CN116850274A - Tumor vaccine for ultrasound-assisted immune activation and preparation method and application thereof - Google Patents
Tumor vaccine for ultrasound-assisted immune activation and preparation method and application thereof Download PDFInfo
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- CN116850274A CN116850274A CN202310769607.5A CN202310769607A CN116850274A CN 116850274 A CN116850274 A CN 116850274A CN 202310769607 A CN202310769607 A CN 202310769607A CN 116850274 A CN116850274 A CN 116850274A
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Abstract
The invention provides an ultrasonic-assisted immune activated tumor vaccine and a preparation method and application thereof, belonging to the field of tumor biological immunotherapy. The tumor vaccine provided by the invention is obtained by taking polylactic acid-glycolic acid copolymer (PLGA) as a carrier and co-loading tumor cell membrane protein, tumor cell total RNA and a sound sensitizer chlorine6, wherein the tumor cell total RNA is subjected to polymerization protection by using polylysine (Poly-L-Lysine, PLL). The invention promotes the escape of tumor vaccine in lysosomes, improves MHCI antigen presenting level, and can promote dendritic cell maturation by generating ROS to enhance T cell immune response. The vaccine provided by the invention has high biological safety, the nano particle size grade is easier to be phagocytized by cells and enter lymph nodes, and the vaccine can be used for preventing the recurrence and metastasis of various tumors after operation.
Description
Technical Field
The invention belongs to the field of tumor biological immunotherapy, and in particular relates to an ultrasonic-assisted immune activated tumor vaccine, and a preparation method and application thereof.
Background
Tumors are a global public health problem threatening human life health, and although tumor treatment has been applied for hundreds of years, the use of various modes of surgery, drug treatment, radiation treatment has greatly reduced the overall mortality of tumors, a significant portion of refractory tumors are insensitive to various modes of treatment, especially recurrent metastasis after surgery often results in death of patients, and thus there is a need to continually develop new tumor treatment methods to address new challenges.
Tumor vaccines are a type of tumor immunotherapy, and a vaccine preparation is prepared by jointly preparing tumor antigens and an immunoadjuvant, so that specific immune response aiming at the tumor antigens is activated in a human body, and T cells are identified and killed by an immune system, so that a tumor therapeutic effect is achieved. Tumor vaccine function requires antigen presentation to T cells while the antigen is expressed in tumor cells. However, currently tumor vaccine antigens are mainly derived from resected tumor tissue of patients, and the existing antigens have hysteresis and cannot cope with new antigens generated by continuous mutation of tumors. And the adjuvant capable of activating cellular immunity is limited, and the problem of biological safety is outstanding. The tumor vaccine studied at present is mainly used for treating advanced tumors, however, the immune function is low in the later stage of tumor development, antigen presenting cells are gradually disabled, and the application effect of the vaccine is poor. The existence of these problems has limited the research and application of tumor vaccines. Therefore, the tumor vaccine should be improved in several aspects such as antigen, immune adjuvant, carrier and application time.
The antigen is one of the important components of the tumor vaccine, and determines whether the activated immunity of the tumor vaccine can accurately identify and kill the tumor. Tumor vaccines developed at present are of various types, and can be largely classified into protein-resistant type, nucleic acid-resistant type and whole cell type according to antigen type. Three major antigen presentation processes are currently thought to exist within dendritic cells, one type of presentation that activates cellular immunity, two types of presentation that activates humoral immunity, and cross presentation, respectively. Where protein-type vaccines activate cellular immunity primarily through cross-presentation, whereas nucleic acid vaccines activate immunity through one type of presentation. Tumor vaccines function primarily by antigen presenting cells, which carry out antigen presentation by dendritic cells, and thus present antigens to cytotoxic T cells. This process is the process by which cellular immunity is activated. The protein antigen vaccine mainly utilizes cross presentation to realize the activation of cell immunity; the nucleic acid vaccine synthesizes corresponding proteins in cytoplasm, and is presented through a presentation path after being degraded by proteasome and a series of physiological processes; whole cell vaccines are mainly in vitro to activate dendritic cells for return to the body, but the complexity of the process is far greater than that of simple antigen vaccines, so that the clinical application has a large limit.
The immune adjuvant can stimulate dendritic cells to mature and improve antigen presenting efficiency. The antigen alone is not effective in stimulating dendritic cell maturation and is susceptible to eliciting immune tolerance, so modern vaccine formulations all contain immunoadjuvant components. Immune adjuvants have been developed for about a hundred years, and because most vaccines use activated humoral immunity to produce antibodies to achieve a prophylactic effect, adjuvants have also been developed primarily for humoral immunity. The total of 6 clinically approved adjuvants are used for activating humoral immunity and have poor cell immunity activation effect on resisting tumors. Adjuvants used in scientific experiments include CpG and poly (I: C), and the like, and although the adjuvants can effectively activate cellular immunity when applied in animals, the effects in human body experiments are controversial, and safety problems such as systemic inflammation and autoimmune reaction are also worth focusing. Aiming at the current situation of lack of the cellular immunity adjuvant, the safe and effective cellular immunity adjuvant needs to be continuously searched to support the development of tumor vaccines.
The vectors used in modern vaccines are of various types such as viruses, bacteria, cells, lipid nanoparticles, polymers and the like, and the selection of reasonable vectors aiming at antigens and immune response types plays an important role in improving the immune response level and the immunogenicity of the antigens. Taking mRNA as an example, the delivery carrier is Lipid Nanoparticle (LNP), and through carrier transportation, the mRNA of the antigen can be additionally protected from degradation, the antigen can be changed into a particle form, and the antigen is easier to be recognized by dendritic cells, so that immune response is induced. Although LNP technology is widely used in mRNA vaccine, LNP has limited stability, needs to be stored at about-80 ℃ and limits application scenes; and LNP cannot encapsulate protein components, and is difficult to apply to the loading of multiple antigen types. Viral vectors, while promoting immune activation, have the disadvantage of reduced safety, complexity of production, and multiple use titers due to limitations imposed by their use. Viral vectors are also difficult to load with multiple antigen types. The polymer such as polylactic acid-glycolic acid copolymer (PLGA) is a biosafety in vivo slow release material, has the advantages of large drug loading capacity, capability of loading hydrophobic and hydrophilic drugs simultaneously, simple preparation process and the like, and is a vaccine carrier type with great development potential. And the carrier is also required to comprehensively consider multiple parameters such as slow release time, vaccine particle size, preparation process and the like to select reasonable PLGA types.
The tumor vaccine which is currently studied or clinically tested is mostly a therapeutic vaccine, namely, the tumor vaccine is used as a therapeutic drug after tumorigenesis, and the anti-tumor immune response in vivo is improved. While this approach may play a role in inhibiting tumor progression, the prophylactic properties that the vaccine itself should possess are lacking. The mechanisms involved in tumor progression and escape are quite complex, and destruction of the immune system and the formation of a locally inhibitory immune microenvironment are one of the causes of immunotherapy failure. Taking immune checkpoint blocking therapy as an example, the related guidelines are gradually advancing the administration time, because even if immune checkpoints are blocked, the T cell sap is difficult to exert killer cell action due to the systemic immunosuppressive state in the late stage of the tumor. The problem of application time is also considered in the application of tumor vaccines. If the vaccine is applied later after the tumor is resected by operation, not only the whole body immunosuppression can affect the action, but also the local immunosuppression immune microenvironment after the tumor is formed can prevent T cell infiltration and the killing effect. Therefore, the tumor vaccine should be applied as early as possible on the premise of reasonable antigen to play a role in prevention.
Disclosure of Invention
Therefore, the invention aims to provide the tumor vaccine for ultrasound-assisted immune activation, which has high presenting efficiency and high biological safety, and can be used for preventing the recurrence and metastasis of various tumors after operation.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an ultrasonic-assisted immune activated tumor vaccine, which is obtained by taking PLGA as a carrier and co-loading tumor cell membrane protein, tumor cell total RNA and chlorine 6; the tumor cell total RNA was protected by polymerization using PLL.
The invention also provides a preparation method of the tumor vaccine, which comprises the following steps: extracting total RNA and cell membrane of tumor cell respectively, mixing RNA solution and PLL solution, adding cell membrane suspension as water phase; mixing PLGA and chlorine6, and adding dichloromethane to obtain oil phase; adding the water phase into the oil phase after the first homogenization treatment, carrying out the second homogenization treatment, pouring the homogenized system into the first PVA aqueous solution, carrying out the third homogenization treatment, dropwise adding the treated system into the second PVA aqueous solution in stirring, and continuing stirring; centrifuging the stirred suspension, and precipitating to obtain the tumor vaccine.
Preferably, the tumor cells are treated with the small molecule compound pladienolide b prior to extraction of total RNA or cell membranes of the tumor cells.
Preferably, in the aqueous phase, the concentration of the PLL solution is 0.1-100 mug/mu l, the concentration of the RNA solution is 0.1-10 mug/mu l, and the mass ratio of the RNA to the PLL is 1:0.0162-6.12; the concentration of protein in the cell membrane suspension is 0.1-10 mug/mu l, and the mass ratio of RNA to cell membrane is 1:300-300:1.
Preferably, in the oil phase, the PLGA concentration is 50-200 mg/mL, and the chlorine6 concentration is 0.1-10 mg/mL.
Preferably, the volume ratio of the water phase to the oil phase is 1:100-1:3.
Preferably, the homogenization is ultrasonic homogenization, the homogenization time is 10-100 s, and the parameters are set to 1% -80% of the ampliude, and 5s is on/5 s off.
Preferably, the concentration of the first PVA aqueous solution is 15-25 mg/mL; the concentration of the second PVA aqueous solution is 2-8 mg/mL.
The invention also provides application of the tumor vaccine or the preparation method in preparation of medicaments for preventing postoperative recurrence or metastasis of tumors.
The invention also provides tumor vaccine freeze-dried powder for ultrasound-assisted immune activation, and the tumor vaccine or the precipitate obtained by the preparation method is resuspended by using a trehalose aqueous solution and freeze-dried to obtain the tumor vaccine freeze-dried powder.
Compared with the prior art, the invention has the following beneficial effects:
the invention takes the total membrane protein and the total RNA of tumor cells as antigens, and compared with the existing mainstream tumor vaccine technology loaded with a few predetermined proteins or nucleic acids as antigens, the antigen spectrum of the vaccine can be enlarged, and the recognition of an unknown antigen by an immune system can be induced; compared with single polypeptide vaccine or nucleic acid vaccine, the method can comprehensively utilize two modes of cross presentation and classical 1 type presentation of dendritic cell presentation antigen, and improves the efficiency of MHC1 type antigen presentation. The protein and RNA use tumor cells per se, belong to autologous antigens for the administration individuals, and have no risk of genome integration, and are safe to use.
Compared with common adjuvants such as CpG, poly (I: C), and the like, the invention uses the sound sensitizer, namely the chlorine6, as an immune adjuvant, and the chlorine6 generates ROS under the ultrasonic action to promote tumor vaccine to escape from endosomes/lysosomes, and promotes MHCI antigen presentation by releasing antigen into cytoplasm. And moderate oxidative stress in cells can induce heat shock protein to generate, so that the degradation efficiency of the intracellular protein in a proteasome is improved, more antigen epitopes are generated, and the protein degradation rate is improved. Meanwhile, oxidative stress can also stimulate dendritic cells to convert to the mature direction, and finally activate antigen-specific T cell immune response.
The invention uses three components of degradable polylactic acid-glycolic acid copolymer (PLGA) loading protein, RNA and chlorine6 to prepare the tumor vaccine, has high biological safety, and the nanometer particle size grade is easier to be phagocytized by cells and enter lymph nodes. The tumor vaccine can be immunized in advance before tumor recurrence and metastasis, and is favorable for exerting the effect of the tumor vaccine in early stage by utilizing a relatively complete immune system.
The tumor vaccine of the invention has a relatively simple preparation mode and can be used for various tumors. The future clinical transformation can be quickly prepared after the treatment of the tumor tissue excised by the operation of the patient, is used for preventing postoperative tumor recurrence and metastasis, and has wide application prospect.
Drawings
Fig. 1: extracting tumor cell membrane and identifying protein;
fig. 2: schematic preparation of tumor vaccine;
fig. 3: agarose gel electrophoresis results of different N/P ratios;
fig. 4: tumor vaccine transmission electron microscopy (left) and scanning electron microscopy (right);
fig. 5: tumor vaccine particle size distribution (left) and Zeta potential analysis (right) results;
fig. 6: loading tumor vaccine dissolution liquid UV-Vis absorbance curves with different components;
fig. 7: co-localization of tumor vaccine with DC2.4 cells;
fig. 8: confocal fluorescence pictures of tumor vaccine distribution in lymph nodes;
fig. 9: in-vivo fluorescence imaging detection of tumor vaccine distribution in lymph nodes;
fig. 10: CCK-8 detection of cellular activity after application of different treatment conditions;
fig. 11: tumor vaccine and lysosome co-localization and ultrasonic irradiation promote endosome escape;
fig. 12: confocal pictures of the tumor vaccine for promoting the generation of ROS in DC2.4 cells by ultrasonic irradiation;
fig. 13: westernblot detects the expression of HSP70 protein in DC2.4 cells;
fig. 14: detecting the presenting effect of a mode antigen SIINFEKL under the condition of ultrasonic assistance by flow cytometry;
fig. 15: growth curve of mouse tumor after preimmunization of the mouse with tumor vaccine;
fig. 16: qPCR detects representative gene alternative splice changes and RNA-seq detects total transcriptome intron and exon ratios;
fig. 17: CCK-8 detects the effect of Pladienolide B treatment on 4T1 cell activity;
fig. 18: tumor vaccine loaded with alternative splicing interfering neoantigen inhibits the mouse 4T1 breast cancer model.
Detailed Description
The invention provides an ultrasonic-assisted immune activated tumor vaccine, which is prepared by taking polylactic acid-glycolic acid copolymer (PLGA) as a carrier and co-loading tumor cell membrane protein, tumor cell total RNA and an acoustic sensitizer chlorine6, wherein the tumor cell total RNA is polymerized and protected by Polylysine (PLL).
The invention adopts a double emulsion method to prepare tumor vaccine, and the preparation method comprises the following steps:
extracting total RNA and cell membrane of tumor cell respectively, mixing RNA solution and PLL solution, adding cell membrane suspension as water phase; mixing PLGA and chlorine6, and adding dichloromethane to obtain oil phase; adding the water phase into the oil phase after the first homogenization treatment, carrying out the second homogenization treatment, pouring the homogenized system into the first PVA aqueous solution, carrying out the third homogenization treatment, dropwise adding the treated system into the second PVA aqueous solution in stirring, and continuing stirring; centrifuging the stirred suspension, and precipitating to obtain the tumor vaccine.
In the invention, which type of tumor vaccine is prepared, the corresponding type of tumor cells are selected for extracting total RNA and cell membranes of the tumor cells. Before the total RNA and cell membrane of the tumor cells are extracted, a small molecular compound Pladienolide B is adopted to treat the tumor cells.
The preparation method adopts DMSO to prepare the Pladienolide B solution, and adds the Pladienolide B solution into a culture medium to culture tumor cells, wherein the volume ratio of the Pladienolide B solution to the culture medium is preferably 1:5000. Before the total RNA of the tumor cells is extracted, adding Pladienolide B solution with the concentration of 1-1000 nM into a culture medium to culture the tumor cells for 0.5-24 h, wherein the concentration is preferably 100-200 nM, and the time is preferably 3-4 h; before extracting the cell membrane of the tumor cell, adding Pladienolide B solution with the concentration of 1-1000 nM into the culture medium to culture the tumor cell for 1-48 h, wherein the concentration is preferably 2.5-3 nM, and the time is preferably 22-24 h.
The phenomenon of alternative splicing disorder exists in the tumor recurrence and metastasis process, and the protein with abnormal splicing can be generated, so that the protein is one of sources of tumor new antigens. The invention uses the small molecular compound Pladienolide B for inhibiting the function of a spliceosome to treat tumor cells, induces tumor splicing disorder in vitro, can generate protein and RNA with abnormal splicing, uses the protein and RNA as antigens to construct tumor vaccine, can ensure that an immune system has the capability of recognizing the antigen in advance before the mutant antigen appears in the tumor, and can ensure that the immune cells recognize and kill the tumor at the first time once the mutant antigen is generated in the tumor recurrence or metastasis process. The invention gives consideration to unknown antigens possibly generated in the future progress of the tumor, and achieves better effect in tumor treatment and prevention application.
The method comprises the steps of fully and uniformly mixing the extracted tumor cell total RNA solution with the PLL solution, and then adding the cell membrane suspension to obtain an aqueous phase system, wherein the aqueous phase system is placed on ice for standby.
The invention uses PLL to carry out polymerization protection on the total RNA of the extracted tumor cells (the amount used for mixing the RNA and the PLL is calculated by the ratio of the amount of ammonium radical carried by the PLL to the amount of phosphate radical substances, namely, the N/P ratio is 0.1-10).
In the aqueous phase system of the invention: the PLL solution is preferably prepared by using ultrapure water, and the concentration of the solution is 0.1-100 mug/mu l, preferably 10-20 mug/mu l; the RNA solution is preferably obtained by dissolving the RNA solution in ultrapure water, and the concentration of the RNA solution is 0.1-10 mug/mu l; the mass ratio of the RNA to the PLL is 1:0.0162-6.12, preferably 1:1.53; the concentration of the protein in the cell membrane suspension is 0.1-10 mug/mu l; the mass ratio of the RNA to the cell membrane is 1:300-300:1, preferably 1:1.
PLGA and chlororine 6 are mixed, and then dichloromethane (analytically pure) is added to prepare a dichloromethane solution containing PLGA and chlororine 6, and the dichloromethane solution is used as an oil phase.
In the oil phase system of the invention, the final concentration of PLGA is 50-200 mg/mL, preferably 100mg/mL; the final concentration of the chlorine6 is 0.1-10 mg/mL, preferably 4mg/mL.
The water phase system is placed on ice, the oil phase system is added after the first homogenization treatment, and the volume ratio of the water phase to the oil phase is 1:100-1:3, preferably 3:10. The obtained mixed system is subjected to a second homogenization treatment, the homogenized system is poured into a first PVA aqueous solution, and a third homogenization treatment is performed, wherein the volume of the first PVA aqueous solution is 2-3 times, preferably 2.5 times, of the volume of the mixed system (total volume of the water phase and the oil phase). The system is added into the second PVA aqueous solution in stirring drop by drop, and stirring is continued for 4 hours; the stirring is preferably light-proof, room temperature, magnetic stirring, and the stirring speed is preferably 800rpm. The invention carries out centrifugation on the suspension obtained by stirring to obtain sediment, namely the tumor vaccine.
In the invention, the homogenization is ultrasonic homogenization and is carried out on ice; the homogenizing time is 10-100 s, the parameters are set to 1% -80% of the parameters of the sample, preferably 40%, and 5s is on/5 s off. The first homogenization treatment is preferably 20 to 25 seconds; the second homogenization treatment is preferably 30-90 s; the third homogenization treatment is preferably 30 to 90 seconds.
In the invention, the aqueous solution of vinyl alcohol (PVA) is prepared by dissolving the PVA in ultrapure water and performing ultrasonic treatment in a water bath. The concentration of the first PVA aqueous solution is 15-25 mg/mL, preferably 20mg/mL; the concentration of the second PVA aqueous solution is 2 to 8mg/mL, preferably 5mg/mL.
In the present invention, the centrifugation condition is preferably 4℃and 12000g is centrifuged for 15min, and the precipitate obtained by the centrifugation is preferably subjected to resuspension washing 2 to 3 times by adding ultrapure water.
The invention also provides the application of the tumor vaccine or the preparation method in preparing the medicine for preventing tumor postoperative recurrence or metastasis, and the tumor vaccine can be immunized in advance before tumor recurrence and metastasis, thereby being beneficial to utilizing the relatively complete immune system to exert the effect of the tumor vaccine in early stage.
The invention also provides tumor vaccine freeze-dried powder for ultrasound-assisted immune activation, which is obtained by re-suspending the tumor vaccine (or centrifugal sediment prepared by the method) by using an aqueous solution of trehalose and freeze-drying. As an alternative embodiment, the invention uses ultrapure water to prepare w/v4% aqueous solution of trehalose, transfers the aqueous solution into a centrifuge tube, freezes the aqueous solution at-80 ℃ overnight, and transfers the tumor vaccine pre-frozen at-80 ℃ into a vacuum freeze dryer for freeze drying, thus obtaining the tumor vaccine freeze-dried powder.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
In the following examples, conventional methods are used unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1
The preparation method of tumor vaccine loaded with tumor cell membrane protein, total RNA and chlorine6 comprises the following steps:
1. total RNA and cell membrane extraction of 4T1 mouse breast cancer cells:
total RNA extraction: using Tripure TM The RNA extract was dissolved in ultrapure water to obtain a total RNA extract, and the concentration was adjusted to 6. Mu.g/. Mu.l by quantifying the ultraviolet absorbance.
Cell membrane extraction:
a. preparing a cell hypotonic buffer solution: 20mM Tris-HCl, pH7.5;10mM KCl;2mM MgCl 2 The method comprises the steps of carrying out a first treatment on the surface of the 1 piece of cOmpleemeiniEDTA-freeprotease inhibitortablet (Roche, 4693159001) was added per 10mL of hypotonic buffer. Preserving at 4 ℃.
b. Pancreatin digestion and collection of 1X 10 8 The 4T1 cells were washed 3 times with 1 XPBS buffer and 200g of the solution at room temperature, the residual PBS buffer was discarded, 20mL (about 10 volumes) of the hypotonic buffer of step a was added and the mixture was left on ice for 30min.
c. A Dounce mill (Kimble, 3110) was assembled, 2mL of cell hypotonic buffer was added and the mill was rinsed using a type B mill bar. 2mL of the cell suspension is sucked each time, the cell suspension is added into a grinder, ground on ice for 40 times, and the ground cell suspension is placed into a new centrifuge tube.
d. The milled cell suspension was centrifuged at 4℃differential speed. Centrifuging at 700g for 10min, discarding the precipitate, and collecting the supernatant to a new centrifuge tube; centrifuging 7000g of supernatant for 30min, discarding precipitate, and collecting supernatant; the supernatant was centrifuged at 100000g for 2 hours, the supernatant was discarded, and the pellet was resuspended in ultrapure water to give a cell membrane fraction containing the protein, and the protein was quantified using BCA and stored at-80℃at a concentration of 6. Mu.g/. Mu.l. The results of cell membrane extraction and protein identification are shown in FIG. 1. In FIG. 1, A is a cell membrane extraction flow; B-C are protein identification results of cell membranes, wherein Cellysate is a cell lysate group; cellmembrane is a group of cell membrane lysates.
2. Vaccine preparation (see FIG. 2 for details of the preparation process)
(1) The tumor vaccine which takes PLGA as a carrier and is loaded with cell membrane protein, total RNA and chlorine6 is prepared by a multiple emulsion method. To avoid destruction of RNA during preparation, the RNA was pre-polymerized using Polylysine (PLL).
(2) The N/P ratio was calculated as PLL residues to nucleotide phosphate groups, different ratio gradients were set and the optimal ratio was analyzed using agarose gel electrophoresis. The final N/P ratio was chosen to be 2.5, converted to reagent concentrations for tumor vaccine preparation (agarose gel electrophoresis results for different N/P ratios are shown in FIG. 3).
The specific method comprises the following steps: a. PLL (sigma-aldrich, P7890) mother liquor was configured with ultrapure water at a concentration of 10 μg/μl; b. mu.g/mu.l RNA was added to a centrifuge tube, placed on ice, then 92. Mu.l PLL stock was added, and the mixture was gently shaken to mix the RNA with the PLL, and placed on ice for 30min.
(3) To the PLL-protected RNA suspension, 100. Mu.l of a cell membrane suspension with a protein amount of 6. Mu.g/. Mu.l was added, and the addition of 8. Mu.l of ultrapure water was continued to make the total volume of the system 300. Mu.l. This part of the system was kept as an aqueous phase on ice for the next step.
(4) Preparing PLGA methylene dichloride solution containing chlorine6 in dark place: PLGA (RESOMER@RG502H, mw=7000-17000, sigma-aldrich 719897) was weighed precisely 100mg to an ampoul, and chlorine6 (Cayman, no. 21684) 4mg was weighed and mixed with PLGA. Dichloromethane (analytically pure) was added to the mixture of PLGA and chlororine 6 and the volume was set to 1mL to prepare a dichloromethane solution containing 100mg/mL lga and 4mg/mL chlororine 6 as an oil phase.
(5) Preparing a polyvinyl alcohol (PVA) aqueous solution: 1g of PVA (Mw=9000-10000, sigma-aldrich 360627) was weighed into a centrifuge tube, ultrapure water was added to a constant volume of 50mL, and the PVA was dissolved by ultrasonic treatment in a water bath to obtain a PVA aqueous solution having a concentration of 20 mg/mL. As the external aqueous phase.
(6) Tumor vaccine prepared by a double emulsion method: a. the system obtained in step (3) was placed on ice and homogenized using a probe-type ultrasonic homogenizer (ColeParmer, ultrasonicHomogenizer4710 series) for 20s with parameters set to sample 40% and 5s on/5 s off.
(7) 1mL of the oil phase system prepared in the step (4) is added to the step (6) in the avoidance mode, ultrasonic homogenization is carried out on ice for 60s, the parameters are amplitude40%, and 5s are on/5 s off.
(8) The homogenized system of step (7) was poured into 3.25mL20mg/mL PVA aqueous solution (PVA aqueous solution 2.5 times the total volume of the internal aqueous phase and the oil phase) in the dark, and homogenized on ice for 60s with parameters of amplitude40% and 5s on/5 s off.
(9) 40mL of 5mg/mL aqueous PVA solution was prepared with 20mg/mL aqueous solution of LPVA and stirred with a magnetic stirrer at 800rpm at room temperature. Dropwise adding the system obtained in the step (8) into the stirred PVA aqueous solution, and stirring for 4 hours in a dark place.
(10) Transferring the stirred suspension into a centrifuge tube, centrifuging at 4 ℃ for 15min with 12000g, discarding the supernatant, adding 50mL of ultrapure water for resuspension, cleaning the precipitate, repeating the centrifugation, cleaning for 3 times, discarding the supernatant, and obtaining the precipitate as the tumor vaccine.
(11) The w/v4% aqueous solution of trehalose (Biyun Tian, ST 1245) was prepared with ultrapure water, and C was resuspended (10) by precipitation with 10mL aqueous solution of trehalose, transferred to a 5mL centrifuge tube, and stored overnight at-80 ℃.
(12) Transferring the tumor vaccine pre-frozen in the step (11) to a vacuum freeze dryer (SCIENTZ, N series) for freeze drying for 48 hours to obtain tumor vaccine freeze-dried powder for subsequent verification.
Example 2
Characterization and identification of tumor vaccine (in lyophilized powder form)
1. And (3) morphological identification of a transmission electron microscope: (1) Weighing 2mg of tumor vaccine freeze-dried powder, adding 1mL of ultrapure water for redissolving, centrifuging at 4 ℃ for 12000g, and cleaning for 3 times; (2) And (3) dripping the tumor vaccine suspension onto a copper mesh, standing for half an hour, and dripping a proper amount of 2% phosphotungstic acid dye liquor for negative dyeing for 15min. Air-drying at 4deg.C overnight; (3) The shape and the size of the tumor vaccine are observed by using a transmission electron microscope, and the tumor vaccine is seen to be spherical particles with the diameter of 150-200 nm. The results are shown in FIG. 4.
2. Scanning electron microscope morphology and surface identification: (1) Dripping tumor vaccine cleaned by ultrapure water on the surface of the tinfoil paper, and airing overnight; (2) surface metal spraying treatment; (3) And observing the surface morphology of the tumor vaccine by using a scanning electron microscope, wherein the tumor vaccine is spherical particles with smooth surfaces, and the diameter is consistent with that of the transmission electron microscope. The results are shown in FIG. 4.
3. Particle size and Zeta potential analysis: the tumor vaccine with proper volume is weighed, 1mLPBS is used for re-suspending and precipitating, 12000g is centrifuged and washed for 3 times, the particle size distribution is analyzed by NanoPlus after PBS with proper volume is re-suspended, and the result shows that the particle size is mainly concentrated to about 150-200 nm. The tumor vaccine with proper volume is weighed, 1mLPBS is used for re-suspending and precipitating, 12000g is centrifuged, the solution is washed for 3 times, and the Zeta potential is analyzed by NanoPlus after the PBS with proper volume is re-suspended, so that the Zeta potential is-26.32 mv, and the dispersibility is good. The particle size distribution of the tumor vaccine and the Zeta potential analysis result are shown in figure 5.
4. Chlorine6 load verification and encapsulation efficiency calculation: (1) 1mg of tumor vaccine is weighed, 30 mu l of DMSO is used for dissolving for 30 minutes, 170 mu l of ultrapure water is added for uniform mixing, and 12000g is centrifuged for 5 minutes; (2) A mixed solution of 30. Mu.l of DMSO and 170. Mu.l of ultrapure water was prepared as a calibration solution; (3) The absorbance of the solution at 404nm was analyzed using a UV-Vis analyzer, and the CM-RNA@Ce6/PLGA group chlorine6 encapsulation rate was calculated to be 15.2%.
5. The UV-Vis absorbance curves of the respective nanoparticles were measured (method same as step 2) by preparing nanoparticles loaded with different components, see FIG. 6 (FIG. 6, blank@PLGA represents nanoparticles with PLGA components only; CM@Ce6/PLGA represents PLGA nanoparticles loaded with cell membrane and chlorine 6; RNA@Ce6/PLGA represents PLGA nanoparticles loaded with RNA and chlorine 6; CM-RNA@Ce6/PLGA represents PLGA nanoparticles loaded with cell membrane, RNA and chlorine6, which are tumor vaccines according to the present invention). In FIG. 6, the UV-Vis absorbance curves are, from bottom to top, blank@PLGA, CM@Ce6/PLGA, RNA@Ce6/PLGA, CM-RNA@Ce6/PLGA, and the absorbance of PLGA nanoparticles loaded with cell membranes, RNA and chloroine 6 is strongest.
6. Protein quantification: (1) preparing hydrolysate: prepared by using ultrapure water, and contains 0.05 percent of NaOH and 0.1 percent of Tween 20 (the range is NaOH0.001-1 percent, and Tween 20 is 0.001-1 percent); (2) Weighing 1mg of tumor vaccine, adding 100 μl of hydrolysate, mixing well, 1h at 90 ℃, and oscillating for 18h at 37 ℃; (3) Centrifuging 12000g for 15min, adding 0.05 MHz in equal volume into supernatant to adjust pH, quantifying protein by BCA method, and calculating protein encapsulation efficiency to 68.3%;
7. and (3) RNA detection: (1) 1mg of tumor vaccine is weighed, added with 30 mu l of DMSO, and then kept stand at room temperature for 30min to dissolve tumorA vaccine; (2) Adding Tripure TM Mixing 500 μl of RNA extract with vaccine solution; (3) 100 μl of chloroform was added and mixed well, and centrifuged at 12000g for 15min at 4deg.C; (4) Taking supernatant to a new centrifuge tube, adding equal volume of isopropanol, uniformly mixing, adding 1 μl of glycogen, and standing at-20 ℃ for 2 hours; (5) RNA precipitate was extracted by centrifugation at 12000g for 15min at 4℃and 20. Mu.l DEPC in water was dissolved, and the RNA amount was quantitatively analyzed by ultraviolet, and the encapsulation efficiency was calculated to be 31.5%.
Example 3
Tumor vaccine delivery efficacy validation
1. Ability of endocytic tumor vaccine: (1) co-culturing the tumor vaccine with DC2.4 cells for 12h; (2) The cell nucleus and the cell skeleton are respectively marked by Hoechst33342 and phalloidin-FITC, the confocal microscope is used for detecting the co-localization condition of the tumor vaccine and the cell, and the condition of the tumor vaccine entering the cell is estimated. FIG. 7 shows that tumor vaccine can be efficiently endocytosed by cells in case of co-localization of tumor vaccine with DC2.4 cells.
2. In vivo delivery of tumor vaccine: 2mg of tumor vaccine was weighed and 100. Mu.l PBS was used to prepare the suspension. The mice were subcutaneously injected, and after 12 hours, lymph nodes near the injection site were prepared into frozen sections, and the nuclei were labeled with Hoechst33342 and the distribution of tumor vaccine in the lymph nodes was examined by confocal microscopy. And adding fluorescent dye DiR into the oil phase in the preparation process of the tumor vaccine to prepare the DiR-marked tumor vaccine. 2mg of DiR-labeled tumor vaccine was weighed and 100. Mu.l of PBS was used to prepare a suspension. Subcutaneous injection, lymph nodes and fat near the injection site were taken after 12h, and fluorescence expression of lymph nodes and fat was detected using an animal in vivo fluorescence imaging system. FIG. 8 is confocal fluorescence of tumor vaccine distribution in lymph nodes; FIG. 9 is a fluorescence imaging assay of tumor vaccine distribution in lymph nodes. The results demonstrate that subcutaneous tumor vaccine can be rapidly enriched into lymph nodes.
Example 4
Verification of antigen presentation effect promoted by ultrasonic irradiation
1. Ultrasonic and Ce6 parameter determination: (1) DC2.4 cells were cultured in 96-well plates to a density of 60%, and cell viability was measured using the CCK-8 method using different intensity sonicationsSex; (2) Determination of 1.0W/cm 2 Performing experiments by intensity ultrasound, wherein cells are incubated with Ce6 with different concentrations, detecting the activity of the cells by using a CCK-8 method after ultrasonic treatment, and determining that the optimal concentration of Ce6 is 2 mug/ml; (3) Cells were treated with Ce6 concentration of 2. Mu.g/ml, different ultrasound parameters were applied and cell activity was again detected using the CCK-8 method. FIG. 10 shows the activity of cells after CCK-8 detection under different treatment conditions, a is the activity of DC2.4 cells under different ultrasonic intensities, b is the activity of DC2.4 cells under different ultrasonic intensities at a Ce6 concentration of 2. Mu.g/ml, c is the ultrasonic intensity of 1.0W/cm 2 DC2.4 cells were active at different Ce6 concentrations. The results in fig. 10 show that the mere sonication of the cells did not significantly affect the cell activity, which significantly decreased with increasing concentration after Ce6 addition. The use of appropriate Ce6 concentrations and ultrasound parameters can effectively reduce the toxic effects on cells.
2. Detecting tumor vaccine intracellular localization and ultrasonic promotion endosome escape conditions: DC2.4 cells were cultured in confocal dishes, tumor vaccine was co-cultured with cells for 6h, after 6h, the medium was replaced with fresh medium without tumor vaccine, culture was continued, lysosomes were labeled with Lysotracker-FITC lysosome fluorescent probes at different time points, and were observed using confocal microscopy. Wherein the sonicated group applied 1W/cm to the cells after 6h of incubation 2 After 1min,24h of intense ultrasonic irradiation, the intracellular distribution of the tumor vaccine was observed using a confocal microscope. FIG. 11 is a confocal microscopy image of tumor vaccine co-localization with lysosomes. The results show that the co-localization of the tumor vaccine and the lysosome is gradually increased along with the time extension, which indicates that the tumor vaccine gradually enters the lysosome after entering cells. Ultrasonic irradiation groups showed scattered tumor vaccine fluorescence in cytoplasm, indicating that ultrasonic treatment promoted escape of tumor vaccine endosomes.
3. Detecting the ability of the tumor vaccine to promote ROS production in cells under ultrasound: (1) Co-culturing tumor vaccine with concentration of 0.5mg/mL and DC2.4 dendritic cells for 12h, changing liquid, and applying ultrasonic treatment with parameters of 1W/cm 2 The time is 1min; (2) Intracellular ROS were detected using DCFH-DA, and confocal microscopy detected changes in intracellular ROS amounts. FIG. 12 is a confocal map of DC2.4 cellsAnd (3) a sheet. The results indicate that tumor vaccines produced reactive oxygen species in cells after sonication.
4. Cellular HSP70 expression assay: DC2.4 cells were cultured in six well plates, co-cultured with cells using 0.5mg/mL tumor vaccine for 12h, and 1.0W/cm was applied after liquid exchange 2 Cells were collected after 1min and 1h of intensive sonication, and the expression of HSP70 protein in the cells was detected by Westernblot. FIG. 13 shows the result of Western blot detection of HSP70 protein expression in DC2.4 cells, which indicates that tumor vaccine increased heat shock protein expression in cells with the assistance of ultrasound. Nanoparticles loaded with different antigen components simultaneously also promoted HSP70 protein production in cells, whereas PLGA empty vector without any loaded components failed to increase HSP70 expression.
5. And (3) verifying antigen presenting effect: (1) In order to study the influence of ultrasonic irradiation on antigen presentation, ovalbumin (OVA) is used as a model protein, and SIINFEKL epitopes in the OVA protein can be presented on the surface of cells through processing treatment in the cells, so that the antigen presentation efficiency is detected. First a tumor vaccine containing OVA antigen was constructed: transfecting an OVA expression vector into HEK293T cells to enable the cells to express OVA protein, and collecting cell membranes and total RNA as antigen components; (2) Constructing a tumor vaccine comprising an OVA antigen using the method described in step 2; (3) Co-culturing 0.5mg/mL of the vaccine with C57 mouse primary bone marrow dendritic cells for 6 hours, and using 1W/cm for ultrasonic treatment group 2 The ultrasonic treatment with the intensity is carried out for 1min, and the culture is continued for 48h. (4) Flow cytometry detects the proportion of dendritic cells presenting SIINFEKL epitopes. The results are shown in FIG. 14. The result shows that after the dendritic cells are co-cultured with OVA antigen tumor vaccine, the cells can present SIINFEKL epitope, and after the ultrasonic irradiation is increased, the proportion of the cells presenting the epitope is obviously improved. It is demonstrated that ultrasound irradiation can increase antigen presentation efficiency.
6. Tumor vaccine inhibition tumor growth effect verification: female BALB/c mice with age of 6-8 weeks are selected as experimental animals, 100 mu l PBS-resuspended freeze-dried tumor vaccine (containing 2mg of freeze-dried powder) is injected subcutaneously for immunization, and 1W/cm is used after 12 hours 2 Is treated for 1min at the site of the adjacent lymph node. Immunization was performed every 5 days, and ultrasonic irradiation treatment was performed after each immunization. A total of 3 immunizations were performed. After the immunization is completedTumor bearing (5X 10) using 4T1 cells in situ in mouse pads 5 Individual cells). The change in tumor volume over time was measured continuously, see fig. 15. The results show that the tumor vaccine co-loaded with membrane proteins and RNAs has the best tumor growth inhibition effect compared to single membrane protein or RNA loaded nanoparticles.
Example 5
Tumor vaccine efficacy validation of loading alternatively spliced interfering neoantigen
1. Pladienolide b affects alternative splicing: (1) Treating 4T1 cells for 4h by using pladienolide B small molecule compounds with different concentrations, and collecting cells to extract RNA; (2) Detecting the expression of the introns of the Dnajb1 and Cdkn1b genes by qPCR; (3) The proportion of introns and exons in total mRNA was detected by second generation transcriptome sequencing using a100 nM concentration of Pladienolide B treated 4T1 cells for 4h. FIG. 16 shows the qPCR assay for intron expression, RNA-seq assay for total transcriptome intron and exon ratios. The results showed that as the concentration of pladienolide b increased, the expression level of the introns of the representative gene increased, indicating that pladienolide b could affect alternative splicing, with the effect being enhanced with increasing concentration. Cell sequencing results further demonstrate the altered ratio of introns and exons in the whole transcriptome by pladienolide b treatment.
2. The inhibition effect of tumor vaccine loaded with new antigen on tumor growth is studied by using a mouse 4T1 breast cancer model:
tumor vaccines containing alternatively spliced interfering neoantigens are prepared. Since high doses of pladienolide b are cytotoxic, the appropriate concentration of pladienolide b needs to be determined first. The effect of different Pladienolide B treatments 24h on 4T1 cell activity was examined using the CCK-8 method, see FIG. 17. The results showed that the majority of cells survived less when the pladienolide b concentration exceeded 2.5nM, so to obtain sufficient cell membrane protein, 4T1 cells were treated at 2.5nM concentration for 24h and the cell membrane was extracted. To obtain more alternatively spliced interfering RNA, 4T1 cells were treated with Pladienolide B at a concentration of 100nM for 4h and the cells were harvested to extract RNA. The extracted cell membrane and RNA are used as antigens.
Small molecule compound pladienolide b treated 4T1 mouse breast cancer cells and extracted RNA and cell membranes:
(1) Different concentrations of pladienolide b (TOCRIS, cat.No.6070) stock were prepared using DMSO.
(2) Mixing Pladienolide B mother solution with 100nM concentration at a volume ratio of mother solution to 1640 medium of 1:5000, culturing 4T1 cells for 4 hr, qPCR detecting expression level of Dnajb1 and Cdkn1b gene introns, and using Tripure TM The RNA extract was dissolved in ultrapure water to obtain a total RNA extract, and the concentration was adjusted to 6. Mu.g/. Mu.l by quantifying the ultraviolet absorbance.
(3) The cell activity was detected by culturing 4T1 cells for 24h using a 2.5nM concentration Pladienolide B stock solution mixed in a ratio of 1:5000 of the volume ratio of stock solution to 1640 medium, collecting cells, extracting cell membranes, and extracting cell membranes by the same method as in step 1 of example 1.
A tumor Vaccine was prepared using the procedure described in step 2 of example 1, designated "PlaB-Vaccine", while a 4T1 prepared without Pladienolide B treatment was designated "4T1-Vaccine" in example 1.
6-8 week female BALB/c mice were subcutaneously injected with 100. Mu.l PBS-resuspended lyophilized tumor vaccine (containing 2mg of lyophilized powder) and after 12 hours 1.0W/cm was used near the lymph node at the injection site 2 Intensity ultrasonic treatment is carried out for 1min. Three vaccine immunizations were performed in total, each 5 days apart. After the immunization process was completed, the cells were isolated with 4T1 cells (5X 10 5 And b) carrying out tumor in situ on a mouse breast pad, monitoring the growth condition of the tumor, detecting the number of infiltrated T cells in tumor tissues by flow cytometry, and taking lung tissues for staining to observe the number of metastasis. The results are shown in FIG. 18, wherein A is the tumor-bearing growth condition of 4T1 cells, B is the quantitative observation of lung tissue staining metastasis, and C is the quantitative detection of infiltrated T cells in tumor tissues. The results show that PlaB-Vaccine has better tumor inhibition effect than tumor Vaccine 4T1-Vaccine loaded with common 4T1 tumor cell antigen, improves the microenvironment inside the tumor, and can effectively reduce the distant metastasis of the tumor in the lung.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. The tumor vaccine for ultrasound-assisted immune activation is characterized in that the vaccine is obtained by taking PLGA as a carrier and co-loading tumor cell membrane protein, tumor cell total RNA and chlorine e 6; the tumor cell total RNA was protected by polymerization using PLL.
2. A method of preparing a tumor vaccine according to claim 1, comprising the steps of:
extracting total RNA and cell membrane of tumor cell respectively, mixing RNA solution and PLL solution, adding cell membrane suspension as water phase; mixing PLGA and chlorine e6, and adding dichloromethane to obtain an oil phase; adding the water phase into the oil phase after the first homogenization treatment, carrying out the second homogenization treatment, pouring the homogenized system into the first PVA aqueous solution, carrying out the third homogenization treatment, dropwise adding the treated system into the second PVA aqueous solution in stirring, and continuing stirring; centrifuging the stirred suspension, and precipitating to obtain the tumor vaccine.
3. The method of claim 2, wherein the tumor cells are treated with a small molecule compound Pladienolide B prior to extraction of total RNA or cell membrane from the tumor cells.
4. The preparation method according to claim 2, wherein the concentration of the PLL solution in the aqueous phase is 0.1 to 100 μg/μl, the concentration of the RNA solution is 0.1 to 10 μg/μl, and the mass ratio of RNA to PLL is 1:0.0162 to 6.12; the concentration of protein in the cell membrane suspension is 0.1-10 mug/mu l, and the mass ratio of RNA to cell membrane is 1:300-300:1.
5. The method according to claim 2, wherein the PLGA concentration in the oil phase is 50 to 200mg/mL and the chlorin e6 concentration is 0.1 to 10mg/mL.
6. The method of claim 2, wherein the volume ratio of the aqueous phase to the oil phase is 1:100 to 1:3.
7. The preparation method according to claim 2, wherein the homogenization is ultrasonic homogenization, the homogenization time is 10-100 s, and the parameters are set to 1% -80% of the ampliude, and 5s is on/5 s off.
8. The method according to claim 2, wherein the first aqueous PVA solution has a concentration of 15 to 25mg/mL; the concentration of the second PVA aqueous solution is 2-8 mg/mL.
9. Use of a tumor vaccine according to claim 1 or a method according to any one of claims 2 to 8 for the preparation of a medicament for the prevention of postoperative recurrence or metastasis of a tumor.
10. An ultrasonic-assisted immune activated tumor vaccine freeze-dried powder, which is characterized in that the tumor vaccine of claim 1 or the precipitate obtained by the preparation method of any one of claims 2 to 8 is resuspended by aqueous trehalose solution and freeze-dried to obtain the tumor vaccine freeze-dried powder.
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