CRT (Cathode Ray Tumor) extroverted CT-26 tumor cell membrane coated drug-loaded peptidoglycan

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a calreticulin ectropion tumor cell membrane wrapped drug-loaded peptidoglycan as well as a preparation method and application of the calreticulin ectropion tumor cell membrane wrapped drug-loaded peptidoglycan. According to the invention, the complete peptidoglycan (WPG) is extracted from bacteria, so that the complete peptidoglycan (WPG) is used as a composite immunologic adjuvant, namely, the complete peptidoglycan (WPG) is used as a carrier and has an inherent immune activation capability. According to the invention, a CRT extroversion CT-26 tumor cell model is constructed, and the cell membrane of the CRT extroversion CT-26 tumor cell model is extracted to coat the surface of the drug-loaded peptidoglycan, so that the uptake of the dendritic cells to the whole particle system and the lymph node drainage capability are effectively improved; animal in-vitro experiments verify the immunotherapy effect of the drug-loaded peptidoglycan treatment system and the combined treatment effect of the drug-loaded peptidoglycan treatment system combined with radiotherapy. The invention provides a safe and efficient immunotherapy drug, and provides a new scheme for realizing effective cancer immunotherapy and combined therapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and relates to a drug-loaded peptidoglycan wrapped in a calreticulin-everted tumor cell membrane and a preparation method thereof, and specifically relates to a drug-loaded peptidoglycan treatment system wrapped in a CRT-everted CT-26 tumor cell membrane and a preparation method and application thereof. Background Art

[0002] It is reported that cancer is still a serious disease that threatens the health of all mankind due to its high mortality and morbidity rates, among which the mortality rate of colorectal cancer ranks second in the world. The prior art discloses that immunotherapy can activate the host's immune system, enabling the body to actively identify and eliminate cancer cells in the body, and has become an effective cancer treatment method other than surgical resection, chemotherapy, and radiotherapy. However, studies have shown that since most tumor microenvironments are in an immunosuppressive state, the patient's response rate to the above-mentioned therapies is low. Therefore, activating the immune system and reversing the immunosuppressive microenvironment are the keys to the success of immunotherapy.

[0003] Studies have shown that bacteria and their derived components can bind to pattern recognition receptors in the innate immune system as pathogen-associated molecular patterns, producing a strong innate immune activation effect. Bacterial peptidoglycan (PG) exists in the cell walls of most bacteria and is composed of disaccharide units, tetrapeptide tails and peptide bridges. When PG enters the body, the host's hydrolases will degrade it into small molecule metabolites, which are then recognized by pattern recognition receptors and play an immune activation role. The shape of bacterial complete peptidoglycan (WPG) is similar to that of bacteria and has a macromolecular reticular structure. In recent years, studies have shown that the cGAS-STING signaling pathway plays an increasingly important role in cancer immunotherapy. SR-717 is a very promising small molecule STING agonist that activates the cGAS-STING signaling pathway, induces the release of type I interferon, and plays an anti-tumor immunotherapy role. However, due to the small molecular weight, poor water solubility and poor bioavailability of SR-717, in addition, studies have shown that when tumor cells are stimulated by external stimuli and undergo immunogenic cell death, the intracellular calreticulin (CRT) will be exposed on the cell surface, and dendritic cells can effectively recognize this signal and take up a large number of tumor cells.

[0004] Based on the foundation and current status of the prior art, the inventors of the present application intend to provide a drug-loaded peptidoglycan therapeutic system wrapped with CRT-everted CT-26 tumor cell membrane and its preparation method and application; in the present invention, WPG is extracted as a composite immune adjuvant, and it is loaded with small molecule immunotherapy drug SR-717 to prepare PW / SR, and the CRT-everted CT-26 cell membrane is coated on the surface of PW / SR, and a new therapeutic system CPW / SR is developed for immunotherapy of colon cancer. Summary of the invention

[0005] The purpose of the present invention is to provide a drug-loaded peptidoglycan therapeutic system wrapped with a CRT-everted CT-26 tumor cell membrane, and its preparation method and application based on the basis and current status of the prior art; in the present invention, WPG is extracted as a composite immune adjuvant, and is loaded with a small molecule immunotherapy drug SR-717 to prepare PW / SR, and the CRT-everted CT-26 cell membrane is coated on the surface of PW / SR, providing a new therapeutic system CPW / SR for the immunotherapy of colon cancer.

[0006] The present invention provides a drug-loaded peptidoglycan therapeutic system CPW / SR wrapped by the membrane of CRT-everted CT-26 tumor cells, evaluates the physicochemical properties and in vivo and in vitro immune activation ability of CPW / SR, and further verifies its immunotherapy and combined therapy effects in a mouse subcutaneous colon cancer tumor model.

[0007] Specifically. The present invention extracts the complete peptidoglycan (WPG) from bacteria to make it a composite immune adjuvant, that is, it has the ability to activate innate immunity while serving as a carrier. This patent selects a non-nucleoside STING agonist SR-717 as an immunotherapy drug, loads it into the porous mesh structure of WPG to form PW / SR, synergistically activates innate immunity and acquired immunity, and reverses the immunosuppressive tumor microenvironment. The present invention constructs a CRT everted CT-26 tumor cell model and extracts its cell membrane to wrap it on the surface of the drug-loaded peptidoglycan, effectively increasing the uptake of the entire particle system by dendritic cells and the lymph node drainage capacity. Through a variety of in vitro experiments, the physicochemical properties and immune activation mechanism of CPW / SR were studied in detail, and finally the immunotherapy effect and the combined treatment effect of radiotherapy were verified through the mouse CT-26 colon cancer subcutaneous tumor model. The present invention combines the functional cell membrane derived from tumor cells and the composite immune adjuvant derived from bacteria to provide a safe and efficient immunotherapy drug, providing a new solution for achieving effective cancer immunotherapy and combined therapy.

[0008] More specifically,

[0009] In the present invention, a trichloroacetic acid method is adopted to extract WPG from bacteria, and the extraction method is as follows: firstly, a bacterial precipitate is dissolved in a 10% TCA solution, and stirred in a water bath to remove teichoic acid; then the precipitate obtained in the previous step is added to a mixed solution of V (sodium acetate): V (chloroform): V (methanol) = 4:5:10, wherein the pH value of the sodium acetate solution is 4.6, and the concentration is 0.02 mol / L, and stirred at room temperature for 24 hours to remove lipid substances; next, the precipitate in the previous step is added dropwise to a Tris-HCl buffer solution (0.1 mol / L, pH 7.5) containing 1 mg / ml trypsin, and the mixture is placed in a 37°C constant temperature shaker, the rotation speed is set to 120 rpm, and the mixture is shaken to remove protein substances; finally, the precipitate is collected by centrifugation, and freeze-dried to obtain WPG freeze-dried powder.

[0010] In the above operation of the present invention: the water bath temperature used for removing teichoic acid in the step is 60°C-80°C, preferably 70°C, and the water bath time is 3h-6h, preferably 4h; the time used for removing protein substances in the step is 10h-16h, preferably 12h.

[0011] In the present invention, the method for preparing PW / SR is as follows: adding SR-717 solution to P.WPG suspension, stirring at room temperature, centrifuging and washing the precipitate, and the precipitate is PW / SR.

[0012] In the above operation: the mass ratio of P.WPG to SR-717 in the steps is 12:1, 11:1, 10:1, 9:1, preferably 10:1, and the stirring time is 12h-48h, preferably 24h.

[0013] In the present invention, the method for preparing CPW / SR is as follows: first, different concentrations of doxorubicin are co-incubated with CT-26 cells to construct CT-26 cells with CRT eversion; then, the cells are scraped and collected from the culture dish using a cell scraper, the cells are lysed by a cell membrane extraction kit and repeatedly extruded, and then the functional cell membrane is separated and collected by differential centrifugation; finally, the functional cell membrane is mixed with PW / SR, and repeatedly extruded through a 1 μm polycarbonate membrane using an extruder to fully fuse PW / SR with the cell membrane to obtain CPW / SR.

[0014] In the above operation: the concentration of doxorubicin in the step is 1 μg / mL, 10 μg / mL, 50 μg / mL, preferably 10 μg / mL, and the administration time is 4h, 12h, 24h, 48h, preferably 12h; the mass ratio of cell membrane (based on the mass of membrane protein contained) to PW / SR in the step is 1:5-5:1, preferably the mass ratio of cell membrane to PW / SR is 1:1; the number of repeated extrusion in the step is 17-25 times, preferably 21 times.

[0015] The present invention extracts the complete peptidoglycan (WPG) from bacteria to make it a composite immune adjuvant, that is, it has the ability to activate inherent immunity while acting as a carrier. The present invention constructs a CRT everted CT-26 tumor cell model and extracts its cell membrane to wrap it on the surface of the drug-loaded peptidoglycan, effectively increasing the uptake of the entire particle system by dendritic cells and the lymph node drainage capacity. The immunotherapy effect of the drug-loaded peptidoglycan treatment system and the combined treatment effect of radiotherapy are verified by animal in vitro experiments. The present invention develops a safe and efficient immunotherapy drug, which provides a new solution for achieving effective cancer immunotherapy and combined treatment.

[0016] Beneficial effects of the present invention:

[0017] (1) We prepared a drug-loaded peptidoglycan CPW / SR wrapped in the tumor cell membrane with calreticulin externalized, which achieved efficient dendritic cell uptake, lymph node drainage, and immune system activation in vivo and in vitro.

[0018] (2) The prepared calreticulin-externalized tumor cell membrane-wrapped drug-loaded peptidoglycan CPW / SR can be used for immunotherapy of colon cancer; in addition, combined with radiotherapy, it achieved 100% tumor recurrence-free rechallenge in a mouse subcutaneous colon cancer tumor model. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 .Preparation and characterization of drug-loaded peptidoglycan CPW / SR wrapped by tumor cell membrane with calreticulin externalization,

[0020] (A) Laser confocal image of CT-26 cells with CRT externalization, scale bar: 100 μm;

[0021] (B) Transmission electron microscopy images of PW / SR and CPW / SR, scale bar: 1 μm;

[0022] (C) Laser confocal image of colocalization of PW / SR and functional cell membrane, scale bar: 1 μm;

[0023] (D) Characterization of surface proteins of CT-26 cells with CRT externalization, cell membrane, PW / SR and CPW / SR; (E) Western-blot protein detection results of calreticulin.

[0024] Figure 2 .In vitro dendritic cell uptake and immune activation study of CPW / SR,

[0025] (A) Flow cytometry results of DC 2.4 cell uptake;

[0026] (B) MFI value of the co-stimulatory molecule CD80 on the surface of BMDC cells;

[0027] (C) MFI value of the co-stimulatory molecule CD86 on the surface of BMDC cells;

[0028] (D) Representative dot plots of flow cytometry analysis of mature BMDC cells.

[0029] Figure 3 .Study on the in vivo distribution of CPW / SR, including:

[0030] (A) Representative fluorescence imaging of the main organs of mice in each group 12 h after subcutaneous injection of different preparations;

[0031] (B) Representative fluorescence imaging of the draining inguinal lymph nodes (ILN) of mice in each group 12 h after subcutaneous injection of different preparations;

[0032] (C) Fluorescence quantitative results of ILN in each group of mice 12 h after subcutaneous injection of different preparations;

[0033] (D) Laser confocal images of the distribution of different preparations in dendritic cells of ILN 12 h after subcutaneous injection of different preparations, scale bar: 50 μm.

[0034] Figure 4 .In vivo immune activation study of CPW / SR,

[0035] (A) CD8 + Representative confocal laser images of T cells, scale bar: 100 μm;

[0036] (B) Representative laser confocal images of Treg cells in tumor tissues, scale bar: 100 μm;

[0037] (C) Representative dot plots of flow cytometry analysis of mature DCs in lymphoid tissues;

[0038] (D) Quantification results of mature DC cells in lymphoid tissues;

[0039] (E) Representative dot plots of flow cytometry analysis of cytotoxic T lymphocytes in tumor tissues;

[0040] (F) Quantification of cytotoxic T lymphocytes in tumor tissues.

[0041] Figure 5 .CPW / SR is used to treat colon cancer in mice and its efficacy in combination with radiotherapy.

[0042] (A) Survival curves of tumor-bearing mice in each treatment group during CPW / SR immunotherapy (n=6);

[0043] (B) Images of in vitro tumor tissues of tumor-bearing mice in each treatment group during CPW / SR immunotherapy (n=6);

[0044] (C) Survival curves of tumor-bearing mice in each treatment group in combined therapy (n=10);

[0045] (D) Inhibition rate of tumor cell re-inoculation in combined therapy (n=6);

[0046] (E) Pictures of mice in each group re-inoculated with tumor cells in the combined treatment (n=6). DETAILED DESCRIPTION

[0047] To facilitate those skilled in the art to understand the contents of the present invention, the technical solutions and corresponding evaluation characterization solutions of the present invention will be further described below in conjunction with specific embodiments, but the following content should not limit the scope of protection requested by the claims of the present invention in any way.

[0048] Example 1. Preparation of drug-loaded peptidoglycan CPW / SR wrapped by tumor cell membrane with calreticulin externalization

[0049] (1) Extract P.WPG using trichloroacetic acid method. Dissolve Pseudomonas aeruginosa sludge with 10% trichloroacetic acid solution and stir in a 70℃ water bath for 4h. Collect the precipitate by centrifugation (12000g, 20min, 4℃) to remove teichoic acid; add the precipitate from the previous step to a mixture of V(sodium acetate):V(chloroform):V(methanol)=4:5:10, where the pH value of the sodium acetate solution is 4.6 and the concentration is 0.02mol / L, stir at room temperature for 24h, and collect the precipitate by centrifugation to remove lipid substances; add the precipitate from the previous step dropwise to a Tris-HCl buffer solution (0.1mol / L, pH 7.5) containing 1mg / ml trypsin, shake in a constant temperature shaker at 37℃ for 12h at a speed of 120rpm, collect the precipitate by centrifugation, and wash the precipitate three times with sterile distilled water in each step; freeze-dry to obtain P.WPG freeze-dried powder.

[0050] (2) According to the mass ratio of P.WPG to SR-717 of 10:1, SR-717 solution was added to the P.WPG suspension, stirred at room temperature for 24 h, centrifuged at 10000 rpm for 10 min, and the precipitate was collected and washed 3 times to obtain PW / SR.

[0051] (3) CT-26 cells were cultured in DMEM medium containing 10% FBS and 1% double antibody, and DOX aqueous solution was added to make the final concentration 10μg / mL. The cells were incubated in a 37℃ incubator for 12h, the medium was removed, and the cells were washed 3 times with pre-cooled PBS. The cells were scraped from the culture dish with a cell scraper and collected in a centrifuge tube. Membrane protein extraction reagent A (1ml) containing 1mM protease inhibitor (PMSF) was added, and the cells were resuspended in an ice bath for 15min. The cell suspension was then repeatedly ground 50 times using a glass grinder to break the cells. The cell fragment suspension was then centrifuged at 700g for 10min at 4℃, and the supernatant was collected to remove the cell nucleus and unlysed cells. Finally, the supernatant was centrifuged at 14,000g for 30min at 4℃ to obtain a cell membrane precipitate. The amount of purified cell membrane protein was determined using a BCA protein quantification kit for subsequent use.

[0052] (4) PW / SR and functional cell membrane were mixed in a ratio of 1:1 by mass, and 1 μm polycarbonate membrane was repeatedly extruded 21 times by a liposome extruder to prepare CPW / SR.

[0053] Example 2. Characterization of the physical and chemical properties of CPW / SR

[0054] (1) Place 1×10 4 CT-26 cells were cultured overnight to allow the cells to adhere to the wall. PBS and 10μg / mL DOX aqueous solution were added to each well, respectively, and incubated in a 37°C incubator for 12 hours. The cells were collected and fixed with 4% paraformaldehyde and blocked with 5% BSA; according to the instructions, appropriate concentrations of anti-CRT primary antibody and fluorescently labeled secondary antibody were added, respectively, and reacted at room temperature and in the dark for 1 hour, and DAPI staining solution was added to stain the nuclei. The cells were washed 3 times and photographed under laser confocal microscope. The experimental results are shown in Figure 2. Figure 1 As shown in A, after the cells were treated with 10 μg / mL DOX, the cells underwent ICD effect, and CRT was externalized to the cell surface, thereby observing significant green fluorescence, indicating that CRT externalized CT-26 cells were successfully constructed.

[0055] (2) Take an appropriate amount of 2 mg / ml PW / SR and CPW / SR suspension and place it on the carbon support film copper grid. After all the water evaporates, take an appropriate amount of 2% phosphotungstic acid to immerse the copper grid for negative staining. After all the water evaporates, observe its morphology with the help of TEM and take pictures. The experimental results are as follows Figure 1 As shown in B, both PW / SR and CPW / SR are rod-shaped structures. The surface of PW / SR is smooth and has clear boundaries, while the outer layer of CPW / SR has an obvious membrane coating structure, indicating the successful coating of functional cell membrane.

[0056] (3) PW / SR was labeled with fluorescent dye Di1, and CMs was labeled with fluorescent dye DiO. Fluorescently labeled CPW / SR was prepared by the method described in Example 1, and the co-localization of the preparation was observed under laser confocal microscope. The experimental results are shown in Figure 1 As shown in C, CMs labeled with fluorescent dye DiO were coated on the surface of PW / SR labeled with fluorescent dye Dil, showing good co-localization, proving the successful coating of the membrane.

[0057] (4) Polyacrylamide gel electrophoresis and western blot were used to detect the surface proteins of CRT externalized CT-26 cells, cell membrane, PW / SR and CPW / SR. Figure 1 As shown in D and 1E, the bands of CT-26 cells were basically consistent with those of the cell membrane and CPW / SR, indicating that the membrane extraction process and repeated extrusion process did not destroy the integrity of the membrane protein. CRT characteristic proteins were detected in both the cell membrane fraction and the CPW / SR fraction, while no bands were detected in P.WPG. The above results indicate that CRT was successfully coated on the CT-26 cell membrane.

[0058] Example 3. In vitro immune activation study of CPW / SR

[0059] (1) P.WPG was labeled with 5 μg / mL fluorescent dye Dil, and flow cytometry was used to compare the ability of DC 2.4 cells to take up P.WPG, PW / SR, and CPW / SR. DC 2.4 cells were plated at 3×10 5 Cells were seeded in a 12-well plate at a density of 1.54 cells / well. After overnight incubation and adherence to the plate, different preparations (WPG concentration was 25 μg / mL) were added to each well and incubated in a 37°C incubator in the dark for 4 h. Flow cytometry was used to quantitatively measure the fluorescence intensity of each group. The experimental results are shown in Figure 2. Figure 2 As shown in A, the uptake in the CPW / SR group was approximately 3.8 times that of the other preparation groups, indicating that the presence of CRT-externalized CT-26 cell membrane can significantly increase the uptake of dendritic cells, thereby more effectively activating innate immunity.

[0060] (2) BMDCs were isolated and cultured from the femur and tibia of mice. The cells were cultured at 3×10 5Cells were seeded in a 12-well plate at a density of 1.5 μg / mL and 2.5 μg / mL, respectively, and SR-717, P.WPG, PW / SR, and CPW / SR were added to each well. The cells were incubated in a 37°C incubator for 24 h. Anti-CD11c, anti-CD80, and anti-CD86 flow cytometry antibodies were added at corresponding concentrations according to the instructions. The cells were reacted in the dark at 4°C for 45 min. Mature BMDC cells (CD11c + CD86 + CD80 + The experimental results are as follows Figure 2 As shown in Figures 2B, 2C, and 2D, the expression levels of CD80 and CD86 on the surface of BMDC cells induced by the CPW / SR group were 2.5 times that of P.WPG, 1.2 times that of PW / SR, and 1.8 times that of the classic adjuvant LPS, respectively. The results showed that CPW / SR exhibited the strongest DC cell activation ability.

[0061] Example 4. In vivo distribution study of CPW / SR

[0062] (1) DiD-labeled P.WPG, PW / SR, and CPW / SR (250 μg P.WPG per mouse) were injected subcutaneously at the tail base of BALB / c mice. The mice were killed by cervical dislocation 12 hours later. The main organs and inguinal lymph nodes of the mice were removed, and the fluorescence intensity of DiD in each tissue and organ was observed and recorded using a small animal in vivo imaging device. The experimental results are shown in Figure 2. Figure 3 As shown in Figures A, 3B, and 3C, compared with other organs, P.WPG, PW / SR, and CPW / SR have a clear tendency to drain lymph nodes. The strongest fluorescence was observed in the CPW / SR group in the draining inguinal lymph nodes of mice, and the fluorescence intensity was 1.6 times and 1.5 times that of the P.WPG group and the PW / SR group, respectively. This indicates that the CRT-everted CT-26 cell membrane can not only increase the uptake of dendritic cells, but also enhance the ability of lymph node drainage, so that it can efficiently activate various immune-related cells in the draining lymph nodes. The activated immune effector cells infiltrate into the tumor tissue and kill cancer cells, which provides a basis for its ability to activate effective immune responses in vivo.

[0063] (2) To study the distribution of different preparations in dendritic cells of ILN, after the mice were given the above-mentioned drugs, their lymph nodes were removed and embedded with OCT, frozen sections were prepared, dendritic cells were labeled with anti-CD11c antibody, and cell nuclei were labeled with DAPI, and then observed under CLSM. Figure 3 As shown in D, CPW / SR is abundantly distributed in the dendritic cells of ILN, indicating that it has the highest accessibility to dendritic cells.

[0064] Example 5. In vivo immune activation study of CPW / SR

[0065] (1) To construct a mouse CT-26 subcutaneous tumor model, 100 μL of CT-26 cell suspension (density 5×10 6 / mL) was inoculated on the lower right back of BALB / c mice. The day of tumor inoculation was counted as Day 0. On Day 7, the tumor volume increased to 50-100mm 3 The drug was administered by subcutaneous injection near the tumor of mice on Day 7, Day 10, and Day 13. The single administration volume for each group was 100 μL (350 μg P.WPG per mouse).

[0066] (2) Tumor-bearing mice were killed 24 h after the last administration (Day 14), and tumor tissues were removed and embedded in OCT. Frozen sections were prepared and CD8α and Foxp3 antibodies were used to detect T cell typing in the tumor microenvironment. Figure 4 As shown in A and 4B, after CPW / SR treatment, CD8 + The number of T cells in tumor tissues increased significantly, while the number of Treg cells in tumor tissues decreased significantly. This result showed that the infiltration of immune active cells increased and the inhibitory cells decreased in the CPW / SR treatment group, which also indicated a good tumor prognosis.

[0067] (3) Tumor-bearing mice were killed 24 h after the last administration (Day 14), and the inguinal lymph nodes were removed under sterile conditions. The tissues were cut into small pieces with scissors and placed in a 40 μm cell strainer. The tissue fragments were gently ground with a 1 mL disposable syringe piston and rinsed with PBS until the tissues were dispersed into single cells. The cells were collected by centrifugation. Anti-CD11c and anti-CD86 flow cytometry antibodies of corresponding concentrations were added according to the instructions, and the reaction was carried out in the dark at 4°C for 45 min. The mature DC cells (CD11c + CD86 + The experimental results are as follows Figure 4 As shown in C and 4D, the number of mature DC cells in the draining lymph nodes of the CPW / SR treatment group was the largest, 3.5 times that of the negative control group. As the most powerful professional antigen-presenting cells, mature DC cells can present tumor antigens to naive T cells, thereby activating T cells and triggering specific anti-tumor immune responses.

[0068] (4) Tumor-bearing mice were killed 24 hours after the last administration (Day 14), and the tumor tissue was removed under sterile conditions. A single cell suspension was prepared and 1640 culture medium containing 10% FBS and 1× cell stimulation mixture (with protein transport inhibitor) was added for stimulation for 12-18 hours. The cell membrane surface CD8 was stained according to the method described in Example 5 (3). The cells were then fixed with 4% paraformaldehyde, and after removing the fixative, 400 μL of membrane permeabilization solution was added to permeabilize the membrane in an ice bath for 5 minutes. According to the instructions, the corresponding concentration of anti-IFN-γ flow cytometry antibody was added, and the cells were finally resuspended with 100 μL of antibody-containing membrane permeabilization solution and reacted in the dark at 4°C for 45 minutes. The number of cytotoxic T lymphocytes (CTL) in the tumor tissue was analyzed by flow cytometry. The experimental results are as follows. Figure 4 As shown in E and 4F, CPW / SR can significantly increase the number of CTL cells in tumor tissues, which is 11.0 times that of the negative control group, thereby effectively killing cancer cells.

[0069] Example 6. Study on the efficacy of CPW / SR in immunotherapy of mouse colon cancer and its combination with radiotherapy

[0070] (1) To study the efficacy of CPW / SR-induced immunotherapy for mouse colon cancer, BALB / c mice were inoculated with 5×10 5 The day of tumor inoculation was counted as Day 0. The mice were subcutaneously injected near the tumor on Day 7, Day 10, and Day 13. The survival curves of the mice were recorded, and the tumor tissues of the mice in each group were removed at the end of the experiment. Figure 5 As shown in A and 5B, the median survival time of mice in each group was ranked as follows: CPW / SR (40d) > PW / SR (31d) > PBS (16d). The results showed that CPW / SR treatment could significantly prolong the median survival time of mice and slow down the tumor growth rate.

[0071] (2) To study the efficacy of CPW / SR combined with radiotherapy for the treatment of mouse colon cancer, BALB / c mice were inoculated with 1×10 6 CT-26 tumor cells were planted on the day of tumor inoculation as Day 0. The mice were first irradiated on Day 7 (absorbed dose = 6 Gy), and then injected subcutaneously near the tumor on Day 7, Day 10, and Day 13 after irradiation. The experimental groups all contained P.WPG (350 μg per mouse), and the RT group was given 100 μL PBS. The survival curve of the mice was recorded. The experimental results are shown in Figure 5As shown in Figure C, the median survival of the RT group and the CPW / SR group was 22 days, while the RT and CPW / SR combined treatment group was able to significantly inhibit tumor growth. On the 30th day, the tumors of 6 mice completely regressed, achieving a complete response (CR) rate of 60%. On the 30th day, the 6 mice that had recovered from the RT and CPW / SR combined treatment were inoculated with a second tumor subcutaneously on the contralateral back. The experimental results are shown in Figure C. Figure 5 As shown in D and 5E, no signs of tumor growth were observed in any mouse on the 30th day after inoculation, achieving a 100% tumor re-challenge inhibition rate, while all 6 blank mice in the control group died on the 12th day after the tumor inoculation. The above results indicate that the combined treatment of RT and CPW / SR can produce an effective anti-tumor immune memory effect, and thus can withstand the re-challenge of the tumor.

[0072] In summary, the present invention prepares a drug-loaded peptidoglycan therapeutic system CPW / SR wrapped with CRT-everted CT-26 tumor cell membrane, which can safely and efficiently activate immune responses in vivo and in vitro, reverse the immunosuppressive tumor microenvironment, and provide a new solution for achieving effective cancer immunotherapy and combined therapy.

Claims

1. A drug-loaded peptidoglycan wrapped by the membrane of CT-26 tumor cells with CRT eversion, characterized in that: The drug-loaded peptidoglycan is a drug-loaded peptidoglycan therapeutic system CPW / SR including a drug, a drug carrier and a functional cell membrane, wherein the drug is a drug for treating cancer; the drug carrier is a bacterial-derived composite immune adjuvant, which is extracted and purified from bacteria by a trichloroacetic acid method, and encapsulates the drug to form an inner core particle system; the functional cell membrane is a CRT-everted CT-26 tumor cell membrane induced by chemotherapy drugs, and the functional tumor cell membrane encapsulates the inner core particle system by extrusion to form a drug-loaded peptidoglycan therapeutic system wrapped by the CRT-everted CT-26 tumor cell membrane.

2. The drug-loaded peptidoglycan wrapped by the membrane of CRT-everted CT-26 tumor cells according to claim 1, characterized in that: The drugs used to treat cancer are chemotherapy drug doxorubicin and immunotherapy drug SR-717.

3. The drug-loaded peptidoglycan wrapped by the membrane of CRT-everted CT-26 tumor cells according to claim 1, characterized in that: The bacterial-derived drug carrier is a complete peptidoglycan (WPG) extracted and purified from bacteria, preferably Staphylococcus aureus WPG (S.WPG), Pseudomonas aeruginosa WPG (P.WPG), Lactobacillus rhamnosus WPG (L.WPG), and more preferably P.WPG.

4. The drug-loaded peptidoglycan wrapped by the membrane of CRT-everted CT-26 tumor cells according to claim 1, characterized in that: The functional CT-26 tumor cell membrane is a mouse colon cancer cell (CT-26 cell) that has been induced by chemotherapy drugs to produce an immunogenic cell death effect and has CRT eversion characteristics. The cells are lysed using a cell lysis solution, and the cells are broken into fragments by repeated grinding. Finally, the CT-26 cell membrane with CRT eversion is separated by differential centrifugation.

5. The method for preparing the drug-loaded peptidoglycan wrapped by the membrane of CRT-everted CT-26 tumor cells according to claims 1-4, characterized in that: The following steps are involved: (1) Extraction of WPG: WPG was extracted using the trichloroacetic acid (TCA) method. First, the bacterial precipitate was dissolved in a 10% TCA solution and stirred in a water bath to remove teichoic acid. Then, the precipitate obtained in the previous step was added to a mixture of V (sodium acetate): V (chloroform): V (methanol) = 4:5:10 and stirred at room temperature to remove lipid substances. Next, the precipitate in the previous step was added dropwise to a Tris-HCl buffer solution (0.1 mol / L, pH 7.5) containing 1 mg / ml trypsin and shaken in a shaker to remove protein substances. Finally, the precipitate was collected by centrifugation and freeze-dried to obtain WPG freeze-dried powder. (2) Preparation of PW / SR: Add SR-717 solution to P.WPG suspension, stir at room temperature, centrifuge and wash the precipitate, the precipitate is PW / SR; (3) Preparation of CPW / SR: Doxorubicin was co-incubated with CT-26 cells to construct CT-26 cells with CRT eversion. The cell membrane was extracted and repeatedly extruded with PW / SR through an extruder to prepare CPW / SR.

6. The preparation method according to claim 5, characterized in that: In the step (1), the water bath temperature for removing teichoic acid is 60° C.-80° C., preferably 70° C., and the water bath time is 3 h-6 h, preferably 4 h.

7. The preparation method according to claim 5, characterized in that: In the step (1), the pH value of the sodium acetate solution in the mixed solution used to remove lipid substances is 4.6, the concentration is 0.02 mol / L, and it is stirred at room temperature for 24 hours.

8. The preparation method according to claim 5, characterized in that: In the step (1), the temperature of the thermostatic shaker used to remove protein substances is set to 37° C., the rotation speed is 120 rpm, and the time is 10 h-16 h, preferably 12 h.

9. The preparation method according to claim 5, characterized in that: In the step (2), the mass ratio of P.WPG to SR-717 is 12:1, 11:1, 10:1, 9:1, preferably 10:1, and the stirring time is 12h-48h, preferably 24h.

10. The preparation method according to claim 5, characterized in that: In the step (3), the concentration of doxorubicin for constructing CRT everted CT-26 cells is 1 μg / mL, 10 μg / mL, 50 μg / mL, preferably 10 μg / mL, and the administration time is 4 h, 12 h, 24 h, 48 h, preferably 12 h.

11. The preparation method according to claim 5, characterized in that: In the step (3), the cell membrane extraction step is as follows: using a cell scraper to scrape the CRT-inverted CT-26 cells from the culture dish and collect them into a centrifuge tube, adding a membrane protein extraction reagent A (1 ml) containing 1 mM protease inhibitor (PMSF), and resuspending the cells in an ice bath for 15 minutes, then repeatedly grinding the cell suspension 50 times using a glass grinder to break the cells, then centrifuging the cell fragment suspension at 700g for 10 minutes at 4°C, collecting the supernatant to remove the cell nucleus and unlysed cells, and finally, centrifuging the supernatant at 14,000g for 30 minutes at 4°C to obtain a cell membrane precipitate, and using a BCA protein quantification kit to determine the quality of the purified cell membrane protein for subsequent use.

12. The preparation method according to claim 5, characterized in that: In the step (3), the mass ratio of the cell membrane (based on the mass of the contained membrane protein) to PW / SR is 1:5-5:1, and preferably the mass ratio of the cell membrane to PW / SR is 1:

1.

13. The preparation method according to claim 5, characterized in that: In the step (3), the extruder is a polycarbonate membrane extruder having a pore size of 1 μm, and the number of repeated extrusions is 17-25 times, preferably 21 times.

Images