×g for 10 minutes at 4°C in an ultrafiltration centrifuge tube (MWCO=100KDa). The solution was washed with PBS several times and then centrifuged at 150,000×g for 3 hours in a centrifuge. The precipitate was resuspended in PBS to obtain a tumor-targeting vector based on lactic acid bacteria vesicles, which was designated as cRGD-CMVs.

[0058] Example 2

[0059] This example is the application of a tumor-targeted carrier based on lactic acid bacteria vesicles, specifically the application of a tumor-targeted carrier based on lactic acid bacteria vesicles in the preparation of a tumor-targeted drug delivery system that simultaneously loads SR-18292 and chemotherapy drugs.

[0060] In this example, the technology used in preparing the targeted tumor drug delivery system loaded with SR-18292 and chemotherapy drugs at the same time is electroporation technology, the chemotherapy drug is doxorubicin, and the prepared targeted drug delivery system loaded with SR-18292 and chemotherapy drug doxorubicin is recorded as cRGD-CMVs@SR / DOX.

[0061] Test Example 1 Identification of CMVs

[0062] 1.1. CMVs yield

[0063] The yield of CMVs is an important evaluation index for evaluating whether it can be used as a vector source. In this test example, a culture medium without penicillin G was used to culture Lactobacillus rhamnosus and extract CMVs as a blank control group. The protein concentration of CMVs extracted from Example 1 and the blank control group was detected using a BCA protein analysis kit. The results are as follows: Figure 2 shown.

[0064] Figure 2 This is the yield result of CMVs extracted in Example 1. Figure 2 It can be seen that the content of CMVs extracted in Example 1 is 1.626 ± 0.049 mg / mL, and the content of CMVs taken in the blank control group is 0.623 ± 0.013 mg / mL, and the difference is statistically significant (****P<0.0001). As can be seen from the above, the addition of penicillin G sodium can increase the yield of CMVs. This is because the addition of antibiotics interferes with the synthesis of the cell wall of Lactobacillus rhamnosus, destroys the integrity of the cell wall, and can promote the release of more CMVs. Therefore, penicillin G sodium was added to increase yield when the CMVs carrier was subsequently extracted.

[0065] 1.2 Particle size, potential, morphology and stability

[0066] The particle size and potential of the CMVs extracted in Example 1 were measured using a laser particle size analyzer. The results are as follows: Figure 3 shown.

[0067] The morphology of CMVs extracted from Example 1 was characterized using a transmission electron microscope (TEM). Figure 4 shown.

[0068] Figure 3 The particle size and potential of CMVs extracted in Example 1 are shown in FIG. Figure 3 It can be seen that the hydrated particle size of CMVs is 97.09±1.01nm (see Figure 3 A), the zeta potential of CMVs was -34.95±0.95 mV (see Figure 3 B), the polydispersity index (PDI) obtained by the instrument was 0.132±0.033. The above results show that the particle size of the CMVs prepared by the present invention is relatively uniform and can be used for subsequent experiments.

[0069] Figure 4 is a TEM morphology of CMVs extracted in Example 1. Figure 4 It can be seen that CMVs have a distinct vesicle structure and a phospholipid bilayer structure. The overall shape is approximately round, and it presents a red blood cell-like structure with one side concave inward. The diameter of CMVs is about 80-100nm, and the particle size distribution is relatively uniform.

[0070] Experimental Example 2 Identification of cRGD-CMVs

[0071] 2.1. Laser confocal microscopy was used to determine the connection of cRGD-CMVs prepared in Example 1

[0072] Since the preparation method of the present invention was intended to demonstrate the successful connection of lactic acid bacteria vesicles (CMVs) with DSPE-PEG2000-cRGD, and DSPE-PEG2000-cRGD could not be labeled with FITC, this experimental example fluorescently labeled the lactic acid bacteria vesicles and DSPE-PEG2000, thereby visually determining whether the vesicles and polypeptides were successfully connected by fluorescence colocalization. The fluorescent labeling process is as follows:

[0073] First, CMVs were incubated with DiD (chemical name: 1,1-octadecyl-3,3,3,3-tetramethylindole dicarbocyanine) at 37°C in the dark for 20 min, and then ultracentrifuged at 4°C, 150,000×g, for 3 h to obtain fluorescently labeled CMVs (DiD-CMVs); chemically synthesized fluorescently labeled DSPE-PEG2000-FITC (synthesized by a biotechnology company) was fully dissolved in PBS to obtain DSPE-PEG2000 with a concentration of 1 mg / mL. 000-FITC solution was mixed with DiD-CMVs at a mass ratio of 1:1 and incubated at 37°C for 4 hours. Then, an ultrafiltration centrifuge tube (MWCO = 100KDa) was used to centrifuge at 4°C at 4000×g for 10 minutes. PBS was repeatedly washed several times to remove free DSPE-PEG2000-FITC. Then, an ultrahigh-speed centrifuge was used to centrifuge at 150000×g for 3 hours. The precipitate was resuspended in PBS to obtain the fluorescently labeled lactic acid bacteria vesicle-based carrier. After obtaining the carrier, a laser confocal microscope was used to observe the fluorescence co-localization in different channels. The results are shown in FIG. Figure 5 shown.

[0074] Figure 5 This is a diagram showing the connection of cRGD-CMVs prepared in Example 1. Figure 5 As can be seen, CMVs are stained with DiD, resulting in red fluorescence under a microscope, while DSPE-PEG2000 is labeled with FITC, resulting in green fluorescence under a microscope. Images of the two fluorescence channels were captured using a laser confocal microscope and then merged to observe colocalization. The images show effective overlap and uniform fluorescence from the two merged channels, demonstrating that DSPE-PEG2000 can attach FITC to the vesicles. Similarly, cRGD-CMVs were successfully constructed.

[0075] 2.2 Analysis of particle size, potential, and TEM morphology changes

[0076] The particle size and potential of cRGD-CMVs were tested using the same method as in Experimental Example 1. Figure 6 shown.

[0077] The cRGD-CMVs prepared in Example 1 were characterized using transmission electron microscopy. Figure 7 shown.

[0078] Figure 6 The graph is the particle size and potential results of cRGD-CMVs prepared in Example 1. Figure 6 The hydrated particle size of cRGD-CMVs was 98.28 ± 1.46 nm, the potential was -35.98 ± 0.88 mV, and the PDI was 0.135 ± 0.035. The attachment of cRGD slightly decreased the potential of the vesicles and slightly increased their particle size, while maintaining good particle dispersibility.

[0079] Figure 7 This is a TEM image of cRGD-CMVs prepared in Example 1. Figure 7 It can be seen that cRGD connection did not affect the structure and morphology of the vesicles, which still showed an inward-concave hemispherical structure, and it was visually found that the particle size did not change significantly.

[0080] Experimental Example 3 Application Effect of Lactobacillus Vesicle-Based Targeted Tumor Carrier Loaded with SR-18292 / DOX (cRGD-CMVs@SR / DOX)

[0081] 3.1. Investigation of drug loading conditions

[0082] In order to fully utilize the carrier and drug, the ratio of carrier to drug added during electroporation was investigated.

[0083] 40 μg of cRGD-CMVs were immobilized and loaded with SR-18292 at mass ratios of 1:1, 1:2, and 2:1, respectively, under electroporation conditions of 40 V and 100 μF. Triplicate samples were prepared for each group. Encapsulation efficiency and drug loading efficiency were determined. Encapsulation efficiency and drug loading efficiency were calculated as follows: Encapsulation efficiency (wt.%) = (loaded drug amount / drug amount) × 100%; drug loading efficiency (wt.%) = [loaded drug amount / (drug amount + carrier amount)] × 100%.

[0084] The mass of cRGD-CMVs was fixed at 40 μg, and the mass of SR-18292 was fixed at 40 μg. According to the combined concentrations of SR-18292 and DOX, which were 20 μM (7.33 μg / mL) and 0.25 μg / mL, the amount of DOX was changed, and cRGD-CMVs and DOX were mixed. The drug loading rate and encapsulation efficiency were measured in the same electroporation method to determine the optimal mass ratio of drug loading. The drug loading rate and encapsulation efficiency detection methods were the same as above, and the results are shown in Table 1. Figure 8 shown.

[0085] Table 1 Investigation of the optimal mass ratio of cRGD-CMVs and SR-18292

[0086]

[0087] Figure 8 This is the result of the best mass ratio of cRGD-CMVs and SR-18292. Figure 8 It can be seen that the encapsulation efficiency and drug loading capacity are the highest when the ratio of cRGD-CMVs to SR-18292 is 1:1. Then, the ratio of cRGD-CMVs to SR-18292 is fixed at 1:1, and the appropriate ratio is set for co-loading of drugs based on the required DOX concentration and loading efficiency. It has been verified that when the ratio of SR-18292 to DOX is 8:1, the SR-18292 encapsulation efficiency is 50.18±1.96% and the drug loading capacity is 33.4±0.87%. The DOX encapsulation efficiency is 12.83±0.96% and the drug loading capacity is 1.58±0.17%, which is basically in line with the dosing concentration. However, the encapsulation efficiency of both drugs decreased, which may be due to the competition between the two drugs when co-loading.

[0088] 3.2 Particle size, potential, and TEM morphology of cRGD-CMVs@SR / DOX

[0089] An appropriate amount of electroporated drug-loaded vesicles (the drug-loaded vesicles were prepared using the aforementioned preferred process conditions: electroporation voltage 40V, capacitance 100μF; cRGD-CMVs:SR:DOX = 1:1:0.125) were taken and the morphology after electroporation and drug loading was characterized using a transmission electron microscope. The particle size and potential of the drug-loaded vesicles were measured using a laser particle size analyzer. The results are shown in Figure 2. Figure 9-10 shown.

[0090] Figure 9 The graph shows the particle size and potential of cRGD-CMVs@SR / DOX prepared in Example 2. Figure 9 It can be seen that the hydrated particle size of cRGD-CMVs@SR / DOX is 172.2±6.52nm, the potential is -40.81±2.20mV, and the PDI is 0.307±0.018.

[0091] Figure 10 This is the TEM morphology of cRGD-CMVs@SR / DOX prepared in Example 2. Figure 10 It can be seen that the structure and morphology of the vesicles were not destroyed after electroporation, but the particle size of some vesicles increased to about 200 nm.

[0092] 3.3 Effect of electroporation on CMV-targeted vesicles

[0093] In order to determine whether electroporation affects the connection between cRGD and CMVs vesicles, laser confocal microscopy was used to observe the co-localization of peptides and vesicles. Specifically, laser confocal microscopy was used to image and determine whether electroporation destroyed the connection between peptides and vesicles based on the presence of fluorescence co-localization. Figure 11 shown.

[0094] Figure 11 This is a diagram showing the effect of electroporation on targeting CMVs vesicles. Figure 11 It can be seen that the green fluorescence is FITC labeled with DSPE-PEG2000-cRGD, and the red fluorescence is the fluorescence after DiD staining CMVs. The two fluorescences overlap, indicating that electroporation does not affect the connection between cRGD and CMVs, indicating that the cRGD-CMVs vector has high stability.

[0095] Experimental Example 4 In vitro cytological study of cRGD-CMVs@SR / DOX

[0096] 4.1. CCK-8 cytotoxicity assay

[0097] 4.1.1. Cytotoxicity of the vector

[0098] After counting the cells of SK-OV-3 cells in good growth condition, they were seeded into 96-well plates at a density of 8,000 cells per well. The concentration of the required vector was calculated to be 14.66 μg / mL at the concentration of the drug. Therefore, the vector concentrations were set to 20, 40, 60, and 80 μg / mL, with 4 replicates for each concentration. A control group and a blank group were also set. When the cell density reached above 80%, the above-prepared drug was added and cultured for 24 hours. The culture medium was discarded, and a blank culture medium containing 10% volume of CCK-8 was added to each well. The cells were incubated in the dark for 1 hour. The absorbance at 450 nm was measured with a microplate reader, and the cell survival rate and inhibition rate were calculated. The inhibition rate calculation formula is: The results are as follows Figure 12 shown.

[0099] 4.1.2 Cytotoxicity of Different Formulations

[0100] For free SR-18292+DOX, CMVs@SR / DOX, and cRGD-CMVs@SR / DOX, the drug concentrations were set to SR-18292 (20 μM) and DOX (0.2 μg / mL). Four replicate wells were set up in each group. After culturing for 24 h, 48 h, and 72 h, the culture medium was discarded, and blank culture medium containing 10% volume of CCK-8 was added to each well. The cells were incubated in the dark for 1 h. The absorbance at 450 nm was measured with a microplate reader, and the cell viability and inhibition rate were calculated using the same formula as in 4.1.1. The results are shown in Figure 4. Figure 12 shown.

[0101] Figure 12 The figure is a graph showing the toxicity test results of the cRGD-CMVs vector prepared in Example 1 and different preparations on ovarian cancer cells. Figure 12 A is a graph showing the toxicity test results of the cRGD-CMVs vector prepared in Example 1 on ovarian cancer cells.

[0102] Figure 12 B is the toxicity test results of different preparations on ovarian cancer cells. Figure 12 As shown in Figure A, cell viability remained above 85% at a concentration of 80 μg / mL for both vectors, and increased as the vector concentration decreased. There was no significant difference in the cytotoxicity of CMVs and cRGD-CMVs. This indicates that the vectors lack significant cytotoxicity at therapeutic concentrations, and that the construction of targeted vectors does not increase vector toxicity.

[0103] Depend on Figure 12B shows that the cytotoxicity of the preparation group is significantly improved compared with the free drug. At 24h, the cell inhibition rate of CMVs@SR / DOX and cRGD-CMVs@SR / DOX is significantly different from that of the free drug (****P<0.0001). The difference decreases with the extension of the drug action time, but it is still statistically significant. It may be because the drug has reached its final inhibitory effect after a certain period of action. There are significant differences in the cell inhibition rate of CMVs@SR / DOX and cRGD-CMVs@SR / DOX at different action times, indicating that the targeted preparation group can increase the cell uptake efficiency of the drug and produce stronger cytotoxicity. In summary, the results show that the vector extracted and constructed in this project has high in vitro safety, the synthesized preparation can increase the targeted uptake of the drug and the anti-tumor effect, and cRGD-CMVs@SR / DOX has both good safety and therapeutic effect.

[0104] 4.2 Qualitative Cell Uptake Assay

[0105] After counting the number of SK-OV-3 cells that were growing well, 9×10 4 The cells were seeded at a density of 100 cells per well into a 12-well plate with a cell slide placed in advance. FITC was used instead of SR-18292 / DOX. FITC group, DiD-CMVs@FITC group, and DiD-cRGD-CMVs@FITC group were set up. The concentration of FITC in each group was 8 μg / mL. The drug was administered when the cell density reached 60% to 70%. After incubation for 1, 2, 4, and 6 hours, the drug-containing culture medium was discarded and the cells were washed 3 times with PBS. The cells were fixed with 4% paraformaldehyde for 15 minutes. After 15 minutes, the paraformaldehyde was removed and washed 3 times with PBS. 1 μg / mL DAPI stain was added in the dark for 15 minutes. After incubation, it was washed 3 times with PBS. The slide was buckled out and inverted on a glass slide with anti-fluorescence attenuation mounting medium. A laser confocal microscope was used to capture images and observe cell uptake. The cell uptake of each group of preparations at different time points was observed and photographed under a laser confocal microscope. The results are shown in the figure. Figure 13-14 shown.

[0106] Figure 13 This is an analysis chart of the qualitative uptake results of different preparations on ovarian cancer cells. Figure 13 The fluorescence intensities of the CMVs@FITC and cRGD-CMVs@FITC groups were higher than those of the free FITC group, with a significant increase at 2 hours and reaching saturation after 4 hours, indicating that the vesicles facilitated drug entry into cells. Furthermore, the fluorescence intensity of cRGD-CMVs@FITC was even higher than that of CMVs@FITC, indicating that the targeting peptide enhanced the efficiency of drug uptake by cells.

[0107] Figure 14 This is a 4-hour qualitative analysis of the uptake of different preparations on ovarian cancer cells. Figure 14 As can be seen, after DiD staining, CMVs exhibited red fluorescence, which was highly colocalized with green fluorescence, demonstrating that the drug was successfully loaded into the vesicles and taken up into the cells through the vesicles. The formulation group showed significantly higher fluorescence intensity than the free FITC group. Furthermore, the fluorescence intensity of DiD-cRGD-CMVs@FITC was higher than that of DiD-CMVs@FITC, indicating a higher efficiency of targeted vesicle uptake. These results demonstrate that cRGD-CMVs effectively encapsulates drugs and delivers them to cells in a targeted manner, achieving better efficacy.

[0108] 4.3 Mitochondrial membrane potential detection (JC-1) test

[0109] SK-OV-3 cells with good growth status were taken for cell counting and the number of cells was 1.5×10 5 The cells were seeded into a 12-well plate at a density of 100 cells and cultured for 24 hours. Five drug-dosing groups were set up: SR-18292, DOX, SR+DOX, CMVs@SR / DOX, and cRGD-CMVs@SR / DOX. The drug concentrations were set to SR-18292 (20 μM) and DOX (0.25 μg / mL). A negative control group was also set up. The JC-1 staining working solution and washing solution were prepared according to the instructions of the JC-1 test kit, and the washing solution was stored in an ice bath. After 24 hours of drug culture, the drug-containing culture medium was aspirated and discarded, and the cells were washed three times with PBS. An equal volume of culture medium and staining working solution was added to each well, and the cells were placed in an incubator in the dark and incubated for 20 minutes. After the staining was completed, the working solution was discarded, and the cells were washed three times with washing solution. The changes in cell fluorescence were observed and photographed under a fluorescence microscope. The results are as follows Figure 15 shown.

[0110] A decrease in mitochondrial membrane potential (MMP) is a hallmark of early apoptosis, and apoptosis can cause changes in MMP. JC-1 is a fluorescent probe used to detect MMP, and the degree of mitochondrial depolarization can be determined by the red / green fluorescence ratio. Figure 15 This is an analysis of the effects of different preparations on the mitochondrial membrane potential of ovarian cancer cells. Figure 15It is known that normal cells exhibit strong red fluorescence and almost no green fluorescence. After cell treatment with drugs, red fluorescence weakened and green fluorescence increased, indicating a partial decrease or loss of MMP. Cells treated with a single drug showed red / green fluorescence co-localization, while the SR+DOX group showed increased green fluorescence and decreased red fluorescence intensity compared to the single drug group, indicating an increase in mitochondrial damage. Compared with the combination drug group, the red fluorescence intensity of the CMVs@SR / DOX and cRGD-CMVs@SR / DOX groups was further reduced, and fluorescence co-localization was reduced, indicating a higher degree of mitochondrial depolarization and that the agent induced more cell apoptosis. In summary, the results show that both SR-18292 and DOX can cause a decrease in cellular MMP. It is speculated that SR-18292 acts on PGC-1α protein, inhibiting mitochondrial biogenesis, thereby leading to mitochondrial damage; while DOX can cause a decrease in MMP by inducing cell apoptosis. The combined use of the two drugs enhanced the effect. CMVs@SR / DOX and cRGD-CMVs@SR / DOX could further enhance the degree of mitochondrial depolarization, indicating that the preparations could increase the cellular uptake of drugs and improve the drug's efficacy.

[0111] 4.4 Reactive oxygen species (ROS) detection test

[0112] SK-OV-3 cells with good growth status were taken for cell counting and the number of cells was 1.5×10 5 The cells were seeded into a 12-well plate at a density of 100 cells and cultured for 24 hours. Five drug-dosing groups were set up, including SR-18292, DOX, SR+DOX, CMVs@SR / DOX, and cRGD-CMVs@SR / DOX. The drug concentrations were set to SR-18292 (20 μM) and DOX (0.25 μg / mL). A negative control group was also set up. After 24 hours of drug culture, the drug-containing culture medium was discarded, the cells were washed three times with PBS, and the 2',7'-dichlorofluorescein diacetate (DCFH-DA) staining working solution was prepared according to the instructions of the ROS kit. 1 mL was added to each well and the cells were placed in an incubator in the dark for 30 minutes. After the staining was completed, the working solution was discarded, the cells were washed three times with PBS, and the intracellular fluorescence was observed and photographed under a fluorescence microscope. The results are as follows Figure 16 shown.

[0113] ROS is involved in the process of cell apoptosis. Increased intracellular ROS levels induce cell apoptosis. The DCFH-DA fluorescent probe detects changes in intracellular ROS levels, and the fluorescence intensity can reflect the level of intracellular ROS. Figure 16 This is an analysis of the effects of different preparations on ROS levels in ovarian cancer cells. Figure 16As shown, both drug-addition groups induced increased ROS production in cells. The combination group exhibited greater green fluorescence compared to the single-drug groups, and the formulation group exhibited an even greater increase. This suggests that the combination of SR-18292 and DOX can induce increased ROS production in cells. It is speculated that SR-18292 may induce mitochondrial ROS (MitoROS) production through mitochondrial damage, though the specific mechanism remains to be verified. cRGD-CMVs@SR / DOX improved the efficiency of drug uptake by cells and enhanced the drug's ability to induce ROS production.

[0114] 4.5 Cell apoptosis assay

[0115] SK-OV-3 cells with good growth status were taken and counted, and 2×10 5 The cells were seeded in a 6-well plate, and the drug treatment was given after the cell concentration reached 60% to 80%. The cells were grouped as follows: SR-18292, DOX, SR+DOX, CMVs@SR / DOX, cRGD-CMVs@SR / DOX, and the drug concentrations were set to SR-18292 (20μM) and DOX (0.25μg / mL). A control group was also set up. After 24 hours of culture, the 6-well plate was taken out, washed three times with PBS, and the cells were digested with EDTA-free trypsin and washed twice with PBS. The cells should be blown gently during the period to avoid mechanical damage. After staining with an apoptosis kit, the flow cytometer was used for detection. The Flowjo software was used to statistically analyze the results and the flow detection results were as follows. Figure 17 shown.

[0116] Flow cytometry was used to further detect the ability of the preparations to induce cell apoptosis. Figure 17 This is the result analysis of the apoptosis ability of different preparations on ovarian cancer cells. Figure 17 The addition of the drug significantly increased the level of apoptosis (***P < 0.001). The combination group experienced a significantly higher rate of apoptosis compared to the single-drug group (****P < 0.0001), indicating a synergistic effect of drug combination in inducing apoptosis. CMVs@SR / DOX and cRGD-CMVs@SR / DOX further increased the rate of apoptosis, with significant differences compared to the combination group (***P < 0.001), indicating that the vesicles facilitate drug entry into cells and exert their effects. A significant difference was observed between cRGD-CMVs@SR / DOX and CMVs@SR / DOX (**P < 0.01), suggesting that the formulation can actively target cells and enhance the drug's ability to induce apoptosis.

[0117] 4.6. Detection of the expression levels of apoptosis-related proteins in cells by Western blotting

[0118] Western blotting was used to detect the expression of apoptosis-related proteins in cells and the expression level of BCL-2 protein to investigate the apoptosis-inducing effect of the preparation. The specific experimental steps are as follows:

[0119] (1) Whole cell protein extraction: SK-OV-3 cells with good growth status were taken for cell counting and 3×10 5 Cells were seeded into 6-well plates at a density of 100 cells / well and cultured for 24 hours. Five drug-addition groups were set up: SR-18292, DOX, SR+DOX, CMVs@SR / DOX, and cRGD-CMVs@SR / DOX. The drug concentrations were set as SR-18292 (20 μM) and DOX (0.25 μg / mL). A negative control group was also set up. After 24 hours of drug culture, the drug-containing culture medium was discarded, the cells were washed three times with PBS, and the cells were trypsinized and centrifuged. 100 μL of lysis buffer (RIPA: PMSF = 100:1) was added to each well. Lysis was carried out on ice for 30 minutes. After lysis, the cells were centrifuged at 12,500 × g for 15 minutes at 4°C. The supernatant was the whole protein extract.

[0120] (2) Protein quantification: After the extracted total protein is quantified according to the instructions of the BCA kit, add one-fourth volume of 5× protein loading buffer to the supernatant and denature at 95°C for 10 min. The sample can then be loaded or temporarily stored at -20°C.

[0121] (3) Electrophoresis: Prepare the gel according to the instructions of the one-step gel preparation kit. Add appropriate amounts of protein marker and sample to the sample wells in sequence. Set the electrophoresis condition to 200V and run for about 50 minutes before transferring to the membrane.

[0122] (4) Transfer: Activate the PVDF membrane in methanol for 5 minutes and then soak it in rapid transfer solution along with the transfer sponge and filter paper. Place the gel and PVDF membrane in the transfer splint in order, remove any bubbles, clamp the splint, and transfer it to the transfer tank containing rapid transfer solution. Set the current to 300 mA and the transfer time to 40 minutes to perform the transfer.

[0123] (5) Antibody incubation: After transfer, remove the PVDF membrane and place it in an antibody incubation box. Wash it three times with TBST, add a rapid blocking solution to block at room temperature for 30 minutes, wash it three times with TBST, add the primary antibody diluted in proportion, incubate it at 4°C overnight, and then wash it three times with TBST. Add the secondary antibody, incubate it at room temperature for 1 hour, and then wash it three times with TBST.

[0124] (6) Development: Prepare ECL luminescent solution according to the instructions, place the PVDF membrane on a black background plate, remove excess TBST with filter paper, and incubate the luminescent solution in the dark for 2 minutes. Place the PVDF membrane in an automatic gel chemiluminescence imager to scan and image. The results are as follows: Figure 18 shown.

[0125] Figure 18 This is the analysis of the expression of apoptosis-related proteins in cells by different preparations. Figure 18 As shown, the addition of the drug downregulated BCL-2 protein expression. The protein downregulation level in the cRGD-CMVs@SR / DOX group was significantly different from that in the control group (**P < 0.01), indicating that the drug can inhibit the anti-apoptotic effect of BCL-2 protein and induce cell apoptosis. The addition of DOX significantly decreased BCL-2 protein levels, and the cRGD-CMVs@SR / DOX group showed a significant decrease in protein levels compared to the SR-18292 group (**P < 0.01), with the lowest protein expression level, indicating that the agent can induce apoptosis in more cells. This overall protein downregulation trend is consistent with the results of the apoptosis experiments in this chapter, further verifying the ability of different agents to induce cell apoptosis at the protein level. These results demonstrate that cRGD-CMVs@SR / DOX can significantly downregulate the expression of the anti-apoptotic protein BCL-2 in cells, further promoting the drug's ability to induce cell apoptosis.

[0126] Experimental Example 5 In vivo pharmacodynamic study of cRGD-CMVs@SR / DOX

[0127] The mice used in this experiment were BALB / c nude mice, SPF grade, purchased from Beijing Sibeifu Biotechnology Co., Ltd., female, 4-6 weeks old, weighing 10-15g. The conditions in the animal breeding room were 20-25°C, 40%-70% relative humidity, and the cycle was alternating between light and dark for 12 hours. SK-OV-3 ovarian cancer cells were cultured and passaged to a density of 80%, digested and centrifuged using trypsin, and the cell pellet was resuspended in PBS and then mixed with matrix gel at a ratio of 1:3. A concentration of 6×10 6 The growth of the tumor at the inoculation site was observed every day. When the tumor volume was 100-200 mm 3 When the results are good, the next step of experimental research can be carried out.

[0128] 5.1. Distribution of the Preparation in Vivo

[0129] In order to investigate the distribution and targeting of the targeted agent in vivo, a small animal in vivo imaging device was used to track the distribution of the agent in nude mice in real time. 3Afterwards, the DiR cell membrane fluorescent probe was used instead of the drug to investigate the in vivo distribution and tumor targeting of the preparation. The nude mice were divided into three groups: free DiR group, CMVs@DiR, and cRGD-CMVs@DiR. The concentration of DiR in the three groups of preparations was 5 mg / kg, and the preparations were injected into the mice through the tail vein, and this time was recorded as 0h. An in vivo imaging device was used to monitor the fluorescence intensity and distribution of the preparations in the mice at different times (2h, 4h, 8h, 10h, 12h, 24h and 48h), and X-ray images and fluorescence images of the mice were collected. By comparing the differences in fluorescence intensity and distribution among different preparation groups, the targeting of the preparations to tumor tissues and the side effects were evaluated.

[0130] The results showed that the preparation was injected into the body via the tail vein and was almost completely distributed in the liver after 2 hours, with no difference between the groups. After 4 hours, the free DIR group began to distribute to other parts of the body, while CMVs@DiR and cRGD-CMVs@DiR began to show a trend of distribution to the tumor site, and cRGD-CMVs@DiR distributed to the tumor site faster than CMVs@DiR. At 12 hours, the fluorescence at the tumor site in the preparation group reached its peak, and CMVs@DiR showed a small amount of fluorescence distributed in the limbs, while the free group had almost no accumulation in the tumor site. After 24 hours, the fluorescence at the tumor site weakened, while the fluorescence of cRGD-CMVs@DiR was stronger than that of CMVs@DiR. After 48 hours, the fluorescence in each group basically disappeared. The above results show that both CMVs@DiR and cRGD-CMVs@DiR have good tumor targeting and accumulation properties, can reduce the distribution of free drugs in non-target sites, and reduce toxic side effects. In addition, cRGD-CMVs@DiR shows faster distribution to the tumor and longer retention time in the tumor site than CMVs@DiR, which can prolong the drug's action time at the tumor location and enhance the drug's therapeutic effect in vivo.

[0131] 5.2 In vivo antitumor activity studies

[0132] When the tumor volume of nude mice reaches 100mm 3After administration, the drug can be administered. Nude mice were randomly divided into five groups, each containing five mice: control (normal saline), SR-18292, DOX, CMVs@SR / DOX, and cRGD-CMVs@SR / DOX. The SR-18292 concentration was 29.32 mg / kg, and the DOX concentration was 4 mg / kg. The CMVs@SR / DOX and cRGD-CMVs@SR / DOX groups were administered with a DOX concentration of 4 mg / kg. Five doses were administered via tail vein injection every other day. Changes in mouse body weight and tumor volume were monitored daily during the dosing period. 48 hours after the last dose, eye bleeding was performed, and plasma and serum were collected for routine blood tests and liver and kidney function biochemical markers. Following sacrifice, the heart, liver, spleen, lung, kidney, and tumor tissues of the mice were dissected and removed. Tumors were weighed and photographed. A portion of the tumor tissue was used for TUNEL staining, and the remainder was temporarily stored at -80°C. The tumor inhibition rate (IR) was calculated as follows:

[0133]

[0134] W c : Average weight of tumor in control group mice, W t : The average weight of the tumor in the mice in the drug administration group, the results are as follows Figure 19-20 shown.

[0135] Figure 19 This is the analysis of the effect of different preparations on the tumor size of tumor-bearing mice. Figure 19 It can be seen that the CMVs@SR / DOX group and cRGD-CMVs@SR / DOX group have more obvious anti-tumor effects. Figure 20 The following is an analysis of the tumor inhibition rate of different preparations on tumor mice. Graph A shows the tumor volume results, and Graph B shows the tumor inhibition rate. Figure 20 As shown in Figure 4, each preparation group slowed down the increase in tumor volume to varying degrees. The CMVs@SR / DOX and cRGD-CMVs@SR / DOX groups significantly inhibited tumor growth compared to the control group (****P<0.0001). The anti-tumor effects of the SR-18292 and DOX groups were weaker, possibly due to the poor targeting of the free drug, which is easily cleared by the body and has reduced efficacy. DOX, as a chemotherapy drug, showed better anti-tumor effects than the SR-18292 group. Figure 20As shown in Figure 2, the inhibition rates of the various groups were SR-18292: IR = 16.8%, DOX: IR = 36.7%, CMVs@SR / DOX: IR = 60.7%, and cRGD-CMVs@SR / DOX: IR = 72.1%. Compared with the control group, tumor weight in the DOX group was significantly reduced (**P < 0.01). The cRGD-CMVs@SR / DOX group showed a further decrease in tumor weight, with a significant difference compared to the DOX group (**P < 0.01), further demonstrating the superior antitumor activity of the cRGD-CMVs@SR / DOX formulation. These results demonstrate that cRGD-CMVs@SR / DOX significantly inhibits tumor growth and exhibits a favorable therapeutic effect. This suggests that cRGD-CMVs effectively encapsulates SR-18292 and DOX, preventing drug clearance before reaching the target site, enhancing tumor targeting of the free drug, and enhancing its antitumor activity.

[0136] 5.3 In vivo pharmacodynamic evaluation

[0137] After the mouse tumor was removed, it was fixed with 4% paraformaldehyde, sectioned in paraffin, and stained with TUNEL. The apoptosis of the tumor was observed under a fluorescence microscope, and the anti-tumor therapeutic effect of the preparation was analyzed. The results are as follows: Figure 21-22 shown.

[0138] The tumor tissue was stained with TUNEL kit to detect the apoptosis level and further investigate the anti-tumor effect of the preparation. Figure 21 This figure shows the effect of different preparations on tumor tissue apoptosis. Blue fluorescence represents cell nuclei, and green fluorescence represents apoptotic tumor cells. The cRGD-CMVs@SR / DOX group showed the largest green fluorescence area, indicating the highest apoptosis rate, which was significantly higher than the DOX group (****P<0.0001). Figure 22 This is a semi-quantitative analysis of TUNEL fluorescence results of different preparations. Figure 22 The percentages of apoptotic area in each formulation group were: Control: 0.52±0.21%, SR-18292: 4.01±0.37%, DOX: 8.15±0.14%, CMVs@SR / DOX: 14.22±0.71%, and cRGD-CMVs@SR / DOX: 17.03±2.23%. These results indicate that cRGD-CMVs@SR / DOX exhibits the best antitumor efficacy, effectively inducing tumor cell apoptosis and achieving the most effective therapeutic effect.

[0139] 5.4. Detection of the expression of related apoptosis proteins by immunoblotting

[0140] The expression levels of related apoptosis proteins BCL-2 and BAX in mouse tumor cells were detected by protein immunoblotting. Weigh the appropriate weight of tumor tissue, rinse with pre-cooled PBS, add an appropriate amount of RIPA lysis buffer (RIPA: PMSF = 100: 1) and place on ice for 30 minutes. After the lysis, add magnetic beads and homogenize twice on a tissue grinder. Centrifuge at 12000 rpm at 4°C for 15 minutes. The supernatant is a total protein extract. After BCA quantification of the supernatant, refer to the operating steps of Section 4.6 of Experimental Example 4 for the test. The results are as follows Figure 23 shown.

[0141] Western blotting was used to further detect the expression levels of related apoptosis proteins in tumor tissues. Figure 23 This is the result analysis of the expression levels of anti-apoptotic protein BCL-2 and pro-apoptotic protein BAX in different preparations. Figure 23 As shown, compared with the control group, the anti-apoptotic protein BCL-2 was significantly downregulated in all formulation groups (****P<0.0001), indicating that the formulation can induce tumor cell apoptosis to varying degrees. The cRGD-CMVs@SR / DOX group further significantly reduced BCL-2 protein expression compared to CMVs@SR / DOX, indicating that cRGD-CMV@SR / DOX has the strongest anti-tumor effect. While BAX protein expression levels did not differ significantly between individual groups, BAX protein levels were significantly increased in the cRGD-CMVs@SR / DOX group compared to the control group (****P<0.0001). These results demonstrate that cRGD-CMVs@SR / DOX can significantly upregulate BAX protein, downregulate BCL-2 protein, induce tumor cell apoptosis, and exhibit promising therapeutic effects.

[0142] 5.5. In vivo safety evaluation of preparations

[0143] The safety of the preparation in vivo was investigated by monitoring the weight of mice, analyzing blood biochemical indicators and changes in organ tissue morphology. During the administration period under "5.2", the weight changes of mice were monitored. Collect whole blood into a sample tube with an anticoagulant added and perform a routine blood test. Collect another 1 mL of blood, place it at room temperature for 2 hours, and then centrifuge to obtain the supernatant, which is plasma. Analyze the levels of creatinine (CREA), urea (UREA), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in the blood sample as indicators of renal and liver function. The heart, liver, spleen, lung, and kidney tissues of the mice were immersed in 4% paraformaldehyde, paraffin-sectioned, and used for H&E staining analysis. The toxicity of the preparation in vivo was investigated by changes in the weight of mice, detection of blood biochemical indicators and morphological changes in tissues and organs, and the safety of the preparation was evaluated. The results are as follows: Figures 24-25 shown.

[0144] The in vivo safety of the formulation is also an important part of the efficacy evaluation. The in vivo toxicity of the formulation was comprehensively assessed by monitoring the changes in mouse body weight during daily dosing, measuring routine blood parameters and serum levels of creatinine (CREA), urea (UREA), aspartate aminotransferase (AST), and alanine aminotransferase (ALT). Hematoxylin and eosin (H&E) staining was performed on heart, liver, spleen, lung, and kidney tissues.

[0145] Figure 24 The figures are analysis graphs of the in vivo safety evaluation results of different preparations, where Panel A shows weight changes (n=5), Panel B shows routine blood parameters (n=3), Panel C shows liver function indicators ALT and AST (n=3), and Panel D shows renal function indicators UREA and CREA (n=3). In the figures, *P<0.05, **P<0.01, ns: no significant difference. Figure 25 These are H&E-stained sections of different preparations.

[0146] observe Figure 24 As can be seen from the body weight curves, except for the DOX group, no significant changes in body weight were observed in all groups. Mice in the DOX group experienced a slow weight loss after administration, suggesting that free DOX has potential toxicity to nude mice at this dose. Routine blood count parameters showed no significant differences among the groups. Liver function indicators ALT and AST were significantly elevated in the DOX group, significantly different from those in the control group (**P < 0.01), suggesting possible liver damage in the DOX group. However, these indicators in the CMVs@SR / DOX and cRGD-CMVs@SR / DOX groups were significantly lower than those in the DOX group (*P < 0.05), approaching those in the control group. This suggests that CMVs@SR / DOX and cRGD-CMVs@SR / DOX can effectively ameliorate DOX-induced liver damage and reduce the drug's toxic side effects. Renal function indicators UREA and CREA showed no significant differences among the groups (P > 0.05), indicating that the formulation had no significant effect on renal function in mice.

[0147] observe Figure 25 The cardiac sections of the DOX group suggest that DOX at this dose may cause myocardial damage. These results indicate that free DOX exhibited some toxicity in nude mice, while drug-encapsulated CMVs effectively reduced the toxic side effects of DOX in vivo and demonstrated high in vivo safety and good biocompatibility.

[0148] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the same. The basic principles and main features of the present invention have been described above using specific implementation schemes. Modifications or substitutions may be made based on the present invention, but such modifications or substitutions do not detract from the essence of the corresponding technical solutions from the scope of protection claimed by the present invention.

Claims

1. A tumor-targeting carrier based on lactic acid bacteria vesicles, characterized in that: The targeted tumor carrier based on lactic acid bacteria vesicles is used to prepare a targeted tumor drug delivery system simultaneously loaded with SR-18292 and doxorubicin; the targeted tumor carrier based on lactic acid bacteria vesicles comprises lactic acid bacteria vesicles and DSPE-PEG2000-cRGD anchored on the surface of the lactic acid bacteria vesicles; the structural formula of the DSPE-PEG2000-cRGD is as follows: ； The method for preparing the tumor-targeting carrier based on lactic acid bacteria vesicles comprises the following steps: mixing a DSPE-PEG2000-cRGD solution with lactic acid bacteria vesicles, incubating, and then centrifuging to obtain the tumor-targeting carrier based on lactic acid bacteria vesicles, which is denoted as cRGD-CMVs; The tumor is ovarian cancer; the loading is carried out using electroporation technology.

2. The tumor-targeting carrier based on lactic acid bacteria vesicles according to claim 1, characterized in that The lactic acid bacteria vesicles are obtained by inoculating and culturing Lactobacillus rhamnosus and concentrating through differential centrifugation-ultrafiltration. The average diameter of the lactic acid bacteria vesicles is 80-100 nm, and the hydrated particle size is 85-105 nm.

3. The tumor-targeting carrier based on lactic acid bacteria vesicles according to claim 1, characterized in that The mass ratio of the DSPE-PEG2000-cRGD to the lactic acid bacteria vesicles is 1-2:1, and the concentration of the DSPE-PEG2000-cRGD solution is 0.5-1.5 mg / mL.

4. The tumor-targeting carrier based on lactic acid bacteria vesicles according to claim 1, characterized in that The incubation temperature is 35-40° C. and the incubation time is 3.5-4.5 hours.

5. The tumor-targeting carrier based on lactic acid bacteria vesicles according to claim 1, characterized in that The centrifugation process is as follows: centrifugation at 3500×g-4500×g for 8-12 minutes at 3-5° C., washing with PBS, and then centrifuging in a centrifuge at 130,000×g-150,000×g for 2.5-3.5 hours.

6. The tumor-targeting carrier based on lactic acid bacteria vesicles according to claim 5, characterized in that The molecular weight cut-off of the ultrafiltration centrifuge tube is 100 KDa.

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