Dendritic cell membrane-based co-loaded nano preparation and application thereof

Abstract

The invention relates to the technical field of biological medicines and nano medicines, and discloses a co-loaded nano preparation based on a dendritic cell membrane and application of the co-loaded nano preparation. The nano preparation is prepared from hollow mesoporous MnO2, si-CTLA4, PD-1alpha and a DCs hybrid membrane, the preparation is prepared by loading the si-CTLA4 through the hollow mesoporous MnO2, coating the DCs hybrid membrane, and coupling the PD-1alpha through EDC / NHS mediated amidation reaction, so that multi-mechanism synergistic immune activation is realized. In-vitro experiments prove that the nano preparation can efficiently promote T cell proliferation and cancer cell killing; in-vivo experiments show that the nano preparation significantly inhibits LS-related CRC growth and has a good anti-tumor effect. The invention provides a new strategy for improving the immune response rate of ICIs, and also provides a theoretical basis for clinical treatment of LS-related CRC.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and nanomedicine, and particularly relates to a dendritic cell membrane-based co-loaded nano preparation and application thereof. Background Art

[0002] Lynch syndrome (LS) is a common hereditary cancer syndrome. Due to germline mutations in DNA mismatch repair genes (MMR genes), patients with this condition are susceptible to a variety of cancers, including colorectal cancer (CRC), gastrointestinal cancer, endometrial cancer (EC), and ovarian cancer. LS is closely associated with the development of CRC, with patients experiencing a lifetime risk of 70-80% for developing CRC. Therefore, there is an urgent need to treat LS, particularly LS-associated CRC. Current conventional treatments for LS-associated CRC include chemotherapy and surgery. However, LS-associated CRCs exhibit high microsatellite instability (MSI-H), leading to a high tumor mutational burden (TMB) and numerous neoantigens. Consequently, they are susceptible to developing resistance to chemotherapy drugs. Repeated surgeries are also difficult to prevent the development of new tumors and cannot completely eliminate tumor cells.

[0003] Studies have reported enhanced T cell responses within the tumor microenvironment of LS patients, suggesting that immunotherapy has potential for LS-related CRC. Numerous studies have reported on immunotherapy for LS-related CRC, particularly immune checkpoint inhibitors (ICIs) targeting immune checkpoints. There are many types of ICIs available on the market, with programmed death ligand 1 (PD-L1) / programmed death 1 (PD-1) immune checkpoint inhibitors being the most widely used. Although PD-1 / PD-L1 show high response rates and durable clinical benefits overall, approximately 50% of LS-related CRC patients still show significant low response to PD-1 / PD-L1 inhibitor monotherapy. Therefore, a synergistic treatment strategy is needed to improve the immune response rate and durability of ICIs. Summary of the Invention

[0004] In response to the above technical problems, the present invention aims to provide a dendritic cell membrane-based co-loaded nanoformulation, which solves the defects of the prior art through the following strategies:

[0005] (1) Dendritic cell (DC) hybrid membrane enhanced antigen presentation: using LS-related CRC antigen (Lynch syndrome colorectal cancer antigen MC38 Mlh1 KD DCs membranes pre-stimulated with cell lysate to activate tumor-specific T cells;

[0006] (2) Dual immune checkpoint blockade: Simultaneously targeting the PD-1 / PD-L1 and CTLA4 (cytotoxic T lymphocyte-associated protein 4) pathways, significantly improving the treatment response rate and synergistically enhancing the anti-tumor immune response.

[0007] The nanoformulation is DCsLipo@MnO2@si-CTLA4@PD-1α, which has a multilayer core-shell structure and includes:

[0008] a. Core layer: hollow mesoporous MnO2 nanoparticles loaded with si-CTLA4;

[0009] b. Middle layer: liposomes, encapsulating PD-1α;

[0010] c. Outer shell: mature DCs membrane stimulated with LS-related CRC antigens;

[0011] Furthermore, the LS-related CRC antigen is the Lynch syndrome colorectal cancer antigen MC38 Mlh1 KD Cell lysate.

[0012] The present invention also provides a method for preparing a dendritic cell membrane-based co-loaded nanoformulation, the preparation method comprising the following steps:

[0013] (1) Preparation of hollow mesoporous MnO2

[0014] a. Preparation of SiO2 NPs: Mix 14 mL of ethanol, 2 mL of deionized water, and 500 μL of ammonia water in a 50 mL round-bottom flask. Stir magnetically in a 50°C oil bath for 5 min. Slowly add 500 μL of ethyl orthosilicate dropwise and continue stirring for 2 h to obtain SiO2 NPs. Wash with ethanol and water and store in water until use.

[0015] b. Preparation of SiO2@MnO2 NPs: 600 mg of KMnO4 was dissolved in 20 mL of water, dispersed evenly, and then 400 mg of SiO2 NPs was added dropwise. After ultrasonic treatment for 1 hour, the mixture was stirred at room temperature overnight. The next day, the mixed solution was washed three times with deionized water and then centrifuged at 14,800 rpm for 15 minutes to obtain SiO2@MnO2 NPs.

[0016] c. Preparation of hollow mesoporous MnO2: 50 mg of SiO2@MnO2 NPs were dispersed in 20 mL of 2M Na2CO3 solution and reacted at 60°C for 12 h; then centrifuged at 12000 rpm for 15 min, washed three times with deionized water and vacuum dried to obtain hollow mesoporous MnO2.

[0017] (2) Preparation of MnO2@si-CTLA4

[0018] 2.5 nmol si-CTLA4 was added to 2 mg MnO2 in 5 mL of deionized water and shaken at 4°C in the dark for 12 h. The resulting solution was then stored at 4°C in the dark.

[0019] (3) Preparation of DCsLipo@MnO2@si-CTLA4

[0020] a. Lynch syndrome colorectal cancer antigen MC38 Mlh1 KD Preparation of cell lysate: pLKO.1-Puro empty vector (sh-NC) and pLKO.1-M1h1 shRNA (sh-M1h1) interference plasmids were transfected into MC38 colon cancer cells using Lipofectamine 2000 and incubated for 24 h. The transfected cells were screened with corresponding antibiotics, and the knockdown of the Mlh1 gene was verified by qPCR. The MC38 cell line with Mlh1 knockdown (MC38 Mlh1 KD ), MC38 cells were treated with cell lysate Mlh1 KD Lynch syndrome colorectal cancer antigen MC38 was obtained Mlh1 KD The cell lysate was frozen at -80°C until use;

[0021] b. Preparation of DCs cell membrane: Using 10 μg protein / mL Lynch syndrome colorectal cancer antigen MC38 Mlh1 KD Mouse bone marrow-derived DCs were stimulated with cell lysate and incubated for 24 h. After DC maturation was confirmed by flow cytometry, the cells were washed with PBS and centrifuged at 300 × g for 5 min. The cell membranes of the stimulated DCs were extracted by differential centrifugation.

[0022] c. Preparation of DCsLipo@MnO2@si-CTLA4: A certain amount of soybean phosphatidylcholine (SPC), cholesterol (CHOL), and N-distearoylphosphatidyl acetamide-PEG-carboxyl (DSPE-PEG-COOH) were dissolved in 5 mL of chloroform solution in a round-bottom flask. The amount of DSPE-PEG-COOH was 2 μmol, and the molar ratio of DSPE-PEG-COOH:CHOL:SPC was 1:5:10, respectively. The solution was then evaporated under reduced pressure at 41°C for approximately 30 min to obtain a solid film. The film was then flushed with nitrogen for 30 min and dried in a vacuum oven for 12 h to remove residual chloroform. To this solution, 2 mL of DCs cell membrane aqueous solution and 2 mL of MnO2@si-CTLA4 aqueous solution were added and ultrasonically hydrated for 10 min to produce a liposome suspension. The vesicle suspension was obtained by extruding through an 800 nm polycarbonate membrane 50 times using a liposome extruder, then through a 400 nm polycarbonate membrane 50 times, and finally through a 200 nm polycarbonate membrane 21 times, and then stored at low temperature.

[0023] (4) Preparation of DCsLipo@MnO2@si-CTLA4@PD-1α

[0024] DCsLipo@MnO2@si-CTLA4 was mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) in 10 mL of PBS at pH 7.4, and then sonicated for 20 min. PD-1α was mixed with the above solution and stirred at 25°C in the dark for 24 h. The product was collected and purified with water to obtain the final product, DCsLipo@MnO2@si-CTLA4@PD-1α.

[0025] Furthermore, in step (3), the concentration of the DCs cell membrane aqueous solution is 2 mg protein / mL, and the concentration of the MnO2@si-CTLA4 aqueous solution is 40 μmol siRNA;

[0026] Furthermore, in step (4), the molar ratio of DCsLipo@MnO2@si-CTLA4:EDC-HCl:NHS is 1:2:2;

[0027] Furthermore, the amount of NHS used in step (4) is 40 μmol;

[0028] Furthermore, the mass ratio of PD-1α to DCsLipo@MnO2@si-CTLA4 in step (4) is 1:100;

[0029] The present invention also provides an application of a dendritic cell membrane-based co-loaded nanoformulation in the preparation of a drug for treating Lynch syndrome-related colorectal cancer.

[0030] Furthermore, the drug can promote the proliferation and activation of T cells in vitro and reduce the mRNA expression of CTLA4 in T cells;

[0031] Furthermore, the drug can promote the proliferation and activation of T cells by combining with PL-1α in vitro to promote MC38 Mlh1 KD Cell apoptosis;

[0032] Furthermore, the drug can inhibit MC38 through immune activation in vivo Mlh1 KD proliferation of tumors in mice with colon cancer;

[0033] Furthermore, the immune activation effect is manifested by decreased CTLA4 expression in tumor tissue, increased perforin (PFP) content, and CD4 + T, CD8 + T, CD3 + CD69 + Increased number of T cells.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The nanoformulation DCsLipo@MnO2@si-CTLA4@PD-1α synthesized in the present invention can effectively promote the infiltration of T cells into tumor tissues and activate their anti-tumor immune response through the synergistic effect of the antigen presentation effect of DCs membrane, PD-1α blocking the PD-1 / PD-L1 pathway, si-CTLA4 silencing immunosuppressive signals, and MnO2 as a carrier drug, and has a good inhibitory effect on LS-related CRC.

[0036] (2) The DCs hybrid membrane prepared by the present invention is a fusion DCs membrane of a mature DC membrane and a liposome membrane loaded with PD-1α. The good tissue compatibility of the DCs hybrid membrane can promote the uptake of therapeutic drugs by T cells, thereby improving the silencing efficiency of si-CTLA4. At the same time, it can enhance the stability of the DCs membrane, which is more conducive to promoting the activation of T cells.

[0037] (3) The nanoformulation DCsLipo@MnO2@si-CTLA4@PD-1α prepared by the present invention has high safety. In vitro hemolysis, CCK-8 and Calcein-AM / PI live / dead cell staining experiments prove that the nanoformulation has better biosafety than MnO2@si-CTLA4. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1:Basic structure characterization and stability testing of nanoformulations: Figure 1 (A) is the expression data of DCs cell membrane CD80 / CD86 detected by flow cytometry; Figure 1 (B) is the fluorescence resonance energy transfer (FRET, FITC / CY3) signal data of FITC / CY3 labeled liposome membrane after hybridization with DCs membrane; Figure 1 (C) Dynamic light scattering (DLS) particle size distribution of DCsLipo@MnO2@si-CTLA4@PD-1α; Figure 1 (D) Transmission electron microscopy (TEM) image of DCsLipo@MnO2@si-CTLA4@PD-1α; Figure 1 (E) Fourier transform infrared (FT-IR) spectra of nanoformulations at each stage; Figure 1 (F) Stability test of nanoformulations at each stage.

[0039] Figure 2 :Biosafety testing of nanoparticles: Figure 2 (A) Hemolysis test to investigate the blood compatibility of different nanoformulations; Figure 2 (B) CCK-8 assay for the cell viability of normal intestinal epithelial cells (MODE-K) and T cells of mice treated with different concentrations of nanoparticles; Figure 2 (C) The live-dead staining results of MODE-K cells after treatment with 50 μM of different materials.

[0040] Figure 3 : Evaluation of nanoformulation uptake by T cells.

[0041] Figure 4 :Study on the ability of nanoformulations to promote T cell activation in vitro: Figure 4 (A) qRT-PCR detection of CTLA4 mRNA expression levels in T cells treated with PBS and different nanoformulations; Figure 4 (B) Flow cytometry analysis of CD3 expression in T cells derived from mouse peripheral blood mononuclear cells (PBMC) after treatment with different nanoparticles. + CD69 + T cell count; Figure 4 (C) Flow cytometry analysis of the proliferation of PBMC-derived T cells after treatment with different nanoformulations; Figure 4 (D) T cells and MC38 treated with different nanoformulations Mlh1 KD Apoptosis levels of cancer cells after co-culture.

[0042] Figure 5 :In vivo tumor killing and immune activation effects of nanoformulations: Figure 5 (A) is the curve of tumor volume changes in tumor-bearing mice after treatment with different drugs; Figure 5 (B) Photographs of mouse tumor tissue in vitro; Figure 5 (C) is the weight of mouse tumor tissue; Figure 5 (D) H&E staining of mouse tumor tissues and major organs (heart, liver, spleen, lung, and kidney); Figure 5 (EG) is the staining of proliferating cell nuclear antigen Ki-67 (Ki67) (E), CTLA4 (F) and PFP / CD4 / CD8 (G) in mouse tumor tissue. Figure 5 (H) Flow cytometry analysis of CD3 in mouse tumor tissue + CD69 + T cell infiltration. DETAILED DESCRIPTION

[0043] The technical solutions of the present invention will be described clearly and completely below with reference to the accompanying drawings. It is obvious that the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort shall fall within the scope of protection of the present invention.

[0044] Example 1: Preparation of DCsLipo@MnO2@si-CTLA4@PD-1α

[0045] (1) Preparation of hollow mesoporous MnO2

[0046] a. Preparation of SiO2 NPs: Mix 14 mL of ethanol, 2 mL of deionized water, and 500 μL of ammonia water in a 50 mL round-bottom flask. Stir magnetically in a 50°C oil bath for 5 min. Slowly add 500 μL of ethyl orthosilicate dropwise and continue stirring for 2 h to obtain SiO2 NPs. Wash with ethanol and water and store in water until use.

[0047] b. Preparation of SiO2@MnO2 NPs: 600 mg of KMnO4 was dissolved in 20 mL of water and uniformly dispersed. Then, 400 mg of SiO2 NPs was added dropwise. After ultrasonic treatment for 1 hour, the mixture was stirred at room temperature overnight. The next day, the mixture was washed three times with deionized water and centrifuged at 14,800 rpm for 15 minutes to obtain SiO2@MnO2 NPs.

[0048] c. Preparation of hollow mesoporous MnO2: 50 mg of SiO2@MnO2 NPs were dispersed in 20 mL of 2M Na2CO3 solution and reacted at 60°C for 12 h; then centrifuged at 12000 rpm for 15 min, washed three times with deionized water and vacuum dried to obtain hollow mesoporous MnO2.

[0049] (2) Preparation of MnO2@si-CTLA4

[0050] 2.5 nmol si-CTLA4 and 2 mg MnO2 were mixed in 5 mL of deionized water and shaken at 4°C in the dark for 12 h. After shaking, the resulting solution was stored at 4°C in the dark.

[0051] (3) Preparation of DCsLipo@MnO2@si-CTLA4

[0052] a. Lynch syndrome colorectal cancer antigen MC38 Mlh1 KD Preparation of cell lysate: pLKO.1-Puro empty vector (sh-NC) and pLKO.1-Mlh1 shRNA (sh-Mlh1) interference plasmid were transfected into MC38 colon cancer cells using Lipofectamine 2000 and incubated for 24 h. The transfected cells were screened with corresponding antibiotics, and the knockdown of Mlh1 gene was verified by qPCR. MC38 cell lines with Mlh1 knockdown were screened (MC38 Mlh1 KD ), MC38 cells were treated with cell lysate Mlh1 KD Lynch syndrome colorectal cancer antigen MC38 was obtained Mlh1 KD The cell lysate was frozen at -80°C until use.

[0053] b. Preparation of DCs cell membrane: Using 10 μg protein / mL Lynch syndrome colorectal cancer antigen MC38 Mlh1 KD Mouse bone marrow-derived DCs were stimulated with cell lysate and incubated for 24 h. After confirming the maturation of DCs by flow cytometry, the cells were washed with PBS and centrifuged at 300 × g for 5 min. The cell membranes of the stimulated DCs were extracted by differential centrifugation.

[0054] c. Preparation of DCsLipo@MnO2@si-CTLA4: A certain amount of SPC, CHOL, and DSPE-PEG-COOH were dissolved in 5 mL of chloroform solution and placed in a round-bottom flask. The amount of DSPE-PEG-COOH was 2 μmol, and the molar ratio of DSPE-PEG-COOH:CHOL:SPC was 1:5:10, respectively. The solution was then evaporated under reduced pressure at 41°C for approximately 30 minutes to obtain a solid film. The film was flushed with nitrogen for 30 minutes and dried in a vacuum oven for 12 hours to remove any residual chloroform. To this solution, 2 mL of an aqueous solution of DCs cell membranes at 2 mg protein / mL and 2 mL of an aqueous solution of MnO2@si-CTLA4 containing 40 μmol of siRNA were added and sonicated for 10 minutes to produce a liposome suspension. The vesicle suspension was obtained by extruding through an 800 nm polycarbonate membrane 50 times using a liposome extruder, then through a 400 nm polycarbonate membrane 50 times, and finally through a 200 nm polycarbonate membrane 21 times, and then stored at low temperature.

[0055] (4) Preparation of DCsLipo@MnO2@si-CTLA4@PD-1α

[0056] DCsLipo@MnO2@si-CTLA4 was mixed with EDC·HCl and NHS (DCsLipo@MnO2@si-CTLA4:EDC·HCl:NHS molar ratio of 1:2:2) in 10 mL of PBS (40 μmol NHS, pH 7.4), and sonicated for 20 minutes. PD-1α was added to the solution at a mass ratio of PD-1α:DCsLipo@MnO2@si-CTLA4 of 1:100, and the mixture was stirred in the dark at 25°C for 24 hours. The product was collected and purified with water to obtain the final product, DCsLipo@MnO2@si-CTLA4@PD-1α.

[0057] Example 2: Preparation of Lipo@MnO2@si-CTLA4@PD-1α

[0058] The difference between this embodiment and embodiment 1 is that in step (3) DCsLipo@MnO2@si-CTLA4 is prepared, the Lynch syndrome colorectal cancer antigen MC38 is used in this embodiment. Mlh1 KD The cell lysate is directly combined with the liposome membrane to synthesize a new hybrid membrane. The specific steps are as follows:

[0059] A certain amount of SPC, CHOL, DSPE-PEG-COOH and MC38 Mlh1 KDThe cell lysate was dissolved in 5 mL of chloroform solution. The amount of DSPE-PEG-COOH was 2 μmol, where the molar ratio of DSPE-PEG-COOH: CHOL: SPC was 1:5:10. The lipid and MC38 Mlh1 KD The weight ratio of cell lysate was 15:2. The mixture was then evaporated under reduced pressure on a rotary evaporator at 41°C for approximately 30 minutes to obtain a solid film. Subsequently, 5 mL of deionized water was added and the mixture was sonicated for 10 minutes to obtain a liposome suspension. This suspension was then extruded 21 times through a polycarbonate membrane using a liposome extruder to obtain a vesicle suspension, which was then stored at low temperatures.

[0060] Example 3: Structural characterization and stability testing of DCsLipo@MnO2@si-CTLA4@PD-1α of Example 1

[0061] (1) Flow cytometry: For the analysis of DCs maturation during the preparation of nanoformulations, MC38 Mlh1 KD DCs treated with lysate were labeled with anti-CD80 and anti-CD86 antibodies, and the maturation of DCs was detected by flow cytometry. Figure 1 As shown in A, MC38 Mlh1 KD The expression of the co-stimulatory molecule CD86 on the DC cell membrane surface was significantly elevated in the lysate-stimulated group, while CD86 expression in the negative control group remained at baseline. This result confirms that the LS-related CRC cell lysate successfully activates DCs and supports the use of activated DC cell membranes as the base material for subsequent hybrid membrane preparation.

[0062] (2) FRET detection: In order to verify the successful preparation of the hybrid membrane, the present invention mixed liposomes and DCs cell membranes in different mass ratios (liposomes: DCs cell membrane = 1:0 / 1 / 2 / 3 / 4), labeled the liposomes with FITC / Cy3, and used FRET to verify whether the hybrid membrane preparation was successful. When the membrane components are relatively single, FITC receives excitation light of 488nm wavelength and emits emission light of 525nm. The emission light of 525nm further activates CY3 through fluorescence resonance energy transfer, and finally CY3 can emit emission light of 570nm wavelength. Figure 1 As shown in Figure B, liposomes and DC cell membranes were mixed and ultrasonically fused. As the ratio of liposomes to DC cell membranes increased, the distance between the two fluorophores increased, resulting in a decrease in the FRET signal. Consequently, as the liposomes and DC cell membranes hybridized, the fluorescence intensity of the hybrid membrane decreased at 570 nm, while the fluorescence signal at 525 nm increased.

[0063] (3) DLS characterization: DLS was used to detect the particle size of DCsLipo@MnO2@si-CTLA4@PD-1α. Figure 1 As shown in C, the particle size distribution was detected by DLS and showed a unimodal symmetrical distribution, indicating that DCsLipo@MnO2@si-CTLA4@PD-1α was uniform in size and the particle size was mainly distributed at 216.7 nm.

[0064] (4) TEM characterization: TEM was used to detect the morphological characteristics of DCsLipo@MnO2@si-CTLA4@PD-1α. Figure 1 As shown in D, transmission electron microscopy shows that DCsLipo@MnO2@si-CTLA4@PD-1α has a uniform morphology and good dispersion, with a typical spherical morphology. In addition, it can be seen that the liposomes encapsulate MnO2 with obvious edge contrast.

[0065] (5) FT-IR characterization: DCsLipo@MnO2@CTLA4@PD-1α was detected using FT-IR instrument. Figure 1 As shown in E, FT-IR results show that at 2850 cm -1 and 3000cm -1 Symmetrical and asymmetric CH stretching vibration peaks were observed between the two groups, which are characteristic absorption peaks of fatty acid chains in liposome components; -1 The stretching vibration peak of the ester carbonyl C=O appeared nearby, confirming the successful formation of the lipid structure of SPC, 1640cm -1 The absorption peak at 1540 cm is attributed to the amide I band, while the absorption peak at 1540 cm -1 The nearby amide II band further confirms that PD-1α is coupled to the nanoparticle surface via an amide bond. These results indicate that the liposomes of the present invention were successfully prepared and PD-1α has been successfully modified onto the surface of DCsLipo@MnO2@CTLA4i@PD-1α nanoparticles.

[0066] (6) Stability test: DCsLipo@MnO2@si-CTLA4@PD-1α was placed in a phosphate buffered saline (PBS) solution containing 10% fetal bovine serum (FBS) and incubated at 37°C for 72 hours. Samples were collected at each time point (2, 4, 6, 8, 12, 24, 48 and 72 hours) and the stability of the nanodrug DCsLipo@MnO2@si-CTLA4@PD-1α was evaluated by measuring the particle size change of DCsLipo@MnO2@si-CTLA4@PD-1α in PBS solution for 72 hours. Figure 1 As shown in F, the results show that the particle size of DCsLipo@MnO2@si-CTLA4@PD-1α detected at each time point in PBS solution is basically stable at the same level, and the particle size does not change significantly until 72 hours, indicating that the nanoformulation has good stability.

[0067] Example 4: Biosafety Testing of Nanoformulations

[0068] (1) Hemolysis experiment: Fresh blood from mice was collected by orbital bleeding and washed with physiological saline. The supernatant was discarded after centrifugation to obtain a red blood cell (RBC) suspension. The RBC suspension was incubated with deionized water (positive control group), physiological saline (negative control group) and different nanoformulations (MnO2@Si-CTLA4, DCsLipo@MnO2@Si-CTLA4, Lipo@MnO2@si-CTLA4@PD-1α, DCsLipo@MnO2@Si-CTLA4@PD-1α) at ​​37°C for 0.5-1h. After incubation, the hemolysis of each treatment group was photographed and recorded, and the sample was centrifuged at 3000rpm for 10min. The supernatant was collected and the absorbance of the supernatant at 540nm was detected by an enzyme-linked microplate reader. Finally, the hemolysis of each treatment group was quantified using the hemolysis rate calculation formula. According to international standards, when the hemolysis rate is less than 5%, it means that the sample has no obvious hemolysis.

[0069] The results are as follows Figure 2 As shown in A, the blood cells in the positive control group (ddH2O-treated group) were severely ruptured, and the RBC suspension showed a distinct red color. The blood cells in the experimental groups MnO2@si-CTLA4, DCsLipo@MnO2@si-CTLA4, Lipo@MnO2@si-CTLA4@PD-1α, and DCsLipo@MnO2@si-CTLA4@PD-1α showed only slight hemolysis, and the hemolysis rates were all lower than the internationally recognized standard (5%), indicating that the nanoformulations prepared in this study have good blood compatibility.

[0070] (2) CCK-8 experiment: Mouse normal intestinal epithelial cells MODE-K and T cells were injected at 5×10 3 Cells were seeded at a density of 100 μg / well in a 96-well plate and incubated in a 37°C, 5% CO2 cell culture incubator for 24 hours. Different concentrations (0, 50, 100, 200, 300, and 400 μM) of MnO2@si-CTLA4, DCsLipo@MnO2@si-CTLA4, Lipo@MnO2@si-CTLA4@PD-1α, and DCsLipo@MnO2@si-CTLA4@PD-1α diluted in culture medium were added to the cells and incubated at 37°C until the drug treatment time. Finally, CCK-8 solution (5%) was added to each well and incubated for a further 2 hours. The absorbance of the samples at 450 nm was measured using a microplate reader.

[0071] The results are as follows Figure 2As shown in Figure 3, different nanoformulation groups, including MnO2@si-CTLA4, DCsLipo@MnO2@si-CTLA4, Lipo@MnO2@si-CTLA4@PD-1α, and DCsLipo@MnO2@si-CTLA4@PD-1α, had no significant effect on the viability of MODE-K and T cells when the concentration was lower than 50 μM. However, when the concentration reached 100 μM, due to the lack of hybrid membrane modification effect of MnO2@si-CTLA4, it had varying degrees of effect on the viability of MODE-K and T cells. However, DCsLipo@MnO2@si-CTLA4, Lipo@MnO2@si-CTLA4@PD-1α, and DCsLipo@MnO2@si-CTLA4@PD-1α had no obvious toxicity to either cell type due to the presence of DCs hybrid membrane or liposome membrane. This indicates that the membrane-coated nanoformulation can significantly reduce the cytotoxicity of the MnO2 core, reflecting the good biocompatibility of the DCsLipo@MnO2@si-CTLA4@PD-1α nanoformulation prepared by the present invention.

[0072] (3) Calcein-AM / PI live / dead cell staining: In order to more intuitively observe the effect of nanoformulations on the viability of the two cell lines, MODE-K cells were treated with 50 μM of different nanoformulations. MODE-K cells were plated at 1×10 4 Each well was seeded in a 96-well plate and cultured at 37°C for 12 hours. 50 μM MnO2@si-CTLA4, DCsLipo@MnO2@si-CTLA4, Lipo@MnO2@si-CTLA4@PD-1α, and DCsLipo@MnO2@si-CTLA4@PD-1α diluted in DMEM were added to each well and cultured for another 5 hours. MODE-K cells were stained for live and dead cells using a Calcein-AM / PI kit and observed under a fluorescence microscope at 470 nm and 560 nm. Live cells appear green, and dead cells appear red.

[0073] The results are as follows Figure 2 As shown in Figure C, the cell live-dead staining results showed a similar trend to the CCK-8 results, indicating that the membrane-coated nanoformulation had good biocompatibility.

[0074] The above results show that the DCsLipo@MnO2@si-CTLA4@PD-1α nanoformulation prepared in the present invention has good biosafety.

[0075] Example 5: Uptake of Nanoformulations

[0076] Cellular uptake is an important factor affecting the effect of nanomedicines and an important indicator for evaluating their bioavailability. Therefore, this application further explored the uptake of different nanoformulations by T cells. The nanoformulations labeled with the fluorescent dye coumarin (C-6) were obtained to obtain nanoformulations with fluorescent signals (Lipo@C-6, DCsLipo@C-6, DCsLipo@PD-1α@C-6). 1×10 5 T cells were seeded in 6-well plates and cultured for 12 hours. The C-6-labeled nanoformulation was then added to the T cells and incubated for another 6 hours. After incubation, the T cells were harvested and washed three times with PBS. T cell uptake was quantified using flow cytometry.

[0077] The results are as follows Figure 3 As shown, compared with the control group (PBS group), the fluorescence intensity of T cells incubated with free C-6, Lipo@C-6, DCsLipo@C-6, and DCsLipo@PD-1α@C-6 increased significantly, indicating that T cells can effectively take up the nanoformulation. Further quantitative analysis showed that the number of positive T cells in the free C-6 treatment group and the Lipo@C-6 treatment group was only about 8.98% and 9.54%, respectively, while the number of positive T cells in the DCsLipo@C-6 and DCsLipo@PD-1α@C-6 treatment groups reached 23.72% and 33.76%, respectively, indicating that the DCs hybrid membrane nanoformulation prepared by the present invention can be effectively taken up by T cells.

[0078] Example 6: Study on the ability of nanoformulations to promote T cell activation in vitro

[0079] Since PD-1α, si-CTLA4 and mature DCs can activate T cells through different mechanisms, thereby exerting a more powerful anti-tumor effect, the present invention preliminarily evaluated in vitro whether the DCsLipo@MnO2@si-CTLA4@PD-1α nanoformulation can effectively inhibit cancer cell growth and activate T cells, mainly through its si-CTLA4 silencing efficiency, T cell activation ability, T cell proliferation ability and in vitro tumor killing effect.

[0080] (1) Silencing efficiency of si-CTLA4: T cells were treated with PBS and different nanoformulations (50 μM MnO2@si-CTLA4, DCsLipo@MnO2@si-CTLA4, Lipo@MnO2@si-CTLA4@PD-1α and DCsLipo@MnO2@si-CTLA4@PD-1α diluted in DMEM medium) for 24 h. Total RNA was extracted with Trizol reagent and reverse transcribed into cDNA using PrimeScriptTM RT reagent Kit. Finally, SYBR TM Green I was used to quantify the target gene RNA. -ΔΔCt The relative expression levels were calculated using the β-actin method, and β-actin was used as an internal reference.

[0081] The results are as follows Figure 4 As shown in Figure A, CTLA4 expression was downregulated to varying degrees after treatment of T cells with different nanoformulations. Treatment of T cells with hybrid membrane-containing DCsLipo@MnO2@si-CTLA4 and DCsLipo@MnO2@si-CTLA4@PD-1α showed a significant downregulation of CTLA4 compared to the MnO2@si-CTLA4 and Lipo@MnO2@si-CTLA4@PD-1α treatment groups without hybrid membranes. This suggests that the good tissue compatibility of DCs membranes can promote T cell uptake of nanoformulations, thereby improving the silencing efficiency of si-CTLA4.

[0082] (2) T cell activation ability: Mouse blood was collected and peripheral blood mononuclear cells (PBMCs) were separated from the mouse blood using Ficoll density gradient centrifugation. The collected PBMC cells were incubated with PBS and different nanoformulations (50 μM MnO2@si-CTLA4, DCsLipo@MnO2@si-CTLA4, Lipo@MnO2@si-CTLA4@PD-1α, DCsLipo@MnO2@si-CTLA4@PD-1α diluted in DMEM medium) for 24 hours, and then the PBMC cells were collected and labeled with anti-CD3 and anti-CD69. Finally, the activated CD3 + CD69 + T cell count.

[0083] The results are as follows Figure 4 As shown in B, compared with the control group (PBS group), the different nanoformulations used in this experiment treated PBMC cells, and the T cell surface activation marker CD69 +The expression of T cell surface activation marker CD69 was upregulated to varying degrees after DCsLipo@MnO2@si-CTLA4@PD-1α treatment. + The expression of mature DCs increased significantly, reaching about 79%, and was better than that of the Lipo@MnO2@si-CTLA4@PD-1α treatment group. This not only shows that mature DCs hybrid membrane can promote the activation of T cells, but also shows that its activation effect on T cells is better than that of liposome membrane containing LS-related CRC antigens.

[0084] (3) T cell proliferation ability: Carboxyfluorescein diacetate succinimidyl ester (CFSE) dye was diluted to 1 μM with PBS and added to activated T cells. The cells were incubated at room temperature for 10 min and free dye was washed away with PBS. CFSE-labeled T cells were seeded into 96-well plates and treated with PBS and different nanoformulations (50 μM MnO2@si-CTLA4, DCsLipo@MnO2@si-CTLA4, Lipo@MnO2@si-CTLA4@PD-1α and DCsLipo@MnO2@si-CTLA4@PD-1α diluted in DMEM medium) for 24 h. The cells were collected by centrifugation at 8000 × g for 5 min, stained with anti-CD3 antibody in the dark, washed twice with PBS, and T cell proliferation was detected by flow cytometry.

[0085] The results are as follows Figure 4 As shown in C, the number of proliferating T cells after DCsLipo@MnO2@si-CTLA4 treatment was 5.02%, which was higher than that of the MnO2@Si-CTLA4 treatment group (2.12%), indicating that mature DCs hybrid membranes can effectively activate T cells and promote their proliferation. It is worth noting that the number of proliferating T cells in the DCsLipo@MnO2@si-CTLA4@PD-1α treatment group was higher than that in the DCsLipo@MnO2@si-CTLA4 group, indicating that PL-1α can further promote the proliferation and activation of T cells. In addition, the number of T cells proliferated in the DCsLipo@MnO2@si-CTLA4@PD-1α treatment group was higher than that in the Lipo@MnO2@si-CTLA4@PD-1α treatment group, which also shows that the DCs hybrid membrane synthesized after pre-treatment of DCs cells with LS-related CRC antigens has a better effect on the proliferation and activation of T cells than the liposome membrane containing LS-related CRC antigens.

[0086] (4) In vitro tumor killing effect: In order to further explore whether DCsLipo@MnO2@si-CTLA4@PD-1α activated T cells can inhibit the development of cancer cells, the present invention treated T cells with different nanomedicines (treatment methods are similar to the experimental steps of the above-mentioned si-CTLA4 silencing efficiency) and MC38Mlh1 KD The cells were directly co-cultured for 48 h and stained with the Annexin V-FITC / PI double staining kit in the dark. The specific staining method was referred to the manufacturer's instructions. After staining, the excess dye was washed away with PBS and the cells were detected by flow cytometry.

[0087] The experimental results are as follows Figure 4 As shown in D, T cells treated with DCsLipo@MnO2@si-CTLA4@PD-1α can induce about 50% of MC38 Mlh1 KD Cells underwent apoptosis, while the number of apoptotic cells in the control group was only 6%. In addition, the effects of promoting cell apoptosis in the other groups showed a positive correlation with the ability of T cells to proliferate and activate in the above experiments, indicating that DCsLipo@MnO2@si-CTLA4@PD-1α can effectively activate the proliferation and activation of T cells, thereby promoting tumor cell apoptosis.

[0088] Example 7: Tumor killing and immune activation effects of nanoformulations in vivo

[0089] Based on the good activation ability of nanoformulation on T cells in vitro, MC38 was further constructed. Mlh1 KD Colon cancer mouse homograft model was used to verify the tumor inhibition effect of DCsLipo@MnO2@si-CTLA4@PD-1α in vivo. Mlh1 KD Construction of homograft tumor model. 100 μL of 2×10 7 MC38 per mL Mlh1 KD The cells were then grown in mice under suitable conditions. When the tumor volume reached 80-100 mm 3 At the same time, the mice were randomly divided into 5 groups, namely PBS (control group), MnO2@si-CTLA4, Lipo@MnO2@si-CTLA4@PD-1α, DCsLipo@MnO2@si-CTLA4@PD-1α(untreated)(untreated), DCsLipo@MnO2@si-CTLA4@PD-1α(treated)(treated), with 5 mice in each group. The mice were treated by intravenous injection once a week. At the same time, the tumor volume of the mice was measured once a week. After 5 weeks of treatment, the mice were euthanized, the tumor tissue was removed, weighed and photographed, and part of the tumor tissue was taken for flow cytometry analysis to explore the activation of T cells in the tumor tissue. The remaining tissue was used for subsequent pathological experiments.

[0090] (1) Evaluation of nanoformulations for tumor killing in vivo

[0091] a. Tumor records: After recording, photographing, and weighing the tumor volumes of mice, we found that during the entire treatment process, the tumors of mice in the control group grew rapidly, while the tumors of mice in the other groups grew relatively slowly. In particular, the tumors of mice in the DCsLipo@MnO2@si-CTLA4@PD-1α(treated) group barely grew ( Figure 5 A). It is worth noting that DCsLipo@MnO2@si-CTLA4@PD-1α(treated) treated with tumor cell lysate showed better tumor suppression effect than DCsLipo@MnO2@si-CTLA4@PD-1α(untreated) and Lipo@MnO2@si-CTLA4@PD-1α groups that were not treated with tumor cell lysate, and the final tumor volume and weight of mice also showed similar trends ( Figure 5 B, C). This indicates that DCsLipo@MnO2@si-CTLA4@PD-1α treated with tumor cell lysate has a better tumor inhibition effect, and hybrid membrane coating has a better tumor inhibition effect than liposome membrane coating nanoformulation.

[0092] bH&E staining: At the end of the experiment, major organs and tumors from tumor-bearing mice were collected and fixed in 4% paraformaldehyde (PFA) at 4°C for 24 hours. The tissues were then embedded in paraffin and cut into 4 μm sections. Hematoxylin and eosin (H&E) staining was used to assess pathological changes in major organs and tumors, as well as tumor immune infiltration.

[0093] The results are as follows Figure 5 As shown in Figure D, compared to the control group, the major organs of the mice in each experimental treatment group showed no significant physiological morphological changes or tissue damage, indicating that the nanoformulation has good tissue compatibility. In addition, after the mice were treated with DCsLipo@MnO2@si-CTLA4@PD-1α (treated), the tumor tissue showed large areas of cell nuclear shrinkage and enlarged intercellular spaces, indicating severe tumor tissue damage.

[0094] c. Immunohistochemical staining: At the end of the experiment, the main organs and tumors of tumor-bearing mice were collected and fixed in 4% paraformaldehyde (PFA) at 4°C for 24 h. The tissues were then embedded in paraffin and cut into 4 μm sections. Immunohistochemical staining was used to detect Ki67 in the tumor tissues.

[0095] The results of Ki67 staining of mouse tumor tissues are as follows Figure 5 As shown in E, the number of proliferating cells in tumor tissue of mice was significantly reduced after treatment with DCsLipo@MnO2@si-CTLA4@PD-1α(treated).

[0096] The above results show that DCsLipo@MnO2@si-CTLA4@PD-1α(treated) has a good tumor inhibitory effect, does not cause tissue damage, and has good tissue compatibility.

[0097] (1) Evaluation of in vivo immune activation of nanoformulations

[0098] In order to evaluate the in vivo immune activation effect of DCsLipo@MnO2@si-CTLA4@PD-1α(treated), the present invention took mouse tumor tissues for immunohistochemical staining and flow cytometry analysis to explore the immune activation of mouse tumor tissues after treatment with different nanomedicines.

[0099] a. Immunohistochemical staining and multiplex immunofluorescence assay: Immunohistochemical staining was used to detect CTLA4 in tumor tissues, and multiplex immunofluorescence assay was used to detect the expression of perforin (PFP), CD4, and CD8 in tumor tissues.

[0100] The results are as follows Figure 5 As shown in F and G, compared with the control group (PBS), the expression of CTLA4 in tumor tissues of mice treated with DCsLipo@MnO2@si-CTLA4@PD-1α(treated) was significantly reduced, while the content of PFP (red fluorescence) was increased. + T cells (orange fluorescence) and CD8 + The number of T (green fluorescence) cells increased significantly, indicating that DCsLipo@MnO2@si-CTLA4@PD-1α (treated) treatment can promote the penetration and activation of T cells into tumor tissues.

[0101] b. Flow cytometry analysis: Flow cytometry was used to detect activated T cells in tumor tissues. The tumors were cut into 1-2 mm pieces and then digested with DMEM medium containing 0.5 mg / mL DNase I and collagenase I at 37°C for 0.5 h. The cells were further ground with a syringe plunger and passed through a 70 μm cell filter to prepare a single-cell suspension. The cells were then labeled with anti-CD3 and anti-CD69 antibodies. Finally, flow cytometry was used to detect activated CD3 in the tumor tissues. + CD69 + T cell count.

[0102] Similar trends to those of immunohistochemistry and multiplex immunofluorescence were also observed in flow cytometry results ( Figure 5 H), Activated CD3 in tumor tissue after nanoparticle treatment + CD69 +T cells increased to varying degrees in all groups, with the DCsLipo@MnO2@si-CTLA4@PD-1α(treated) group showing the highest number of activated T cells, reaching 20%. This suggests that the DCsLipo@MnO2@si-CTLA4@PD-1α(treated) group can effectively activate T cell proliferation and activation in mice.

[0103] In summary, the DCsLipo@MnO2@si-CTLA4@PD-1α nanoformulation prepared in the present invention can effectively promote the infiltration of T cells into tumor tissues and activate their anti-tumor immune response, and has a good inhibitory effect on LS-related CRC.

[0104] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and do not impose any form of limitation on the implementation methods of the technology of the present invention. Any person skilled in the art may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0105] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression, there are objectively infinite specific structures. For ordinary technicians in this technical field, without departing from the principles of this application, they can also make several improvements, modifications or changes, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A dendritic cell membrane-based co-loaded nanoparticle preparation, characterized in that: include: hollow mesoporous MnO2 nanoparticle core; si-CTLA4 adsorbed on the MnO2 surface; The hybrid membrane coated on the outside of the core is formed by the fusion of liposomes loaded with PD-1α and mature DCs membrane, and the DCs membrane is derived from Lynch syndrome colorectal cancer antigen MC38 Mlh1 KD Dendritic cells stimulated with cell lysate.

2. A method for preparing a dendritic cell membrane-based co-loaded nanoformulation, characterized in that: The following steps are involved: (1) Preparation of hollow mesoporous MnO2 a. Preparation of SiO2NPs: 14 mL of ethanol, 2 mL of deionized water, and 500 μL of ammonia water were mixed in a 50 mL round-bottom flask. The mixture was magnetically stirred in a 50°C oil bath for 5 min. 500 μL of ethyl orthosilicate was slowly added dropwise and stirred for 2 h to obtain SiO2NPs. The NPs were washed with ethanol and water and then stored in water until further use. b. Preparation of SiO2@MnO2 NPs: 600 mg of KMnO4 was dissolved in 20 mL of water, dispersed uniformly, and then 400 mg of SiO2 NPs was added dropwise. The mixture was ultrasonicated for 1 h and stirred at room temperature overnight. The next day, the mixed solution was washed three times with deionized water and then centrifuged at 14,800 rpm for 15 min to obtain SiO2@MnO2 NPs. c. Preparation of hollow mesoporous MnO2: 50 mg of SiO2@MnO2 NPs were dispersed in 20 mL of 2 M Na2CO3 solution and reacted at 60°C for 12 h. The mixture was then centrifuged at 12,000 rpm for 15 min, washed three times with deionized water, and dried under vacuum to obtain hollow mesoporous MnO2. (2) Preparation of MnO2@si-CTLA4 2.5 nmol si-CTLA4 was added to 2 mg MnO2 in 5 mL of deionized water and shaken at 4°C in the dark for 12 h. After the end, the resulting solution was stored at 4°C in the dark. (3) Preparation of DCsLipo@MnO2@si-CTLA4 a. Lynch syndrome colorectal cancer antigen MC38 Mlh1 KD Preparation of cell lysate: pLKO.1-Puro empty vector and pLKO.1-Mlh1 shRNA interference plasmid were transfected into MC38 colon cancer cells using Lipofectamine 2000 and incubated for 24 h. The transfected cells were screened with corresponding antibiotics and the knockdown of Mlh1 gene was verified by qPCR. The MC38 cell line with Mlh1 knockdown was screened, namely MC38 Mlh1 KD MC38 cells were then treated with cell lysate. Mlh1 KD Lynch syndrome colorectal cancer antigen MC38 was obtained Mlh1 KD The cell lysate was frozen at -80°C until use; b. Preparation of DCs cell membrane: Using 10 μg protein / mL Lynch syndrome colorectal cancer antigen MC38 Mlh1 KD Mouse bone marrow-derived DCs were stimulated with cell lysate and incubated for 24 h. After DC maturation was confirmed by flow cytometry, the cells were washed with PBS and centrifuged at 300 × g for 5 min. The cell membranes of the stimulated DCs were extracted by differential centrifugation. c. Preparation of DCsLipo@MnO2@si-CTLA4: SPC, CHOL, and DSPE-PEG-COOH were dissolved in 5 mL of chloroform solution and placed in a round-bottom flask. The amount of DSPE-PEG-COOH was 2 μmol, and the molar ratio of DSPE-PEG-COOH:CHOL:SPC was 1:5:10, respectively. Then, the film was evaporated under reduced pressure at 41°C for about 30 min to obtain a solid film. The film was flushed with nitrogen for 30 min and dried in a vacuum oven for 12 h to remove residual chloroform. 2 mL of DCs cell membrane aqueous solution and 2 mL of MnO2@si-CTLA4 aqueous solution were added and ultrasonically hydrated for 10 min to produce a liposome suspension. The liposome suspension was extruded 50 times through an 800 nm polycarbonate membrane using a liposome extruder, then extruded 50 times through a 400 nm polycarbonate membrane, and finally extruded 21 times through a 200 nm polycarbonate membrane to obtain a vesicle suspension, which was stored at low temperatures. (4) Preparation of DCsLipo@MnO2@si-CTLA4@PD-1α DCsLipo@MnO2@si-CTLA4 was mixed with EDC·HCl and NHS in 10 mL PBS at pH 7.4, and then sonicated for 20 min. PD-1α was mixed with the above solution, stirred at 25°C in the dark for 24 h, and the product was collected and purified with water to obtain the final product DCsLipo@MnO2@si-CTLA4@PD-1α.

3. The preparation method according to claim 2, wherein In step (3), the concentration of the DCs cell membrane aqueous solution is 2 mg protein / mL, and the concentration of the MnO2@si-CTLA4 aqueous solution is 40 μmol siRNA.

4. The preparation method according to claim 2, wherein In step (4), the molar ratio of DCsLipo@MnO2@si-CTLA4:EDC·HCl:NHS is 1:2:2, wherein the amount of NHS used is 40 μmol.

5. The preparation method according to claim 2, wherein The mass ratio of PD-1α to DCsLipo@MnO2@si-CTLA4 in step (4) is 1:

100.

6. Application of a dendritic cell membrane-based co-loaded nanoformulation in the preparation of drugs for the treatment of Lynch syndrome-related colorectal cancer.

7. The use according to claim 6, characterized in that The drug can promote the proliferation and activation of T cells in vitro and reduce the mRNA expression of CTLA4 in T cells.

8. The use according to claim 6, characterized in that The drug can promote the proliferation and activation of T cells by combining with PL-1α in vitro to promote the Mlh1 KD Cell apoptosis.

9. The use according to claim 6, characterized in that The drug can inhibit MC38 through immune activation in vivo Mlh1 KD Proliferation of colon cancer tumors in mice.

10. The use according to claim 6, characterized in that The immune activation effect is manifested by decreased CTLA4 expression in tumor tissue, increased PFP content, and CD4 + T, CD8 + T, CD3 + CD69 + Increased number of T cells.

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