CN110124018B - Calcium phosphate-lipid nano vaccine simulating necrotic tumor cells and application thereof - Google Patents
Calcium phosphate-lipid nano vaccine simulating necrotic tumor cells and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of vaccines, relates to a bionic nano vaccine preparation, and particularly relates to a calcium phosphate-lipid nano vaccine simulating necrotic tumor cells and application thereof. The calcium phosphate in the inner layer of the vaccine is hydrophobic nanoparticles which effectively entrap oligonucleotide substances; the outer lipid structure is a material which highly simulates the fluidity of cell membranes and can load natural cell membrane proteins and HSP70 active peptide fragments. The nano vaccine preparation can overcome many limitations of the traditional tumor vaccine, has the core advantage of high integration capability, and loads 3 natural multivalent tumor cell membrane antigen proteins (CM) as active ingredients, a heat shock protein HSP70 active peptide segment (alpha HSP70p) modified by alpha helix, and adjuvant oligonucleotide (CpG) into calcium phosphate nanoparticles (CaP) and simultaneously delivers the calcium phosphate nanoparticles (CaP) to antigen presenting cells, thereby activating innate immunity and adaptive immunity. Has higher potential application value and clinical transformation prospect.
Description
Technical Field
The invention belongs to the field of vaccines, relates to a bionic nano vaccine preparation, and particularly relates to a calcium phosphate-lipid nanoparticle with a core-shell structure, in particular to a calcium phosphate-lipid nano vaccine simulating necrotic tumor cells and application thereof. The calcium phosphate in the inner layer of the vaccine is hydrophobic nanoparticles capable of efficiently encapsulating oligonucleotide substances; the outer lipid structure is a material highly simulating the fluidity of cell membranes, and can load natural cell membrane proteins and HSP70 active peptide fragments.
Background
The data show that malignant tumor is the first killer threatening human health, and the conventional treatment means comprises surgical treatment, radiotherapy and chemotherapy, but practice shows that the treatment schemes have limited effect, the fundamental reason is that the metastasis and recurrence of the tumor in the body cannot be inhibited, and meanwhile, the treatment process has various toxic and side effects. Research reports that immunotherapy, as a novel anti-tumor therapy mode, can stimulate the immune system of an organism to effectively remove tumor cells, and brings new hopes for treating malignant tumors. Since 2011, the immunotherapy of tumors passes through a rapid development stage, the Science journal also ranks the immunotherapy of tumors as a ten-major breakthrough in Science in 2013, the effect of the PD/PDL-1 antibody as an immune checkpoint inhibitor on malignant melanoma also has a remarkable effect, and the survival time of patients is remarkably prolonged; however, immune checkpoint inhibitor therapy has a low response rate, and most of the reasons are that infiltration of immune cells caused by insufficient tumor mutation load in a tumor microenvironment is low, so that constructing a new comprehensive treatment means by other means is especially important for improving the clinical effect of checkpoint blockade therapy.
Tumor vaccines are currently the first choice for synergistic therapy with checkpoint blockade therapy. The tumor vaccine can activate infiltration of peripheral T cells of CD4+/CD8+ and the like at a tumor part by directly stimulating dendritic immune cells, thereby activating potential immune functions of a human body upstream. Ideally, the vaccine-stimulated CD8+ T cells have good activity, can recognize polypeptide-MHC I antigen peptide complexes on tumor cells, and have better recognition for specific markers of the tumor cells. But its design is a very challenging task, including three factors to consider: (1) selecting a suitable tumor-associated antigen, intended to elicit a specific T cell response; (2) selecting a suitable immunostimulatory adjuvant intended to activate pattern recognition receptors to enhance innate immunity; (3) simultaneously, the antigen and the adjuvant are efficiently delivered into the same dendritic cell. Practice shows that the tumor vaccine with a single antigen component is most easy to realize clinical transformation, and a plurality of ongoing clinical tests exist, but the breakthrough is not realized due to the high heterogeneity of tumors and the influence of immune resistance; personalized tumor vaccines developed in recent years pay more attention to tumor heterogeneity and antigen difference, and obtain some good effects, for example, Ott, P.A. and the like screen 20 self neoantigens of patients through computer algorithms and laboratory high-throughput sequencing, and the prepared mixture of 20 polypeptides and adjuvants stimulates the strong immune response of the patients, thereby preliminarily proving the feasibility of the personalized tumor vaccine based on cancer; however, this method suffers from drawbacks such as complicated preparation process, low efficiency of cell entry of the components of the free mixture, and the like. Although tumor lysate ex vivo stimulation of dendritic cell tumor vaccine Provenge has been approved for the treatment of prostate cancer, the effect is still to be improved due to the complex components in tumor lysate and the problem of immune tolerance, so the development of tumor vaccine is an urgent problem to be solved.
In recent years, research has revealed that the subcellular components of tumor cells have respective immunogenicity, such as cell nucleus, cytoplasm, cell membrane components and the like; first, in some tumors, such as melanoma, the cell membrane contains a large amount of tumor-associated antigen (TAA), which can be used as a potentially effective antigen material, while the tumor cell membrane derived from the patient himself may be one of the ideal materials for precise treatment, but the natural tumor cell membrane component is not sufficient to effectively activate dendritic cells, and the addition of a suitable immunoadjuvant component is essential for the therapeutic effect. Research has revealed that low-dose chemotherapy, radiotherapy and drastic environmental changes can mediate tumor cell necrosis (necrosis), thereby releasing a large amount of strongly immunogenic substances, namely danger signaling molecules (DAMPs), such as heat shock proteins (hsps), GRP78 bips, calreticulin and the like, which can be used as a novel immunoadjuvant material. The heat shock protein HSP70 is a heat stress protein with the molecular weight of about 70kDa, has the functions of molecular chaperone, is found to be expressed in various tumors, and induces and enhances the anti-tumor immune response of the body; HSP70 has a special structure capable of forming a complex with a polypeptide, binding with a receptor associated with the surface of an antigen presenting cell, and simultaneously stimulating innate immunity and specific immunity; relevant clinical tests for treating granulocytic leukemia are in progress, but the natural HSP70 polypeptide compound separated from a patient body has complex technology, low extraction efficiency and higher preparation difficulty.
Based on the current state of the art; the inventor of the application intends to provide a nano drug delivery platform of 'artificially simulated necrotic tumor cells', in particular to a calcium phosphate-lipid nanoparticle with a core-shell structure, wherein the nano drug delivery platform can simultaneously deliver tumor cell membrane proteins and HSP70 active peptide fragments into dendritic cells, and can potentially better activate the innate immunity and adaptive immunity of a human body.
Disclosure of Invention
The invention aims to provide a calcium phosphate-lipid nanoparticle with a core-shell structure based on the current situation and the foundation of the prior art, in particular to a calcium phosphate-lipid nano vaccine simulating necrotic tumor cells and application thereof.
The invention provides nanoparticles (alpha HSP70p-CM-Cap) of 'artificially simulated necrotic tumor cells' designed based on a bionics principle, wherein a natural multivalent tumor antigen protein and an HSP70 active peptide segment (adjuvant) are loaded in an artificial lipid material, and the nanoparticles are prepared by taking calcium phosphate as a material and used as a core and are coated with another adjuvant, namely oligonucleotide CpG ODN: on one hand, the new antigen component in the tumor cell membrane protein is utilized to cause anti-tumor specific immunity; on the other hand, two adjuvant components of oligonucleotide CpG and alpha HSP70p are utilized to activate the inherent immune function of human body and comprehensively play the role of anti-tumor. The artificial simulated necrotic tumor cell (alpha HSP70p-CM-Cap) nanoparticle overcomes the limitations of the traditional tumor vaccine, has the core advantage of high integration capability, and is prepared by loading 3 natural multivalent tumor cell membrane antigen proteins (CM) as active ingredients, an alpha helix modified heat shock protein HSP70 active peptide segment (alpha HSP70p) and adjuvant oligonucleotide (CpG) into calcium phosphate nanoparticles (Cap) and simultaneously delivering the calcium phosphate nanoparticles (Cap) to an antigen presenting cell, thereby activating innate immunity and adaptive immunity.
Specifically, the invention provides a calcium phosphate-lipid nano vaccine simulating necrotic tumor cells, wherein the calcium phosphate at the inner layer of the vaccine is a hydrophobic nanoparticle capable of efficiently encapsulating oligonucleotide substances; the outer layer of lipid structure is a material which highly simulates the fluidity of cell membranes and can load natural cell membrane protein and HSP70 active peptide; in the invention, tumor cell membrane protein from natural sources and an HSP70 active peptide segment modified by alpha helix are combined into an artificially constructed flowing lipid membrane material, and oligonucleotide CpG is efficiently encapsulated by the inner calcium phosphate nanoparticles.
The calcium phosphate is adopted to construct the nanoparticle inner core, and the oligonucleotide drug CpG entrapped has higher stability and entrapment efficiency; the preparation method is a reverse microemulsion method, the method is used for encapsulating siRNA, and the prepared nanoparticles are uniformly dispersed into an oil phase and have a certain slow release effect.
The adjuvant component, namely the unmethylated oligodeoxynucleotide (CpG ODN for short), is an artificially synthesized DNA sequence with immunostimulating effect and containing unmethylated CpG oligodeoxynucleotide; CpG ODN can directly activate B cells and dendritic cells to cause direct Th1 type immune response by being combined with Toll-like receptor-9 (TLR9) (CpG ODN is generally accepted as immune adjuvant of vaccine and will realize commercial application), and is encapsulated in the nano-carrier, so that a certain slow release effect can be realized, and a synergistic effect can be realized with an alpha HSP70p active peptide segment, and the peptide segment is effectively taken by the dendritic cells.
The particle construction method adopted by the invention specifically comprises the following steps: firstly, preparing a calcium phosphate nanoparticle core carrying CpG ODN by using an inverse microemulsion method, wherein the inverse microemulsion is prepared by dispersing an aqueous solution into a cyclohexane oil phase solution containing nonylphenol polyoxyethylene ether; obtaining tumor cell membranes from B16OVA tumor cells cultured in vitro through ultracentrifugation, dissolving and separating cell membrane proteins through a proper detergent, and then further obtaining cell membrane protein solution through ultracentrifugation; preparing an outer layer by a film hydration method, and adding mixed solution of membrane protein and alpha HSP70p polypeptide for constant temperature hydration after a pre-prepared calcium phosphate nano core and a lipid membrane material are formed into a film together; thereby preparing self-assembled calcium phosphate-lipid nanoparticles.
In the invention, the lipid material comprises a class of lipids with a choline phosphate structure and cholesterol, and is added according to a certain proportion, namely DPPC: DSPC: DOPC: cholesterol was 5:1:3: 1.
The sequence of the alpha HSP70 active polypeptide is as follows:
the stability and drug-loading capacity of the active peptide segment of the biological material adjuvant HSP70p in the artificial lipid material can be enhanced by adding an alpha helical sequence AC-FAEKFKEAVKDYFAKFWD to AC-FAEKFKEAVKDYFAKFWD-GSG-TKDNNLLGRFELSG, and the artificial lipid material can be constructed in an artificial synthesis mode.
According to the invention, the nanoparticle inner core constructed by calcium phosphate and the lipid layer are combined through hydrophobic effect to form nano-scale bionic particles with a core-shell structure;
the average particle size of the calcium phosphate nanometer inner core is 30-40nm, the final particle size of the particles is about 50nm, compared with micron-scale cells, the calcium phosphate nanometer inner core has a larger specific surface area, and can more efficiently play a role of a membrane structure.
The invention adopts a plurality of in vitro and in vivo techniques and evaluation methods, uses dendritic cells (BMDC) from mouse bone marrow as a model to evaluate the in vitro induction DC cell entrance characteristic and the induction maturation function, and adopts the imaging flow type technique to quantitatively evaluate the expression condition of the antigen-MHC complex, wherein the two evaluation methods are both recognized and known in the field.
The invention describes a preparation scheme of the drug delivery system and provides the results of in vitro and in vivo immune activation and pharmacodynamic evaluation of the system.
According to the invention, the dendritic cells (BMDC) derived from mouse bone marrow are used as a model, the functions of taking up and inducing the maturation of the preparation in the dendritic cells are evaluated, and the in-vivo Th1 type cell immunity can be remarkably induced; flow experiment results prove that the preparation can obviously improve the content of specific CD8+ T cells of IFN gamma + in a mouse body; the lung metastasis model nude mouse living body imaging experiment proves that the preparation can be specifically accumulated to a drainage lymph node; pharmacodynamic evaluation and monitoring in vivo show that compared with a free mixture, the drug delivery system can obviously inhibit the formation of metastasis; the content of memory T cells can be remarkably improved after the preparation is immunized; the bionic nano vaccine preparation is combined with a PD-1 antibody for application, the progress of subcutaneous tumor is obviously inhibited, a large number of effector memory T cells can be detected after treatment is finished, and comprehensive results prove that the bionic nano vaccine preparation has an obvious anti-tumor effect and effectively prevents tumor recurrence.
In the invention, the prepared alpha HSP70p-CM-CaP has the grain diameter of about 30nm, and can effectively activate the maturation of mouse bone marrow-derived dendritic cells (BMDC) and promote the presentation of antigen in vitro; in vivo imaging experiments of small animals show that the nanoparticles injected subcutaneously selectively accumulate in draining lymph nodes in mice, and the maintenance time exceeds 24 hours; in sensitization experiments, T cells for identifying various antigen components and killer T cells are detected in a mouse body; the evaluation results of lung metastasis and subcutaneous tumor efficacy models show that the combination treatment scheme of the alpha HSP70p-CM-CaP and the PD-1 monoclonal antibody has obvious inhibition effect, and the existence of memory T cells is continuously monitored for a period of time after the treatment is finished. Furthermore, the vaccine can be combined with the PD-1 antibody for application, and has a higher clinical transformation prospect.
The bionic particle platform has unique advantages in the aspect of anti-tumor immunotherapy:
enriching tumor-associated antigens with high immunogenicity on cell membranes, and building a nano drug delivery platform for precise treatment; secondly, alpha HSP70p is constructed by adopting an alpha helical peptide fusion technology, and the loading capacity of a lipid membrane structure to HSP70 polypeptide is improved, so that synergistic Th1 response to two immunologic adjuvants is realized; thirdly, the calcium phosphate molecules can be highly combined with the DNA skeleton components of the CpG ODN, so that the encapsulation capacity of the calcium phosphate nanoparticles to the CpG ODN is improved; the nano-particle size effect is obvious, the average particle size of about 50nm can improve the uptake efficiency of the particles on dendritic cells, and the delivery of various active ingredients on the same dendritic cell is realized.
Drawings
FIG. 1. characterization of. alpha. HSP70p-CM-CaP, in which,
(A) a calcium phosphate-lipid nanoparticle (alpha HSP70p-CM-CaP) pattern diagram, wherein the artificial lipid membrane structure is lipid and cholesterol with a choline phosphate structure, and the lipid and cholesterol are added according to a certain proportion, namely DPPC: DSPC: DOPC: cholesterol is 5:1:3: 1;
(B) transmission electron micrographs of four nanoparticles, of which (a) the calcium phosphate nanoparticle control group coated with an artificial lipid membrane (without CM and with α HSP70p), referred to as LCP, (B) α HSP70p-CM-CaP, (c) the calcium phosphate nanoparticle control group coated with an artificial lipid membrane loaded with CM (CM-CaP), (d) the B16OVA tumor cell membrane vesicle control group (CMV);
(C) the particle size and zeta potential of four kinds of nanometer particle LCP, alpha HSP70p-CM-Cap, CMV measured in solution;
(D) prescription optimization experiments, namely adding alpha HSP70p, CM and CpG into nano preparation particles according to different proportions, taking the encapsulation rate (%) of the final active ingredients as an evaluation index (n is 3), and selecting the optimal active ingredients as a subsequent experiment prescription;
(E) coomassie brilliant blue staining experimental results;
(F) westernblot results of several key immune activating molecules.
FIG. 2. alpha HSP70p-CM-Cap induces BMDC maturation and promotes antigen-presenting function, wherein,
(A) the in vitro induction effect of alpha HSP70p-CM-Cap on BMDC maturation;
(B) results of quantitative analysis of several cell types in panel a;
(C) activation of antigen presentation by α HSP70p-CM-CaP on BMDC cells;
(D, E) MHC-antigen peptides on the surface of BMDC in Panel CCompound (SIINFEKL-H-2K) b Red) are shown as fluorescence plots and semi-quantitative analysis, and the data were analyzed as mean ± SD (n-3) with significant differences<0.001。
FIG. 3 Selective accumulation of nanoparticles in draining lymph nodes in mice and sensitization to T cells of different phenotypes, wherein,
(A) results of small animal in vivo imaging experiments: (a) alpha HSP70p-CM-Cap-Cy5, (b) alpha HSP70p-CM (Fluorescein) -CaP；
(B) Alpha HSP70p-CM-Cap nanoparticles for identifying CD8 of different antigenic phenotypes + Activation of T cells;
(C) CD8 in Panel B + Quantitative analysis of T cell activation results, data were analyzed as mean ± SD (n ═ 3), and significant differences were observed compared to mice injected with α HSP70p-CM-CaP<0.05,***p<0.001。
FIG. 4. alpha HSP70p-CM-Cap mediate activation of the adaptive immune system in C57BL/6 mice, where (A) alpha HSP70p-CM-Cap immunized IFN-gamma-in the spleen of C57BL/6 mice + CD8 + T cell flow cytogram;
(B) OVA-specific IFN-. gamma.secreting CD8 in Panel A + T cell assay quantification;
(C) ELISA results of cytokines (IFN- γ, IL-4, IL-2, IL-10) in peripheral blood of C57BL/6 mice, data analyzed as mean ± SD (n ═ 3), were significantly different with p <0.01 and p <0.001 compared to mice injected with α HSP70 p-CM-CaP.
FIG. 5 anti-tumor effect in mice immunized with α HSP70p-CM-CaP, wherein,
(A) the inhibition experiment result of the preparation on a mouse lung metastasis model;
(B) quantitative analysis of lung metastasis foci in mice, data analyzed as mean ± SD (n ═ 3), with significant differences compared to mice injected with α HSP70p-CM-CaP, { p } 0.01, { p } 0.001;
(C) shown in the figure is the dosing regimen for the B16OVA mouse subcutaneous tumor model;
(D) post-treatment tumor growth curves, data analyzed as mean ± SD (n ═ 6), were significantly different as compared to mice injected with α HSP70p-CM-CaP + PD-1 mab, × <0.001, × < 0.01;
(E) mouse lymph node effector memory T cell subset (T) 40 days after immunization EM ,CD3 + CD8 + CD62L – CD44 + )；
(F) T in graph E EM The quantitative analysis result shows that compared with mice injected with the alpha HSP70p-CM-CaP + PD-1 monoclonal antibody, the monoclonal antibody has significant difference<0.001。
Detailed Description
Example 1
Preparing an artificial lipid membrane coated calcium phosphate nanoparticle control group (LCP), alpha HSP70p-CM-Cap, an artificial lipid membrane coated calcium phosphate nanoparticle control group (CM-Cap) carrying CM, a B16OVA tumor cell membrane vesicle control group (CMV) according to the method in the claims of the invention, measuring the particle size of the nanoparticles by using a Malvern particle size tester, and observing the form of the nanoparticles by using a transmission electron microscope;
the results show that: the CpG-loaded calcium phosphate nanoparticles are used as an inner core, nanoparticles of 'artificial lipid membranes' embedded with alpha HSP70p polypeptide and cell membrane protein (CM) are coated outside the inner core and are dispersed in an aqueous solution after being successfully prepared, and DLS results show that the average particle sizes of LCP, alpha HSP70p-CM-CaP and CMV are respectively 18.7nm, 31.4nm, 24.7nm and 196.7 nm. Alpha HSP70p-CM-CaP has a slightly increased particle size compared to LCP. The potentials for LCP, α HSP70p-CM-CaP and CMV were-17 mV, -22mV, and-32.3 mV.
An electron microscope image shows that the surface of the polymer nanoparticle is covered with a layer of lipid component, which indicates that the alpha HSP70p-CM-CaP is successfully constructed. The four nanoparticles have uniform size and round shape;
after CM, alpha HSP70p and CpG are respectively marked by different dyes NHS-fluorescein, FITC and Cy5, the encapsulation rate of 3 active ingredients is measured, and the optimal formula alpha HSP70p is obtained: CM: when the weight ratio of CpG is 5:50:2, the encapsulation rates of the three active ingredients are 31.4%, 42.5% and 35.6% respectively;
the method comprises the following steps of (1) carrying out equal-quantity loading on a plurality of groups of samples including B16OVA tumor cells, CMV, CM and alpha HSP70p-CM-CaP, treating the samples by using Coomassie brilliant blue staining solution after electrophoresis is finished, and displaying that western blots of the samples are basically consistent by using a polyacrylamide gel electrophoresis chart (SDS-PAGE), which indicates that cell membrane protein is successfully transferred to the surface of a nano preparation;
western blot results show that key immune activation molecules such as human malignant melanoma associated antigen (gp100), melanocyte tyrosinase associated protein-2 (Trp-2), molecular chaperones (GRP78BiP, calreticulin) and the like are completely retained on B16OVA cells, CMV, CM and alpha HSP70 p-CM-Cap.
Example 2
Immature BMDC (mouse somatic cell death factor) extracted from mice at a rate of 1X 10 6 The density of cells was plated in 12-well plates and cultured at 37 ℃ for 7 days, and the culture medium was changed every other day. The cell culture medium was then incubated with a negative and positive control containing 100ng of alpha HSP70p, 40ng/mL of CpG, 1. mu.g/mL of CM mixed solution and 0.5. mu.g/mL of LPS solution for 12,24 and 48 hours, 37 ℃ respectively. Subsequently, BMDCs were incubated with CD16 antibody at room temperature followed by CD11c, CD80, CD86 and MHCII antibody on ice, and cells were analyzed by flow cytometry after one wash with PBS for CD11c + CD80+, CD11c + CD86+, CD11c + MHC ii + cells. To further verify the antigen presentation function of BMDCs, BMDCs incubated with each of the formulations were incubated with SIINFEKL/H-2K b Antibody incubation, using the same treatment protocol as above for analysis;
the results show that: FIG. 2A shows that the MHCII molecules of BMDC surface CD80 and CD86 molecules incubated with alpha HSP70p-CM-CaP are significantly improved compared with CM-CaP, which indicates that the alpha HSP70p-CM-CaP can significantly improve the maturation of BMDC cells, and the quantitative result in B also proves the conclusion. Data are analyzed as mean ± SD (n ═ 3), p <0.001, p <0.01, p < 0.05;
to further confirm whether OVA antigen components are presented, SIINFEKL-H-2K was labeled with the fluorescent monoclonal antibody APC-25-D1.16 b Complexes, shown in FIG. 2C as a result of imaging flow, BMDC and Fluorescein Fluorescein (Green) labeled groups of reagents, α HSP70p-CM (Fluorescein) -CaP,CM (Fluorescein) V and free CM (Fluorescein) + CpG incubation after 48 hours, different fractions were directed to MHC-antigenic peptide complexes (SIINFEKL-H-2K) on the surface of BMDCs b Red) expression induction. Incubate alpha HSP70p-CM (Fluorescein) Fluorescein and APC signals were strongest in BMDC cells with reagents of-CaP, quantitative results in 2D showed, with CM (Fluorescein) -CaP,CM (Fluorescein) V, and free CM (Fluorescein) + CpG component comparison, α HSP70p-CM (Fluorescein) -CaP-induced fluorescence intensity of BMDC complexes increased 62.3%, 128%, and 116% respectively. Indicating that the uptake of the nano-formulation is enhanced and the MHC-antigenic peptide complex expression is highest due to the entrapment of the active ingredient;
experimental results show that the protein and polypeptide active ingredients simulating the necrotic tumor cells can obviously enhance the uptake of nanoparticles and stimulate the maturation of BMDCs.
Example 3
In vivo imaging experiment of small animals, C57BL/6 mice were randomly divided into two groups, one group was inoculated with nanoparticle alpha HSP70p-CM subcutaneously at the tail of C57BL/6 mice (Fluorescein) -CaP (mean each dosed at 25ng α HSP70p,250ng CM,10ng CpG) and another group was pre-inoculated subcutaneously with nanoparticulate α HSP70p-CM-CaP-Cy5 in mice cauda. Detecting a fluorescence signal in a living body imaging system of a small animal, and for in-vitro research, separating a lymph node of the mouse and taking a picture;
the results show that: as shown in FIG. 3, mice were injected with separate injections of alpha HSP70p-CM (Fluorescein) Signals appeared in the draining lymph nodes after 24 hours and after 24 hours, the signals were significantly enhanced after-CaP and alphahsp 70p-CM-CaP-Cy56 hours, indicating that the nanoparticles could significantly prolong the accumulation time of CM and CpG active ingredients in the lymphatic system, whereas no significant signal was detected in the free active ingredient injected formulation groups during both time periods. Lymph nodes were removed after 6 and 24 hours, respectively, for semiquantitative analysis, and the results showed that the fluorescence signal values of tumor antigen components and CpG were significantly higher than 6 hours in 24 hours in lymph nodes.
Example 4
In T cell in vivo sensitization experiments, C57BL/6 mice were inoculated subcutaneously with equal amounts of HSP70p-CM-Cap, free CM + CpG, LCP, CM-Cap, CMV and PBS 3 times. Taking animal peripheral blood and analyzing PE-H-2K by flow cytometry result b OVA and PE-H-2K b Various subtypes of CD8 alpha labeled with Trp2 tetramer + The number of T cells;
the results show that: OVA and Trp2 specific CD8 in mice immunized with alpha HSP70p-CM-CaP and CM-CaP + The T cell content is obviously higher than that of mice immunized by the CMV group, the LCP group and the free group, which shows that adaptive immunity can be activated by simultaneously delivering CM and CpG to a lymphatic system, and the content of specific T cells is increased, wherein the content of OVA specific T cells in the mice immunized by the alpha HSP70p-CM-CaP is 5 times of that of the free group, the content of Trp-2 specific T cells is 7.2 times of that of the free group, and the alpha HSP70p-CM-CaP greatly improves the antigen presentation function of DC cells, so that the subsequent adaptive immunity is efficiently activated.
Example 5
In OVA-specific killer T cell activation experiments, T cells in the kidney of C57BL/6 mice immunized with each group of the preparation were removed at 1X 10 6 (ii) one/well Density in well plates, incubation of 20. mu.M antigen OVA polypeptide for 24 hours, further incubation with BFA-containing uptake inhibitor for 6 hours, followed by labeling with CD3e-Percp-Cy5.5 antibody, and determination of OVA-specific IFN-. gamma. + CD8 + T cell levels (approximately 25ng of alphahsp 70p,250ng of CM,10ng of CpG per mouse were administered). Treating blood of the same mouse by an ELISA method, and detecting the contents of IFN-gamma, IL-4, IL-2 and IL-10;
the results show that: IFN-gamma secreting CD8 was directly measured in C57BL/6 mice inoculated with alpha HSP70p-CM-CaP + T cell content was 5.51,0.37 and 10.29 times higher than in the free CM + CpG solution group, CM-CaP group and LCP group. The fact that the capacity of T cells for secreting IFN-gamma is the strongest under the condition that antigen, alpha HSP70p and CpG are delivered simultaneously indirectly proves that the entrapment of the alpha HSP70p has a certain promotion effect on the activation of the overall killing effect. Activation of downstream killer T cells without leaving helper TThe secretion levels of a plurality of cytokines of h1 and Th2 cells can reflect the activation level of helper T cells, experiments show that the IL-2 and IFN-gamma levels of mice immunized by alpha HSP70p-CM-CaP are respectively 3.9 and 123 times of those of PBS group, and the IL-10 and IL-4 levels are not obviously different from those of PBS group, which indicates that the nanoparticle promotes the differentiation of downstream killer T cells by triggering Th1 type reaction, and the secretion levels of IL-2 and IFN-gamma in mice immunized by CM-CaP are stronger than free CM + CpG, which indicates that the combined delivery of CM and CpG can induce Th1 type immune reaction to a certain extent.
Example 6
In an evaluation test on a tumor metastasis lesion model, mice were immunized 3 times with each group of formulations within 20 days (each mouse was dosed with approximately 25ng of α HSP70p,250ng of CM,10ng of CpG). After 20 days, each mouse was adjusted to 5X 10 5 B16OVA cells are injected to establish a metastatic focus model in a mouse lung metastasis model. And taking the lung of the mouse after another 20 days for observation, and detecting the number of lung metastasis focuses. To evaluate the combined therapeutic effect of the vaccine and PD-1, on day 0, 2X 10 s.c. was previously injected in the mouse subcutaneous tumor suppression experiment 5 B16OVA cells, after which mice were immunized 3 times on days 10,17,23, respectively, with the same formulation and dose, injected with PD-1 antibody (100 μ g per mouse) after each immunization, the size of the tumor was recorded every other day, measured with a vernier caliper, and the tumor size was calculated:
the results show that: as shown in FIG. 5A, in the mouse lung metastasis experiment, mice immunized by alpha HSP70p-CM-CaP and CM-CaP receive tumor cell injection again, only a trace amount of metastasis is generated, and other PBS (phosphate buffer solution) groups, free preparation groups, CMV (cytomegalovirus) groups and LCP (liquid Crystal Polymer) groups all have macroscopic metastasis, which indicates that the two preparations can possibly inhibit the formation of the lung metastasis of the mice, and the alpha HSP70p-CM-CaP effect is better than that of the CM-CaP group after artificial counting, and the number of the metastasis is reduced by 77%. The carrier band of the alpha HSP70p can obviously inhibit the formation of final metastatic lesions, the thought of the simulated necrosis signal provides a new way for preventing tumors and shows the superiority of treatment, the experiment continues to adopt the therapeutic effect of a mouse weighing preparation of a subcutaneous tumor treatment model, as shown in figure 5D, compared with a PBS (phosphate buffer solution) group, the free preparation group can not obviously slow down the growth of the tumors, after the treatment by combining a PD-1 antibody, the final free group + PD-1 monoclonal antibody combination can reduce the tumor growth by 46.2 percent, and particularly, after the combined treatment of the alpha HSP70p-CM-CaP and the PD-1 monoclonal antibody, the tumor inhibition rate is 89 percent. The result well indicates that the bionic nano preparation has strong inhibition effect on melanoma.
Example 7
Detection of memory T cell production in the assay, 40 days after the PD-1 mab combination therapy was completed, the lymph nodes were harvested, the connective tissue was removed by gentle abrasion on cell filters, the lymphocytes were extracted after PBS washing, and the cells were purified by anti-CD3e-Percp-Cy5.5(eBioscience,48-0031-82), anti-CD8a-Alexa
647(Biolegend,100724), anti-CD44-FITC (ebioscience,11-0441-82) and anti-CD62L (L-Selectin) -PE (ebioscience,12-0621-82), and detecting on a flow cytometer;
the results show that: CD8 in mice immunized with alpha HSP70p-CM-CaP + The two types of memory T cell content were different, as shown in FIG. 5E&F, effector memory T cells (T) EM Cell, CD3 + CD8 + CD62L – CD44 + ) The highest content accounts for lymph node immune cell CD8 + The number of T cells is 40 percent and is obviously higher than that of the free preparation group; central memory T cells (T) CM Cell, CD3 + CD8 + CD62L + CD44 + ) At the lowest level, the cells are not as effective as T in long-term protective function EM The cellular importance indicates that the alpha HSP70p-CM-CaP has long-term protection effect and prevents the recurrence of the tumor.
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Claims (12)
1. A calcium phosphate-lipid nano vaccine simulating necrotic tumor cells is characterized in that the nano bionic particles are of a core-shell structure, a nano inner core constructed by calcium phosphate and a lipid bilayer shell artificially synthesized are combined through hydrophobic effect, the calcium phosphate of the inner core is hydrophobic nano particles efficiently encapsulating oligonucleotide substances, and the shell is an artificial lipid material highly simulating the fluidity of cell membranes and is loaded with natural or genetically engineered cell membrane protein and HSP70 active peptide segments.
2. The necrotic tumor cell-mimicking calcium phosphate-lipid nanoball of claim 1, wherein the artificial lipid material of the outer shell is loaded with natural multivalent tumor cell membrane antigen protein and alpha helix modified HSP70 active peptide fragment (alpha HSP70 p); the inner core takes calcium phosphate (CaP) as a material to prepare nanoparticles and entraps oligonucleotide CpG serving as an adjuvant;
the vaccine adopts oligonucleotide CpG and HSP70p as adjuvant components.
3. The calcium phosphate-lipid nanoball simulating necrotic tumor cells of claim 1, wherein the non-methylated oligodeoxynucleotides (CpG ODN for short) as adjuvant components used in the inner core of calcium phosphate are artificially synthesized DNA sequences containing non-methylated CpG oligonucleotides with immunostimulating effect, and the CpG ODN sequence is CpG ODN 1826 (5'-TCCATGACGTTCCTGACGTT-3').
4. The nano calcium phosphate-lipid vaccine mimicking necrotic tumor cells of claim 1, wherein the lipid of said outer shell has an adjuvant component of alpha helix modified HSP70 active peptide (alpha HSP70 p).
5. The nano calcium phosphate-lipid vaccine mimicking necrotic tumor cells of claim 1, wherein said cell membrane protein is a multivalent tumor cell membrane protein component selected from the group consisting of mouse-derived B16OVA cells, a genetically engineered mouse melanoma cell line stably expressing OVA model antigens obtained by in vitro amplification and extraction.
6. The nano calcium phosphate-lipid vaccine mimicking necrotic tumor cells of claim 1, wherein said artificial lipid membrane material is lipid and cholesterol of phosphocholine structure, including DPPC, DSPC, DOPC and cholesterol.
7. The calcium phosphate-lipid nanoball simulating necrotic tumor cells of claim 1, wherein the inner core comprises nucleic acid drugs selected from the group consisting of adjuvant oligonucleotide CpG ODN, siRNA, and DNA.
8. The calcium phosphate-lipid nanoball simulating necrotic tumor cells of claim 3, wherein the adjuvant component CpG ODN, has strong innate immune stimulation effect, and is mediated by TLR9 to directly activate innate immune system and activate various immune cells to induce B cell differentiation and T cell differentiation.
9. The calcium phosphate-lipid nanoball simulating necrotic tumor cells of claim 4, wherein the alpha-helix modified HSP70 active peptide fragment (alpha HSP70) has the sequence AC-FAEKFKEAVKDYFAKFWD-GSG-TKDNNLLGRFELSG, which is prepared by adding the alpha-helix sequence AC-FAEKFKEAVKDYFAKFWD to HSP70 active peptide fragment TKDNNLLGRFELSG by lipid insertion technique.
10. The calcium phosphate-lipid nanoball simulating necrotic tumor cells of claim 4, wherein the alpha helix modified HSP70 active peptide fragment (alpha HSP70) is the active component of HSP70, which can directly activate CD49 molecules on dendritic cells and Natural Killer (NK) cells.
11. The nano calcium phosphate-lipid vaccine for simulating necrotic tumor cells as in claim 1, wherein the nano biomimetic particles with core-shell structure have an average particle size of 30-40nm for the nano calcium phosphate core and a final particle size of about 50nm after coating with artificial lipid membrane.
12. Use of the calcium phosphate-lipid nanobvaccine mimicking necrotic tumor cells of claim 1 for the preparation of an agent for inhibiting the growth and metastasis of tumors in vivo by subcutaneous administration route, local targeting to dendritic cells in draining lymph nodes after immunization of mice, serial downstream immune activation after in vivo activation, induction of Th1 type cytokine response, production of killer CD8+ T Cells (CTLs), and production of helper CD4+ cells.
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| CN110898215A (en) * | 2019-12-06 | 2020-03-24 | 郑州大学 | Preparation method and application of anti-tumor vaccine based on cell microvesicles |
| CN114306623B (en) * | 2021-12-10 | 2023-08-22 | 浙江大学杭州国际科创中心 | Core-shell calcium phosphate polypeptide composite nano vaccine and preparation method thereof |
| CN114306244B (en) * | 2022-01-14 | 2023-04-07 | 苏州尔生生物医药有限公司 | Micron-sized lipid compound and preparation and application thereof |
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