Gallium indium liquid metal nano vaccine deformed under driving of near-infrared light as well as preparation method and application of gallium indium liquid metal nano vaccine

Abstract

The invention discloses a near-infrared light driven deformed gallium-indium liquid metal nano vaccine as well as a preparation method and application thereof, and belongs to the technical field of biomedical composite materials. The preparation method comprises the following steps: (1) adding 80-120mg of gallium indium liquid metal and 50-70mg of methoxy polyethylene glycol-sulfydryl into 10-20mL of absolute ethyl alcohol, carrying out ultrasonic treatment for 30-50 minutes, uniformly mixing, and centrifuging twice to obtain a precipitate, namely liquid metal nanoparticles; and (2) blending 450-550 [mu] g of liquid metal nanoparticles, 80-120 [mu] g of an antigen Adpgk and 80-120 [mu] g of a CpG1826 adjuvant in double distilled water, uniformly mixing, and centrifuging to obtain a precipitate, namely the liquid metal nano vaccine. The liquid metal nano-vaccine prepared by the invention deforms through light driving and can be used for inhibiting postoperative recurrence and metastasis of tumors, and a new thought is provided for developing nano-vaccine-mediated postoperative treatment of tumors.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical composite materials, and in particular relates to a near-infrared light-driven deformation gallium-indium liquid metal nanovaccine and a preparation method and application thereof. Background Art

[0002] The morbidity and mortality of colorectal cancer have ranked fourth and second among malignant diseases in my country. Although colorectal cancer treatment strategies have been continuously optimized in recent years, 30-40% of patients still experience metastasis after surgery, leading to poor prognosis. Compared with traditional chemotherapy and radiotherapy, immunotherapy has lower toxicity and can mobilize the body's immune system to target and kill tumors, which is more conducive to clearing residual lesions after surgery. Unlike immune checkpoint blockade therapy and chimeric antigen receptor T cell therapy, tumor vaccines can not only activate tumor-killing CD8 by introducing antigens into the patient's body, but also activate tumor-killing CD8 + T cells, inhibit tumor progression, and are expected to form anti-tumor immune memory and inhibit tumor recurrence in the long term. Therefore, it is very promising to use tumor vaccines to inhibit the recurrence of colorectal cancer after surgery.

[0003] The research on tumor vaccines is becoming increasingly popular, but there are still challenges in lymph node targeted transport and dendritic cell activation. Compared with traditional tumor vaccines, emerging nanovaccines based on polymers, liposomes, biomimetic vesicles, etc. have been significantly improved in antigen loading rate, stability, and intracellular uptake efficiency. More importantly, nanovaccines can improve the lymph node transport efficiency of drugs through targeted modification; combined with adjuvants or as self-adjuvants to enhance dendritic cell activation. However, most antigens will be degraded in endosomes / lysosomes after entering dendritic cells and cannot be effectively delivered to CD8 + T cells greatly limit the efficacy and clinical application of tumor vaccines. In order to improve the efficiency of cross-presentation of antigens, a variety of hypotheses have been proposed, such as using cationic polymers as carriers to induce the "proton sponge effect", using lipid nanoparticles to promote the fusion of carriers with endosomal membranes, or carrying cell-penetrating peptides to promote vaccine escape from endosomes. However, cationic polymers and lipid nanoparticles are prone to drug leakage and may have potential toxicity, so new cytoplasmic delivery strategies are still needed.

[0004] Deformable nanovaccines are becoming a new research direction, which promote the intracellular delivery of drugs by mechanically destroying the endosomal membrane. Gallium-indium liquid metal nanoparticles are easy to prepare, have high drug loading efficiency, and have strong photothermal properties, and have gradually been used in tumor treatment. Summary of the invention

[0005] In our previous work, we have demonstrated that gallium-indium liquid metal nanoparticles can undergo significant deformation under near-infrared light (NIR) irradiation. The liquid metal nanovaccine prepared based on this feature can drive the vaccine to deform by external near-infrared light irradiation to achieve lysosomal escape. Compared with the reported pH-responsive deformation nanovaccine, it has better controlled release characteristics, lower off-target effects and toxic side effects. At the same time, subcutaneously injected nanovaccines are easy to drain to the lymph nodes, and the deformation of liquid metal nanovaccines in the lymph nodes can prolong the retention time, which is conducive to the recognition and activation of dendritic cells. Therefore, liquid metal nanovaccines are expected to enhance the lysosomal escape of antigens and prolong the lymph node retention of vaccines through controllable deformation, promote the activation of anti-tumor immune responses, and inhibit the recurrence of colorectal cancer.

[0006] Based on this, the applicant used ice-water bath ultrasound combined with gradient centrifugation to prepare gallium-indium liquid metal nanoparticles, and then efficiently loaded antigen polypeptides and nucleic acid adjuvants through electrostatic adsorption to prepare liquid metal nanovaccines. This spherical nanovaccine has light-driven deformation properties, and can efficiently aggregate and deform under near-infrared light, and increase to a spindle shape. Subcutaneously injected liquid metal nanovaccines can be drained to the inguinal lymph nodes of mice and taken up by dendritic cells. Subsequently, by externally applying near-infrared light to the mouse lymph nodes, the vaccine can be promoted to deform within the cells, escape from lysosomes, and enhance subsequent antigen cross-presentation. At the same time, the deformation promotes the retention of the vaccine in the lymph nodes, further promoting the maturation and activation of dendritic cells. The dual effects induced by vaccine deformation significantly enhanced the downstream CD8 + The activation of T cells significantly inhibited tumor recurrence in situ and synchronous metastasis in the incomplete tumor resection model and bilateral tumor model.

[0007] The liquid metal nanoparticles in the existing patent (202110667385.7) are a liquid metal core combined with an outer shell of an antigen-adsorbing surfactant. The nanoparticles kill tumors by mediating photothermal effects locally in the tumor, and then adsorb antigens released by dead tumor cells to form an in situ tumor vaccine, activating dendritic cells in the local tumor to activate anti-tumor immunity. However, intravenously injected liquid metal nanoparticles are difficult to enrich to a sufficient concentration in the local tumor to mediate photothermal effects only by high permeability and retention effects. At the same time, the in situ vaccine formed by the adsorption of tumor antigens by liquid metal nanoparticles in the lesion is limited by endosomal / lysosomal degradation and immunosuppressive microenvironment, making it difficult to efficiently activate dendritic cells. Therefore, based on the light-driven deformation characteristics discovered, we proposed another controllable deformation liquid metal nanovaccine, which is different in three points: ① Different administration methods: The original patent is delivered to the local tumor, but the passive transport efficiency of nanoparticles is poor, and off-target toxicity is prone to occur. The liquid metal nanovaccine of the present invention is injected subcutaneously and is easily drained to the lymph nodes, which are rich in dendritic cells and T cells, providing a basis for subsequent immune activation. ② Different working principles: The nanoparticles of the original patent adsorb antigens locally in the tumor to activate dendritic cells, but it is difficult to cope with lysosomal degradation. The nanovaccine of the present invention has near-infrared light-driven characteristics, and can be artificially irradiated with near-infrared light to drive the vaccine to deform and escape from lysosomes in dendritic cells, thereby enhancing antigen cross-presentation. At the same time, the deformed nanovaccine is more likely to be retained in the lymph nodes, further promoting the maturation and activation of dendritic cells. ③ Different application backgrounds: The nanoparticles of the original patent are applied to tumors before surgery, but are limited by the tumor immunosuppressive microenvironment, and it is difficult to efficiently activate anti-tumor immune responses. Surgical resection of tumor foci can greatly alleviate immunosuppression, so the nanovaccine of the present invention is applied to postoperatively, which is easier for anti-tumor immune activation and clinical application.

[0008] The present invention provides a gallium-indium liquid metal nanovaccine that undergoes light-driven deformation to inhibit the recurrence and metastasis of CRC after surgery. Gallium-indium liquid metal (75% gallium, 25% indium) and methoxypolyethylene glycol-thiol are added to anhydrous ethanol and ultrasonically mixed to form liquid metal nanoparticles, and then the liquid metal nanoparticles are mixed with Adpgk (MC38 neoantigen) and CpG adjuvant in double distilled water to form a liquid metal nanovaccine. After the gallium-indium liquid metal nanovaccine is inoculated into the lymph nodes, the nanovaccine aggregation and deformation in the lymph nodes are driven by giving the draining lymph nodes near-infrared light, thereby enhancing the retention of the vaccine in the lymph nodes; at the same time, light-driven deformation can also promote the escape of the vaccine from the dendritic cell endosomes, thereby enhancing the cross-presentation of tumor antigens, and ultimately activating downstream specific immune responses and immune memory, inhibiting the recurrence and liver and lung metastasis of colorectal cancer after surgery.

[0009] The liquid metal nano-vaccine proposed in the present invention can be used to inhibit the postoperative recurrence and metastasis of CRC by light-driven deformation. The implementation and proposal of the present invention provide a new idea for the development of nano-vaccine-mediated postoperative treatment of CRC.

[0010] In order to achieve the above technical purpose, the present invention adopts the following specific implementation scheme:

[0011] A method for preparing a near-infrared light-driven deformation-induced gallium-indium liquid metal nanovaccine, the preparation method comprising the following steps:

[0012] (1) Preparation of liquid metal nanoparticles

[0013] 80-120 mg of gallium-indium liquid metal and 50-70 mg of methoxy polyethylene glycol-thiol were added to 10-20 mL of anhydrous ethanol and ultrasonicated for at least 30 minutes. After mixing, the suspension was heated at 2 × 10 3 -2.2×10 3 Centrifuge at a speed of g for at least 4 minutes, take the supernatant and centrifuge at 1.1 × 10 4 -1.3×10 4 g for at least 4 min, and the resulting precipitate is the liquid metal nanoparticles;

[0014] (2) Preparation of liquid metal nanovaccine

[0015] 450-550 μg of liquid metal nanoparticles were mixed with 80-120 μg of antigen Adpgk and 80-120 μg of CpG1826 adjuvant in double distilled water. After mixing evenly, the suspension was diluted with 1.1×10 4 -1.3×10 4 g for at least 4 minutes, and the resulting precipitate is the liquid metal nanovaccine;

[0016] The amino acid sequence of the antigen Adpgk is ASMTNMELM;

[0017] The sequence of CpG1826 is shown in SEQ ID NO.1.

[0018] Preferably, the amount of gallium-indium liquid metal used in step (1) is 100 mg.

[0019] More preferably, the amount of methoxypolyethylene glycol-thiol used in step (1) is 60 mg.

[0020] More preferably, the volume of anhydrous ethanol in step (1) is 15 mL.

[0021] More preferably, the ultrasonic treatment time in step (1) is 40 minutes.

[0022] More preferably, the amount of liquid metal nanoparticles used in step (2) is 500 μg.

[0023] More preferably, the amount of antigen Adpgk used in step (2) is 100 μg.

[0024] More preferably, the amount of CpG1826 used in step (2) is 100 μg.

[0025] A gallium-indium liquid metal nano-vaccine prepared by the above preparation method.

[0026] The use of the above-mentioned gallium-indium liquid metal nanovaccine in the preparation of anti-tumor products, wherein the tumor is colon cancer or rectal cancer.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1) Gallium-indium liquid metal nanoparticles have good biosafety, high antigen loading rate and variable size.

[0029] 2) Gallium-indium liquid metal nanovaccine has a strong retention capacity in lymph nodes: the nanovaccine can aggregate and deform under near-infrared light, transforming from a spherical shape with a diameter of 50nm to a spindle shape with a long diameter of 500nm. After the nanovaccine is drained to the lymph nodes of mice, near-infrared light can promote the retention of the vaccine in the lymph nodes.

[0030] 3) Gallium-indium liquid metal nanovaccine can enhance antigen cross-presentation: After the nanovaccine is taken up by dendritic cells, it can promote the escape of tumor antigens from endosomes / lysosomes through light-driven deformation, thereby enhancing antigen cross-presentation.

[0031] 4) The subcutaneously injected gallium-indium liquid metal nanovaccine can effectively activate the anti-tumor immune response after drainage to the body's lymph nodes, inhibiting postoperative in situ recurrence of CRC, liver and lung metastasis, and distant tumor growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 A is the transmission electron microscopy image of LM and LMV and the statistical diagram of hydrated particle size.

[0033] Figure 1 B is the degradation rate curve of LMV in pH 7.4 and pH 5.0 PBS.

[0034] Figure 1 C is the release rate curve of antigen and adjuvant of LMV in pH 7.4 and PBS (pH 5.0).

[0035] Figure 1 D is a schematic diagram of the aggregation and deformation of LMV under NIR.

[0036] Figure 1E is a fluorescence microscopy image of LMV aggregation deformation under NIR.

[0037] Figure 1 F is the transmission electron microscopy image of LMV aggregation deformation under NIR.

[0038] Figure 1 G is the long and short diameter bar chart of FLM and FLMV under transmission electron microscope.

[0039] Figure 1 H is a line graph showing the enrichment and degradation rate of LMV in lymph nodes with or without light irradiation.

[0040] Figure 1 I is the transmission electron microscope elemental analysis image before and after LMV illumination.

[0041] Figure 2 Representative confocal images of BMDC uptake of three vaccines (free vaccine, Montanide vaccine, and deformed LMV).

[0042] Figure 3 A is the live and dead cell staining fluorescence image of DC2.4 cells with different concentrations of liquid metal nanovaccine combined with or without NIR.

[0043] Figure 3 B is Figure 3 A's bar graph.

[0044] Figure 4 A is the flow cytometric analysis and bar graph of the CpG / Adpgk uptake ratio of BMDC.

[0045] Figure 4 B is the average fluorescence intensity of BMDCs after treatment with different vaccines for 24 hours.

[0046] Figure 4 C is Figure 4 Pearson correlation coefficient bar chart of liquid metal nanovaccine fluorescence and lysosome fluorescence in D.

[0047] Figure 4 D is the fluorescence image of liquid metal nanovaccine and lysosome in DC2.4 cells observed by confocal microscope at two time points: 2 hours and 7 hours. Adpgk-FITC was used to label LMV, and Lyso-Tracker was used to label lysosome. The scale bar is 10 μm.

[0048] Figure 4 E is a biological electron microscopy image of DC2.4 cells treated with the deformed LMV (LMV+NIR) group for 7 hours. The image on the right (scale bar: 1 μm) is an enlarged image of the corresponding position of the image on the left (scale bar: 200 nm).

[0049] Figure 4 F is the flow cytometry analysis of CD11C in each group 24 hours after vaccine treatment + BMDC surface CD80 + CD86 + Proportional bar chart.

[0050] Figure 4 G is the ELISA test of TNF-α and IL-6 levels in the supernatant of BMDCs after 24 hours of vaccine treatment in each group.

[0051] Figure 5 A is the immunofluorescence image of the inguinal lymph nodes of mice in the LMV+NIR group, and the image on the right (scale bar: 200 μm) is an enlarged view of the corresponding position of the image on the left (scale bar: 20 μm).

[0052] Figure 5 B is a biological electron microscope image of the inguinal lymph nodes of mice in the LMV+NIR group. The image on the right (scale bar: 1 μm) is an enlarged view of the corresponding position of the image on the left (scale bar: 200 nm).

[0053] Figure 5 C is the in vivo fluorescence image of mice in each group at different time points, and the red circle is the draining lymph node area.

[0054] Figure 5 D is the fluorescence image of the isolated lymph nodes of mice in each treatment group at different time points. Each group of vaccines was labeled with CpG-Cy5.

[0055] Figure 5 E is Figure 5 C is a line graph of the mean fluorescence intensity of mouse lymph node regions.

[0056] Figure 5 F is Figure 5 D is a line graph of the mean fluorescence intensity of lymph nodes in each group.

[0057] Figure 5 G is the flow cytometry analysis of CD80 on the surface of dendritic cells in the lymph nodes of each group of mice + CD86 + proportion.

[0058] Figure 6 A is a schematic diagram of the incomplete tumor resection model.

[0059] Figure 6 B is a line graph of the primary tumor volume in mice with incomplete tumor resection model.

[0060] Figure 6 C is the primary tumor volume curve of each group of mice in the incomplete tumor resection model and the proportion of mice in each group with complete tumor cure.

[0061] Figure 6D is the survival time of mice in each group of the incomplete tumor resection model.

[0062] Figure 6 E is the tumor infiltrating CD8 + Flow cytometry analysis of T cell proportion.

[0063] Figure 6 F is Figure 4 E. The tumor infiltrating CD8 + Histogram of T cell proportions.

[0064] Figure 6 G is a schematic diagram of the bilateral tumor model.

[0065] Figure 6 H is the gross image of the primary tumor and the line graph of tumor volume in bilateral tumor model mice.

[0066] Figure 6 I is the gross image of the distal tumor and the line graph of tumor volume in the bilateral tumor model mouse.

[0067] Figure 6 J is the CD8 in the tumor microenvironment of the distal tumor in the bilateral tumor model mouse + T cell infiltration ratio.

[0068] Figure 7 A is a line graph of mouse body weight in the incomplete resection model.

[0069] Figure 7 B is a line graph of mouse body weight in the bilateral tumor model.

[0070] Figure 8 A is the H&E staining image of the main organs of mice in different treatment groups.

[0071] Figure 8 B is the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (Urea) and creatinine (Crea) in mice in different treatment groups.

[0072] Fig. 9 Schematic diagram of the working process of liquid metal nanovaccine, including (A) the synthesis roadmap of liquid metal nanovaccine, which can be deformed into a spindle shape under the drive of near-infrared light; (B) subcutaneous injection of nanovaccine, the deformation promotes the retention of the vaccine in the lymph nodes and prolongs the action time with dendritic cells; (C) the deformation promotes the endosomal escape of the vaccine and subsequent antigen cross-presentation; (D) the nanovaccine activates immune memory to inhibit the recurrence of colorectal cancer after surgery. DETAILED DESCRIPTION

[0073] Example 1 Preparation of Light-Driven Deformed Gallium-Indium Liquid Metal Nanovaccine

[0074] 1. Preparation of liquid metal nanoparticles

[0075] 100 mg of gallium-indium liquid metal (purchased from Alfa Esha, containing 75 wt% gallium and 25 wt% indium) and 60 mg of methoxypolyethylene glycol-thiol were added to a 50 mL centrifuge tube containing 15 mL of anhydrous ethanol and ultrasonicated for 40 minutes. 3 g for 5 minutes, and the supernatant was collected and centrifuged at 1.24 × 10 4 g for 5 min, and the resulting precipitate is the liquid metal nanoparticles.

[0076] 2. Preparation of liquid metal nanovaccine

[0077] 500 μg of liquid metal nanoparticles were mixed with 100 μg of antigen Adpgk (MC38 cell neoantigen) and 100 μg of CpG1826 adjuvant in double distilled water and stirred at room temperature for 1 hour. 4 The mixture was centrifuged at 1000 g for 5 minutes, and the resulting precipitate was the liquid metal nanovaccine.

[0078] The amino acid sequence of Adpgk (MC38 cell neoantigen) is: ASMTNMELM; the sequence of CpG1826 (hereinafter referred to as CpG) is: 5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO.1).

[0079] Example 2 Characterization of Light-Driven Deformation Gallium-Indium Liquid Metal Nanovaccine

[0080] 1. Microstructure

[0081] High-resolution transmission electron microscopy was used to characterize the morphology and analyze the elemental composition of the nanovaccine. Bio-electron microscopy was used to detect the light-driven deformation properties of the nanovaccine in mouse lymph nodes. A particle size meter was used to characterize the hydration particle size and electric potential of the nanovaccine.

[0082] The experimental results are as follows Figure 1 As shown, spherical gallium-indium liquid metal nanoparticles (liquid metal, LM) were first prepared by ice-water bath ultrasonic method ( Figure 1 A), and further electrostatically adsorbed peptide antigen Adpgk and CpG1826 adjuvant to prepare liquid metal nanovaccine (liquid metal vaccine, LMV) ( Figure 1 A), where each 100 μg of liquid metal nanoparticles can adsorb 5.5 μg of antigen and 3.9 μg of CpG. Hydration particle size analysis showed that the particle sizes of LM and LMV were 54.5 nm and 67.2 nm, respectively ( Figure 1A). The team previously discovered (Biomaterials.2019,7:76-88.) that LM can deform under near-infrared light (NIR), and here we found that LMV also has this property ( Figure 1 D) Fluorescence microscopy of LMVs shows that LMVs can aggregate under NIR ( Figure 1 E), further transmission electron microscopy images confirmed that after NIR irradiation, LMV can be transformed from a spherical aggregate with a diameter of about 50nm to a spindle-shaped liquid metal nanovaccine (Fusiform LMV, FLMV) with a long diameter of 480nm and a short diameter of 280nm ( Figure 1 FG).

[0083] 2. Loading efficiency and antigen release efficiency of liquid metal nanovaccine

[0084] The BCA protein detection kit was used to detect the loading efficiency of the nanovaccine on the polypeptide antigen. Nanodrop was used to detect the loading efficiency of the nanovaccine on the CpG adjuvant. The nanovaccine was placed in phosphate buffer with different pH values, and the supernatant was collected on different days. The degradation rate of the nanovaccine and the release rate of each component were characterized by atomic absorption spectrometry.

[0085] The experimental results are as follows Figure 1 As shown in Figure 2, LMV has good stability and only degrades by about 5% in PBS at pH 5.0 and 7.4 for 7 days ( Figure 1 B). LMV released 30.4% of Adpgk and 33.4% of CpG in PBS at pH 7.4 over 7 days, while in PBS at pH 5.0, they released 44.8% and 49.0%, respectively. Figure 1 C).

[0086] We then detected the enrichment and degradation of LMV in the lymph nodes. The specific steps were as follows: 50 μL LMV (200 μg) was injected subcutaneously at the base of the mouse tail. The mice in the LMV combined with light group were irradiated with near-infrared light (808 nm) for 4 minutes at 6, 30, and 72 hours after vaccination, with a power of 1 W / cm 2 The mice were euthanized at different time points and the lymph nodes were removed. The gallium ion content in the lymph nodes was detected by inductively coupled plasma mass spectrometry. The results showed that the gallium ion content in the lymph nodes of the mice in the LMV combined with NIR group reached a peak on the 7th day and then gradually decreased, and was higher than that in the LMV group at each time point (no infrared light irradiation was performed, and the rest of the operations were the same as those in the LMV combined with light group), suggesting that light deformation can promote the retention of LMV in the lymph nodes ( Figure 1 H). Further transmission electron microscopy element analysis results showed that LMV was enriched with P and S elements before and after illumination, indicating that the vaccine can effectively load CpG adjuvant (containing P element) and peptide Adpgk (containing S element) ( Figure 1I).

[0087] The above results show that the applicant has successfully prepared a liquid metal nanovaccine (LMV) with light-driven deformation properties. The deformation properties of this vaccine also provide a theoretical basis for subsequent lysosomal escape and lymph node retention studies.

[0088] Example 3 Detection of intracellular uptake efficiency and cytotoxicity of gallium-indium liquid metal nanovaccine

[0089] 1. Detection of the vaccine uptake efficiency by dendritic cells:

[0090] Liquid metal nanovaccine was prepared using fluorescent label adjuvant CpG-Cy3, and the process was the same as in Example 1.

[0091] Preparation of vaccines for each group: 200 μg of LMV adsorbed with 11.0 μg of ADPGK and 7.8 μg of CpG was dissolved in 50 μL of PBS as the LMV group. The LMV group was further given 1 W / cm 2 The fusiform vaccine (FLMV) group was prepared by inducing deformation by near-infrared light (808 nm). 11.0 μg ADPGK and 7.8 μg CpG were dissolved in 50 μL PBS as the free vaccine group (V). 11.0 μg ADPGK and 7.8 μg CpG were dissolved in a mixture of 25 μL PBS and 25 μL Montanide ISA-51 as the Montanide vaccine group (Montanide-V).

[0092] An appropriate amount of DC2.4 cells (cultured in 1 mL of DMEM medium) were plated in a 6-well plate or confocal dish, and 10 μL of each group of fluorescently labeled nanovaccines was added. The LMV combined with NIR (LMV+NIR) group was given 1 W / cm2 for 4 minutes at the 6th hour. 2 At 12 hours, DC2.4 cell nuclei were stained with Hoechst 33258, and then the uptake efficiency of DC2.4 cells for nanovaccine was detected by flow cytometry or confocal imaging.

[0093] The experimental results are as follows Figure 2 , Figure 4 As shown in AB, the confocal microscopy images showed that compared with the free vaccine group (10 μL) and the Montanide vaccine group (10 μL), the bone marrow-derived dendritic cells (BMDC) had the highest uptake efficiency for the LMV combined with NIR group ( Figure 2 More importantly, the LMV combined with NIR group had the highest efficiency in delivering antigen and adjuvant to BMDCs simultaneously, which was 2.26 times that of the Montanide vaccine group ( Figure 4A). Flow cytometry analysis showed that at 24 hours, there was no significant difference in the uptake efficiency of BMDC for the three groups of vaccines: FLMV, LMV, and LMV+NIR ( Figure 4 B).

[0094] 2. Detection of the cytotoxicity of the vaccine

[0095] Appropriate amount of DC2.4 cells were plated in 48-well plates, and different concentrations of LMV were given after adherence, with or without light treatment. After 24 hours of culture, the supernatant in the wells was discarded and washed with PBS, and then 150 μL of CalceinAM / PI detection working solution was added to each well, incubated at 37℃ in the dark, and observed and photographed under a fluorescence microscope after 30 minutes.

[0096] The experimental results are as follows Figure 3 As shown in the live-dead cell staining experiment, different concentrations of LMV, with or without light, had no obvious toxicity to DC2.4 cells ( Figure 3 AB).

[0097] The results of this section show that the liquid metal nanovaccine LMV has high intracellular uptake efficiency and low cytotoxicity, providing a basis for subsequent cell experiments.

[0098] Example 4 Evaluation of the efficacy of the vaccine in inducing dendritic cell maturation and activation and lymph node retention

[0099] 1. Cellular level

[0100] Bone marrow-derived dendritic cells were extracted from 6-8 week old C57BL / 6j mice, induced to mature and plated. After the cells adhered to the wall, 10 μL of each group of vaccines (same as in Example 3) were given respectively. After 24 hours, the cells were collected for flow staining (CD11C, CD80, CD86) to evaluate the maturation and activation of dendritic cells. The culture supernatant was collected and the secretion of cytokines such as interleukin 6 (IL-6) was detected by ELISA.

[0101] The experimental results are as follows Figure 4 As shown in CG, three treatment groups were set up: pre-illuminated fusiform vaccine group (FLMV), liquid metal nanovaccine group (LMV), and LMV combined with NIR group (LMV+NIR). Subsequent confocal images showed that at 2 hours, all three groups of nanovaccines were highly co-localized with DC2.4 cell lysosomes; while at 7 hours, the co-localization of nanovaccines and lysosomes in the LMV+NIR group (illuminated at the 6th hour) decreased significantly, and the Pearson correlation coefficient decreased from 0.79 to 0.33 ( Figure 4C, D), indicating that this group of vaccines effectively escaped from lysosomes. Further biological electron microscopy photography of DC2.4 cells in the LMV+NIR group revealed three stages of LMV deformation: before deformation, during aggregation, and after deformation into a spindle shape and escape from lysosomes ( Figure 4 E), confirming that LMV can escape from lysosomes by NIR-driven deformation. Subsequent flow cytometry analysis results showed that compared with the PBS group, the CD80 + CD86 + The proportion of BMDC in the LMV+NIR group increased by 82.1%, 168.9% and 141.9%, respectively, while that in the LMV+NIR group increased by 236.5% ( Figure 4 F). ELISA results also showed that the LMV+NIR group could most effectively stimulate BMDC to secrete pro-inflammatory cytokines TNF-α and IL-6, which were increased by 22.8% and 71.8% respectively compared with the LMV group ( Figure 4 G).

[0102] The results in this section prove that the liquid metal nanovaccine can escape from lysosomes through light-driven deformation after entering the cell, thereby promoting dendritic cell maturation and cross-presentation of tumor antigens.

[0103] 2. In vivo level

[0104] Experimental method: The right abdomen of 6-8 week old female C57BL / 6j mice was depilated and given different treatments: free vaccine (V) group, Montanide vaccine (Montanide-V) group, liquid metal nano vaccine (LMV) group, pre-illuminated fusiform liquid metal nano vaccine (FLMV) group and liquid metal nano vaccine combined with illumination (LMV+NIR) group. The above groups were set up as in Example 3. At 0 hour, 50 μL of each vaccine was injected subcutaneously at the base of the tail of each group of mice. The mice in the combined illumination group were irradiated with near infrared light (808 nm) for 4 minutes at 6 hours, 30 hours and 72 hours after the injection of the vaccine, with a power of 1 W / cm 2 . To evaluate the enrichment of the vaccine in the lymph nodes, each group of vaccines was labeled with CpG-Cy5, and the inguinal lymph nodes of mice were taken for frozen sections and fluorescent staining on the second day. To evaluate the level of vaccine retention in the lymph nodes, each group of vaccines was labeled with CpG-Cy5, and the four groups of mice were anesthetized at different time points and subjected to in vivo fluorescence imaging, and the average fluorescence intensity of the draining lymph node area was calculated; in addition, the four groups of mice were killed at different time points, the draining lymph nodes were taken out and ex vivo fluorescence imaging was performed, and the average fluorescence intensity of the draining lymph nodes was calculated. To evaluate the efficacy of the vaccine on the activation of lymph node dendritic cells, the inguinal draining lymph nodes of mice were collected after the fifth day. After the lymph nodes were ground and dispersed into single cells, flow staining (CD11C, CD80, CD86) was performed to evaluate the maturation and activation of dendritic cells in the lymph nodes.

[0105] The experimental results are as follows Figure 5 As shown in the figure, fluorescent staining of frozen sections showed that LMVs were scattered in the lymph nodes and, similar to DCs, were more distributed in the cortical area. Fluorescence co-localization of DCs and LMVs was also detected ( Figure 5 A). Further, the lymph nodes were taken for biological electron microscopy, and LMV in lysosomes and FLMV that escaped were observed ( Figure 5 B). The above experiments show that subcutaneous inoculation of LMV at the tail base can effectively drain to the inguinal lymph nodes of mice, providing a basis for the subsequent activation of anti-tumor immunity in mice.

[0106] In vivo fluorescence imaging showed that the draining lymph nodes of mice in group V had no fluorescence at all time points, indicating that it was difficult for the vaccine alone to drain to the lymph nodes; compared with the LMV and FLMV groups, the fluorescence in the lymph nodes of mice in the LMV+NIR group was the strongest, and the fluorescence intensity was highest at 30 hours, and then gradually decreased ( Figure 5 C, E). The draining lymph nodes of each group of mice at different time points were taken for in vitro fluorescence imaging, and the results were similar. The fluorescence of LMV+NIR was stronger than that of the other three groups at each time point ( Figure 5 D, F). Flow cytometry analysis of lymph node immune cells showed that compared with the PBS group, the CD80 expression on the surface of lymph node dendritic cells in the Montanide vaccine group, FLMV group, and LMV group was higher than that in the PBS group. + CD86 + The proportions increased by 71.3%, 68.4% and 134.0% respectively, and the LMV+NIR group had the largest increase of 254.6% ( Figure 5 G)

[0107] The results of this section indicate that the liquid metal nanovaccine can enhance lymph node retention through light-driven deformation, thereby activating the body's anti-tumor immune response and is expected to inhibit the recurrence of colorectal cancer after surgery.

[0108] Example 5 Evaluation of the efficacy of gallium-indium liquid metal nanovaccine in inhibiting postoperative recurrence of colorectal cancer

[0109] Experimental method: 6-8 week old C57BL / 6j mice were selected for the following animal experiments. To establish an incomplete tumor resection model: 3×10 5 MC38 cells, when the tumor grows to about 100 mm 3 At 1 pm, the mice were anesthetized, the tumors were surgically removed and about 1% was left to simulate clinical tumor recurrence in situ after surgery. At the same time, 50 μL of the vaccine of each group (same as in Example 3) was subcutaneously inoculated at the base of the tail. The mice in the combined illumination group were irradiated with near-infrared light (808 nm) for 4 minutes at 6 hours, 30 hours and 72 hours after the injection of the vaccine, with a power of 1 W / cm 2The tumor recurrence and survival period of mice in each group were recorded to evaluate the efficacy of the vaccine in inhibiting postoperative in situ recurrence.

[0110] The experimental results are as follows Figure 6 As shown in the figure, we constructed an incomplete tumor resection model, that is, when the subcutaneous tumor grows to 100mm 3 Surgical resection was performed, leaving only 1% of the tumor lesions to simulate in situ recurrence, and then different treatments were given to evaluate the efficacy of each group of vaccines in inhibiting tumor recurrence ( Figure 6 A). The results showed that the free vaccine group (V) and the free vaccine combined with light irradiation group (V+NIR) had no tumor inhibition effect, and all mice in the two groups died on day 42; the Montanide vaccine group (Montanide-V) and the FLMV group only slightly delayed tumor progression, and the 60-day survival rate of the two groups of mice was 28.6%; the LMV group could effectively delay tumor recurrence, and the 60-day survival rate of the mice in this group was 42.8%; and the LMV combined with NIR group (LMV+NIR) could most effectively inhibit tumor recurrence, and 4 out of 9 mice were completely cured, with a 60-day survival rate of 66.7% ( Figure 6 BD). Flow cytometry analysis was performed on the recurrent tumors of mice in each group. The results showed that compared with the PBS group, the tumor infiltrating CD8 + The proportion of T cells increased by 70.9%, 116.9% and 216.4% respectively, while the proportion of tumor-infiltrating CD8 + The proportion of T cells was the highest, increasing by 327.5% ( Figure 6 EF).

[0111] To evaluate whether the nanovaccine can inhibit simultaneous metastasis, we further constructed a bilateral tumor model using MC38 cells, that is, two tumors, one large and one small, were inoculated on both sides of the mouse abdomen. 3 Incomplete surgical resection was performed, and subsequent vaccination and light treatment were the same as the incomplete resection model above. The efficacy of each group of vaccines in inhibiting distal tumors was then evaluated ( Figure 4 G). The results were similar to those in the previous study, that is, the LMV combined with NIR group could most effectively inhibit the progression of bilateral tumors, with the primary tumor and distal tumor being only 29.7% and 34.5% of those in the PBS group ( Figure 6 In addition, CD8 + The proportion of T cell infiltration was significantly higher than that of other groups ( Figure 6 J).

[0112] The above animal experimental results preliminarily show that the liquid metal nanovaccine combined with near-infrared light (LMV+NIR) can effectively activate the body's anti-tumor immunity and promote CD8 +The infiltration of T cells in the tumor ultimately inhibits the progression of the in situ tumor and distal tumors.

[0113] Example 6 Biosafety Evaluation of Gallium-Indium Liquid Metal Nanovaccine

[0114] 1. Mouse weight evaluation

[0115] The body weight changes of the incomplete resection model mice were recorded every 4 days, and the body weight changes of the bilateral tumor model mice were recorded every 3 days.

[0116] like Figure 7 As shown, there was no significant change in body weight of the vaccinated mice during the treatment period.

[0117] 2. Pathological analysis of major organs

[0118] Three weeks after the mice were inoculated with different vaccines, their main organs were taken for H&E staining analysis after euthanasia. Figure 8 As shown in A, H&E staining analysis showed that no obvious organ lesions occurred in the vaccinated mice.

[0119] 3. Blood biochemical analysis

[0120] Three weeks after the mice were inoculated with different vaccines, serum was collected for analysis of major biochemical markers (alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine (Cr)) after euthanasia.

[0121] like Figure 8 As shown in B, blood biochemical analysis showed that vaccination did not affect the secretion levels of major blood biochemical markers in mice.

[0122] In summary, this liquid metal nanovaccine has good biosafety.

[0123] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a near-infrared light-driven deformation-deformed gallium-indium liquid metal nanovaccine, characterized in that: The preparation method comprises the following steps: (1) Preparation of liquid metal nanoparticles 80-120 mg of gallium-indium liquid metal and 50-70 mg of methoxy polyethylene glycol-thiol were added to 10-20 mL of anhydrous ethanol and ultrasonicated for at least 30 minutes. After mixing, the suspension was heated at 2 × 10 3 -2.2×10 3 g for at least 4 minutes, and the supernatant was collected and centrifuged at 1.1 × 10 4 -1.3×10 4 g for at least 4 min, and the resulting precipitate is the liquid metal nanoparticles; (2) Preparation of liquid metal nanovaccine 450-550 μg of liquid metal nanoparticles were mixed with 80-120 μg of antigen Adpgk and 80-120 μg of CpG1826 adjuvant in double distilled water. After mixing evenly, the suspension was diluted with 1.1×10 4 -1.3×10 4 g for at least 4 minutes, and the resulting precipitate is the liquid metal nanovaccine; The amino acid sequence of the antigen Adpgk is ASMTNMELM; The sequence of CpG1826 is shown in SEQ ID NO.

1.

2. The preparation method according to claim 1, characterized in that: The amount of gallium-indium liquid metal used in step (1) is 100 mg.

3. The preparation method according to claim 2, characterized in that: The amount of methoxypolyethylene glycol-thiol used in the step (1) is 60 mg.

4. The preparation method according to claim 3, characterized in that: The volume of anhydrous ethanol in step (1) is 15 mL.

5. The preparation method according to claim 4, characterized in that: The ultrasonic treatment time in step (1) is 40 minutes.

6. The preparation method according to claim 5, characterized in that: The amount of liquid metal nanoparticles used in step (2) is 500 μg.

7. The preparation method according to claim 6, characterized in that: The amount of antigen Adpgk used in step (2) is 100 μg.

8. The preparation method according to claim 7, characterized in that: The amount of CpG1826 used in step (2) is 100 μg.

9. Gallium-indium liquid metal nanovaccine prepared by the preparation method described in any one of claims 1 to 8.

10. The use of the gallium-indium liquid metal nanovaccine according to claim 9 in the preparation of anti-tumor products, characterized in that: The tumor is colon cancer or rectal cancer.

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