CN116271002A - Immune coactivated recombinant bionic anti-tumor nano vaccine and preparation method and application thereof - Google Patents
Immune coactivated recombinant bionic anti-tumor nano vaccine and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an immune coactivated recombinant bionic anti-tumor nano vaccine, a preparation method and application thereof, wherein the nano vaccine consists of an immune activation functional hybrid peptide modified recombinant exosome carrier shell and a nano-form drug core, and the recombinant exosome is prepared by recombining ginsenoside and exosome. The preparation method provided by the invention has the advantages of simple and mild conditions, low cost, simplicity in operation, high endogenous property, focus part targeting property, high-efficiency drug load, strong-efficiency immunity activation, high biosafety and the like. The recombinant bionic anti-tumor nano vaccine can simultaneously realize direct tumor killing and coactivation of powerful innate immunity and adaptive immunity, and has considerable clinical application prospect in inhibiting growth and recurrence of various malignant tumors.
Description
Technical Field
The invention belongs to the technical field of nano preparations, and particularly relates to an immune coactivated recombinant bionic anti-tumor nano vaccine, and a preparation method and application thereof.
Background
Chemotherapy, radiation therapy, surgical treatment are conventional treatments for tumors, but their overall therapeutic effect is limited and resistance to drugs or treatments is easily developed. Malignant tumor has biological characteristics of strong invasiveness and easy recurrence, and tumor tissue has extremely complex immunosuppressive microenvironment, so that an individualized treatment scheme with stronger targeting, smaller toxic and side effects and capability of improving the immune microenvironment needs to be designed. With the rapid development of tumor genomics and immunology, immunotherapy has become the most potential tumor treatment. The current tumor immunotherapy approaches mainly include adoptive cell immunotherapy, immune checkpoint blockade, chimeric antigen receptor T cell therapies and tumor vaccines. The tumor vaccine can powerfully overcome the immunosuppression state in the tumor microenvironment, enhance immunogenicity, activate the immune system of a patient, and induce the organism to generate more powerful immune response so as to achieve the aim of controlling or eliminating tumors due to the fact that tumor related antigens, such as tumor cell lysate, tumor related proteins or antigen polypeptides, related genes for expressing tumor antigens and immune adjuvants are directly introduced. Currently, tumor vaccines have become a research hotspot in the field of tumor therapy.
Dendritic Cells (DCs) are known to be the most powerful professional antigen presenting cells and are the key cells to elicit an anti-tumor immune response. Since 2005, research and construction of DC vaccines have been carried out. Most tumor vaccines today focus on enhancing the degree of specific T cell activation by engineering the DCs with loaded antigens, adjuvants to induce powerful specific immunity. However, innate immunity may also play an important role in tumor immunotherapy. The natural immune system is the first line of defense of the body against pathogen invasion and infection, and is capable of recognizing pathogen-associated molecular patterns (pathogen associated molecular patterns, PAMPs) common to different pathogens, such as flagellin, lipopolysaccharide, and viral nucleic acids, etc., through a variety of different pattern recognition receptors (pattern recognition receptors, PRRs). PRRs in the body mainly include Toll-like receptors (Toll like receptor, TLR), RIG-I-like helicase Receptors (RLH), NOD-like receptors (NLR) and cGAS-STING. Second, innate immune cells such as natural killer cells (natural killer cells, NKs) can be activated and lyse tumor cells directly, or secrete cytokines and chemokines to inhibit proliferation of tumor cells. It follows that designing an innate + specific "immune co-activation" tumor vaccine is of great importance for inducing a strong immune response.
Although DCs serve as the most important, powerful professional antigen presenting cells with a key role in inducing immunity, in reality, DCs in the tumor microenvironment infiltrate less and are mostly in a non-functional state. Thus, the in vitro cultivation and amplification of autologous DCs carrying tumor antigens is an effective way to improve the anti-tumor immune response of the organism by generating a large amount of DC tumor vaccines. However, the DC cells have complex components, more contents and very limited loading capacity, are not easy to store, and severely restrict the application development and clinical transformation of the DC cells as carriers.
Exosomes (exosomes) are lipid bilayer membrane vesicles 40-160 nm in diameter, widely distributed in various body fluids, and secreted by almost all living cells including stem cells, immune cells, tumor cells, etc. Exos has the same topology as cells and is rich in proteins, lipids, nucleic acids and glycocomplexes. Exos contains a series of membrane-associated higher order oligomeric protein complexes, exhibiting pronounced molecular heterogeneity, produced by budding on plasma and endosomal membranes. Upon release, exos can be taken up by distant or nearby cells, thereby exerting intercellular communication functions. This intercellular vesicle transport pathway plays an important role in many aspects of human health and disease, including development, immunity, tissue homeostasis, cancer, and neurodegenerative diseases, among others. Exos has a robust phospholipid bilayer structure that protects its contents from long-term presence in the extracellular environment without degradation or dilution. The outer surface of DC-Derived Exosomes (DE) presents a plurality of DC-related cytoplasmic proteins, participate in a plurality of immune-related biological effects, and also have high-efficiency immune activation function: (1) The member of the DE membrane surface HSP70 protein family forms immunogenicity of DE together with the membrane surface HSP90 family, and participates in activation of tumor-associated immune cells; (2) The DE surface is rich in ICAM-1, MHC-I molecules, CD86, which can induce T cell activation and immune response. As a 'Cell-free' carrier, DE inherits the immune activation capability of parent DCs, has higher safety and stability, is more beneficial to drug transportation and can be stored for a long time without losing the immunotherapeutic activity; and the DE, serving as an inert vesicle, has resistance to the regulation of tumor-associated factors compared with DC, and can overcome tumor-mediated immunosuppression. However, the construction of the vaccine by using DE as a carrier still has the problems of short body circulation time, limited in-vivo stability and the like, and is unfavorable for the occurrence of long-acting immune activation of the vaccine.
Therefore, there is no report on how to use the exosomes derived from dendritic cells and to recombine them to construct an innate + specific "immune co-activated" tumor vaccine to induce potent immune development.
Disclosure of Invention
The invention aims to: aiming at the technical problems, the invention aims to provide an immune co-activated recombinant bionic anti-tumor nano vaccine, a preparation method and application thereof, wherein the bionic anti-tumor nano vaccine retains all physicochemical properties of exosomes derived from endogenous immune cells, contains a large amount of marked immune characteristic proteins derived from parent cells, and can be identified as a DC vaccine cell-free substitute for preliminary construction of a tumor vaccine system. Simultaneously, the antigen hybridized peptide is loaded on the surface of the exosome, so that the specific immune activation intensity is further enhanced; and ginsenoside is selected to recombine exosomes, so that the in vivo circulation time is further prolonged. Meanwhile, the hydrophilic space inside the carrier is fully utilized, and the innate immunity activating component is loaded inside. Thus solving the problems of high cost, difficult storage, limited immune activation capability and the like of the existing dendritic vaccine, realizing the effects of directly killing tumor and inducing organism to generate strong double immunity.
The technical scheme is as follows: in order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the bionic anti-tumor vaccine consists of an immune activation functional hybrid peptide modified recombinant exosome carrier shell and a nano-form drug core, wherein the recombinant exosome is formed by recombining ginsenoside and exosome.
Preferably, the immune activation functional hybrid peptide is composed of an antigen peptide and a biological peptide, wherein the antigen peptide is selected from OVA 257-264 、OVA 323-339 、gp 10025-33 One or more of MUC1 glycosylated peptides, but not limited to these; the biological peptide is selected from one or more of alpha-Mel, iRGD, NAP, angiopep-2 and Tf, but is not limited to the substances.
Preferably, the exosomes are derived from autologous cells of the patient, and are selected from one or more of tumor cells, dendritic cells, macrophages, neutrophils and mesenchymal stem cells, but are not limited to these substances.
Preferably, the ginsenoside is selected from one or more of 20 (S) -Rg3, 20 (R) -Rg3, 20 (S) -Rh2 and 20 (R) -Rh 2.
Preferably, the recombinant exosomes are obtained by ultrasonic crushing of the extracted exosomes and then co-incubation with ginsenoside; preferably, the mass ratio of the recombinant exosomes to the ginsenoside is 1, based on the mass of the ginsenoside, based on the protein content: (1-2); preferably, the ultrasonication conditions are as follows: the ultrasonic crushing time is 5-10min, the power is 250-350W, and the ultrasonic frequency is 1-2s/4-6s on/off; the co-incubation is performed at 32-40℃for 0.5-2h.
Preferably, the nano-form medicine core contains a medicine which is an innate immunity activating substance, and the nano-form medicine core is selected from nano-particles formed by one or more of manganese-albumin complex, manganese colloid, cpG and aluminum hydroxide, but is not limited to the substances.
Preferably, the particle size of the immune co-activated bionic anti-tumor nano vaccine is 100-140nm.
The preparation method of the immune co-activation bionic anti-tumor nano vaccine comprises the following steps:
(1) Extracting and separating exosomes, performing ultrasonic crushing, and then incubating with ginsenoside to obtain recombinant exosomes;
(2) Preparing a drug core in a nano form;
(3) Mixing the recombinant exosome obtained in the step (1) with the medicine core obtained in the step (2), and incubating to obtain medicine-carrying exosome solution;
(4) Adding a biological peptide solution into the drug-carrying exosome solution under stirring, emulsifying for 1-2h, and then adding a solution of immune activation functional hybrid peptide, emulsifying for 1-2h;
(5) Filtering to remove free medicine.
Preferably, in the step (1), the exosome is obtained by adopting a combination of iodixanol density gradient centrifugation and ultracentrifugation.
Preferably, in the step (3), the medicine core is calculated according to the medicine mass, the exosome is calculated according to the protein content, and the mass ratio of the medicine to the recombinant exosome is 1: (1-4).
Preferably, in the step (3), the recombinant exosomes obtained in the step (1) and the medicine inner cores obtained in the step (2) are mixed, electroporation medium solution is added first, electric shock is carried out, and incubation is carried out for 0.5-2h at 32-40 ℃ after the electric shock is finished.
Preferably, in the step (4), when the solid mass is mg, the liquid mass is 1 part of the drug-carrying exosome solution and 1-2 parts of the immune activation functional hybrid peptide in terms of mL.
Preferably, the incubation time in step (4) is 60-120 min.
The invention also provides application of the immune co-activated recombinant bionic anti-tumor nano vaccine in preparation of anti-tumor drugs. The tumor comprises lung cancer, breast cancer, brain glioma, ovarian cancer, gastric cancer, colorectal cancer, osteosarcoma, prostatic cancer, cervical cancer, malignant pleural effusion or melanoma, etc. When the nano vaccine is applied, physiological saline, phosphate buffer or 5% glucose solution is added for dissolution, intravenous injection, intramuscular injection or oral administration is adopted, the nano vaccine can automatically load antigen peptide in a self-assembled mode and efficiently load innate immunity activator, the bioavailability and in-vivo transportation efficiency of two substances are improved, and the immunogenicity is improved, so that the innate immunity and the specific immune system are awakened to jointly inhibit the growth of tumors.
The invention reserves the immune activation function of DC as antigen presenting cells by extracting diseased animal DC cells and inducing the differentiation of exosomes, and the exosomes as acellular bionic carriers solve the key problems of complicated production process, harsh storage conditions, difficult mass production and the like of the traditional DC vaccine.
The invention selects DC cell source exosome as vaccine carrier and recombines it, and selects ginsenoside to replace original cholesterol component, further prolonging its body circulation time; meanwhile, hydrophilic drugs are loaded by virtue of the double-layer lipid structure of the amphiphilic hybrid polypeptide, and the amphiphilic hybrid polypeptide is modified on the surface of the amphiphilic hybrid polypeptide, so that the drug loading space of the amphiphilic hybrid polypeptide is fully utilized, and the high-efficiency drug loading is realized. The amphiphilic hybrid polypeptide endows the nano vaccine with direct tumor killing and specific immune activation functions; the hydrophilic medicine endows the nano vaccine with an innate immunity activating function, and effectively overcomes the defects of poor in vivo stability, short in vivo circulation time, low in vivo activity, limited immune response intensity and the like of the traditional vaccine.
The invention utilizes autologous cells of patients to derive exosomes, antigenic peptides and biological peptides, constructs the powerful immune activation bionic nano vaccine by electroporation and ultrasonic disruption, loads innate immune activation components, and can improve the problems of poor stability, effective bioavailability, low response efficiency, insufficient immune activation and the like of the traditional nano vaccine. It has the following advantages:
(1) Highly endogenous: the exosomes are generated by utilizing autologous cells, so that the physical and chemical characteristics and biological characteristics of parent cells can be completely reserved, and the exosomes have high endogenous property;
(2) Biosafety: the bionic nano vaccine has high biocompatibility, is easy to biodegrade, has low toxic and side effects and good safety effect;
(3) The drug loading capacity is strong: the structural characteristics of exosome double-layer phospholipids are fully utilized, amphiphilic functional polypeptides are modified in the amphiphilic phospholipid layers, hydrophilic drugs are loaded in the inner core hydrophilic space, the drug loading space of the carrier is fully utilized, and the drug loading capacity and encapsulation rate of the drugs are improved;
(4) Strong penetration ability: the particle size of the nano vaccine is in the nano range, so that the nano vaccine can easily pass through a plurality of layers of tissues and a plurality of physiological barriers to reach a focus area, the tumor infiltration degree is high, and a foundation is laid for fully exerting the drug effect;
(5) Highly immunogenic: the exosomes derived from autologous cells of the diseased animals are used as carriers, so that the exosomes are endowed with high immunogenicity, and the immune activation capacity of the exosomes is further improved after the exosomes are modified by antigen hybridization peptides, so that a foundation is laid for activating strong in-vivo immunity;
(6) High in vivo Activity: the nano carrier is used for delivering the immune activating substance, so that the problems of easy decomposition, poor efficacy and the like of the traditional vaccine preparation are overcome; the immune activating substance is delivered by the recombinant bionic nano-carrier, so that the defect that the traditional nano-preparation is easy to be identified by an endothelial reticulation system to be rapidly cleared is further overcome, the bioavailability of the medicine is improved, and the in vivo activity and the utilization efficiency of the vaccine are improved;
(7) Complementary treatment mechanism and multiple treatment means: the antigen hybrid peptide is modified and immune activating substances are delivered, so that the effect of direct tumor killing, dual immune co-activation of innate immunity and specific immunity is realized, the bottleneck problems of limited efficacy, low patient responsiveness, poor protection efficiency and the like of the nano vaccine are completely broken, the tumor treatment effect is fully improved, and long-acting protection is generated.
The immune co-activated recombinant bionic anti-tumor nano vaccine provided by the invention can be used for completing the efficient in-vivo delivery of specific immune activated polypeptide antigen peptide and innate immune activator, the nano preparation has high endogenous property, biological safety, strong penetrability and high immunogenicity, the efficient load and delivery of two physicochemical property medicaments further fully utilize the advantage of the drug delivery mode of the bionic medicament carrier to be diversified, and the innate + specific immune co-activated recombinant bionic nano vaccine is successfully constructed, so that the immune co-activated recombinant anti-tumor nano vaccine has high in-vivo activity, in-vivo stability, long in-vivo circulation time and tumor treatment efficiency, accords with the development trend of tumor treatment, meets the clinical requirements of tumor treatment, provides a model for the efficient accurate treatment of tumors and the construction of a long-acting protection platform, and has wide application prospect and clinical transformation potential.
Drawings
FIG. 1 is a representation of the surface characteristic proteins of the "immune co-activated" recombinant biomimetic anti-tumor nanovaccine of example III 1.1;
FIG. 2 is a graph showing the particle size and Zeta potential of the "immune co-activated" recombinant bionic anti-tumor nanovaccine according to example III 1.2;
FIG. 3 is a storage stability study of the "immune co-activated" recombinant biomimetic anti-tumor nanovaccine of example III 1.3;
FIG. 4 is an in vitro hemolytic assay of the "immune co-activated" recombinant biomimetic anti-tumor nanovaccine of example III 1.4;
FIG. 5 is a DC cell uptake study of the "immune co-activated" recombinant biomimetic anti-tumor nanovaccine of example III 1.5.1;
FIG. 6 is a tumor cell uptake study of the "immune co-activated" recombinant biomimetic anti-tumor nanovaccine of example III 1.5.2;
FIG. 7 is a cell uptake dependency study of the "immune co-activated" recombinant biomimetic anti-tumor nanovaccine of example III 1.5.3;
FIG. 8 is a cytotoxicity study of a "immune co-activated" recombinant biomimetic anti-tumor nanovaccine according to example three 1.5.4;
FIG. 9 is a DC activation ability study of the "immune co-activation" recombinant bionic anti-tumor nano vaccine in example III 1.6;
FIG. 10 is a T cell activation ability study of the "immune co-activated" recombinant biomimetic anti-tumor nanovaccine of example III 1.7;
FIG. 11 is a serum pharmacokinetic study of the "immune co-activated" recombinant biomimetic anti-tumor nanovaccine of example III 1.8.
Detailed Description
The invention is further illustrated by the following examples. These examples are given solely for the purpose of illustration and are not to be construed as limitations of the present invention. The invention is further described below with reference to the drawings and examples.
Embodiment one: preparation of BSA/Mn nanocores
Bovine serum albumin BSA (20 mg) was weighed, 2mL of ultrapure water was added thereto, and the mixture was dissolved by vortexing to prepare a 10mg/mL BSA solution, which was transferred to a penicillin bottle. 15mg of MnCl is weighed 2 ·4H 2 O, 1mL of ultrapure water was added thereto and dissolved by vortexing to prepare a mother liquor of 15 mg/mL. 100 mu L of MnCl is sucked under magnetic stirring 2 ·4H 2 The O solution was added drop wise to the penicillin bottle. After 5min, the pH was adjusted to 11 by adding an appropriate amount of 1M NaOH. Magnetically stirring for 2h at room temperature. Free drug was removed by centrifugal ultrafiltration (4500 r/min, 8 min) in a 30k Da ultrafiltration tube. PBS constant volume weight suspension is used for diluting BSA/Mn kernel solution, and the BSA/Mn kernel solution is refrigerated at 4 ℃ for standby.
Embodiment two: preparation process of DC source exosome carrying BSA/Mn and modifying antigen hybridized peptide
GL261 cells in logarithmic growth phase were taken and resuspended and dispersed (6X 10) in PBS buffer (pH 7.4) 7 and/mL), and stored on ice. C57BL/6 mice were anesthetized with 1% pentobarbital (40 mg/kg) and fixed with a small animal brain stereotactic apparatus. The head of the mouse is disinfected by 75% alcohol, a longitudinal incision is made along the junction of the inner canthus connecting line and the sagittal midline of the head, a small amount of 3% hydrogen peroxide is dipped on a cotton stick to destroy the surface tissue of the skull, and the skull is separated and exposed. The periosteum is stripped, the bregma is exposed, the needle insertion part is positioned at the position 1.5mm behind the bregma and 2mm on the right side, and the bone drill is perforated at the position. GL261 cell suspension (30X 10) was aspirated by microinjector 4 And 5 mu L), the needle depth is 3mm, the injection is slowly performed for 5min, the cell suspension stays for 5min after being completely injected into the brain of the mouse, the injector is slowly pulled out, and the medical suture is used for suturing the scalp. After 8d of tumor growth, the mice are killed by cervical vertebra removal, and brains of the mice are dissected and separated to determine success of tumor implantation.
Sterilizing the surgical instrument with 75% alcohol, and sterilizing with ultraviolet irradiation for 30min. The mice were soaked at 75% for 5min and sterilized. Sterile 12-well plates were poured into PBS. The femur and tibia of the mice were removed by aseptic manipulation in an ultra clean bench, transferred to a PBS well plate, rinsed to remove hair, and medium was aspirated with a 1mL needle, repeatedly rinsing the bone marrow cavity until the bone became white. The obtained bone marrow cells were centrifuged (1200 r/min, 5 min) and the supernatant was discarded. Adding erythrocyte lysate, standing for 3min, adding 1640 complete culture medium to stop digestion, centrifuging (1200 r/min, 5 min), and discarding supernatant. The cell concentration was adjusted to 106/mL and resuspended in fresh medium for cultivation, and the medium was replaced every two half daily. After 6 days, the loosely adherent cells were collected as dendritic cells by gently blowing the walls. The supernatant was removed by centrifugation (1200 r/min, 5 min) and resuspended in RPMI 1640 serum-free medium. Centrifuging (1200 r/min, 5 min) after 48h to collect supernatant, adding exosome extraction reagent to collect exosome, and obtaining DE solution.
Rh 2.5 mg was weighed and dissolved in 100. Mu.L of DMSO. Taking the DE solution obtained in the second example, adding the Rh2 solution dropwise after ice bath ultrasonic crushing (the mass ratio of the exosomes to the ginsenoside is 1:1 based on the mass of Rh2 based on the mass of protein), and incubating at 37 ℃ for 1h to obtain RDE. Taking the BSA/Mn solution prepared in the item of the example I, dropwise adding RDE, and incubating (37 ℃ C., 1 h) after electroporation to prepare RDE/Mn (the medicine core is calculated by medicine mass, the exosomes are calculated by protein content, and the mass ratio of Mn to recombinant exosomes is 1:2). The alpha-Mel solution was added under magnetic stirring to emulsify for 1h, and then the alpha-Mel-OVA (purchased from the pharmaceutical industry of GuoPing) solution was added to emulsify for 1h (RDE/Mn- -1 part, alpha-Mel-OVA- -1 part). Filtering with 0.8 μm filter membrane, centrifuging with 30k Da ultrafiltration tube (5000 r/min, 10 min), removing free medicine, and re-suspending with PBS buffer solution to obtain MORDE/Mn. MODE/Mn (where DE does not recombine with Rh 2), MDE/Mn (where DE does not recombine with Rh2 and no antigenic peptide is included in the hybrid peptide), DE/Mn (where DE does not recombine with Rh2 and no hybrid peptide modification is used) were prepared in the same manner.
Embodiment III: property investigation of bionic anti-tumor nano vaccine for immune coactivation
1.1 MODE/Mn surface characterization protein expression
Taking DE and MORDE obtained in the second step, re-suspending exosomes by PBS, sequentially adding CD81, CD63, CD9 and negative control thereof according to the amount of protein, incubating for 15min at room temperature in the absence of light, transferring to a flow tube, and measuring by a flow cytometer. The results are shown in FIG. 1, and CD9, CD63 and CD81 in DE and MORDE are positive, which proves that the extracted vesicle is exosome and the polypeptide modification does not influence the expression of exosome characteristic protein.
1.2 MODE/Mn particle size and potential investigation
The particle size and Zeta potential of the preparation were measured by a Markov laser particle sizer, and the results are shown in FIG. 2. The Zeta potentials of DE/Mn, MDE/Mn, MODE/Mn and MODE/Mn are all lower than BSA/Mn, and the particle sizes are gradually increased, so that the antigen hybrid peptide is successfully modified and Mn is successfully loaded.
1.3 examination of the shelf stability of MODE/Mn
Preparing MORDE/Mn, placing in PBS, storing at 4deg.C in dark, and performing particle size, PDI and Zeta potential every day after preparation, and examining its placement stability. MODE/Mn is prepared, placed in PBS and stored in dark at 4deg.C, particle size, PDI and Zeta potential are carried out every day after preparation is completed, and the placement stability is examined. The results are shown in FIG. 3. Compared with MODE/Mn, the MODE/Mn has better stability after Rh2 is introduced, and the PDI and the particle size are obviously changed within 5 days.
1.4 in vitro hemolytic examination of MODE/Mn
The Mel, α -Mel-OVA solutions were dissolved in ultrapure water to prepare a solution having a maximum concentration of 50. Mu.M. The prepared MODE was diluted in PBS buffer to give a maximum concentration of 50. Mu.M in alpha-Mel. The three groups of sample solutions were diluted 7 times at concentrations of 50. Mu.M, 25. Mu.M, 12.5. Mu.M, 7.5. Mu.M, 3.75. Mu.M, 1.875. Mu.M, 0.9375. Mu.M, 0.46875. Mu.M. Fresh mouse blood was collected and centrifuged at 5000rpm for 6min to remove plasma, and the lower blood cells were resuspended by gentle shaking with physiological saline. The operation was repeated until the upper liquid did not appear red. Blood cells were diluted to a concentration of 2% and added to the medicated EP tube and incubated for 3h at 37 ℃. Centrifuge at 5000rpm/min for 6min. The supernatant was taken and added to a 96-well plate. Absorbance was measured at 540 nm.
Hemolysis ratio (%) = (As-Ab)/(Ac-Ab) ×100%
As is the average of experimental well absorbance values, ac is the average of Triton well absorbance, ab is the absorbance of physiological saline wells.
The experimental results are shown in FIG. 4. From the figure, after MODE is constructed, alpha-Mel is deeply buried in phospholipid layer to shield positive charge, reduce hemolysis, and prove hemolysis safety.
1.5 examination of cellular uptake of MODE/Mn
DC uptake investigation of 1.5.1MORDE/Mn
DiI was dissolved in DMSO to prepare a 10mM stock solution. And sucking DiI 5 mu L, mixing with MODE, and performing ultrasonic crushing for 5min to obtain MODE/DiI. Control groups Mel Lipos/DiI, DE/DiI, MDE/DiI, MODE/DiI and MODE/DiI were set. DC2.4 cells were resuspended in RPMI 1640 complete medium (1X 10) 4 One per mL), in a 96-well plate, 3 multiple wells per group, 100. Mu.L per well, at 37℃in 5% CO 2 Incubate overnight. After cell attachment, the culture was removed, and Mel Lipos/DiI, DE/DiI, MDE/DiI, MODE/DiI and MODE/DiI were added to the drug-delivery wells, respectively, and serum-free medium was added to the blank wells. After 2h incubation, the liquid was removed and washed 3 times with PBS buffer. The PBS was removed by blotting and stained with Hoechst dye for 15min, and the dye was removed by blotting and washed 3 times with PBS buffer. Shooting under an inverted microscope (DiI: λex/λem=549/565 nm).
DiI was dissolved in DMSO to prepare a 10mM stock solution. And sucking DiI 5 mu L, mixing with MODE, and performing ultrasonic crushing for 5min to obtain MODE/DiI. Control groups Mel Lipos/DiI, DE/DiI, MDE/DiI, MODE/DiI and MODE/DiI were set. DC2.4 cells were resuspended in RPMI 1640 complete medium (1X 10) 4 One per mL), in a 96-well plate, 3 multiple wells per group, 100. Mu.L per well, at 37℃in 5% CO 2 Incubate overnight. After cell attachment, the culture was removed, and Mel Lipos/DiI, DE/DiI, MDE/DiI, MODE/DiI and MODE/DiI were added to the drug-delivery wells, respectively, and serum-free medium was added to the blank wells. After 2h incubation, the liquid was removed and washed 3 times with PBS buffer. Cells were lysed by addition of DMSO and the fluorescence intensity was measured by a microplate reader (DiI: diI: λEx/λEm=549/565 nm).
As shown in the experimental results in FIG. 5, the exosome DE secreted by BMDC has the capability of targeting parent cells, and the uptake capacity of BMDC is further improved after alpha-Mel modification.
Investigation of GL261 uptake Capacity and mechanism of 1.5.2MORDE/Mn
DiI was dissolved in DMSO to prepare a 10mM stock solution. And sucking DiI 5 mu L, mixing with MODE, and performing ultrasonic crushing for 5min to obtain MODE/DiI. DC cells in logarithmic growth phase were resuspended in DMEM complete medium (1X 10) 4 One per mL), in a 96-well plate, 3 multiple wells per group, 100. Mu.L per well, at 37℃in 5% CO 2 Incubate overnight. After the cells are attached to the wall, removing and culturing, adding DE/DiI, D4F-DE/DiI, MDE (-)/DiI, rh2-DE (-)/DiI, MODE/DiI and MODE/DiI into the drug administration holes respectively. Wherein MDE (-)/DiI is pre-treating GL261 cells with D4F (0.5. Mu.M) solution for 12h to saturate SR-BI receptor; rh2-DE (-)/DiI was prepared by pre-treating GL261 cells with WZB-117 solution (40. Mu.M) for 12h to saturate the glucose transporter. Serum-free medium was added to the blank wells. After 2h incubation, the liquid was removed and washed 3 times with PBS buffer. The PBS was removed by blotting and stained with Hoechst dye for 15min, and the dye was removed by blotting and washed 3 times with PBS buffer. Shooting under an inverted microscope (DiI: λex/λem=549/565 nm).
DiI was dissolved in DMSO to prepare a 10mM stock solution. And sucking DiI 5 mu L, mixing with MODE, and performing ultrasonic crushing for 5min to obtain MODE/DiI. DC cells in logarithmic growth phase were resuspended in DMEM complete medium (1X 10) 4 One per mL), in a 96-well plate, 3 multiple wells per group, 100. Mu.L per well, at 37℃in 5% CO 2 Incubate overnight. After the cells are attached to the wall, removing and culturing, adding DE/DiI, D4F-DE/DiI, MDE (-)/DiI, rh2-DE (-)/DiI, MODE/DiI and MODE/DiI into the drug administration holes respectively. Wherein MDE (-)/DiI is pre-treating GL261 cells with D4F (0.5. Mu.M) solution for 12h to saturate SR-BI receptor; rh2-DE (-)/DiI was prepared by pre-treating GL261 cells with WZB-117 solution (40. Mu.M) for 12h to saturate the glucose transporter. Serum-free medium was added to the blank wells. After 2h incubation, the liquid was removed and washed 3 times with PBS buffer. Cells were lysed by addition of DMSO and the fluorescence intensity was measured by a microplate reader (DiI: λEx/λEm=549/565 nm).
The experimental results are shown in FIG. 6, where D4F-DE uptake is significantly higher than in DE, demonstrating that D4F-dominated SR-BI receptor binding can promote DE tumor cell uptake; MDE group uptake is higher than D4F-DE, and MDE (-) group uptake is reduced compared with MDE after saturation of SR-BI receptor, and it is considered that melittin membrane potential in alpha-Mel mediates cell binding and can remarkably improve uptake of tumor cells to the preparation. After saturation of the glucose transporter, rh2-DE uptake decreased significantly, and glucose transporter mediated endocytosis was considered to be one of the important pathways for enhancing tumor cell uptake as well. Taken together, the cellular uptake of MODE is significantly higher than DE, thought to be the result of membrane potential mediated cell binding, SR-BI mediated receptor binding, and glucose transporter mediated endocytosis. Wherein the membrane potential mediates or plays a major role in cell binding.
Investigation of uptake dependence of 1.5.3MORDE/Mn
GL261 cells and DC2.4 cells in logarithmic growth phase were resuspended in complete medium (1X 10) 4 individual/mL) was inoculated into a 96-well plate, each group of 3 multiplex wells, 100 μl of each well was placed at 37deg.C, 5% CO 2 Incubate overnight. After the cells are attached, removing the culture medium, adding MODE liquid medicine for co-incubation, and respectively examining the ingestion conditions at time points of 30, 60, 90, 120 and 150 min. The liquid was aspirated at the above time point, washed 3 times with PBS buffer, and cells were lysed by adding DMSO, and the fluorescence intensity was measured by an enzyme-labeled instrument (DiI: λEx/λEm=549/565 nm).
The experimental results are shown in fig. 7, in which tumor cells have higher uptake for MODE than BMDC and have time dependence.
1.5.4MORDE/Mn cytotoxicity investigation
GL261 cells in logarithmic growth phase were resuspended in DMEM complete medium (1X 10) 4 Individual/well) was seeded in 96-well plates at 100 μl per well. Placing at 37deg.C, 5% CO 2 Is incubated overnight in an incubator at constant temperature. After cell attachment, the medium was aspirated, the Control group was added with 100. Mu.L of serum-free medium, and the dosing group was added with Mel, mel Lipos, DE/Mn, MDE, MODE and MODE. Culturing for 12 hr, sucking off culture medium, adding PBS to dissolve diluted LIVE/DEAD mixed dye solution (Calcium AM concentration 2 μm, ethD-1 concentration 4 μm), incubating at 37deg.C for 20min, and standing in fluorescent displayAnd observing under a micro mirror.
DC2.4 cells were resuspended in RPMI 1640 complete medium (1X 10) 4 One per mL), in a 96-well plate, 3 multiple wells per group, 100. Mu.L per well, at 37℃in 5% CO 2 Incubate overnight. After cell attachment, the medium was aspirated, 100. Mu.L of serum-free medium was added to the Control group, and Mel, mel Lipos, DE/Mn, MDE, MODE and MODE were added to the dosing wells. Culturing for 12h, sucking out the culture medium, adding PBS to dissolve diluted LIVE/DEAD mixed dye solution (Calcium AM concentration 2. Mu.M, ethD-1 concentration 4. Mu.M), incubating for 20min at 37 ℃ per well and observing under a fluorescence microscope.
The experimental result is shown in figure 8, the free Mel has stronger cell killing effect, and the positive charge of the modified Mel is shielded, so that the cell killing effect is greatly reduced. However, since GL261 cells have significantly higher uptake of the preparation than DC, the degree of apoptosis is significantly greater than DC.
1.6 examination of the DC cell activation Capacity of MODE/Mn
GL261 cells in logarithmic growth phase were taken and resuspended in complete medium (20X 10 4 Individual/well) was inoculated into 24-well plates at 500 μl per well. Placing at 37deg.C, 5% CO 2 Is incubated overnight in an incubator at constant temperature. The medium was removed, 300. Mu.L of serum-free medium was added to the Control group, and BSA/Mn, DE/Mn, MDE/Mn, MODE/Mn were added to the dosing wells. Placing at 37deg.C, 5% CO 2 Incubated for 24 hours in a constant temperature incubator, and the supernatant and cells were collected. Dendritic cells were extracted and resuspended in complete medium (20X 10 4 Individual/well) was inoculated into 24-well plates at 500 μl per well. Placed at 37 ℃ and 5% CO 2 Is incubated overnight in an incubator at constant temperature. Tumor supernatant and cells previously collected were added, cultured for 36h and the cells were collected by centrifugation. The addition of CD11c, CD80, CD86 was incubated for 15min in the dark for flow assay.
The experimental results are shown in FIG. 9, and the expression of CD80 and CD86 is obviously increased after administration. Compared with DE/Mn, the MDE expression level is obviously increased, and the alpha-Mel has the DC cell activating capability. And after introducing the antigen peptide OVA, the maturation of the DC is increased again, which proves that the tumor-associated antigen peptide is important for the activation and maturation of the DC.
1.7 investigation of T cell activation Capacity by MODE/Mn
The drug-stimulated DCs were incubated with mice spleen total lymphocytes (DCs: total lymphocytes = 1:10) for an additional 36h following the procedure described under "1.6". Cells from each well were then aspirated and centrifuged (1000 r/min, 5 min), and supernatants were collected and subjected to ELISA to determine TNF- α, IL-6, IL-10, IFN- γ and IL-12 expression. The well plate was washed 2 times with PBS, the cell pellet was resuspended in 500. Mu.L of PBS, 5. Mu.L of flow antibody (CD 3, CD4, CD8 a) was added to stain for 15min away from light, and the fraction of cell subsets of CD3+, CD4+, CD8a+ was analyzed by flow cytometry and the results were analyzed by Kaluza software.
The experimental results are shown in fig. 10, the expression of cd8+ T cells is obviously increased after administration, wherein the activation degree of T cells is obviously increased after introducing tumor-associated antigen peptide OVA, which proves that strong specific immunity is successfully induced; TNF-alpha, IL-6, IL-12 and IFN-gamma expression levels are obviously increased, and IL-10 is obviously reduced, so that the preparation has obvious immune activation effect and improves immune suppression microenvironment.
Serum pharmacokinetic investigation of 1.8 MODE/Mn
The C57BL/6 mice were randomly divided into four groups, lipos/DiI, rh2-Lipos/DiI, DE/DiI and MORDE/DiI, respectively. The tail vein injection is used for administration. After 5min, 10min, 15min, 30min, 1h, 2h, 4h, 6h, 8h, 12h, 24h, blood was collected from the eyeballs, centrifuged at 6000rpm for 6min, serum was collected, and fluorescence intensity was measured by an enzyme-labeled instrument (DiI: λEx/λEm=549/565 nm).
The experimental results are shown in FIG. 11, which shows that MODE/DiI and DE/DiI are not significantly different. Compared with Lipos/DiI, rh2-Lipos/DiI blood circulation time is prolonged; the blood circulation time of MODE/DiI and DE/DiI is longer than that of Rh2-Lipos/DiI, and the endogenous vector is considered to be capable of further avoiding the recognition of endothelial reticulate phagocytosis system, so that the half life of the drug is prolonged; after modification of Rh2, MODE/DiI has a longer half-life, which is believed to be the result of Rh2 co-acting with DE.
Claims (10)
1. The immune coactivated recombinant bionic anti-tumor nano vaccine is characterized by comprising an immune activation functional hybrid peptide modified recombinant exosome carrier shell and a nano-form drug core, wherein the recombinant exosome is formed by recombining ginsenoside and exosome.
2. The 'immune co-activated' recombinant bionic anti-tumor nano vaccine according to claim 1, wherein the immune activated functional hybrid peptide is composed of an antigen peptide and a biological peptide, and the antigen peptide is selected from OVA 257-264 、OVA 323-339 、gp 10025-33 One or more of MUC1 glycosylated peptides; the biological peptide is selected from one or more of alpha-Mel, iRGD, NAP, angiopep-2 and Tf.
3. The 'immune co-activation' recombinant bionic anti-tumor nano vaccine according to claim 1, wherein the exosomes are derived from autologous cells of the patient, and the autologous cells are selected from one or more of tumor cells, dendritic cells, macrophages, neutrophils and mesenchymal stem cells.
4. The 'immune co-activation' recombinant bionic anti-tumor nano vaccine according to claim 1, wherein the ginsenoside is one or more selected from 20 (S) -Rg3, 20 (R) -Rg3, 20 (S) -Rh2, 20 (R) -Rh 2.
5. The immune coactivated recombinant bionic anti-tumor nano vaccine according to claim 1, wherein the recombinant exosomes are obtained by subjecting extracted exosomes to ultrasonic disruption and then co-incubating with ginsenoside; preferably, the mass ratio of the recombinant exosomes to the ginsenoside is 1, based on the mass of the ginsenoside, based on the protein content: (1-2); preferably, the ultrasonication conditions are as follows: the ultrasonic crushing time is 5-10min, the power is 250-350W, and the ultrasonic frequency is 1-2s/4-6s on/off; the co-incubation is performed at 32-40℃for 0.5-2h.
6. The "immune co-activated" recombinant biomimetic anti-tumor nanovaccine of claim 1, wherein the drug core in the nano form comprises a drug that is an innate immune activating substance; preferably, the nano-form medicine core is selected from nano-particles formed by one or more of manganese-albumin complex, manganese colloid, cpG and aluminum hydroxide.
7. The method for preparing the immune co-activated recombinant bionic anti-tumor nano vaccine according to claim 1, which is characterized by comprising the following steps:
(1) Extracting and separating exosomes, performing ultrasonic crushing, and then incubating with ginsenoside to obtain recombinant exosomes;
(2) Preparing a drug core in a nano form;
(3) Mixing the recombinant exosome obtained in the step (1) with the medicine core obtained in the step (2), and incubating to obtain medicine-carrying exosome solution;
(4) Adding a biological peptide solution into the drug-carrying exosome solution under stirring, emulsifying for 1-2h, and then adding a solution of immune activation functional hybrid peptide, emulsifying for 1-2h;
(5) Filtering to remove free medicine.
8. The method for preparing the immune co-activated recombinant bionic anti-tumor nano vaccine according to claim 7, wherein in the step (3), the medicine inner core is calculated by the medicine mass, the exosome is calculated by the protein content, and the mass ratio of the medicine to the recombinant exosome is 1: (1-4); in the step (4), when the solid mass is mg, the liquid mass is calculated by mL, 1 part of the drug-carrying exosome solution and 1-2 parts of the immune activation functional hybrid peptide; the emulsification time is 60-120 min.
9. The use of the 'immune co-activation' recombinant bionic anti-tumor nano vaccine in claim 1 in the preparation of anti-tumor drugs.
10. The use according to claim 9, wherein the tumor comprises lung cancer, breast cancer, brain glioma, ovarian cancer, gastric cancer, colorectal cancer, osteosarcoma, prostate cancer, cervical cancer, malignant pleural effusion or melanoma.
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