Endoplasmic reticulum membrane coated PLGA-poly T / GPC3 mRNA vaccine and preparation method thereof

Abstract

The invention discloses an endoplasmic reticulum membrane coated PLGA-poly T / GPC3 mRNA vaccine and a preparation method thereof, and the preparation method comprises the following steps: step 1, dissolving dioleoyl lecithin, 1, 2-dioleoyl-sn-glycerol-3-phosphate-L-serine, GPC3 mRNA and PLGA-poly T with an organic solvent, and then carrying out reduced pressure rotary evaporation to remove the organic solvent to obtain a lipid membrane; and 2, adding ultrapure water to hydrate the lipid membrane, then adding the endoplasmic reticulum membrane, carrying out ultrasonic treatment, centrifuging, and taking the supernatant, thereby obtaining the endoplasmic reticulum membrane coated PLGA-poly T / GPC3 mRNA vaccine P-(H)-m (at) EMLN. GPC3 mRNA specifically expressed in liver cancer tissue is selected, based on a unique ploy A tail structure of mRNA, a safe material PLGA approved by FDA is subjected to poly T modification, and safe entrapment of GPC3 mRNA is achieved through hydrogen-bond interaction generated by a zipper type structure of basic group interworking between A and T; meanwhile, the endoplasmic reticulum membrane is extracted for carrier coating so as to enhance the immunogenicity of the carrier and avoid the degradation of the endosome of the vaccine, so that the endoplasmic reticulum membrane coated bionic GPC3 mRNA nano vaccine (P-(H)-m (at) EMLN) is constructed for specific immunotherapy of liver cancer, is high in safety and has wide clinical transformation potential.

Description

Technical Field

[0001] This invention belongs to the field of vaccines, specifically relating to an endoplasmic reticulum membrane-coated PLGA-poly T / GPC3 mRNA vaccine and its preparation method. Background Technology

[0002] Traditional treatments for liver cancer, such as surgery, radiotherapy, and chemotherapy, have achieved some success, but face limitations such as drug resistance, toxic side effects, and tumor heterogeneity. In recent years, mRNA vaccines have shown great promise as an emerging anti-tumor approach. The core mechanism of mRNA vaccines lies in delivering mRNA molecules encoding specific antigens into host cells via a delivery system. The host cells then synthesize the target antigen protein through their translation mechanisms, thereby triggering an adaptive immune response. Compared to traditional vaccine technologies, this vaccine system represents a revolutionary breakthrough: by reconstructing the host cell's biosynthetic mechanism, it enables the autonomous expression of antigen proteins, simultaneously stimulating both humoral and cellular immune responses, and inducing the formation of immune memory cells, thus establishing a long-lasting immune protective barrier. mRNA vaccines have demonstrated enormous potential in cancer treatment. Personalized mRNA vaccines developed by companies such as Moderna have entered clinical trials for cancers such as melanoma, with preliminary results showing that they can induce strong T-cell responses, and tumor shrinkage or stabilization in some patients. However, the development of mRNA vaccines also faces many challenges, such as the difficulty of mRNA crossing in vivo barriers and its easy degradation, and the need for specific vaccines for different cancers. To overcome the challenges associated with delivering mRNA into the body, researchers have explored the use of liposomes as a potential solution. However, the use of these materials is also limited by concerns related to cytotoxicity and instability. For example, cationic liposomes (such as DOTAP and DC-Chol), being positively charged, can interact strongly with negatively charged cell membranes, potentially leading to cell membrane disruption, mitochondrial damage, and inflammatory responses. Summary of the Invention

[0003] To address the above problems, this invention discloses an endoplasmic reticulum membrane-coated PLGA-poly T / GPC3 mRNA vaccine and its preparation method.

[0004] To achieve the above objectives, the technical solution of the present invention is as follows:

[0005] A method for preparing an endoplasmic reticulum membrane-coated PLGA-poly T / GPC3 mRNA vaccine includes the following steps:

[0006] Step 1: Dissolve dioleoyl lecithin, 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine, GPC3 mRNA and PLGA-polyT in an organic solvent, and then remove the organic solvent by rotary evaporation under reduced pressure to obtain a lipid membrane;

[0007] Step 2: Add ultrapure water to hydrate the lipid membrane, then add endoplasmic reticulum membrane and sonicate, then centrifuge and collect the supernatant to obtain the endoplasmic reticulum membrane-coated PLGA-poly T / GPC3 mRNA vaccine P-(H)-m@EMLN.

[0008] In a further improvement, the mass ratio of the dioleoyl lecithin, 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine, GPC3 mRNA, PLGA-polyT, and endoplasmic reticulum membrane is 3.32 mg:1.84 mg:5 μg:12.5 μg:20 μg.

[0009] In a further improvement, the organic solvent comprises chloroform and methanol; the volume ratio of chloroform to methanol is 2:1.

[0010] In a further improvement, in step one, the organic solvent is removed by rotary evaporation under reduced pressure at 37°C.

[0011] In a further improvement, in step two, after adding ultrapure water, the mixture is hydrated at 37°C or below for 5 minutes; after adding the endoplasmic reticulum membrane, it is sonicated for 2 minutes; and the centrifugation speed is 10,000 rpm for 10 minutes.

[0012] A further improvement is made to the preparation method of the PLGA-polyT as follows:

[0013] The thymine CNA monomer and mercapto-PLGA were dissolved in N,N-dimethylformamide (DMF) to obtain a DMF solution. The DMF solution was then mixed with 2,2-dimethoxy-2-phenylacetophenone (DMPA) under an argon atmosphere and irradiated for 10 h at 365 nm (20 mW / cm²) to obtain a reaction solution. The reaction solution was transferred to a 3500 MWCO dialysis bag and dialyzed in methanol for 2, 12, 16, and 20 h. Then, it was dialyzed in ethyl acetate for 2, 4, and 8 h. The solvent was removed by rotary evaporation to obtain PLGA-polyT.

[0014] Further improvements were made to the surge ratio of thymine CNA monomer, mercapto PLGA, and 2,2-dimethoxy-2-phenylacetophenone DMPA to 169.8 mg: 5 kDa: 5 mg.

[0015] Further improvements were made, and the structure of PLGA-poly T was identified using 1H NMR spectroscopy. The chemical shift of PLGA-poly T was consistent with the theoretical values ​​of the main functional groups of the target product. The degree of polymerization of poly T was calculated by the integral ratio of hydrogen.

[0016] Further improvements were made by detecting the synthesis and molecular weight using gel permeation chromatography (GPC), and observing the overall shift direction of the elution curves of PLGA and PLGA-poly T, which further confirmed the synthesis of PLGA-poly T.

[0017] A further improvement is made to the preparation method of the endoplasmic reticulum membrane, as follows:

[0018] Four-week-old male C57BL / 6J mice were selected. After disinfecting the skin of both axillae with 75% ethanol, 200 μL of PBS suspension containing 6 million Hepa 1-6 cells was injected subcutaneously into each side. When the average tumor volume reached 1000 mm³, the mice were sacrificed by cervical dislocation. The tumor tissue was quickly dissected and obtained. The endoplasmic reticulum membrane was then extracted using the English Special Animal Cell / Tissue Endoplasmic Reticulum Enrichment Kit.

[0019] An endoplasmic reticulum membrane-coated PLGA-polyT / GPC3 mRNA vaccine comprises dioleoyl lecithin, 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine, GPC3 mRNA, PLGA-polyT, and endoplasmic reticulum membrane.

[0020] In a further improvement, the mass ratio of the dioleoyl lecithin, 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine, GPC3 mRNA, PLGA-polyT, and endoplasmic reticulum membrane is 3.32 mg:1.84 mg:5 μg:12.5 μg:20 μg;

[0021] The poly A length in the GPC3 mRNA is 100-150, and the poly T length in the PLGA-polyT is 25-30.

[0022] Advantages of this invention:

[0023] 1. The P-(H)-m@EMLN nanovaccine of the present invention exhibits good stability under different physiological environments. Western blot (WB) experiments and Coomassie brilliant blue staining confirmed that the extracted endoplasmic reticulum membrane retained its biological characteristics and was successfully coated onto the nanovaccine.

[0024] 2. The in vivo anti-hepatocellular carcinoma immune effect of P-(H)-m@EMLN nanovaccine was investigated using a Hepa1-6 hepatocellular carcinoma xenograft C57BL / 6J mouse model. The results showed that the nanovaccine significantly inhibited tumor growth within 21 days, with a tumor inhibition rate as high as 99.8%. Flow cytometry results showed that P-(H)-m@EMLN could significantly induce the maturation of dendritic cells (DCs) in lymph nodes and increase CD8+. + CD4 + The P-(H)-m@EMLN nanovaccine significantly increased the reserve of T-cell central memory cells (T-cells) in the spleen, reducing T-reg cells and the infiltration of exhausted T-cells. Hematologic and endothelial growth (H&E) in major organs indicated that the nanovaccine possessed good biocompatibility. Furthermore, the P-(H)-m@EMLN nanovaccine significantly increased the reserve of T-cell central memory cells in the spleen, inducing long-term immunity; mice with tumor regression after treatment were unable to form tumors even after re-injection of tumor cells within 42 days.

[0025] 3. This invention utilizes hydrogen bonding to achieve efficient mRNA encapsulation, constructing a biomimetic GPC3 mRNA vaccine coated on the endoplasmic reticulum membrane. This vaccine has high safety and can achieve highly efficient and specific immunotherapy for liver cancer, providing a new strategy for clinical liver cancer treatment and possessing broad clinical translational potential.

[0026] 4. The FDA-approved safe material PLGA is modified with poly T to achieve safe mRNA encapsulation through hydrogen bonding generated by the base pairing zipper structure between AT.

[0027] 5. Lysosomal escape function of the endoplasmic reticulum membrane reduces in vivo degradation of mRNA vaccines.

[0028] 6. Tumor-derived endoplasmic reticulum membrane, as an exogenous antigen, can activate immunity and synergistically enhance the immunotherapeutic effect of mRNA vaccines. Attached Figure Description

[0029] Figure 1 This is the 1H NMR spectrum of PLGA-poly T.

[0030] Figure 2 This is PLGA-poly T gel permeation chromatography.

[0031] Figure 3 P-(H)-m@EMLN particle size distribution.

[0032] Figure 4 The potential diagram is for P-(H)-m@EMLNZeta.

[0033] Figure 5 Transmission electron microscopy image of P-(H)-m@EMLN nanovaccine.

[0034] Figure 6Agarose gel electrophoresis images of different nanoparticles.

[0035] Figure 7 To validate characteristic protein maps of the endoplasmic reticulum membrane using Coomassie Brilliant Blue.

[0036] Figure 8 The particle size change of the P-(H)-m@EMLN nanovaccine over 25 days.

[0037] Figure 9 The particle size and PDI of the P-(H)-m@EMLN nanovaccine under different physiological conditions are shown. Data are expressed as mean ± standard deviation, n = 3.

[0038] Figure 10 IVIS fluorescence imaging of isolated inguinal lymph nodes (mCherry mRNA, 10 μg) from C57BL / 6J mice at 0 h, 12 h, 24 h and 48 h after administration of the P-(H)-m@EMLN nanovaccine.

[0039] Figure 11 IVIS fluorescence imaging of isolated inguinal lymph nodes from C57BL / 6J mice 24 h after administration of free mCherry mRNA, P-(H)-m and P-(H)-m@EMLN.

[0040] Figure 12 The images show the in vivo anti-hepatocellular carcinoma effect of the P-(H)-m@EMLN nanovaccine, including (a) photographs of tumors removed after different treatments; (b) mean growth curves of Hepa1-6 tumors in tumor-bearing mice after different treatments; (c) body weight change curves of tumor-bearing mice after different treatments; (d) mean tumor weight removed on day 21 in each group of mice; and (e) tumor inhibition rate calculated based on tumor weight in each group of mice, where tumor inhibition rate = (1 - tumor weight in treatment group / tumor weight in control group) × 100%. All data are expressed as mean ± SD, n = 5, *P < 0.05, **P < 0.01, ***P < 0.001; ns: no statistical significance.

[0041] Figure 13 For the analysis of tumor tissue sections by H&E staining and TUNEL immunofluorescence staining. Scale bar: 50 μm.

[0042] Figure 14 H&E staining analysis was performed on tissue sections from the heart, liver, spleen, lung, and kidney. Scale bar: 50 μm.

[0043] Figure 15Photographs and volumetric images of inguinal lymph nodes isolated after different treatments: (a) left inguinal lymph node, (b) right inguinal lymph node. Scale bar: 1 cm. All data are expressed as mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001; ns: no statistical significance.

[0044] Figure 16 Flow cytometry analysis and quantification of the maturity ratio of dendritic cells (DCs) in inguinal lymph nodes. All data are expressed as mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001; ns: no statistical significance.

[0045] Figure 17 To analyze and quantify the proportions of tumor-infiltrating CD8+ T and CD4+ T cells using flow cytometry. All data are expressed as mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001; ns: no statistical significance.

[0046] Figure 18 Immunofluorescence analysis of tumor tissue sections was performed to determine the number of tumor-infiltrating CD8+ T and CD4+ T cells. Scale bar: 100 μm.

[0047] Figure 19 To analyze and quantify the proportion of Treg cells in tumor tissues using flow cytometry. All data are expressed as mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001; ns: no statistical significance.

[0048] Figure 20 To analyze and quantify the proportion of exhausted T cells in tumor tissues using flow cytometry. All data are expressed as mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001; ns: no statistical significance.

[0049] Figure 21 To analyze and quantify the proportion of M1 macrophages in tumor tissues using flow cytometry. All data are expressed as mean ± SD, n = 4, *P < 0.05, **P < 0.01, ***P < 0.001; ns: no statistical significance.

[0050] Figure 22To analyze and quantify the proportion of T cells in the spleen using flow cytometry. All data are expressed as mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001; ns: no statistical significance.

[0051] Figure 23 To analyze and quantify the proportion of CD4+ Tcm in spleen T cells using flow cytometry. All data are expressed as mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001; ns: no statistical significance.

[0052] Figure 24 For flow cytometry analysis and quantification of CD8 in spleen T cells + Tcm proportion. All data are expressed as mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001; ns: no statistical significance.

[0053] Figure 25 The concentrations of serum cytokines TNF-α (a), IL-6 (b), and IFN-β (c) in different drug-treated groups were detected by enzyme-linked immunosorbent assay (ELISA). All data are expressed as mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001; ns: no statistical significance.

[0054] Figure 26 In vivo IVIS fluorescence imaging of C57BL / 6J mice after tumor seeding with luciferase-labeled Hepa1-6 cells. Tumors were resected on day 9 in the Operation group and Vaccination + Operation group, while the Vaccination group started treatment at week 1. All groups were seeded with luciferase-labeled Hepa1-6 cells again at week 3.

[0055] Figure 27 For flow cytometry analysis and quantification of CD4 in spleen T cells + Tcm proportion. All data are expressed as mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001; ns: no statistical significance.

[0056] Figure 28 For flow cytometry analysis and quantification of CD8 in spleen T cells + Tcm proportion. All data are expressed as mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001; ns: no statistical significance.

[0057] Figure 29 This is a schematic diagram of the overall principle of the present invention. Detailed Implementation

[0058] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0059] I. Experimental Methods:

[0060] 1.1 Synthesis of PLGA-poly T

[0061] Weigh out 169.8 mg of thymine CNA monomer, 1.20 mmol of 5 kDa thioglycolic acid PLGA (PLGA-SH), 100 mg of PLGA, 0.04 mmol of 2,2-dimethoxy-2-phenylacetophenone (DMPA) (0.5% w / v). Use 1 mL of N,N-dimethylformamide (DMF) as solvent and DMPA as photoinitiator. Irradiate at 365 nm (20 mW / cm2) for 10 h.

[0062] DMPA was placed in a Shrek tube and sealed with a rubber stopper. Thymine CNA monomer and PLGA-SH were dissolved in DMF and set aside. The Shrek vial was then connected to a double-row tubing, ensuring the tubing in use was open while the others were closed. After evacuating for 2 minutes, the argon gas valve was opened. Once bubbles appeared on the bubble counter, the double-row tubing was filled with argon gas with the tip pointing downwards. The evacuation and argon filling were repeated three times each. The DMF solution was injected into the Shrek vial using a syringe. Finally, the argon gas valve was opened wide, the rubber stopper was opened, and the Shrek vial cap was quickly tightened. The vial was irradiated at 365 nm (20 mW / cm²) for 10 hours.

[0063] After the reaction was complete, the reaction solution was transferred to a 3500 MWCO dialysis bag and dialyzed in methanol for 2, 12, 16, and 20 h. Then, it was dialyzed in ethyl acetate for 2, 4, and 8 h, and the solvent was removed by rotary evaporation.

[0064] like Figure 1 As shown in Table 1, the chemical shift of PLGA-poly T matches the theoretical values ​​of the main functional groups of the target product. The degree of polymerization of poly T can be calculated to be 25-30 using the hydrogen integral ratio. The length of PolyA is generally 30-250 mm, preferably 100-150 mm. Figure 2 As shown, GPC results indicate that, compared to the elution curve of PLGA, the elution curve of PLGA-poly T shifts overall towards higher molecular weights, further demonstrating the successful synthesis of PLGA-poly T.

[0065] Table 1 shows the molecular weight and molecular weight distribution data of PLGA-SH and PLGA-poly T.

[0066] 1.2 Preparation of PLGA-polyT nanoparticles

[0067] Solvent evaporation method: The block copolymer PLGA5k-Tn was dissolved in dichloromethane at a concentration of 10 mg / mL, and then added dropwise to an appropriate amount of 10 mM NaCl (pH 7.4) and 100 mM phosphate buffer solution while stirring. The mixture was stirred at 37°C for 2 hours, then the temperature was raised to 45°C and stirred for another hour to remove the organic solvent before being slowly cooled to room temperature. The nanoparticles were collected by centrifugation at 16000 rpm for 5 min and washed three times with PBS to obtain PLGA-poly T nanoparticles.

[0068] 1.3 Preparation of P-(H)-m nanoparticles

[0069] Take an appropriate amount of 100 mg / mL PLGA-poly T nanoparticle stock solution and mix it with mRNA at a mass ratio of 500:1 and incubate at 4℃ for 30 min to obtain PLGA-(H)-mRNA nanoparticles, abbreviated as P-(H)-m.

[0070] 1.4 Extraction of endoplasmic reticulum

[0071] Four-week-old male C57BL / 6J mice were selected. After disinfecting the skin of both axillae with 75% ethanol, 200 μL of PBS suspension containing 6 million Hepa 1-6 cells was subcutaneously injected into each axilla. When the average tumor volume reached 1000 mm³ (approximately 14 days post-inoculation), the mice were sacrificed by cervical dislocation. The tumor tissue was quickly dissected, and after removing peripheral blood vessels and connective tissue, it was stored at -80°C. Subsequent analysis used an English-specific animal cell / tissue endoplasmic reticulum enrichment kit, as follows:

[0072] 1. Pre-cool the centrifuge column and receiving tube on ice.

[0073] 2. Place 30-35 mg of frozen tumor tissue sample (frozen tumor tissue should be completely thawed at room temperature; fresh tissue is not recommended due to lower final yield and purity) on a centrifuge column, add 200 μL of buffer A, and vigorously grind with a plastic rod for 2-3 minutes. Then add 350 μL of buffer A to the centrifuge column. (Note: The plastic grinding rod is reusable; wipe it with 75% alcohol or rinse it with distilled water.)

[0074] 3. Cover the container, invert it several times to mix, and centrifuge at 16000X g for 30 seconds. (This step requires rapid centrifugation; reaching 16000X g within 10 seconds will improve the yield.)

[0075] 4. Discard the centrifuge column, vortex vigorously to resuspend the precipitate for 10 seconds, and centrifuge at 2000X g for 5 minutes (the precipitate contains cell nuclei, large cell fragments, and some unruptured cells).

[0076] 5. Transfer the supernatant from the centrifuge tube to a pre-chilled 1.5 mL centrifuge tube. When transferring, the pipette tip should be inserted vertically into the lower layer of the liquid to avoid contamination by the lipid layer of the tube wall. Then centrifuge at 16000X g, 4℃ for 30 min. After centrifugation, carefully aspirate 400 μL of the supernatant and transfer it to another new 1.5 mL centrifuge tube. (The precipitate obtained from centrifugation mainly consists of large cell debris, mitochondria, lysosomes, and cell membranes.)

[0077] 6. Add 200 μL of buffer B to the centrifuge tube containing the 400 μL supernatant, and vortex to mix the solution (the ratio of buffer B to supernatant is 1:2). Incubate at 4°C for 30 min.

[0078] 7. Centrifuge at 16000X g, 4℃ for 10 min, and discard the supernatant completely. Resuspend the precipitate in 200 μL PBS, pipetting 40-50 times and vortexing for 20 s. Incubate at room temperature for 30 min, vortexing every 5 min to mix. Centrifuge at 2000X g for 5 min and transfer the supernatant to another 1.5 mL centrifuge tube. Add 200 μL of buffer C to the supernatant (1:1 volume ratio of supernatant to buffer C). Incubate at 4℃ for 20 min.

[0079] 8. Centrifuge at 10000X g for 4℃ for 10 min and discard the supernatant. Centrifuge again at 10000X g for a few seconds and completely discard the residual liquid.

[0080] 9. The resulting precipitate is the endoplasmic reticulum, mainly consisting of rough endoplasmic reticulum. When storing endoplasmic reticulum samples, add a protease inhibitor to the lysis solution and freeze the sample at -80°C.

[0081] 1.5 Preparation of P-(H)-m@EMLN nanovaccine

[0082] The DOPC / DOPS lipid polymer hybrid nanovaccine (P-(H)-m@EMLN) modified with endoplasmic reticulum membrane and encapsulating PLGA-(H)-mRNA was prepared by thin-film dispersion.

[0083] Accurately weigh 3.32 mg of dioleoyl lecithin, 1.84 mg of 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine (sodium salt), 5 μg of pre-prepared mRNA, and 12.5 μg of PLGA-polyT. Dissolve them completely in 1 ml of chloroform and 0.5 ml of methanol. Remove the solvent by rotary evaporation under reduced pressure at 37 °C to obtain a homogeneous lipid membrane. Add 500 μg of ultrapure water and hydrate at below 37 °C for 5 minutes. Add 20 μg of endoplasmic reticulum membrane and sonicate for 2 minutes to obtain the final product. Centrifuge at 10,000 rpm for 10 minutes to separate the core P-(H)-m that does not encapsulate the endoplasmic reticulum liposome hybrid membrane. Use the supernatant for subsequent experiments.

[0084] Characterization of 1.6P-(H)-m@EMLN nanovaccine

[0085] 1.6.1. Dynamic Light Scattering Characterization and Screening

[0086] The potential and particle size of the P-(H)-m@EMLN nanovaccine were characterized by dynamic light scattering (DLS). The nanoparticles were placed in a sample cell, and the glass surface of the sample cell was gently wiped clean. The average hydrated particle size and zeta potential of the sample solution were measured by DLS. After measurement, the sample was aspirated, the sample cell was rinsed with ultrapure water, and dried with lens paper.

[0087] 1.6.2. TEM Characterization

[0088] Take 10 μL of the P-(H)-m@EMLN nanovaccine sample stock solution and drop it onto a copper grid with a membrane. Let it stand for 10 min. Blot dry with the side of filter paper and carefully wash the copper grid three times with distilled water, 1 min each time. Add 1% phosphotungstic acid for staining for 10 min. Blot dry with the side of filter paper and carefully wash the copper grid three times with distilled water, 1 min each time. Dry the copper grid and observe it through a transmission electron microscope (TEM).

[0089] 1.3.3. Agarose gel electrophoresis

[0090] Weigh 0.35 g of agarose powder into an Erlenmeyer flask, add 35 mL of TBE to dissolve, microwave for 1 min, shaking every 10-15 s until small bubbles appear. Cool with cold water until slightly warm to the touch, add 3.5 μL of nucleic acid dye (10000X) and shake well. Place the basket in the mold, insert the comb, pour in the mixture, and let stand for 30-60 min to solidify. Place the basket in the electrophoresis tank, with the wells near the negative electrode, add buffer to the sample according to the ratio, and load the sample vertically. Set the electrophoresis parameters to an initial voltage of 80 V for 3 minutes, then adjust to 100 V and continue electrophoresis for 15 minutes. Finally, transfer the agarose gel to a gel imaging system for imaging analysis in the dark.

[0091] 1.6.4. Coomassie Brilliant Blue Staining Experiment

[0092] Assemble the electrophoresis glass plate and pour in approximately 7 mL of 10% separating gel. After the gel solidifies, add approximately 3 mL of 5% stacking gel. Insert the electrophoresis comb, and after the gel solidifies, remove the comb. Add the extracted endoplasmic reticulum and the protein from the P-(H)-m@EMLN nanovaccine. Electrophore the stacking gel at 80 V for 30 min and the separating gel at 120 V for 50 min. After electrophoresis, remove the gel and wash it three times in ultrapure water for 5 min each time. Discard the ultrapure water, add three gel thicknesses of Coomassie Brilliant Blue rapid staining buffer, shake for 1–2 h, recover the staining buffer, and rinse with ultrapure water until the blue background color of the gel disappears. Then add clean ultrapure water and record the experimental results.

[0093] 1.6.5. Stability Test

[0094] To assess the stability of the P-(H)-m@EMLN nanovaccine, samples were stored at -4°C. Samples were taken at the same time every 25 days and placed in particle size dishes. Particle size and PDI changes were measured using DLS. Samples were recovered after measurement, and the effect of storage time on particle size stability was observed. To assess the stability of the P-(H)-m@EMLN nanovaccine in physiological environments, the P-(H)-m@EMLN nanovaccine was added to RPMI-1640 medium containing 10% fetal bovine serum, blank RPMI-1640 medium, PBS, or physiological saline, and incubated at 37°C for 24 h. Particle size and PDI changes were measured using DLS.

[0095] 1.7 Statistical Analysis

[0096] This study used GraphPad Prism 9.1.2 software for statistical analysis. One-way ANOVA was employed to determine whether there were statistically significant differences between groups, and post-hoc tests were performed. P < 0.05 was considered statistically significant.

[0097] II. Detection Results and Discussion of P-(H)-m@EMLN Nanoparticle Vaccine

[0098] 2.1 Dynamic light scattering characterization of P-(H)-m@EMLN nanovaccine

[0099] like Figure 3 and Figure 4 As shown, DLS revealed that the average particle size of the P-(H)-m@EMLN nanovaccine was approximately 160 nm, with a Zeta potential of -25 mV. According to previous results from our research group, the potential of the P-(H)-m nanovaccine was -44.5 mV, while the potential of the endoplasmic reticulum membrane was -23 mV, indicating that the endoplasmic reticulum liposome hybrid membrane successfully encapsulated the P-(H)-m core.

[0100] 2.2 TEM characterization

[0101] like Figure 5 As shown, the physical morphology of the P-(H)-m@EMLN nanovaccine was observed by TEM. The nanoparticles are uniformly spherical with clear and bright circular edges around their surfaces. The particle size of the P-(H)-m@EMLN nanovaccine is around 100 nm, which is significantly smaller than the result of DLS measurement. This may be because DLS measures the hydrated particle size, while TEM shows the particle size under dry conditions.

[0102] 2.3 Agarose gel electrophoresis

[0103] Agarose gel electrophoresis separates nucleic acid molecules based on differences in their migration rates in an electric field. This experiment can be used to examine the encapsulation of mRNA. Naked mRNA migrates towards the positive electrode in an electric field, and its migration rate depends on the molecular size and charge. Figure 6 Experimental results show that mRNA can be loaded under the following conditions: the mass ratio of mRNA to PLGA-poly T is 1:500, the mass ratio of endoplasmic reticulum membrane to lipid membrane is 1:5, and the mass ratio of endoplasmic reticulum lipid hybrid membrane to kernel is 10:1.

[0104] 2.4 Coomassie Brilliant Blue Staining Experiment

[0105] The endoplasmic reticulum (ER), the most widely distributed membrane system within cells, is not only the core site of protein synthesis, folding, and lipid metabolism, but also a key hub in the vesicle transport network. Its membrane is enriched with a variety of bioactive proteins, which, through dynamic regulation of vesicle formation, transport, and targeted fusion, endow modified carriers (such as exosomes and synthetic nanoparticles) with new biological functions. The ER has close structural and functional connections with the cell membrane and nuclear membrane, resulting in a high degree of similarity in its protein composition to these membrane systems. However, the ER membrane contains more vesicle transport-related proteins (mainly SNARE family proteins, 10–40 kDa) and resident proteins (75–100 kDa, 48–60 kDa). Figure 7 As shown, the presence of endoplasmic reticulum characteristic proteins on the P-(H)-m@EMLN nanovaccine was verified by Coomassie brilliant blue staining experiment, indicating that the preparation of the nanovaccine did not result in the loss of endoplasmic reticulum characteristic proteins.

[0106] 2.5 Stability Test

[0107] The storage stability of nanoparticles (i.e., their ability to maintain their physicochemical properties during storage and use) is crucial to their application effectiveness. We used DLS to detect the particle size changes of nanoparticles at 1, 2, 3, 5, 7, 10, 15, 20, and 25 days, such as... Figure 8 The results showed that the particle size of the P-(H)-m@EMLN nanovaccine did not change significantly, and its physicochemical properties were relatively stable. We also tested the stability of the P-(H)-m@EMLN nanovaccine under different physiological environments, such as... Figure 9 The results showed that the nanoparticles did not exhibit significant changes in particle size or PDI in RPMI-1640 medium containing 10% fetal bovine serum, blank RPMI-1640 medium, PBS, and physiological saline.

[0108] III. Evaluation of in vivo anti-hepatocellular carcinoma immune response and therapeutic efficacy of P-(H)-m@EMLN nanovaccine:

[0109] 3.1 Establishment of Hepa1-6 mouse hepatocellular carcinoma xenograft model

[0110] Hepa1-6 mouse liver cancer cells were digested and collected, resuspended in physiological saline, and their concentration was adjusted to 8 × 10⁻⁶ cells / mL. 6 Cells were subcutaneously injected into the right buttock of male C57BL / 6J mice. After tumor growth, the length (L) and width (W) of the tumor were measured using calipers. The tumor volume was calculated using the formula (L×W²) / 2. 8×10⁶ cells / mouse were subcutaneously injected into the right buttock of male C57BL / 6J mice. After tumor growth, the length (L) and width (W) of the tumor were measured using calipers. The tumor volume was calculated using the formula (L×W²) / 2.

[0111] 3.2 Lymph node accumulation in the body

[0112] We used ex vivo lymph node imaging to investigate the in vivo lymph node accumulation effect of the P-(H)-m@EMLN nanovaccine. Hepa1-6 cells were inoculated subcutaneously in the right buttock of male C57BL / 6J mice at 6-8 weeks of age. When the tumor volume reached approximately 400 mm... 3 At that time, P-(H)-m@EMLN nanovaccine containing mCherry mRNA was injected subcutaneously into the right inguinal region of C57BL / 6J mice, with each mouse receiving 10 μg of mRNA. The fluorescence intensity of mCherry in the inguinal lymph nodes was detected at 0 h, 12 h, 24 h, and 48 h using a small animal in vivo imaging system.

[0113] Hepa1-6 cells were injected subcutaneously into the right buttock of male C57BL / 6J mice aged 6-8 weeks. Tumors were inoculated when the tumor volume reached approximately 400 mm. 3 At that time, C57BL / 6J mice were subcutaneously injected with Free mRNA, P-(H)-m nanovaccine, and P-(H)-m@EMLN nanovaccine in the right groin, respectively. The mice were sacrificed 24 h later, and the right inguinal lymph nodes were isolated. The fluorescence intensity of the inguinal lymph nodes was monitored using a small animal in vivo imaging system.

[0114] 3.3 Evaluation of in vivo anti-liver cancer efficacy

[0115] According to 8×10 6 A liver cancer xenograft model was established by subcutaneously inoculating the right buttock of male C57BL / 6J mice with Hepa1-6 cells at 5 weeks of age. The xenograft was established when the tumor reached approximately 100 mm. 3 At the time of administration, mice were subcutaneously injected with saline, PP@EMLN (empty vector), P-(H)-m nanovaccine, P-(H)-m@LNP nanovaccine, and P-(H)-m@EMLN nanovaccine, respectively, at a dose of 10 μg mRNA per mouse, for a total of 3 injections, with an interval of 7 days. Mouse body weight and tumor volume were recorded every 2 days. On day 21 after the start of drug administration, mice were sacrificed by cervical spondylosis, and lymph nodes, heart, liver, spleen, lungs, kidneys, and tumors were collected, and tumor weight was recorded. Major organs were fixed in 4% paraformaldehyde solution and sectioned for hematoxylin-eosin (H&E) or TUNEL staining.

[0116] 3.4 Flow cytometry

[0117] After the anti-tumor experiment, tumor tissue, spleen, and inguinal lymph nodes were collected from mice. Fresh tissue was washed and ground in pre-cooled PBS solution through a 200-mesh sieve to prepare a cell suspension. The suspension was centrifuged at 1200 rpm for 5 min, and the supernatant was discarded. Subsequently, erythrocyte lysis buffer was added for 3 min, followed by centrifugation at 1200 rpm for 5 min, and the supernatant was discarded. The cells were washed once with PBS. The suspension was resuspended in 1% BSA PBS and filtered again through a 200-mesh sieve. To detect the content of infiltrating CD8+ and CD4+ T lymphocytes in the tumor tissue, CD45, CD3, CD4, and CD8a antibodies were added to the tumor single-cell suspension. After incubation for 30 min, the staining was detected by flow cytometry. Tumor tissue sections were labeled with DAPI, CD8-Cy3, and CD4-FITC fluorescent antibodies for immunofluorescence analysis. To detect the content of regulatory T cells in tumors, cells were first stained with CD4 and CD25 fluorescent antibodies for 30 min on the cell surface, then fixed, permeabilized, and stained with Foxp3 fluorescent antibody, followed by flow cytometry analysis. To detect tumor-associated macrophages, tumor cell suspensions stained with CD11b, F4 / 80, CD80, and CD206 fluorescent antibodies for 30 min were analyzed by flow cytometry. To detect the content of exhausted T cells in tumors, tumor cell suspensions stained with CD3, PD-1, CD8, and Tim-3 fluorescent antibodies for 30 min were analyzed by flow cytometry. To study the content of T cells and memory T cells in the spleen, spleen single-cell suspensions stained with CD8a, CD4, CD44, and CD62L fluorescent antibodies were analyzed by flow cytometry. To assess the maturation level of dendritic cells, inguinal lymph node single-cell suspensions labeled with CD11c, CD86, and CD80 fluorescent antibodies were analyzed by flow cytometry.

[0118] Evaluation of long-term immune memory induced by 3.5 nanometer vaccines

[0119] To verify the strength of the immunological memory effect of the nanovaccine, we re-injected tumor cells after tumor regression in the vaccine group and observed their re-tumor formation efficiency. To more directly demonstrate that the drug administration inhibited tumor development and progression, we included a surgical tumor resection group (tumor size ≤ 500 mm). 3 Left and right excision), and vaccination + surgery group (for tumors 300mm in size). 3 Dosage, 500 mm 3(Left and right excisions) were used as controls. Luciferase-labeled Hepa1-6 tumor-bearing mice in the surgery group and the vaccination + surgery group underwent tumor resection on day 9. C57BL / 6J mice in the vaccination + surgery group received subcutaneous injections of P-(H)-m@EMLN nanovaccine (GPC3 mRNA, 10 μg) on ​​days 7, 14, and 21. The vaccination group received P-(H)-m@EMLN nanovaccine (GPC3 mRNA, 10 μg) on ​​days 0, 7, and 14 after tumor development. All groups received a second subcutaneous injection of luciferase-labeled Hepa1-6 cells on day 21.

[0120] 3.6 Statistical Analysis

[0121] This study used GraphPad Prism 9.1.2 software for statistical analysis. One-way ANOVA was employed to determine whether there were statistically significant differences between groups, and post-hoc tests were performed. P < 0.05 was considered statistically significant.

[0122] IV. Results and Discussion of In Vivo Immune Response and Therapeutic Effect of P-(H)-m@EMLN Nanovaccine on Liver Cancer

[0123] 4.1 Lymph node accumulation in the body

[0124] A large proportion of the body's immune cells reside in hundreds of lymph nodes, where lymphocytes accumulate, activate, and proliferate. Direct drug delivery to lymph nodes could address a range of local and systemic immune challenges and offers a promising pathway for diseases regulated by the adaptive immune system. Therefore, we used fluorescence imaging to detect the fluorescence intensity of mCherry cells in inguinal lymph nodes at different time points after vaccination. Figure 8 The results showed that no strong fluorescence was observed in the inguinal lymph nodes at 0 h, but fluorescence appeared at 12 h, indicating that the mRNA had reached the lymph nodes. This accumulation peaked at 24 h, and the fluorescence gradually weakened at 48 h. These results indicate that the vaccine can accumulate in the inguinal lymph nodes, with the strongest accumulation at 24 h and weakening after 48 h. We then evaluated the ability of the P-(H)-m nanovaccine and the P-(H)-m@EMLN nanovaccine to accumulate in the inguinal lymph nodes at 24 h. Figure 11 The results showed that the P-(H)-m@EMLN nanovaccine group exhibited stronger fluorescence intensity at 24 h compared to the Free mRNA group and the P-(H)-m nanovaccine group. This may be due to the presence of many proteins on the endoplasmic reticulum membrane that facilitate active uptake, such as Calnexin, which enhances the lymph node targeting effect of the nanovaccine.

[0125] 4.2 Evaluation of in vivo anti-liver cancer efficacy

[0126] We then evaluated the anti-hepatocellular carcinoma immune response of the P-(H)-m@EMLN nanovaccine in a Hepa1-6 subcutaneous xenograft model. A xenograft model was established by inoculating C57BL / 6J mice with Hepa1-6 cells in the right buttock. The xenograft was established when the right-sided tumor volume reached approximately 100 mm². 3 Mice were randomly divided into 5 groups of 8 each, and administered saline, PP@EMLN (empty vector), P-(H)-m nanovaccine, P-(H)-m@LNP nanovaccine, and P-(H)-m@EMLN nanovaccine subcutaneously to both groins, respectively, for a total of 3 injections, with an interval of 7 days. Tumor tissue, inguinal lymph nodes, and spleen were collected 72 hours after the last administration. Flow cytometry analysis was used to detect mature dendritic cells (DCs) in the inguinal lymph nodes, infiltrating T cells and macrophages in the tumor tissue, and the content of T cells and central memory cells in the spleen. Seven days after the last administration, the mice were sacrificed, and lymph nodes, heart, liver, spleen, lungs, kidneys, and tumors were collected, and tumor weight was recorded.

[0127] Figure 12 The results showed that, compared with the Saline group, all GPC3 mRNA-based vaccines exhibited effective tumor suppression. While the PP@EMLN treatment group (empty vector group) showed some tumor suppression, the significant differences in tumor volume within the group indicated that this PLGA endoplasmic reticulum lipid hybrid membrane carrier could also induce some degree of organismal recognition and tumor killing, possibly possessing immunogenicity and activating innate immunity. Compared to the P-(H)-m nanovaccine treatment group, the P-(H)-m@LNP nanovaccine group, which was only encapsulated in lipid material, showed worse efficacy. This may be because the initial amount of GPC3 mRNA in different formulations was consistent, but the amount of P-(H)-m@LNP nanovaccine mRNA was somewhat lost during the liposome preparation process. Compared with all other groups, the P-(H)-m@EMLN nanovaccine group could more effectively inhibit tumor growth and even induce tumor regression to some extent. The P-(H)-m@EMLN nanovaccine group showed a tumor inhibition rate of up to 99.8%, indicating that the nanovaccine has excellent anti-tumor effects.

[0128] 4.3 H&E and TUNEL staining of tumor tissue

[0129] H&E staining and TUNEL staining are commonly used methods for detecting apoptosis. H&E staining clearly displays tissue structure and cell morphology (such as nuclear pyknosis, increased cytoplasmic eosinophilicity, and apoptotic body formation), making it suitable for initial screening of apoptosis. TUNEL staining, on the other hand, can directly detect apoptotic cells by labeling DNA fragments (characteristic markers of apoptosis), offering high accuracy and the ability to detect early apoptotic cells (even when morphological changes are not obvious). Figure 13 As shown, the results of H&E and TUNEL staining demonstrated that the tumor tissue apoptosis rate in the P-(H)-m@EMLN nanovaccine treatment group was higher than that in other groups, further confirming the effective tumor-killing effect of the P-(H)-m@EMLN nanovaccine.

[0130] 4.4 In vivo safety

[0131] Hematoxylin and eosin (H&E) staining is the most commonly used staining method in histology, frequently used to assess tissue structure and cell morphology. In in vivo safety evaluations, H&E staining is an important tool for detecting the tissue toxicity, inflammatory response, and pathological changes of drugs, nanomaterials, or other therapeutic agents. Figure 14 As shown, the H&E staining results indicate that there were no significant differences in the morphology of major organs such as the heart, liver, spleen, lungs, and kidneys in the group treated with the nanovaccine compared with the saline group, further demonstrating the safety of the nanovaccine.

[0132] 4.5 Inguinal lymph node hyperplasia

[0133] Lymph nodes are important habitats for adaptive immune-related lymphocytes. Activated T cells and B cells within lymph nodes respond to vaccination, thereby activating the lymph nodes, causing them to swell, regulating the adaptive immune response against foreign antigens, and forming a potential long-term immune response. Lymphoid hyperplasia is a reactive change in lymph nodes in response to immune stimulation, usually triggered by factors such as infection, inflammation, autoimmune diseases, or tumors. Essentially, it is a manifestation of immune system activation and is closely related to immune function. The results of examination of isolated inguinal lymph nodes in mice are as follows... Figure 15 As shown, the P-(H)-m@EMLN nanovaccine treatment group significantly induced lymph node enlargement on both the tumor-affected side (right) and the contralateral side (left), indicating that the P-(H)-m@EMLN nanovaccine can effectively activate the body's immunity.

[0134] 4.6 Mature dendritic cells in inguinal draining lymph nodes

[0135] The maturation of dendritic cells (DCs) in lymph nodes is a crucial step in initiating and regulating adaptive immune responses. Mature DCs can activate T cells and regulate immune response types through antigen presentation, co-stimulatory signals, and cytokine secretion. Mature DCs highly express MHC molecules, co-stimulatory molecules (such as CD80 and CD86), and cytokines (such as IL-12), exhibiting strong antigen-presenting capabilities. In the tumor microenvironment, DC maturation and function may be inhibited; promoting DC maturation can enhance anti-tumor immune responses and activate tumor-specific T cells. Figure 16 As shown, the results of flow cytometry analysis of the proportion of mature dendritic cells (DCs) in mouse inguinal lymph nodes were similar to those of in vitro BMDC maturation experiments. The proportion of mature DCs in the inguinal lymph nodes of the empty vector group was 1.67 times that of the Saline group, while the proportion of mature DCs in the P-(H)-m@EMLN nanovaccine group was 1.65 times that of the empty vector group. This indicates that both the vector and GPC3 mRNA themselves can improve the proportion of mature DCs in inguinal lymph nodes to some extent. Compared with the P-(H)-m@LNP nanovaccine group, the proportion of mature DCs in the P-(H)-m@EMLN nanovaccine group increased by 9.52%, which may be attributed to the improved delivery efficiency of GPC3 mRNA and the immunogenicity of the nanovaccine due to the addition of the endoplasmic reticulum membrane.

[0136] 4.7 Tumor-infiltrating T cells

[0137] Tumor-infiltrating T cells (TILs) are T lymphocytes that migrate from peripheral blood into the tumor microenvironment (TME). TILs play a crucial role in anti-tumor immune responses, and their number and functional status are closely related to tumor prognosis and treatment efficacy. Among them, CD8+ TILs... + T cells, CD4 + T cells and Treg cells are key T cell subsets in anti-tumor immunity. CD8+ T cells, also known as cytotoxic T cells (CTLs), can directly kill tumor cells by recognizing the tumor antigen-MHC I complex and releasing perforin and granzymes to induce tumor cell apoptosis. CD4+ T cells, also known as helper T cells (Th), include subsets such as Th1, Th2, Th17, and Treg. They can activate macrophages, NK cells, and B cells by secreting cytokines (such as IFN-γ and IL-2), thereby enhancing the anti-tumor immune response. Regulatory T cells (Tregs) can suppress the anti-tumor immune response, maintain immune tolerance, and prevent excessive immune responses by secreting inhibitory cytokines (such as IL-10 and TGF-β) and expressing inhibitory molecules (such as CTLA-4 and PD-1). Figures 17-20As shown, flow cytometry analysis of tumor tissue revealed that, compared to other treatment groups, the P-(H)-m@EMLN nanovaccine treatment group showed a significant increase in the number of tumor-infiltrating CD8+ T and CD4+ T cells, while the number of Treg cells decreased. Immunofluorescence sectioning of tumor tissue further confirmed that the P-(H)-m@EMLN nanovaccine treatment group showed a significant increase in the number of CD8+ T and CD4+ T cells compared to other treatment groups. + T, CD8 + T cell infiltration was significantly increased. Exhausted T cells exhibited decreased proliferative capacity, reduced cytokine secretion, and weakened cytotoxic function, while simultaneously expressing high levels of inhibitory receptors (such as PD-1, TIM-3, and LAG-3), which is one of the important reasons why the immune system cannot effectively clear chronic infections and tumors. We used flow cytometry to detect the content of exhausted T cells in tumor tissue. The results showed that the content of exhausted T cells in the P-(H)-m@EMLN nanovaccine treatment group was significantly reduced, which may be due to GPC3 being a highly specific antigen for liver cancer. In summary, the P-(H)-m@EMLN nanovaccine can induce a stronger anti-liver cancer response and a lower immunosuppressive response.

[0138] 4.8 Tumor tissue macrophages

[0139] Tumor macrophages (TAMs) are among the most important immune cells in the tumor microenvironment (TME). Derived from monocytes, they exhibit high functional plasticity and are primarily regulated by tumor microenvironment signals. TAMs play a crucial role in tumor growth, invasion, metastasis, and immune escape. Based on their phenotype and function, TAMs can be classified into M1 and M2 types. M1 TAMs possess anti-tumor activity, killing tumor cells through the secretion of cytokines and reactive oxygen species (ROS). M2 TAMs promote tumor growth, invasion, and metastasis by inhibiting T cell function and promoting angiogenesis, thus supporting tumor progression. Figure 19 Flow cytometry results showed that the P-(H)-m@EMLN nanovaccine treatment group had a significantly increased M1 macrophage infiltration compared with other treatment groups.

[0140] 4.9 T cells and central memory T cells in the spleen

[0141] The spleen is a vital organ of the immune system, playing a crucial role not only in innate and adaptive immunity but also in the formation and maintenance of immune memory. Central memory T cells (Tcm), primarily located in secondary lymphoid organs, are an important subset of memory T cells. They possess long-term survival, self-renewal capabilities, and rapid effector differentiation potential, playing a key role in anti-tumor immunity, infection defense, and vaccine design. Upon re-encountering the same antigenic stimulus, they rapidly proliferate and differentiate into effector T cells, directly killing pathogens or regulating immune responses, thus exerting a powerful anti-tumor effect. Figures 22-24Flow cytometry results from mouse spleens showed that the P-(H)-m@EMLN nanovaccine treatment group had CD8 + and CD4 + The content of central memory T cells was significantly increased, which also indicates that the P-(H)-m@EMLN nanovaccine can trigger long-lasting immune memory.

[0142] 4.10 Enzyme-linked immunosorbent assay (ELISA)

[0143] Cytokines are synthesized and released by heterogeneous cell populations such as immune cells (e.g., T lymphocytes, macrophages), stromal cells, and epithelial cells. They are polypeptide molecules with broad regulatory functions on cell function. Enzyme-linked immunosorbent assay (ELISA) is a cornerstone technology for immunoassay, widely used in disease diagnosis, scientific research, and industry. We used ELISA experiments to investigate the levels of cytokines in the serum of mice treated with different nanovaccines, such as... Figure 23 The results showed that the highest concentrations of interleukin-6 (IL-6), interferon-β (IFN-β), and tumor necrosis factor-α (TNF-α) were detected in the P-(H)-m@EMLN nanovaccine treatment group.

[0144] 4.11 Evaluation Protocol for Long-Term Immunological Memory Induced by Nanoparticle Vaccines

[0145] Previous experimental results showed that the tumor almost completely regressed on day 21 after the first administration of the P-(H)-m@EMLN nanovaccine. To verify the strength of its immune memory effect, we re-injected tumor cells after tumor regression and observed their re-tumor formation efficiency. To more intuitively demonstrate that the drug can inhibit tumor development and progression, we established a luciferase-labeled Hepa1-6 liver cancer xenograft model. We also set up a surgical tumor resection group (tumor size ≤ 500 mm). 3 Left and right excision), and surgical excision plus drug administration group (for tumors 300 mm in size). 3 Dosage, 500 mm 3 (Left and right excisions were used as controls). Tumor-bearing mice in the surgery group and the vaccination + Operation group underwent tumor resection on day 9. The vaccination + surgery group received subcutaneous injections of C57BL / 6J mouse P-(H)-m@EMLN nanovaccine (GPC3 mRNA, 10 μg) on ​​days 7, 14, and 21. The vaccination group received P-(H)-m@EMLN nanovaccine (GPC3 mRNA, 10 μg) on ​​days 1, 7, and 14 after tumor development. All groups received a second subcutaneous injection of luciferase-labeled Hepa1-6 cells on day 21. Results are as follows: Figure 24As shown in the in vivo imaging results, compared with the surgical group, the group receiving treatment in the early stage of tumor not only exhibited a better tumor inhibition rate, but also showed an extremely low tumorigenesis rate within 42 days after re-injection of tumor cells. Furthermore, even when treatment was administered in the late stage of tumor development, the recurrence rate was significantly lower compared with surgical treatment alone, indicating that the nanovaccine not only exhibits good tumor inhibition effects but also activates the body's strong immune memory.

[0146] To further verify its immune memory effect, we used flow cytometry to detect the content of CD4 and CD8 central memory T cells in the spleen. Figure 27 and Figure 28 The results showed that the levels of CD4 and CD8 central memory T cells in the spleen were significantly higher in the treated group than in the surgical group. In conclusion, the P-(H)-m@EMLN nanovaccine exhibits excellent inhibitory effects on the development and progression of both early and late-stage liver cancer, and effectively resists tumor recurrence.

[0147] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. Other modifications can be easily made by those skilled in the art. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and shown herein.

Claims

1. A method for preparing an endoplasmic reticulum membrane-coated PLGA-poly T / GPC3 mRNA vaccine, characterized in that, Includes the following steps: Step 1: Dissolve dioleoyl lecithin, 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine, GPC3 mRNA and PLGA-polyT in an organic solvent, and then remove the organic solvent by rotary evaporation under reduced pressure to obtain a lipid membrane; Step 2: Add ultrapure water to hydrate the lipid membrane, then add endoplasmic reticulum membrane and sonicate, then centrifuge and collect the supernatant to obtain the endoplasmic reticulum membrane-coated PLGA-poly T / GPC3 mRNA vaccine P-(H)-m@EMLN.

2. The method for preparing the endoplasmic reticulum membrane-coated PLGA-poly T / GPC3 mRNA vaccine as described in claim 1, characterized in that, The mass ratio of the dioleoyl lecithin, 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine, GPC3 mRNA, PLGA-polyT, and endoplasmic reticulum membrane is 3.32 mg:1.84 mg:5 μg:12.5 μg:20 μg.

3. The method for preparing the endoplasmic reticulum membrane-coated PLGA-poly T / GPC3 mRNA vaccine as described in claim 1, characterized in that, The organic solvents include chloroform and methanol; the volume ratio of chloroform to methanol is 2:

1.

4. The method for preparing the endoplasmic reticulum membrane-coated PLGA-poly T / GPC3 mRNA vaccine as described in claim 1, characterized in that, In step one, the organic solvent is removed by rotary evaporation under reduced pressure at 37°C.

5. The method for preparing the endoplasmic reticulum membrane-coated PLGA-poly T / GPC3 mRNA vaccine as described in claim 1, characterized in that, In step two, after adding ultrapure water, hydrate at 37°C or below for 5 minutes; after adding the endoplasmic reticulum membrane, sonicate for 2 minutes; centrifuge at 10,000 rpm for 10 minutes.

6. The method for preparing the endoplasmic reticulum membrane-coated PLGA-poly T / GPC3 mRNA vaccine as described in claim 1, characterized in that, The preparation method of the PLGA-polyT is as follows: The DMF solution was prepared by dissolving thymine CNA monomer and mercapto-PLGA in N,N-dimethylformamide (DMF). The DMF solution was then mixed with 2,2-dimethoxy-2-phenylacetophenone (DMPA) under an argon atmosphere, and then subjected to a 365 nm atmosphere at 20 mW / cm². 2 The reaction solution was obtained by irradiation under light for 10 h. The reaction solution was transferred to a 3500 MWCO dialysis bag and dialyzed in methanol for 2, 12, 16 and 20 h. Then, it was dialyzed in ethyl acetate for 2, 4 and 8 h. The solvent was removed by rotary evaporation to obtain PLGA-polyT.

7. The method for preparing the endoplasmic reticulum membrane-coated PLGA-poly T / GPC3 mRNA vaccine as described in claim 6, characterized in that, The surge ratio of thymine CNA monomer, mercapto PLGA and 2,2-dimethoxy-2-phenylacetophenone DMPA is 169.8 mg: 5 kDa: 5 mg.

8. The method for preparing the endoplasmic reticulum membrane-coated PLGA-poly T / GPC3 mRNA vaccine as described in claim 1, characterized in that, The method for preparing the endoplasmic reticulum membrane is as follows: Four-week-old male C57BL / 6J mice were selected. After disinfecting the skin of both axillae with 75% ethanol, 200 μL of PBS suspension containing 6 million Hepa 1-6 cells was injected subcutaneously into each side. When the average tumor volume reached 1000 mm³, the mice were sacrificed by cervical dislocation. The tumor tissue was quickly dissected and obtained. The endoplasmic reticulum membrane was then extracted using the English Special Animal Cell / Tissue Endoplasmic Reticulum Enrichment Kit.

9. An endoplasmic reticulum membrane-coated PLGA-poly T / GPC3 mRNA vaccine, characterized in that, It includes dioleoyl lecithin, 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine, GPC3 mRNA, PLGA-polyT, and endoplasmic reticulum membrane.

10. The endoplasmic reticulum membrane-coated PLGA-poly T / GPC3 mRNA vaccine as described in claim 9, characterized in that, The mass ratio of dioleoyl lecithin, 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine, GPC3 mRNA, PLGA-polyT, and endoplasmic reticulum membrane is 3.32 mg: 1.84 mg: 5 μg: 12.5 μg: 20 μg; The poly A length in the GPC3 mRNA is 100-150 bp, and the poly T length in the PLGA-polyT is 25-30 bp.

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