Targeting tumor carrier based on lactic acid bacteria vesicles as well as preparation method and application of targeting tumor carrier

Abstract

The invention belongs to the technical field of medicinal carriers for targeting tumors, and particularly relates to a targeted tumor carrier based on lactic acid bacteria vesicles as well as a preparation method and application of the targeted tumor carrier. The targeted tumor carrier based on the lactic acid bacteria vesicles comprises the lactic acid bacteria vesicles and DSPE-PEG2000-cRGD anchored on the surfaces of the lactic acid bacteria vesicles. The targeted tumor carrier disclosed by the invention can effectively and actively deliver SR-18292 and adriamycin into tumor cells so as to effectively inhibit apoptosis of the tumor cells and remarkably inhibit migration and invasion of the tumor cells, so that a remarkable anti-tumor treatment effect is achieved. In addition, the tumor drug delivery system provided by the invention has good biological safety.

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 the hidden nature of the disease and the ineffectiveness of treatment, it has become the malignant tumor with the highest mortality rate among gynecological diseases. Most cases of ovarian cancer are diagnosed in the advanced stage. The current treatment methods mainly include surgery, radiotherapy and chemotherapy. The first-line chemotherapy drugs for ovarian cancer are platinum and paclitaxel, but the efficacy is limited, and the disease recurrence and drug resistance are prone to occur. Among them, the chemotherapy resistance of platinum drugs is a major challenge in the clinical treatment of ovarian cancer. In order to overcome the disease recurrence caused by drug resistance in patients with advanced ovarian cancer and prolong the survival of patients, it is necessary to explore new treatment methods and improve the clinical treatment effect of ovarian cancer. Some emerging treatment methods for ovarian cancer include immunotherapy, gene therapy, targeted therapy, etc., which have achieved positive results in the treatment of ovarian cancer with their respective advantages. Combination with chemotherapy may achieve better efficacy. With the in-depth study of the mechanism of chemotherapy resistance of cancer cells, actively exploring new targets to resist chemotherapy resistance is also an effective method to improve the quality of life of patients with advanced ovarian cancer. In recent years, the development and maturity of nanotechnology has become a valuable alternative to improve drug delivery efficiency and reduce drug toxicity and side effects. Nanoparticles can precisely deliver drugs to cancer cells, allowing the drugs to accumulate at higher concentrations in 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 the drug resistance of chemotherapy drugs, and thus improve the prognosis of patients with advanced ovarian cancer.

[0003] Nanocarriers can improve the bioavailability of drugs in tumor sites through passive or active targeting. The passive targeting of nanoparticles depends on the enhanced permeability and retention effect (EPR effect) in tumor sites. Compared with normal tissues, solid tumor tissues have abnormal blood vessel proliferation, wide wall gaps, and lack of lymphatic return. They have selective high permeability and retention for some macromolecular substances and tend to accumulate in tumor tissues. Actively targeted nanocarriers further modify the surface of nanoparticles with ligands or antibodies that have specific interactions with tumor cells, so that nanoparticles can actively target tumor cells and promote the selective enrichment of drugs in tumor sites. Nano delivery carriers have always been a hot topic in tumor targeted therapy, and some have been transformed into clinical practice. Nanomaterials are diverse, with the characteristics of adjustable shape and size, easy synthesis and modification, etc. They can be designed and functionalized according to the properties of the materials themselves to improve the delivery efficiency of drugs. Currently, lipid nanoparticles, inorganic nanoparticles, polymer nanoparticles, etc. are widely used to achieve effective targeted drug delivery, but they have disadvantages such as high toxicity, poor stability and metabolism. As more new nanomaterials are being developed and applied in anti-tumor drug delivery, the toxicity of the carrier and the safety of clinical transformation are one of the challenges that need to be faced. Therefore, it is still necessary to find a nanocarrier 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] The 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 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.

[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 method for preparing 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 recorded 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 time is 3.5-4.5h.

[0017] Furthermore, the centrifugation process is: at 3-5°C, centrifuge in an ultrafiltration centrifuge tube at a speed of 3500×g-4500×g for 8-12 minutes, wash with PBS, and then centrifuge in a centrifuge at a speed of 130000×g-150000×g 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 targeted tumor carrier based on lactic acid bacteria vesicles is used to prepare a targeted tumor 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 constructs a targeted tumor carrier based on lactic acid bacteria vesicles. The test results show 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, significantly inhibiting tumor cell migration and invasion, and thus exerting a significant anti-tumor therapeutic effect. The results of in vivo imaging show that cRGD-CMVs can be concentrated at the tumor site and have good tumor targeting. And according to the weight changes, blood routine and liver and kidney function indicators of mice and 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 It is a schematic diagram of the process of CMVs extraction and cRGD-CMVs construction in Example 1;

[0025] Figure 2 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 the TEM morphology of CMVs extracted in Example 1;

[0028] Figure 5 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 best mass ratio of cRGD-CMVs and SR-18292;

[0032] Fig. 9 This is a graph showing the particle size and potential results of cRGD-CMVs@SR / DOX prepared in Example 2;

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

[0034] Fig.11 It is a diagram showing the effect of electroporation on targeted CMVs vesicles;

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

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

[0037] Fig.14 This is a qualitative analysis of the uptake results of different preparations on ovarian cancer cells over 4 hours;

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

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

[0040] Fig.17 This is the result analysis diagram of the apoptosis ability of different preparations on ovarian cancer cells;

[0041] Fig.18 This is the result analysis diagram of the expression ability of different preparations on related apoptosis proteins in cells;

[0042] Fig.19 This is the result analysis chart of the effect of different preparations on the tumor size of tumor mice;

[0043] Fig. 20 It is the result analysis diagram of tumor inhibition rate of tumor mice by different preparations; Figure A is the result diagram of tumor volume, and Figure B is the diagram of tumor inhibition rate;

[0044] Fig.21 This is the result analysis diagram of the effect of different preparations on the apoptosis level of tumor tissue;

[0045] Fig. 22 It is the analysis diagram of TUNEL fluorescence semi-quantitative results of different preparations;

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

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

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

[0049] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, but does not constitute a limitation on the protection scope of the present invention.

[0050] In the following examples, the preparation of DSPE-PEG2000-cRGD was customized by Nanjing Taopu Biotechnology Co., Ltd. according to the requirements of the inventor; 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.; DID kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd. Other reagents and materials not specifically specified are conventional materials that can be obtained from commercial channels.

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

[0054] The Lactobacillus rhamnosus (LGG strain) frozen in a -80℃ refrigerator was thawed in warm water at 37℃, inoculated into the plate by the plate streak method, cultured at 30℃ for 36h, and a single colony was selected and cultured for 20h for activation. Then it was inoculated into a blank MRS liquid culture medium at a volume ratio of 1:100 to expand the culture. The blank culture medium was used as a control. The OD600 value of the bacterial culture solution was detected by an enzyme marker. When the OD value of the bacterial solution reached 0.5, 0.5μg / mL of penicillin G sodium was added. When the OD600 of the bacterial solution was 1, the bacterial solution was first centrifuged at 4℃, 5000×g for 30min to remove most of the bacteria. The supernatant was filtered through 0.45μm and 0.22μm microporous membranes in turn to remove residual bacteria. Ultrafiltration centrifuge tubes (MWCO=100kDa) were used for centrifugation at 4℃, 4000×g for 10min for concentration. Filtered with a 0.22μm filter to remove impurities generated during concentration. Then centrifuge at 4°C, 150,000×g for 3h in an ultra-high speed centrifuge, discard the supernatant, resuspend in PBS, transfer to an ultrafiltration centrifuge tube, wash and concentrate repeatedly with PBS to purify CMVs, and filter and sterilize with a 0.22μm filter. Finally, centrifuge at 4°C, 150,000×g for 3h, resuspend the precipitate in PBS, and obtain lactic acid bacteria vesicles for later use.

[0055] The tumor targeting carrier of lactic acid bacteria vesicles in this embodiment is to connect DSPE-PEG2000-cRGD to lactic acid bacteria vesicles by a biological connection method. After connection, laser confocal microscopy is used to perform fluorescence co-localization of lactic acid bacteria vesicles to determine whether the lactic acid bacteria vesicles and 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 As shown, 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 150000×g for 3 hours in a centrifuge. The precipitate was resuspended in PBS to obtain a targeted tumor vector based on lactic acid bacteria vesicles, which was recorded as cRGD-CMVs.

[0058] Example 2

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

[0060] In this embodiment, 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. Yield of CMVs

[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 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 1 is the yield result diagram of CMVs extracted in Example 1. Figure 2 It can be seen that the content of CMVs extracted by Example 1 is 1.626 ± 0.049 mg / mL, and the content of CMVs taken by 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 the yield when the CMVs carrier was subsequently extracted.

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

[0066] The particle size and potential of 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 is the particle size and potential diagram of CMVs extracted in Example 1. 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 is 0.132±0.033. The above results show that the particle size of 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. Using laser confocal microscopy to determine the connection status of cRGD-CMVs prepared in Example 1

[0072] Since it is necessary to verify that the preparation method of the present invention can successfully connect lactic acid bacteria vesicles (CMVs) with DSPE-PEG2000-cRGD, and DSPE-PEG2000-cRGD cannot be labeled by FITC, the lactic acid bacteria vesicles and DSPE-PEG2000 were fluorescently labeled in this test example, so as to visually judge whether the vesicles and the polypeptide are successfully connected by fluorescence co-localization. The fluorescence labeling process is as follows:

[0073] First, CMVs were incubated with DiD (chemical name: 1,1-octadecyl-3,3,3,3-tetramethylindolyl 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); the 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 a speed of 4000×g for 10 minutes. The free DSPE-PEG2000-FITC was removed by repeated washing with PBS several times. Then, an ultra-high-speed centrifuge was used to centrifuge at a speed of 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 is a diagram showing the connection of cRGD-CMVs prepared in Example 1. Figure 5 It can be seen that CMVs are stained with DiD, so they show red fluorescence under a microscope, and DSPE-PEG2000 is labeled with FITC, showing green fluorescence under a microscope. After taking pictures of different fluorescence channels with a laser confocal microscope, the pictures of the two fluorescence channels are merged to observe whether they are co-localized. It can be seen from the figure that the fluorescence of the two channels after the merger of the present invention effectively overlaps, and the fluorescence display is uniform, indicating that DSPE-PEG2000 can connect FITC to the vesicles. Similarly, cRGD-CMVs are 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 particle size and potential results of cRGD-CMVs prepared in Example 1 are shown in FIG. Figure 6 It can be seen that the hydrated particle size of cRGD-CMVs is 98.28±1.46nm, the potential is -35.98±0.88mV, and the PDI is 0.135±0.035. The connection of cRGD slightly reduces the potential of the vesicles and slightly increases the particle size, but the particle size dispersibility is still good.

[0079] Figure 7 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 presented an inwardly concave hemispherical structure, and it was visually found that the particle size did not change significantly.

[0080] Experimental Example 3 Application effect of targeted tumor carrier based on lactic acid bacteria vesicles 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 the drug, the ratio of the carrier to the drug added during electroporation was investigated.

[0083] The mass of cRGD-CMVs was fixed at 40 μg. Under the electroporation conditions of 40 V and 100 μF, SR-18292 was loaded into cRGD-CMVs at a mass ratio of 1:1, 1:2, and 2:1, respectively. Three samples were prepared in parallel for each group. The encapsulation efficiency and drug loading rate were determined. The encapsulation efficiency and drug loading rate were calculated as follows: encapsulation efficiency (wt.%) = (amount of loaded drug / amount of drug input) × 100%; drug loading rate (wt.%) = [amount of loaded drug / (amount of drug input + amount of carrier input)] × 100%.

[0084] The mass of cRGD-CMVs was fixed at 40 μg, and the mass of SR-18292 was fixed at 40 μg. The combined concentrations of SR-18292 and DOX were 20 μM (7.33 μg / mL) and 0.25 μg / mL, respectively. The amount of DOX was changed, and cRGD-CMVs and DOX were mixed. The drug loading rate and encapsulation rate were measured to determine the optimal mass ratio of drug loading. The drug loading rate and encapsulation rate 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 rate and drug loading 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 according to the required concentration and loading efficiency of DOX. It has been verified that when the ratio of SR-18292 to DOX is 8:1, the SR-18292 encapsulation rate is 50.18±1.96%, the drug loading is 33.4±0.87%, the DOX encapsulation rate is 12.83±0.96%, and the drug loading is 1.58±0.17%, which basically meets the dosing concentration, but the encapsulation rates of both drugs have decreased, which may be due to the competitive relationship between the two drugs when they are co-loaded.

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

[0089] Take appropriate amount of drug-loaded vesicles after electroporation (the drug-loaded vesicles were prepared using the above-mentioned preferred process conditions, electroporation voltage 40V, capacitance 100μF; cRGD-CMVs:SR:DOX=1:1:0.125), and use transmission electron microscopy to characterize the morphology after electroporation and drug loading. Use a laser particle size analyzer to measure the particle size and potential of the drug-loaded vesicles. The results are as follows Figure 9-10 shown.

[0090] Fig. 9 The particle size and potential results of cRGD-CMVs@SR / DOX prepared in Example 2 are shown in FIG. Fig. 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] Fig.10 is a TEM image of cRGD-CMVs@SR / DOX prepared in Example 2. Fig.10 It can be seen that the structure and morphology of the vesicles were not destroyed after electroporation, but the particle size of some of them increased to about 200 nm.

[0092] 3.3 Effect of electroporation on CMVs-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 for imaging, and the presence of fluorescence co-localization was used to determine whether electroporation would destroy the connection between peptides and vesicles. The results are shown in Figure 2. Fig.11 shown.

[0094] Fig.11 Figure 2 is a graph showing the effect of electroporation on targeting CMVs vesicles. Fig.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 will 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 state, the cells were inoculated into 96-well plates at a density of 8,000 cells per well. The concentration of the required carrier was calculated to be 14.66 μg / mL at the concentration of the drug, so the carrier concentrations were set to 20, 40, 60, and 80 μg / mL, 4 replicates were set for each concentration, and a control group and a blank group were set. When the cell density reached more than 80%, the above-prepared drugs were 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, incubated in the dark for 1 hour, and the absorbance at 450 nm was measured by an enzyme reader to calculate the cell survival rate and inhibition rate. The inhibition rate calculation formula is: The results are as follows Fig.12 shown.

[0099] 4.1.2 Cytotoxicity of different preparations

[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, and the absorbance at 450 nm was measured by an ELISA instrument. The cell survival rate and inhibition rate were calculated using the same calculation formula as in 4.1.1. The results are shown in Figure 2. Fig.12 shown.

[0101] Fig.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. Fig.12 A is a graph showing the toxicity test results of the cRGD-CMVs vector prepared in Example 1 on ovarian cancer cells.

[0102] Fig.12 B is the toxicity test results of different preparations on ovarian cancer cells. Fig.12 A shows that the cell survival rate is still above 85% when the concentration of the two vectors is 80 μg / mL, and the cell survival rate increases as the vector concentration decreases. There is no significant difference in the cytotoxicity of CMVs and cRGD-CMVs. This shows that the vector has no obvious cytotoxicity at the therapeutic concentration, and the construction of the targeted vector does not increase the toxicity of the vector.

[0103] Depend on Fig.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, which may be due to the fact that 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. The above 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 cells of SK-OV-3 cells that were growing well, 9 × 10 4 The cells were inoculated at a density of 100 cells 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 8μg / 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 washed with PBS three times. The cells were fixed with 4% paraformaldehyde for 15 minutes. After 15 minutes, the paraformaldehyde was removed and washed with PBS three times. 1μg / mL DAPI dye solution was added to stain for 15 minutes in the dark. After incubation, it was washed with PBS three times. The slide was removed and inverted on a 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] Fig.13 This is an analysis of the qualitative uptake results of different preparations on ovarian cancer cells. Fig.13 It can be seen that the fluorescence intensity of the CMVs@FITC and cRGD-CMVs@FITC groups is higher than that of the free FITC group, and there is a significant increase at the 2h time point, and it basically reaches saturation after 4h, indicating that the vesicles can promote the entry of drugs into cells. In addition, the fluorescence intensity of cRGD-CMVs@FITC is higher than that of CMVs@FITC, indicating that the targeting peptide increases the efficiency of cell uptake of drugs.

[0107] Fig.14 This is a qualitative analysis of the uptake results of different preparations on ovarian cancer cells over 4 hours. Fig.14 It can be seen that after CMVs were stained with DiD, they showed red fluorescence, which was highly co-localized with green fluorescence, proving that the drug could be successfully loaded into the vesicles and taken up into the cells through the vesicles. The preparation group showed a fluorescence intensity significantly higher than that of the free FITC group. At the same time, the fluorescence intensity of DiD-cRGD-CMVs@FITC was higher than that of DiD-CMVs@FITC, which also showed that the targeted vesicle uptake efficiency was higher. The above results show that cRGD-CMVs can effectively encapsulate drugs and deliver them to cells in a targeted manner, thereby better exerting their efficacy.

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

[0109] SK-OV-3 cells with good growth status were selected for cell counting and 1.5×10 5 The cells were inoculated into a 12-well plate at a density of 100 cells per well 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 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 plates were placed in an incubator and incubated in the dark for 20 minutes. After the staining was completed, the working solution was discarded, and the cells were washed three times with the washing solution. The changes in cell fluorescence were observed and photographed under a fluorescence microscope. The results are as follows Fig.15 shown.

[0110] The decline of mitochondrial membrane potential (MMP) is a hallmark event in the early stage of 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. Fig.15 This is an analysis of the effects of different preparations on the mitochondrial membrane potential of ovarian cancer cells. Fig.15It is known that normal cells show strong red fluorescence and almost no green fluorescence. After the cells were treated with drugs, the red fluorescence weakened and the green fluorescence increased, indicating a decrease or loss of some MMPs. The cells treated with a single drug showed red / green fluorescence co-localization, while the green fluorescence increased and the red fluorescence intensity decreased in the SR+DOX group compared with the single drug group, indicating an increase in the degree of mitochondrial damage. Compared with the combination drug group, the red fluorescence intensity of the CMVs@SR / DOX and cRGD-CMVs@SR / DOX groups further decreased, and the fluorescence co-localization decreased, indicating that the degree of mitochondrial depolarization was higher and the preparation 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, inhibits mitochondrial biogenesis, and thus causes mitochondrial damage; while DOX can cause a decrease in MMP by inducing cell apoptosis. The effects of the two drugs were enhanced after combined use. 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 efficacy of drugs.

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

[0112] SK-OV-3 cells with good growth status were selected for cell counting and 1.5×10 5 The cells were inoculated into a 12-well plate at a density of 100 cells per well and cultured for 24 hours. Five drug-addition 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, and the cells were washed three times with PBS. 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 incubated in an incubator in the dark for 30 minutes. After the staining was completed, the working solution was discarded, and the cells were washed three times with PBS. The intracellular fluorescence was observed and photographed under a fluorescence microscope. The results are shown in the figure. Fig.16 shown.

[0113] ROS is involved in the process of cell apoptosis. The increase of intracellular ROS level induces cell apoptosis. DCFH-DA fluorescent probe detects the change of intracellular ROS level, and the fluorescence intensity can reflect the level of intracellular ROS. Fig.16 This is an analysis of the effects of different preparations on ROS levels in ovarian cancer cells. Fig.16It can be seen that the drug-added groups can induce cells to produce more ROS, the combination group has more green fluorescence than the single drug group, and the green fluorescence of the preparation group is further increased. This shows that the combination of SR-18292 and DOX can induce cells to produce more ROS. It is speculated that SR-18292 may cause the generation of mitochondrial ROS (MitoROS) through mitochondrial damage, and the specific mechanism needs to be verified. cRGD-CMVs@SR / DOX improves the efficiency of cell uptake of drugs and can enhance the ability of drugs to induce ROS production.

[0114] 4.5 Cell apoptosis assay

[0115] SK-OV-3 cells with good growth status were selected and counted, and 2×10 5 The cells were seeded in a 6-well plate, and the drugs were 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 removed, washed three times with PBS, and the cells were digested with EDTA-free trypsin and washed twice with PBS. The cells should be gently blown during this period to avoid mechanical damage. After staining with an apoptosis kit, the flow cytometer was used for detection. The results were statistically analyzed using Flowjo software. The results are shown in the figure. Fig.17 shown.

[0116] Flow cytometry was used to further detect the ability of the preparations to induce cell apoptosis. Fig.17 This is the result analysis of the effect of different preparations on the apoptosis of ovarian cancer cells. Fig.17 It can be seen that the addition of drugs significantly increased the level of cell apoptosis (***P<0.001), and the proportion of cell apoptosis in the combination group was significantly higher than that in the single drug group (****P<0.0001), indicating that the combination of drugs has a synergistic effect of inducing cell apoptosis. CMVs@SR / DOX and cRGD-CMVs@SR / DOX further increased the proportion of cell apoptosis, which was significantly different from the combination group (***P<0.001), indicating that the vesicles can promote the entry of drugs into cells to exert their effects. There is a significant difference between cRGD-CMVs@SR / DOX and CMVs@SR / DOX (**P<0.01), indicating that the preparation can actively target cells and enhance the effect of drug-induced cell apoptosis.

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

[0118] The expression of related apoptosis proteins in cells was detected by Western blotting, the expression level of BCL-2 protein was detected, and the apoptosis-inducing effect of the preparation was investigated. 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 The cells were inoculated into 6-well plates at a density of 100 cells and cultured for 24 hours. Five drug-addition 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, washed three times with PBS, digested with trypsin and centrifuged, and 100 μL of lysis solution (RIPA: PMSF = 100: 1) was added to each well. Lysed on ice for 30 minutes, and centrifuged at 12500×g for 15 minutes at 4°C after lysis. 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 supernatant 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 conditions to 200V, and perform the electrophoresis for about 50 minutes before transferring to the membrane.

[0122] (4) Transfer: After activating the PVDF membrane in methanol for 5 min, soak it in the rapid transfer solution together with the transfer sponge and filter paper for balance. Place the gel and PVDF membrane in the transfer clamp in order, remove the bubbles and clamp them, transfer them to the transfer tank containing the rapid transfer solution, set the current to 300 mA, and the time to 40 min for transfer.

[0123] (5) Antibody incubation: After transfer, take out 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 min, 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 h, 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, remove excess TBST with filter paper, and incubate the luminescent solution in the dark for 2 min. Place the PVDF membrane in an automatic gel chemiluminescence imager for scanning and imaging. The results are as follows: Fig.18 shown.

[0125] Fig.18 This is the result analysis of the expression ability of different preparations on related apoptosis proteins in cells. Fig.18 It can be seen that the addition of drugs downregulated the expression of BCL-2 protein, among which the protein downregulation level of the cRGD-CMVs@SR / DOX group was significantly different from that of 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 caused a significant downward trend in the BCL-2 protein level, and the protein level of cRGD-CMVs@SR / DOX was significantly downregulated compared with the SR-18292 group (**P<0.01), with the lowest protein expression level, indicating that the preparation can induce apoptosis in more cells. The overall trend of protein level downregulation is consistent with the trend of the results of the cell apoptosis experiment in this chapter, and the ability of different preparations to induce cell apoptosis was verified again at the protein level. In summary, the results show that cRGD-CMVs@SR / DOX can significantly downregulate the expression of the anti-apoptotic protein BCL-2 in cells, and better promote the drug to play a role in inducing 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 level, 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 darkness for 12 hours. SK-OV-3 ovarian cancer cells were cultured and subcultured until the density reached 80%, digested and centrifuged with trypsin, and the cell pellet was resuspended in PBS and then mixed with matrix gel at a ratio of 1:3. The concentration of 6×10 was inoculated in the right axilla of each nude mouse. 6 The tumor growth at the inoculation site was observed every day. When the tumor volume was 100-200mm 3 The next step of experimental research can be carried out.

[0128] 5.1. Distribution of the drug 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. The fluorescence intensity and distribution of the preparations in the mice at different times (2h, 4h, 8h, 10h, 12h, 24h and 48h) were monitored using an in vivo imager, and the X-ray images and fluorescence images of the mice were collected. By comparing the differences in fluorescence intensity and distribution of different preparation groups, the targeting and side effects of the preparations on tumor tissues were evaluated.

[0130] The results showed that the preparation was injected into the body through the tail vein, and after 2 hours, it was basically distributed in the liver, with no difference between the groups. After 4 hours, the free DIR group showed distribution in 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 of the preparation group reached the strongest, 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, and the fluorescence of cRGD-CMVs@DiR was stronger than that of CMVs@DiR. The fluorescence of each group basically disappeared after 48 hours. The above results show that both CMVs@DiR and cRGD-CMVs@DiR have good tumor targeting and accumulation, 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 in the tumor site than CMVs@DiR, which can prolong the drug's action time at the tumor location and enhance the drug's in vivo therapeutic effect.

[0131] 5.2 In vivo antitumor activity studies

[0132] When the tumor volume of nude mice reaches 100mm 3After that, the drug can be administered. The nude mice were randomly divided into five groups, with five mice in each group, and divided into the following groups: Control group (normal saline), SR-18292 group, DOX group, CMVs@SR / DOX group, and cRGD-CMVs@SR / DOX group. The concentration of SR-18292 was 29.32 mg / kg, the concentration of DOX was 4 mg / kg, and the concentration of DOX in the CMVs@SR / DOX group and the cRGD-CMVs@SR / DOX group was 4 mg / kg. Tail vein injection was performed every other day, and a total of five doses were administered. During the administration period, the changes in the weight and tumor volume of the mice were monitored every day. 48 hours after the last administration, the mice were bled from the eyeballs, and plasma and serum were collected for blood routine and liver and kidney function biochemical indicators. After being killed, the heart, liver, spleen, lung, kidney and tumor tissues of the mice were dissected and removed. After the tumors were weighed and photographed, part of the tumor tissue was taken for TUNEL staining, and the rest was frozen in a -80℃ refrigerator for temporary storage. 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 drug-treated group of mice. Figure 19-20 shown.

[0135] Fig.19 This is an analysis of the effects of different preparations on the tumor size of tumor-bearing mice. Fig.19 It can be seen that the CMVs@SR / DOX group and cRGD-CMVs@SR / DOX group had obvious anti-tumor effects. Fig. 20 The following is an analysis of the tumor inhibition rate of different preparations on tumor mice. Figure A is the tumor volume result diagram, and Figure B is the tumor inhibition rate diagram. Fig. 20 A shows that each preparation group slowed down the increase of tumor volume to varying degrees. Among them, CMVs@SR / DOX group and cRGD-CMVs@SR / DOX group significantly inhibited tumor growth compared with the Control group (****P<0.0001). The anti-tumor effects of SR-18292 and DOX groups were weaker, which may be due to the poor targeting of free drugs, which are easily cleared by the body and have reduced efficacy. DOX, as a chemotherapeutic drug, showed better anti-tumor effects than the SR-18292 group. Observation Fig. 20B shows that the inhibition rates of each group are SR-18292: IR = 16.8%, DOX: IR = 36.7%, CMVs@SR / DOX: IR = 60.7%, cRGD-CMVs@SR / DOX: IR = 72.1%. Compared with the Control group, the tumor weight of the DOX group was significantly reduced (**P < 0.01), and the tumor weight of the cRGD-CMVs@SR / DOX group was further reduced, and there was a significant difference compared with the DOX group (**P < 0.01), which further illustrates that the preparation cRGD-CMVs@SR / DOX has a better anti-tumor effect. In summary, the results show that cRGD-CMVs@SR / DOX can significantly inhibit tumor growth and has a good therapeutic effect, indicating that cRGD-CMVs can effectively encapsulate SR-18292 and DOX, prevent the drug from being cleared by the body before reaching the target site, increase the tumor targeting of the free drug, and better exert the anti-tumor effect of the drug.

[0136] 5.3 In vivo pharmacodynamic evaluation

[0137] After the mouse tumor was removed, it was fixed with 4% paraformaldehyde, sectioned with paraffin, stained with TUNEL, and the apoptosis of the tumor was observed under a fluorescence microscope to analyze the anti-tumor therapeutic effect of the preparation. 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. Fig.21 This is the result analysis of the effect of different preparations on the apoptosis level of tumor tissue. Blue fluorescence represents the cell nucleus, and green fluorescence represents apoptotic tumor cells. The cRGD-CMVs@SR / DOX group has the largest green fluorescence area, showing the highest apoptosis ratio, which is significantly higher than the DOX group (****P<0.0001). Fig. 22 This is a semi-quantitative analysis of TUNEL fluorescence results of different preparations. Fig. 22 It can be seen that the percentage of apoptosis area in each preparation group is Control: 0.52±0.21%, SR-18292: 4.01±0.37%, DOX: 8.15±0.14%, CMVs@SR / DOX: 14.22±0.71%, cRGD-CMVs@SR / DOX: 17.03±2.23%. The above results show that cRGD-CMVs@SR / DOX has the best anti-tumor efficacy, can effectively induce tumor cell apoptosis, and has the best therapeutic effect.

[0139] 5.4. Detection of expression of related apoptosis proteins by western blotting

[0140] The expression levels of related apoptosis proteins BCL-2 and BAX in mouse tumor cells were detected by protein immunoblotting. Weigh an 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 lysis, add magnetic beads and homogenize twice on a tissue grinder. Centrifuge at 12000rpm at 4℃ 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 Fig.23 shown.

[0141] Western blotting was used to further detect the expression levels of related apoptosis proteins in tumor tissues. Fig.23 This is the result analysis of the expression levels of anti-apoptotic protein BCL-2 and pro-apoptotic protein BAX in different preparations. Fig.23 It can be seen that compared with the Control, the anti-apoptotic protein BCL-2 in the preparation group was significantly downregulated (****P<0.0001), indicating that the preparation can induce tumor cell apoptosis to varying degrees. The cRGD-CMVs@SR / DOX group further significantly reduced the expression of BCL-2 protein compared with CMVs@SR / DOX, indicating that cRGD-CMV@SR / DOX has the strongest anti-tumor effect. There was no significant difference in the expression level of BAX protein between individual groups, but the level of BAX protein in the cRGD-CMVs@SR / DOX group was significantly higher than that in the Control group (****P<0.0001). The above results show that cRGD-CMVs@SR / DOX can significantly upregulate BAX protein, downregulate BCL-2 protein, induce tumor cell apoptosis, and has a good tumor treatment effect.

[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 indices, and changes in organ and 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 it to obtain the supernatant, i.e., plasma. The levels of creatinine (CREA), urea (UREA), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in the blood sample were analyzed as indicators of kidney and liver function. The heart, liver, spleen, lung, and kidney tissues of the mice were immersed in 4% paraformaldehyde, and paraffin sections were made 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 indices, and changes in the morphology of tissues and organs, and the safety of the preparation was evaluated. The results are as follows: Figure 24-25 shown.

[0144] The in vivo safety of the preparation is also an important part of the efficacy evaluation. The in vivo toxicity of the preparation is comprehensively evaluated by monitoring the changes in the weight of mice during the administration period, measuring the blood routine parameters of the mice and the levels of creatinine (CREA), urea (UREA), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in the serum, and performing H&E staining sections on the heart, liver, spleen, lung, and kidney tissues.

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

[0146] observe Fig.24 It can be seen from the weight change curve that except for the DOX group, there was no significant change in the weight of each group. The mice in the DOX group slowly lost weight after administration. It is speculated that free DOX has certain potential toxicity to nude mice at this dose. There was no significant difference in the parameters of blood routine tests among the groups. The liver function indicators ALT and AST increased significantly in the DOX group, which was significantly different from the normal group (**P<0.01). This indicates that the DOX group may have liver damage, while the indicators of the CMVs@SR / DOX and cRGD-CMVs@SR / DOX groups were significantly lower than those of the DOX group (*P<0.05), and the levels were close to those of the normal group, indicating that CMVs@SR / DOX and cRGD-CMVs@SR / DOX can effectively improve DOX-induced liver damage and reduce the toxic side effects of the drug. There was no significant difference in the renal function indicators UREA and CREA among the groups (P>0.05). This shows that the preparation has no significant effect on the renal function of mice.

[0147] observe Fig.25 It can be seen that the heart slices of the DOX group suggest that DOX may cause myocardial damage at this dose. The above results show that the free DOX group showed certain toxicity in nude mice, while CMVs encapsulated drugs effectively reduced the toxic side effects of DOX in vivo, showed high in vivo safety, and had good biocompatibility.

[0148] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. On the basis of the present invention, some modifications or replacements may be made thereto, but these modifications or replacements do not make the essence of the corresponding technical solutions deviate 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 invention comprises lactic acid bacteria vesicles and DSPE-PEG2000-cRGD anchored on the surface of the lactic acid bacteria vesicles.

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 structural formula of the DSPE-PEG2000-cRGD is as follows:

4. The method for preparing a tumor-targeting carrier based on lactic acid bacteria vesicles according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: mixing the DSPE-PEG2000-cRGD solution with the lactic acid bacteria vesicles, incubating, and then centrifuging to obtain a tumor targeting carrier based on the lactic acid bacteria vesicles, which is recorded as cRGD-CMVs.

5. The method for preparing a tumor-targeting carrier based on lactic acid bacteria vesicles according to claim 4, 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.

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

7. The method for preparing a tumor-targeting carrier based on lactic acid bacteria vesicles according to claim 4, characterized in that: The centrifugation process is as follows: centrifuge at 3500×g-4500×g for 8-12 min at 3-5° C., wash with PBS, and then centrifuge at 130000×g-150000×g for 2.5-3.5 h in a centrifuge.

8. The method for preparing a tumor-targeting carrier based on lactic acid bacteria vesicles according to claim 7, characterized in that: The molecular weight cut-off of the ultrafiltration centrifuge tube is 100 KDa.

9. The use of the tumor targeting carrier based on lactic acid bacteria vesicles according to any one of claims 1 to 3, characterized in that: Preparation of a tumor-targeted drug delivery system loaded with SR-18292 and chemotherapeutic drugs simultaneously.

10. The use of the tumor targeting carrier based on lactic acid bacteria vesicles according to claim 9, characterized in that: The tumor is ovarian cancer; the loading adopts electroporation technology; and the chemotherapy drug is doxorubicin.

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