CN117599158A - 3D printing vaccine containing cell membrane vesicles, preparation method and application thereof - Google Patents
3D printing vaccine containing cell membrane vesicles, preparation method and application thereof Download PDFInfo
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- CN117599158A CN117599158A CN202410098524.2A CN202410098524A CN117599158A CN 117599158 A CN117599158 A CN 117599158A CN 202410098524 A CN202410098524 A CN 202410098524A CN 117599158 A CN117599158 A CN 117599158A
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Abstract
The invention discloses a 3D printing vaccine containing cell membrane vesicles, a preparation method and application thereof. The 3D printing vaccine is prepared by 3D printing for carrying the target vaccine on the biological ink. The bio-ink comprises PD1 cell membrane vesicles and GelMA hydrogel. By applying the technical scheme of the invention, the PD1 cell membrane vesicles extracted from methacrylic anhydride modified gelatin (GelMA), target vaccine (e.g. dendritic cells) and gene editing cells are printed into the 3D printing vaccine by a 3D printing technology. The GelMA hydrogel porous scaffold has good biocompatibility and provides a growing microenvironment for a target vaccine. The pores of the scaffold promote substance exchange, further improving survival of the vaccine of interest.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a 3D printing vaccine containing cell membrane vesicles, a preparation method and application thereof.
Background
Malignant tumors have a broad impact on public health and global economy. Surgery is the primary treatment option for solid tumors. In clinic, most cancer patients develop a recurrence of metastatic disease after initial tumor resection due to hidden microcosmic and circulating tumor cells. Chemotherapy and radiotherapy are commonly used in adjuvant therapy after tumor surgery, and although chemotherapy and radiotherapy have made rapid progress in effectively controlling distant tumor recurrence after surgery, the toxic side effects of treatment remain a major challenge in clinic. Dendritic Cell (DC) vaccines are a safe, post-operative, anti-tumor therapeutic approach with minimal side effects. Even in patients with advanced disease and weaker immune responses, DC vaccines can elicit adaptive anti-tumor immune responses in many patients. DC vaccines exhibit good clinical efficacy, particularly in combination with immune checkpoint therapy. In the flow, DCs of a tumor patient are extracted from the body, amplified and cultured in vitro, and then the anti-tumor in the body of the patient is infused back; mechanically, mature DCs migrate to lymphoid organs, bind to T lymphocytes, and enhance the antitumor effect of T cells. However, after intravenous or lymph node injection of DCs, the duration of action of DCs in the lymph nodes is short, severely affecting the therapeutic effect. To solve this problem, it has recently been proposed to load DCs on hydrogels which sustain slow release of DCs migration to lymph nodes and extend the duration of action of DCs at lymph nodes. Therefore, the continued migration of DCs stored in vivo to lymph nodes after surgery is one of the ways to enhance the antitumor effect of DC vaccines.
The hydrogel is a soft material with a three-dimensional network structure, and has good flexibility and biocompatibility. And the water-holding capacity and network structure of the hydrogel are similar to those of extracellular matrix, so that the hydrogel can be widely applied to cell delivery and tissue engineering application. However, conventional hydrogels can only provide a limited living environment for cells, and for a long time, most cells die because of the inability to efficiently ingest nutrients. Hydrogels do not maintain the viability of the DC if stored for extended periods of time, resulting in limited therapeutic efficacy. In addition, hydrogels have limited applications due to their poor mechanical properties, high storage costs, and post-operative handling difficulties.
In summary, hydrogel sustained release delivery cells for extended periods of time achieve desirable therapeutic effects and a wide range of applications require new technologies to achieve.
Disclosure of Invention
The invention aims to provide a 3D printing vaccine containing cell membrane vesicles, a preparation method and application thereof, so as to prolong the treatment time of the vaccine.
To achieve the above object, according to one aspect of the present invention, there is provided a bio-ink for 3D printing vaccine. The biological ink comprises PD1 cell membrane vesicles and GelMA hydrogel; preferably, the mass ratio of the PD1 cell membrane vesicles to the GelMA hydrogel is 1: 50-1:200; more preferably 1: 130-1:150; further preferably 150.
According to another aspect of the invention, a 3D printed vaccine is provided. The 3D printing vaccine is prepared by 3D printing for the target vaccine for carrying the biological ink.
Further, the vaccine of interest is one or more of dendritic cell vaccine, T cell vaccine, NK cell vaccine or macrophage vaccine.
Further, the mass ratio of the bio-ink to the target vaccine is 80: 1-30: 1.
further, the GelMA hydrogel contains a photoinitiator, and 3D printing is photo-curing 3D printing.
According to still another aspect of the present invention, there is provided a method for preparing the above 3D printing vaccine. The preparation method comprises the following steps: preparing PD 1-extracellular vesicles, gelMA solution and target vaccine; mixing PD 1-extracellular vesicles, gelMA solution and target vaccine to obtain mixed solution; and preparing the mixed solution through 3D printing to obtain the 3D printing vaccine.
Further, preparing the GelMA hydrogel includes: dissolving gelatin in PBS solution under continuous stirring to form Gel solution; mixing methacrylic anhydride with PBS solution to prepare MA solution; dripping the MA solution into the Gel solution, and reacting in the dark, wherein the preferable reaction temperature is 50-55 ℃, the reaction time is 3-3.5 hours, and the pH of a reaction system is 8.0-9.0; adding deionized water to terminate the reaction, dialyzing, and lyophilizing to obtain GelMA dry powder; adding GelMA dry powder into PBS solution, oscillating, and adding photoinitiator after ultrasonic treatment to obtain GelMA hydrogel; preferably, the photoinitiator is a photoinitiator LAP.
Further, the target vaccine is one or more of dendritic cell vaccine, T cell vaccine, NK cell vaccine or macrophage vaccine; preferably, the dendritic cell vaccine is obtained by extraction of bone marrow primary cell culture.
Further, preparing PD1 cell membrane vesicles includes: suspending 4T1 cells of gene editing PD1 in PBS solution, destroying by a homogenizer, centrifuging, and collecting cell membrane vesicles; washing the cell membrane vesicles with PBS mixed with a protease inhibitor; after the cell membrane vesicles were subjected to ultrasonic treatment, PD1 cell membrane vesicles were obtained by stepwise extrusion through 400 and 200 nm-pore polycarbonate membranes on a micro extruder.
Further, the 3D printing is photo-curing 3D printing.
According to still another aspect of the invention, the application of the 3D printing vaccine in preparing a medicine for inhibiting postoperative tumor recurrence is provided.
The bio-ink comprises PD1 cell membrane vesicles and GelMA hydrogel. By applying the technical scheme of the invention, the PD1 cell membrane vesicles extracted from methacrylic anhydride modified gelatin (GelMA), target vaccine (e.g. dendritic cells) and gene editing cells are printed into the 3D printing vaccine by a 3D printing technology. The GelMA hydrogel porous scaffold has good biocompatibility and provides a growing microenvironment for a target vaccine. The pores of the scaffold promote substance exchange, further improving survival of the vaccine of interest. Preferably, when the vaccine of interest is a dendritic cell, the PD1 cell membrane vesicles act as immunomodulators, stimulating maturation of DCs, binding PDL1 on the surface of DCs, promoting activation of T cells by DCs. In addition, the PD1 cell membrane vesicles can bind PDL1 on the surface of tumor cells, and further help the vaccine to play an anti-tumor role. The mechanisms work together to provide a simple, safe and robust strategy for anti-tumor immunity after tumor operation.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. In the drawings:
FIG. 1 shows a schematic diagram of a 3D printed DC vaccine and its anti-tumor mechanism according to an embodiment of the invention;
FIG. 2 shows a synthetic and characterization diagram of a 3D printed DC vaccine according to an embodiment of the invention;
FIG. 3 shows a graph of cell activity and lymphatic migration capacity results of DCs in a 3D porous scaffold in accordance with an embodiment of the invention;
FIG. 4 is a graph showing experimental results of PD1 cell membrane vesicles as immunomodulators for BMDCs according to an embodiment of the invention;
FIG. 5 is a graph showing experimental results of potent anti-tumor immune responses induced by 3D printed DC vaccine according to an embodiment of the present invention; and
fig. 6 shows a graph of experimental results of suppression of postoperative tumor recurrence by a 3D printed DC vaccine according to an embodiment of the present invention.
Detailed Description
It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other. The invention will be described in detail below with reference to the drawings in connection with embodiments.
Noun interpretation:
PD-1: programmed death receptor 1.
PDL-1: apoptosis-ligand 1.
PD1 cell membrane vesicles: programmed death receptor 1-cell membrane vesicles.
DC: dendritic cells.
The 3D printing technology is an emerging technology and has the advantages of high precision, high yield, low cost and the like. Meanwhile, extracellular vesicles have been widely studied as potential therapies for treating tumor diseases due to their non-immunogenicity and low cytotoxicity.
According to an exemplary embodiment of the present invention, a bio-ink for a 3D printing vaccine is provided. The bio-ink comprises PD1 cell membrane vesicles and GelMA hydrogel. Preferably, in one embodiment of the present application, the mass ratio of PD1 cell membrane vesicles to GelMA hydrogel is 1: 50-1:200; more preferably 1: 130-1:150; further preferably 150 (a standard mouse (6-8 weeks, 18-22 mg), the usual dose of PD1 cell membrane vesicles is 200-300. Mu.g, the dose of GelMA hydrogel is 15-40mg according to the tumor size of the mouse, and the mass ratio of PD1 cell membrane vesicles to GelMA hydrogel is 1:50 to 1:200 according to the above data). Further, the bio-ink is prepared by mixing PD1 cell membrane vesicles with GelMA hydrogel.
The GelMA hydrogel porous scaffold has good biocompatibility and provides a growing microenvironment for a target vaccine. The pores of the scaffold promote substance exchange, further improving survival of the vaccine of interest. PD1 cell membrane vesicles act as immunomodulators, stimulating maturation of DCs, binding PDL1 on the DC surface, promoting DC activation of T cells. In addition, the PD1 cell membrane vesicles can bind PDL1 on the surface of tumor cells, and further help the vaccine to play an anti-tumor role.
According to an exemplary embodiment of the present invention, a 3D printing vaccine is provided. The 3D printing vaccine is prepared by taking PD1 cell membrane vesicles and GelMA hydrogel as biological ink to carry the target cell vaccine through 3D printing.
By applying the technical scheme of the invention, methacrylic anhydride modified gelatin (GelMA), dendritic cells and target vaccine (for example, the target vaccine is one or more of dendritic cell vaccine, T cell vaccine, NK cell vaccine or macrophage vaccine, preferably PD1 cell membrane vesicle extracted by gene editing cells) are printed into 3D printing DC vaccine by a 3D printing technology. The GelMA hydrogel porous scaffold has good biocompatibility and provides a growing microenvironment for a target vaccine. The pores of the scaffold promote substance exchange, further improving survival of the vaccine of interest. Preferably, when the vaccine of interest is a PD1 cell membrane vesicle, the PD1 cell membrane vesicle acts as an immunomodulator, stimulating maturation of DCs, binding PDL1 from the surface of DCs, promoting DC activation of T cells. In addition, the PD1 cell membrane vesicles can bind PDL1 on the surface of tumor cells, and further help the vaccine to play an anti-tumor role. The mechanisms work together to provide a simple, safe and robust strategy for anti-tumor immunity after tumor operation.
In a preferred embodiment of the invention, PD1 cell membrane vesicles, gelMA hydrogels and DC vaccine of interest (100 ul GelMA hydrogels entrap 100 ug PD1 cell membrane vesicles and 2X 10) 5 Individual DCs). Typically, gelMA hydrogels contain photoinitiators and 3D printing is photo-cured 3D printing. In another preferred embodiment of the invention, the mass ratio of the bio-ink to the vaccine of interest is 80:1 to 30:1 (one standard mouse (6-8 weeks, 18-22 mg), the amount of the bio-ink is 15-40mg according to the tumor size of the mouse, the number of the used dendritic cells is 20 ten thousand, about 0.5mg, and the mass ratio of the bio-ink to the target vaccine is 80:1-30:1 according to the data.
In a typical embodiment of the present invention, as shown in fig. 1, a bio-ink DC vaccine was developed by 3D printing a porous scaffold (called DCs/PD1 cell membrane vesicle @ scaffold) composed of DCs, PD1 cell membrane vesicles and GelMA (see a in fig. 1). Porous 3D-printed GelMA scaffolds can significantly improve survival of DCs and release DCs continuously for migration to lymph nodes to extend T cell activation time. PD1 cell membrane vesicles can promote maturation of DCs, bind to DCs and PD-L1 that is highly expressed on the surface of tumor cells, thereby better activating T cells to kill tumors (see B in fig. 1). In addition, based on 3D printing technology, personalized vaccines can be filled at specific sites after tumor resection. These mechanisms work together to promote anti-tumor effects after vaccine surgery.
Specifically, fig. 1 a illustrates the preparation of a 3D printed DC vaccine; FIG. 1B shows that incubation of DCs with PD1 cell membrane vesicles, which bind PD-L1 signals on the surface of DCs and stimulate maturation. Subsequently, the released mature DCs activate CD8 in lymph nodes + T cells. Finally, released PD1 cell membrane vesicles from scaffolds target tumors, with activated CD8 + T cells synergistically inhibit tumors.
According to an exemplary embodiment of the present invention, a method for preparing the 3D printing vaccine is provided. The preparation method comprises the following steps: preparing PD1 cell membrane vesicles, gelMA hydrogel and a target vaccine; mixing PD1 cell membrane vesicles, gelMA hydrogel and a target vaccine to obtain a mixed solution; and preparing the mixed solution through 3D printing to obtain the 3D printing vaccine. The preparation method of the 3D printing vaccine is simple, and the preparation process is safe and easy to implement.
Preferably, preparing the GelMA hydrogel comprises: dissolving gelatin in PBS solution under continuous stirring to form Gel solution; mixing methacrylic anhydride with PBS solution to prepare MA solution; dripping the MA solution into the Gel solution, and reacting in the dark, wherein the preferable reaction temperature is 50-55 ℃, the reaction time is 3-3.5 hours, and the pH of a reaction system is 8.0-9.0; adding deionized water to terminate the reaction, dialyzing, and lyophilizing to obtain GelMA dry powder; adding GelMA dry powder into PBS solution, oscillating, and adding photoinitiator after ultrasonic treatment to obtain GelMA hydrogel; typically, the photoinitiator is the photoinitiator LAP.
Live cell-based vaccines may be used in the present invention, preferably the vaccine of interest is one or more of a dendritic cell vaccine, a T cell vaccine, an NK cell vaccine or a macrophage vaccine; preferably, the dendritic cell vaccine is obtained by extraction of bone marrow primary cell culture.
Preferably, preparing the PD1 cell membrane vesicle comprises: suspending 4T1 cells of gene editing PD1 in PBS solution (e.g., PBS solution, sieimer's fly), disrupting by a homogenizer, centrifuging, and collecting cell membrane vesicles; washing the cell membrane vesicles with PBS mixed with a protease inhibitor; after the cell membrane vesicles were subjected to ultrasonic treatment, PD1 cell membrane vesicles were obtained by stepwise extrusion through 400 and 200 nm-pore polycarbonate membranes on a micro extruder.
According to an exemplary embodiment of the present invention, there is provided a use of a 3D printing vaccine for the preparation of a medicament for inhibiting postoperative tumor recurrence. Wherein the tumor comprises solid tumors such as breast cancer, osteosarcoma, melanoma, liver cancer, etc.
In a preferred embodiment of the invention, the 3D printing vaccine comprises:
preparation of GelMA hydrogel: 10.0 The Gel (Gel) was dissolved in 100 ml of PBS solution at 55deg.C under continuous stirring to form Gel solution. In addition, 8.5. 8.5 ml Methacrylic Anhydride (MA) was mixed with 20 ml PBS to prepare a MA solution. While stirring, the MA solution was added dropwise to the Gel solution, and the reaction between MA and gelatin was carried out at 55℃for 3 hours in the dark. The pH of the reaction solution was maintained at about 8.5 throughout the process. Deionized water (ddH) was distilled by addition of 600 ml 2 O) stopping the reaction. Subsequently, the diluted reaction solution was subjected to ddH 2 Dialysis in O for three days. After dialysis, the solution was freeze-dried to obtain GelMA. 150 mg GelMA dry powder was added to 1 ml PBS, vortexed, and then sonicated for 10 minutes. And then adding 5 mg photoinitiator LAP, uniformly mixing and standing for 1 hour to obtain the GelMA hydrogel.
Preparation of DCs: will be 1X 10 6 The bone marrow cells were added to a 10 cm bacterial culture dish in 10ml complete medium (RPMI 1640 containing glutamine, penicillin, streptomycin all from Invitrogen)]) 10% heat-inactivated fetal bovine serum (Invitrogen) and GM-CSF (20 ng/ml, peprotech). On day 3, 10ml of the above medium was added to each dish. On days 6 and 8, half of the medium was removed and replaced with 10ml of the above medium. On day 10, non-adherent cells and easily adherent cells were collected from the culture supernatant, gently washed with PBS and mixed for flow analysis, indicating successful DCs extraction.
Preparation of PD1 cell membrane vesicles: the 4T1 cells of gene editing PD1 were first suspended in hypotonic solution and then disrupted by Dounce homogenizer. The solution was centrifuged at 3000 g for 15 min at 4 ℃. The supernatant was collected and centrifuged at 10000 g for 30 minutes at 4 ℃. The supernatant was then centrifuged again at 100000 g for 30 minutes. The particles were collected, washed three times with protease inhibitors in PBS, sonicated for 5 minutes, and then extruded through 400nm and 200nm nanopore polycarbonate membranes using a mini-extruder (Avanti Polar Lipids). NTA showed PD1 cell membrane vesicle particle size 120nm and transmission electron microscopy showed cupped phospholipid structure. Western blot analysis and flow analysis showed that PD1 cell membrane vesicles contained PD1, further demonstrating that PD1 cell membrane vesicles were successfully prepared.
Finally, gelMA, DCs and PD1 cell membrane vesicles were mixed and printed out by photocuring 3D printing to produce a multilayer, porous 3D DC vaccine. Scanning electron microscopy revealed that DCs were encapsulated by the scaffold.
The advantageous effects of the present invention will be further described below with reference to examples.
Examples
1. Experimental materials
1. Animals
Mouse BALB/c strain (female, 6-10 weeks old) was obtained from gembarmatech co. Animal experiments have been approved by the ethical review Committee of the Shenzhen Bay laboratory and are conducted in accordance with the guidelines of the animal experiment protection.
2. Cells
The mouse breast cancer 4T1 cell line was obtained from the American Type Culture Collection (ATCC). Cells at 5% CO 2 The culture was carried out under the condition that 10% Fetal Bovine Serum (FBS), 100U/mL penicillin and 100. Mu.g/mL streptomycin were added to Dulbecco's modified chick pea culture medium (DMEM) (Invitrogen). Bone marrow derived dendritic cells (BMDCs) were prepared as follows: 1X 10 6 Bone marrow cells were added to a 10cm dish in 10ml complete medium (RPMI 1640 containing glutamine, penicillin and streptomycin all from Invitrogen]) 10% heat-inactivated fetal bovine serum (Invitrogen) and GM-CSF (20 ng/ml, peprotech). On day 3, 10ml of the above medium was added to each dish. On days 6 and 8, half of the medium was removed and replaced with 10ml of the medium described above. On day 10, the non-adherent cells and loosely adherent cells collected gently in the culture supernatant were washed with PBS and mixed for experiments.
2. Experimental method
1. Preparation and characterization of GelMA hydrogels
10.0 g of gelatin (Gel) was dissolved in 100 ml of PBS solution at 55℃with continuous stirring to obtain a gelatin solution. 8.5 ml of Methacrylic Anhydride (MA) was mixed with 20 ml of PBS, respectively, to prepare MA solutions. While stirring, dripping MA solution into Gel solution, between MA and gelatinThe reaction was carried out in the dark at 55℃for three hours. The pH of the reaction solution was maintained at about 8.5 throughout the process. By adding 600 ml distilled deionized water (ddH 2 O) stopping the reaction. Subsequently, the diluted reaction solution was dialyzed for three days. After dialysis, the solution was freeze-dried to obtain GelMA. Using 1 H NMR (Bruker 400 MHz Advance, switzerland) at D 2 GelMA was checked in O (heavy water) to confirm the methacryloyl modification. The microstructure of the lyophilized GelMA hydrogel was examined using a Scanning Electron Microscope (SEM) (FEI Quanta 200, FEI Company, czech Republic). Rheological properties were analyzed using a TA-DHR-2 rheometer (TA Instruments, united States).
2. Construction and characterization of genetically engineered cells
To construct 4T1 cells that overexpress PD1, mouse PD-1 was first cloned into pCMV6 mammalian expression vector, bearing DsRed tag at the C-terminus (Sino Biological). The PD-1 plasmid was confirmed by DNA sequencing and 4T1 cells were transfected using liposome 2000 (Invitrogen). To establish stable cells, 4T1 cells were transfected with pCMV6-OFR-PD-1 and screened using homomycin B. To confirm the presence of PD1 on the cells, the parent cell 4T1 cells with PD1 were cultured on glass dishes. After overnight incubation, cells were incubated with 1 μg/mL of mouse PD 1-human IgG fusion protein (ab 280864, abcam) for 30 min at 25 ℃ and then with Alexa Fluor 647 labeled rabbit anti-mouse IgG H & L secondary antibody (ab 150075, abcam) for 20 min at 4 ℃. Cells were stained with 4', 6-diamino-2-phenylindole (DAPI) and observed under a confocal microscope system. Flow cytometry was also used to verify PD1 variants on engineered cells. Dead cells were excluded using Fixable Viability Stain 510 (BD Biosciences). The PD 1-carrying plasmid contained mCherry, and primary and engineered cells were analyzed using mCherry channel of CytoFLEX flow cytometer (Beckman Coulter) and data were analyzed using FlowJo software (Tree Star).
3. Preparation and characterization of cell membrane vesicles
The collected 4T1 tumor cells were subjected to sonication for 2 minutes, followed by RNase treatment in solution. Subsequently, centrifugation was performed for 30 minutes at 4000 g, and the supernatant was extracted and sonicated again for 5 minutes. Finally, cell membrane vesicles were obtained using mini-extruders (Avanti polar lipids) of polyethylene terephthalate porous membranes with dimensions of 400, 200 and 100 nanometers. The size and electrostatic potential of the cell membrane vesicles were measured using dynamic light scattering, and the morphology of the cell membrane vesicles was observed using a Transmission Electron Microscope (TEM). The concentration of cell membrane vesicles was determined using BCA protein assay kit (KeyGEN BioTECH).
4. DCs/PD1 vesicle/GelMA bio-ink 3D bio-printing
The 3D bioprinting test was performed using a Digital Light Processing (DLP) printer (nanoArch S240, BMF Material Technology Inc, china) and 405 nm laser. The Solidworks software was used to create a 3D CAD model of the design shape, PD1 vesicles and GelMA hydrogels were used as bio-ink-loaded DCs (in this example, the mass ratio of PD1 cell membrane vesicles to GelMA hydrogels was 1:150, and the mass ratio of bio-ink to vaccine of interest was 1:50). After bioprinting, the printed samples were examined using a digital microscope (Dino-Lite, anMo Electronics Corporation) and after 3 days of incubation, the samples were stained using the fluorochromes calcein-AM and PI and examined using a Confocal Laser Scanning Microscope (CLSM). In addition, cell attachment and morphology in the bio-ink scaffolds after lyophilization were observed using a Scanning Electron Microscope (SEM).
5. Western blot
The sample was denatured and loaded into 10-12% polyacrylamide gel. The protein samples in the gel were then transferred to polyvinylidene fluoride (PVDF) membranes and blocked with milk at 25 ℃ for 1 hour. The membranes were incubated overnight at 4℃with a primary antibody directed against PD1 (ab 214421; dilution 1:1000; abcam), na+/K+ -ATPase (ab 76020; dilution 1:1000; abcam) and GAPDH (14C 10; dilution 1:1000;Cell Signaling Technology). Subsequently, PVDF membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) and then detected using the West Pico PLUS chemiluminescent substrate kit (Thermo Fisher Scientific).
6. Flow cytometry
To verify the change in immune cells, we stained cells with fluorescent antibodies, the procedure was as follows: cells were stained with the appropriate antibody-fluorochrome conjugate in cold Phosphate Buffered Saline (PBS) containing 2% Fetal Calf Serum (FCS). To exclude dead cells, cells were stained using 1 μg/ml fixable active stain 510 (BD Biosciences). Cells were then blocked with rat anti-mouse CD16/CD32 (clone 2.4G2) at 4 ℃ for 5 min, then stained with the following fluorescently labeled antibodies: anti-CD 8 (clone 53-6.7), anti-CD 4 (clone GK 1.5), anti-CD 45 (clone 30-F11), anti-CD 11C (clone HL 3), anti-CD 80 (clone 16-10A 1), anti-CD 86 (clone GL 1), anti-CD 40 (clone 3/23), anti-PDL 1 (clone 10 F.9G2), anti-Granzyme B (clone QA16A 02), anti-MHC-II I-A/I-E (clone M5/114.15.2), anti-CD 3 (clone 145-2C 11). For intracellular staining, cells were fixed and permeabilized using the CytoFix/Cytoperm kit (BD). Multiparameter analysis was performed on a CytoFLEX flow cytometer (Beckman Coulter) and data analysis was performed using FlowJo software (Tree Star). To verify mouse tumors various antibodies were used, including for intracellular staining, cells were fixed and permeabilized using the CytoFix/Cytoperm kit (BD).
7. Immunohistochemistry
Tumor sections were de-waxed, rehydrated, and antigen repaired using sodium citrate, then endogenous peroxidase activity was blocked. Sections were incubated with CD8 antibody (1:1000 dilution), CD86 antibody (1:1000 dilution, both from Cell Signaling Technology), and then stained with ABC reagent (Vector Laboratories). The sections were scanned using a pannarac MIDI scanner (3 DHISTECH).
8. In vitro matured bone marrow derived dendritic cells (BMDCs)
On day 10 of BMDCs culture, BMDCs were collected and grown at 1X 10 6 The individual cells/ml concentration was re-seeded into 12-well plates. Each group was treated with PBS, 0.1. Mu.g/ml LPS, 50. Mu.g/ml 4T1 vesicles or 50. Mu.g/ml PD1 cell membrane vesicles, respectively. After 18 hours, BMDCs were collected for flow cytometry analysis.
9. In vivo sustained release and targeting
By mixing 1X 10 6 The individual BMDCs and 200 μg PD1 cell membrane vesicles were stained and mixed with GelMA hydrogel, 3D printed into scaffolds, and then transplanted subcutaneously into mice. Release of BMDCs and PD1 cell membrane vesicles in mice was assessed by in vivo imaging using an IVIS imaging system (Perkin Elmer). In addition, to study the migration of BMDCs to lymph nodes and the targeting effect of PD1 cell membrane vesicles to tumors, lymph nodes and tumors were collected on days 4 and 7 for flow cytometry and immunofluorescence analysis.
10. In vivo tumor model and treatment
For tumor prevention model, 1×10 will be 6 The BMDCs were mixed with 400 μg PD1 cell membrane vesicles and GelMA hydrogel, then 3D printed into scaffolds and transplanted subcutaneously into the left flank of mice. On day 7 after stent implantation, 6×10 injections were subcutaneously injected into the right flank of mice 5 4T1 tumor cells. On day 14 after cell injection, tumor tissue was surgically resected for downstream analysis. For the individualized postoperative recurrence prevention model, 6×10 was subcutaneously injected into the left flank of mice 5 4T1 tumor cells. On day 14 after tumor cell injection, tumors were resected. According to the size and shape of the tumor (which is similar to the size and shape of the tumor), designing a stent model, performing 3D printing, and then transplanting to the tumor excision part. On day 7 after stent implantation, 6×10 injections were subcutaneously injected into the right flank of mice 5 4T1 tumor cells.
11. Cytokine detection
On day 14 after cell injection, tumor tissue was resected for downstream analysis. On day 7 post-inoculation, BALB/c mouse serum was collected and IL-6, CXCL10 and TNF-a levels were measured using the corresponding ELISA kit (all from Invitrogen) according to the manufacturer's instructions.
12. Biocompatibility assessment
To investigate the in vivo toxicity of DCs/PD-1-cell membrane vesicles/GelMA scaffolds, PBS was injected subcutaneously or stented into BALB/c mice. On day 30 after transplantation, mice were euthanized, and blood samples were collected for whole blood examination and blood biochemical analysis using a blood biochemical analyzer (7080, hitachi, japan). The major organs were routinely processed into sections as described above and subjected to hematoxylin and eosin (H & E) staining for histological examination.
13. Statistical analysis
All results are presented as mean ± standard deviation (s.d.). A unpaired two-tailed t-test was used for both sets of comparisons, and a common one-way (or two-way) analysis of variance (ANOVA) plus Tukey's test (or Dunnett's test) was used for the sets of comparisons. The survival benefit of mice was determined using a log rank (Mantel-Cox) test. All statistical analyses were performed using Prism 9.0 software (GraphPad).
3. Experimental results
GelMA has good biocompatibility, low cost and photocrosslinking structure, making it an attractive hydrogel suitable for drug or cell loading. GelMA was converted from sol to gel under 405 nm flash light, demonstrating successful synthesis of GelMA hydrogels (see FIG. 2A). The photo-cured hydrogel was then freeze-dried and a Scanning Electron Microscope (SEM) was used to observe its morphology and structure. According to the results of the study, the freeze-dried hydrogels had a rich pore structure, including macropores and micropores, which made it easier for nutrients to pass through and for metabolic waste products to exit the cells (see B in fig. 2). In addition, in the case of the optical fiber, 1 The new peak "c=c" in the H NMR spectrum indicates that MA was successfully conjugated to Gel (see C in fig. 2). Dynamic time-scanning rheometry analysis showed that at 30 mW/cm exposure 2 After the 405 nm laser shot dose of (c), gelMA may be photo-cured in less than 50 seconds (see D in fig. 2). In addition, the viscosity of GelMA varies little over time (see E in fig. 2).
The inventors have further explored the 3D printing properties of bio-inks consisting of physically mixed GelMA hydrogels and bone marrow derived dendritic cells (BMDCs). Whereas GelMA is photocurable, this embodiment uses a projection micro stereolithography (pμsl) printer to fabricate hydrogel products. Based on the CAD design model, the DC bio-ink can be printed into various high-precision brackets. For 3D porous scaffolds, the bio-ink was printed layer by layer according to the design model (see F in fig. 2). Finally, the printed 3D porous scaffold exhibits a high fidelity multi-layered structure (see G in fig. 2). In addition, the 3D freeze-dried scaffold still maintains the characteristics of a rich pore structure with different pore sizes (see H, I in fig. 2). Since 3D printing provides the advantages of strong reproducibility and rapid production speed, the present solution enables one-time mass production of scaffolds for subsequent experiments (see J in fig. 2). In addition, these printed structures showed good morphology and cell viability after three days of culture in cell culture medium (see K in fig. 2). These findings indicate that GelMA hydrogels have great potential in 3D bioprinting. More importantly, the morphology of the DCs in the bio-ink scaffolds was observed by scanning electron microscopy. Experimental results indicate that DCs are tightly attached to scaffolds, indicating that scaffolds have good biocompatibility and promote cell survival (see K in fig. 2).
Specifically, fig. 2 a shows the sol-gel transition of GelMA under 405 nm nm flash irradiation. FIG. 2B shows a scanning electron microscope image of a GelMA hydrogel; scale, 10 mu m. FIG. 2C shows a GelMA hydrogel 1 H nuclear magnetic resonance spectroscopy. FIG. 2D shows a GelMA hydrogel at 30 mW cm 2 Dynamic time-scanning rheology under 405 nm laser irradiation. The viscosity change of the GelMA hydrogels is shown in fig. 2E. FIG. 2F shows a stent model of a software design; scale bar, 2 mm. A G photo-cured printed 3D porous scaffold is shown in fig. 2; scale bar, 2 mm. An image of a 3D freeze-dried scaffold is shown at H in fig. 2 and a scanning electron microscope image thereof is shown at I in fig. 2; h, scale bar, 1 mm in fig. 2; i in fig. 2, scale, 50 μm. The mass production and stability of the 3D porous scaffold is shown at J in fig. 2; scale bar, 4 mm. K in FIG. 2 shows 3D bioprinting DC scaffolds of different complex designs and their scanning electron microscope images; scale, 20 mu m.
To verify the advantages of the porous scaffold, mature BMDCs were mixed with GelMA hydrogels and printed into a 3D porous scaffold (see a in fig. 3). The number of surviving DCs on the 3D scaffold was higher on day 7 compared to the hydrogel (see B in fig. 3). Furthermore, as shown in fig. 3B, BMDCs began to migrate toward the edges of the 3D scaffold, which also suggests that the 3D porous scaffold maintained higher DC viability than conventional hydrogels.
To observe the homing ability of DCs in 3D scaffolds, hydrogels, DCs @ hydrogels, and DCs @3D scaffolds were transplanted subcutaneously into mice. On day 7, lymph nodes were surgically excised for flow analysis. It is exciting that DCs@3D scaffold mice have CD11c in the lymph nodes + CD86 + The proportion of DCs increases significantly (see C in figure 3). Consistently, CD11c in lymph nodes of DCs@3D stent mice compared to DCs@hydrogel mice + CD80 + DCs and MHC II + The proportion of DCs increased significantly (see D in figure 3). To determine if mature DCs in lymph nodes were derived from DCs in 3D scaffolds, DCs were stained with 1, 1-behenyl-3, 3-tetramethylindole tricarboxylic acid iodide (DID). The results showed that DID in DCs@3D scaffold mice + The proportion of DCs was significantly higher on both day 4 and day 7 than the DCs @ hydrogel mice (see E, F in figure 3). Furthermore, as shown in FIG. 3G, DID in lymph nodes of DCs@3D stent mice + The percentage of DCs was comparable to day 7 and day 4, suggesting that 3D scaffolds may extend the lymph node homing capacity of DCs. Immunofluorescence also demonstrated DID in lymph nodes of DCs@3D stent mice + The proportion of DCs increases significantly (see H in figure 3). Furthermore, DID + CD86, CD40 and MHC II expression of DCs was significantly higher than DID - DCs (see I-K in FIG. 3). This suggests that exogenous DCs homing to the lymph nodes remain highly mature. More importantly, maturity does not change with group type and time (see L in fig. 3). In addition, to observe the antitumor effect of DCs in 3D scaffolds, healthy mice were treated with hydrogel, dcs@hydrogel and dcs@3d scaffolds, respectively, followed by injection of 4T1 tumor cells. The results show that the dcs@3d scaffolds significantly inhibited tumor growth compared to dcs@hydrogels (see M in fig. 3). In addition, dcs@3d scaffolds prolonged survival of mice (see N in fig. 3). These results indicate that the 3D porous scaffold significantly improves the survival rate of DCs, promotes migration of more DCs to lymph nodes, and exerts an antitumor effect.
Specifically, FIG. 3A shows an illustration of the experimental designIntent. FIG. 3B shows a live and dead cell staining analysis of DCs in 3D scaffolds and hydrogels; scale bar, 800 [ mu ] m. FIG. 3C shows CD86 in draining lymph nodes + Flow cytometry analysis of DCs (from left to right, representing mice treated with blank Hydrogel (hydro gel), DCs in non-porous Hydrogel (dcs@hydro gel) and DCs in 3D porous scaffold (dcs@3D scaffold), respectively). FIG. 3D shows CD80 in draining lymph nodes + DCs and MHC II + Ratio of DCs. FIG. 3E shows DID in day 4 DCs + In FIG. 3, F shows DID in day 7 DCs + Is shown in FIG. 3, G shows DID + Percent DCs. FIG. 3H shows DID in draining lymph nodes + Immunofluorescence images of DCs; scale, 20 mu m. In FIG. 3I shows DID + DCs and DIDs - CD86 in DCs + Flow cytometry analysis and percentages of (a). J in FIG. 3 shows DID + DCs and DIDs - CD40 in DCs + In FIG. 3, K shows the DID + DCs and DIDs - MHC II in DCs + Is a ratio of (2). FIG. 3L shows DID on days 4 and 7, G2 and G3 + CD86 in DCs + And MHC II + Is a percentage of (c). Tumor growth curves for mice treated with hydro el, dcs@hydro el and dcs@3d scaffold are shown in fig. 3 at M. The survival curves of mice after different treatments are shown at N in fig. 3. All data are expressed as mean ± standard deviation (n=4). Statistical significance analysis was performed by either a common one-way anova and Dunnett's test (C, D) or a unpaired two-sample t test (G, I, J, K, L) or a two-way anova and Tukey's test (M) or a log-rank (Mantel-Cox) test (N). ns represents no significance; * P (P)< 0.05；**P < 0.01；***P < 0.001；****P < 0.0001。
To improve the antitumor effect of DCs, the inventors loaded PD1 cell membrane vesicles into scaffolds. Based on its excellent biocompatibility, intrinsic stability, rapid production and nano-size, PD1 cell membrane vesicles can be used to activate DCs and synergistically improve their antitumor effect. In this example, the use of lentiviral vectors to transfect PD1 mutants into 4T1 tumor cells, immunofluorescence and flow cytometry analysis showed successful expression of PD1 on 4T1 cells (see A, B in FIG. 4). To obtain cell membrane fractions, hypotonic lysis, sonication, and gradient centrifugation were performed to remove the cell contents. The vesicles were then collected by passing 400 nm, 200 nm and 100 nm nanoporous membranes sequentially through the cell membrane using a microfluidic extruder. Nanoparticle Tracking Analysis (NTA) showed that the size of the vesicles was about 120 nm (see C in fig. 4). Transmission electron microscopy confirmed that the vesicles had a golgi cup-shaped membrane vesicle structure and expressed PD-1, successfully producing PD1 cell membrane vesicles (see D, E in fig. 4).
BMDCs were treated with Phosphate Buffered Saline (PBS), lipopolysaccharide (LPS), 4T1 vesicles and PD1 cell membrane vesicles, respectively, and analyzed by flow cytometry after 18 hours. After incubation with vesicles, CD80 and CD86 expression of BMDCs was up-regulated (see G, H in fig. 4). Furthermore, vesicles significantly up-regulated the expression of major histocompatibility complex II (MHC II) and CD40 compared to PBS-cultured BMDCs (see I, K in fig. 4). Flow cytometry showed that vesicles were effective in stimulating DCs maturation and antigen presentation. PD-L1 expression on the surface of DCs is a major cause of DC innate immunity suppression, and the expression level increases with the activation degree. As shown by L in fig. 4, PD-L1 expression of LPS and 4T1 vesicle-treated BMDCs was significantly up-regulated. Meanwhile, PD-L1 expression of DCs cultured with PD1 cell membrane vesicles was down-regulated compared to LPS and 4T1 vesicles (see L in fig. 4). To further demonstrate that PD1 cell membrane vesicles can block PD-L1 on BMDC surfaces, DID-stained PD1 cell membrane vesicle 4T1 vesicles were co-cultured with DCs for 18 hours. Flow cytometry showed significant up-regulation of DID expression of DCs co-cultured with DID-stained PD1 cell membrane vesicles. When the PD-L1 antibody was added, the expression of DID was decreased (M, N in FIG. 4).
Specifically, FIG. 4A shows immunofluorescence images of primordial and engineered cells; scale, 10 mu m. Typical flow cytometry analysis results for PD1 in both primordial and engineered cells are shown in fig. 4B. Fig. 4C shows a graph of nanoparticle trace analysis results and fig. 4D shows a transmission electron microscope image of PD1 cell membrane vesicles; scale bar, 100 nm. FIG. 4E shows 4T1 cells, 4Na in T1-PD1 cells and 4T1-PD1 vesicles (PD 1 cell membrane vesicles) + /K + Typical Western blot analysis results plots for ATPase and PD 1. FIG. 4F shows a schematic representation of the co-culture of BMDCs with PBS, LPS, 4T1 vesicles and PD1 cell membrane vesicles. FIG. 4G shows a typical flow cytometry analysis of DC maturation in BMDCs, and FIG. 4H shows a quantitative analysis of percentages. FIG. 4I shows MHC II in DCs + Is shown in figure 4, J shows a proportional quantitative analysis. FIG. 4 shows CD40 in DCs for K and L, respectively + Typical percentages of pdl1+. M and N in FIG. 4 show the DID in DCs, respectively + Is described, and MFI. All data are expressed as mean ± standard deviation (n=3). Statistical significance analysis was performed by common one-way anova and Dunnett's test (H, J, K, L, N). * P < 0.01；***P < 0.001；****P < 0.0001。
To verify the antitumor effect of the vaccine, healthy BALB/c mice were subcutaneously implanted with DCs/PD1 cell membrane vesicle @ scaffold, DCs/4T1 vesicle @ scaffold, DCs @ scaffold or scaffold alone 7 days prior to 4T1 tumor cell inoculation, and other mice were subcutaneously injected with PBS. On day 0, 6×10 mice were injected on the dorsal side 5 The tumors were removed from 4T1 cells for downstream analysis (see a in fig. 5). As shown in fig. 5B, there was no significant difference in tumor growth between PBS and stent-only groups; mice treated with scaffolds alone were not effective in inhibiting tumor growth. In contrast, mice treated with dcs @ scaffolds showed inhibition of tumor growth. The DCs/4T1 vesicle @ stent group did not show significant tumor suppression compared to the DCs @ stent group. On the other hand, DCs/PD1 cell membrane vesicle @ scaffolds were able to significantly and more significantly inhibit tumor growth than DCs @ scaffolds (see C, D in fig. 5). This suggests that PD1 cell membrane vesicles may stimulate and synergistically enhance DCs to inhibit tumor growth; mice receiving DCs/PD1 cell membrane vesicle @ scaffold treatment experienced significant tumor weight inhibition (see E in fig. 5). During the treatment period, the body weight of the mice in the treatment group did not significantly decrease (see F in fig. 5). In addition, CD3 in DCs/PD1 cell membrane vesicles @ scaffold tumors + T cellMiddle CD8 + The proportion of T cells was about 5-fold higher than in the other groups, e.g. the PBS group (see G, H in fig. 5). In contrast, CD4 in DCs/PD1 cell membrane vesicles @ scaffold tumors + The proportion of T cells was lower than in the PBS group (see I in fig. 5). Granzyme B + CD8 + The proportion of T cells in DCs/PD1 cell membrane vesicles @ scaffold tumors was about 3-fold that of PBS group (see J in fig. 5). Spleen CD8 + T cells, granzyme B + CD8 + The ratio of T cells to mature DCs was higher in the DCs/PD1 cell membrane vesicle @ scaffold group than in the other groups (see K-M in fig. 5). The results indicate that the anti-tumor effect is systemic in mice and is not limited to tumor sites. Cytokine testing showed that the expression of IL-6, CXCL10 and TNF- α was significantly increased in serum of DCs/PD1 cell membrane vesicles @ scaffold compared to PBS group (see N in fig. 5), revealing that PD1 cell membrane vesicles exert potent anti-tumor effects in synergy with DCs.
Specifically, fig. 5 a shows a schematic diagram of the experimental design. In fig. 5B shows tumor growth curves of mice treated with PBS, scaffolds (Sca), dcs@3d scaffolds (dcs@scaffolds), DCs/4T1 vesicles@3d scaffolds (DCs/4T 1 vesicles@scaffolds) and DCs/PD1 cell membrane vesicles@3d scaffolds (DCs/PD 1 cell membrane vesicles@scaffolds). The average tumor growth curve is shown at C in fig. 5. The physical size of the tumor is shown at D in fig. 5; scale bar, 2 cm. The tumor weights for each group are shown in fig. 5E. The average body weight curve is shown at F in fig. 5. In FIG. 5, G and F show CD3 in tumors, respectively + CD8 in T cells + Typical flow cytometry analysis and quantitative analysis of the proportion of T cells. FIG. 5I shows CD3 in tumors + CD4 in T cells + T cell ratio. FIG. 5J shows CD8 in tumor + Granzyme B in T cells + Quantitative analysis of the percentage of (a). K, L and M in FIG. 5 show CD8 in spleen, respectively + Ratio of Granzyme B + Quantitative analysis of the ratio and DC maturity of (C). The concentrations of IL-6, CXCL-10 and TNF-alpha in the serum of mice after different treatments are shown at N in FIG. 5. All data are expressed as mean ± standard deviation (n=5). By two-factor analysis of variance and Tukey's test (C) or by a common one-factor analysis of varianceAnd Dunnett's test (E, H-N) for statistical significance analysis. ns represents no significance; * P (P)< 0.05；**P < 0.01；***P < 0.001；****P < 0.0001。
To better expand the application of 3D printing, a 3D stent is used in this embodiment to fill the post-operative tumor surgical incision. In this example, DCs/PD1 cell membrane vesicle @ scaffolds suitable for surgical defects were printed, simulating clinical post-operative breast cancer prosthetic filling. The flow chart is shown in fig. 6 a. First, a tumor implanted in the back of the mouse was excised and scanned using software to design a 3D model. BMDCs were then printed as 3D scaffolds using PD1 cell membrane vesicles/GelMA bio-ink and implanted into the surgical defect site. Finally, 4T1 tumor cells were subcutaneously injected on the contralateral back of mice (B in fig. 6). As shown in fig. 6C, these models were designed and printed into stents according to the shape of the tumor. After stent implantation, mice treated with DCs/PD1 cell membrane vesicles @ stent were able to significantly and more significantly inhibit tumor growth (see D, E in fig. 6) and had a longer survival (see F in fig. 6). During the treatment period, the body weight of the mice in the treatment group did not significantly decrease (see G in fig. 6). CD86 in DCs/PD1 cell membrane vesicle @ scaffold + DC and CD80 + The proportion of DC increases significantly (see H, I in fig. 6). Furthermore, tumor apoptosis was significantly increased in mice treated with DCs/PD1 cell membrane vesicles @ scaffolds, CD8 in tumors + T cell expression was also higher (see J, K in fig. 6). These results indicate that DCs/PD1 cell membrane vesicles @ scaffolds can be used for both post-operative wound packing and inhibition of distant tumor growth.
Specifically, fig. 6 a shows a schematic diagram of a tumor model after surgery. A schematic of the experimental design is shown in fig. 6B. The personalized wound packing design process is shown at C in fig. 6. The tumor growth curves of mice after different treatments are shown in fig. 6D. The average tumor growth curve is shown in fig. 6E. The survival and body weight of mice after different treatments are shown in fig. 6 as F and G, respectively. FIG. 6H and I show CD86+ and CD80, respectively, in spleen + Quantitative analysis of the percentage in DCs. FIG. 6J shows immunofluorescence of tumor cell apoptosis after various treatmentsA light image; scale, 50 mu m. FIG. 6K shows CD8 in tumor + Immunofluorescence image of T cells; scale, 50 mu m. All data are expressed as mean ± standard deviation (n=5). Statistical significance analysis was performed by two-factor analysis of variance and Tukey's test (E in FIG. 6) or log-rank (Mantel-Cox) test (F in FIG. 6) or common one-factor analysis of variance and Dunnett's test (H, I). ns represents no significance; * P (P) < 0.05；**P < 0.01；***P < 0.001；****P < 0.0001。
From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the 3D printing DC vaccine is simple to prepare and safe. In animal models, 3D printed DC vaccines can fill post-operative defects and inhibit distant tumor growth, prolonging mouse survival. Mechanistically, porous scaffolds significantly improved long-term survival of DCs from 16% to 70% and promoted migration of DCs to lymph nodes, relative to traditional hydrogels. Meanwhile, the PD1 cell membrane vesicle is used as an immunomodulator of DCs, can stimulate the DCs to mature and bind PDL1 on the surface of the DCs, and effectively promotes the combination of DCs and T cells. In the mouse 4T1 tumor model, 3D-printed DC vaccine significantly inhibited tumor growth, intratumoral CD8 + The T cell fraction was 55.9%, significantly higher than 11.7% of the control group. In addition, the survival time of the mice is also prolonged obviously.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (16)
1. A bio-ink for a 3D printing vaccine, the bio-ink comprising PD1 cell membrane vesicles and GelMA hydrogel.
2. The bio-ink according to claim 1, wherein the mass ratio of the PD1 cell membrane vesicles to the GelMA hydrogel is 1: 50-1:200.
3. The bio-ink according to claim 2, wherein the mass ratio of the PD1 cell membrane vesicles to the GelMA hydrogel is 1: 130-1:150.
4. A 3D printing vaccine, wherein the 3D printing vaccine is prepared by 3D printing with the bio-ink according to any one of claims 1 to 3.
5. The 3D printed vaccine of claim 4, wherein the vaccine of interest is one or more of a dendritic cell vaccine, a T cell vaccine, an NK cell vaccine, or a macrophage vaccine.
6. The 3D printing vaccine of claim 5, wherein the mass ratio of the bio-ink to the vaccine of interest is 80: 1-30: 1.
7. the 3D printing vaccine of claim 5, wherein the GelMA hydrogel contains a photoinitiator, and the 3D printing is a photo-cured 3D printing.
8. A method for preparing a 3D printing vaccine, comprising the steps of:
preparing PD 1-extracellular vesicles, gelMA solution and target vaccine;
mixing the PD 1-extracellular vesicles, the GelMA solution and the target vaccine to obtain a mixed solution;
and preparing the 3D printing vaccine from the mixed solution through 3D printing.
9. The method of preparing according to claim 8, wherein preparing the GelMA hydrogel comprises:
dissolving gelatin in PBS solution under continuous stirring to form Gel solution;
mixing methacrylic anhydride with PBS solution to prepare MA solution;
dripping the MA solution into the Gel solution, and reacting in the dark;
adding deionized water to terminate the reaction, dialyzing, and lyophilizing to obtain GelMA dry powder;
and adding the GelMA dry powder into PBS solution, oscillating, and adding a photoinitiator after ultrasonic treatment to obtain the GelMA hydrogel.
10. The preparation method according to claim 9, wherein the MA solution is added dropwise to the Gel solution, wherein the reaction temperature is 50-55 ℃, the reaction time is 3-3.5 hours, and the pH of the reaction system is 8.0-9.0.
11. The method of claim 9, wherein the photoinitiator is a photoinitiator LAP.
12. The method of claim 8, wherein the vaccine of interest is one or more of a dendritic cell vaccine, a T cell vaccine, an NK cell vaccine, or a macrophage vaccine.
13. The method of claim 12, wherein the dendritic cell vaccine is obtained by extraction of bone marrow primary cell culture.
14. The method of preparing of claim 8, wherein preparing the PD1 cell membrane vesicle comprises:
suspending 4T1 cells of gene editing PD1 in PBS solution, destroying by a homogenizer, centrifuging, and collecting cell membrane vesicles;
washing the cell membrane vesicles with PBS cocktail protease inhibitor;
and (3) after ultrasonic treatment, gradually extruding the cell membrane vesicles on a micro extruder through a polycarbonate membrane with 400 and 200 nanometer holes to obtain the PD1 cell membrane vesicles.
15. The method of manufacturing according to claim 8, wherein the 3D printing is photo-curing 3D printing.
16. Use of the 3D printing vaccine of any of claims 4 to 7 in the manufacture of a medicament for inhibiting postoperative tumor recurrence.
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