CN114053433A - Stimulus response type nano material and application thereof in preparing in-situ tumor vaccine - Google Patents
Stimulus response type nano material and application thereof in preparing in-situ tumor vaccine Download PDFInfo
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Abstract
本公开涉及一种刺激响应型纳米材料，可与冷冻消融产生的抗原联合形成纳米原位肿瘤疫苗，能够实现淋巴结靶向、体内肿瘤碎片吸附、靶向活化抗原呈递细胞、PH响应药物控释、载药等功能，为肿瘤免疫治疗提供新方法与新技术。一方面冷冻消融产生的肿瘤碎片保留着更多的抗原信息，可更高效的活化抗原呈递系统；另一方面刺激响应型纳米材料有效吸附肿瘤碎片，同时可搭载模式识别受体激动剂或药物，从而增强靶向及活化抗原提呈细胞能力。本公开制备的刺激响应型纳米原位肿瘤疫苗通过负载冷冻消融产生的肿瘤抗原，弥补了冷冻消融对远端病灶及游离肿瘤细胞杀伤不足，有望在对局部病灶产生杀伤的同时诱导全身免疫治疗。The present disclosure relates to a stimuli-responsive nanomaterial, which can be combined with an antigen produced by cryoablation to form a nano-in situ tumor vaccine, which can achieve lymph node targeting, tumor debris adsorption in vivo, targeted activation of antigen-presenting cells, pH-responsive drug controlled release, Drug loading and other functions provide new methods and new technologies for tumor immunotherapy. On the one hand, the tumor fragments generated by cryoablation retain more antigen information, which can activate the antigen presentation system more efficiently; on the other hand, the stimuli-responsive nanomaterials can effectively adsorb tumor fragments, and can carry pattern recognition receptor agonists or drugs at the same time. Thereby enhancing the ability to target and activate antigen-presenting cells. The stimuli-responsive nano-in situ tumor vaccine prepared by the present disclosure makes up for the insufficient killing of remote lesions and free tumor cells by cryoablation by loading tumor antigens produced by cryoablation, and is expected to induce systemic immunotherapy while killing local lesions.
Description
Technical Field
The disclosure belongs to the field of high polymer materials, and particularly relates to a stimulus-responsive nano material and a nano in-situ tumor vaccine formed by loading an antigen generated by cryoablation, and a preparation method and application thereof.
Background
In recent years, development of tumor treatment means represented by surgical resection, radiotherapy and chemotherapy is a bottleneck, and immunotherapy, as an emerging treatment mode, has shown a higher application prospect in treatment of various tumors. At present, tumor immunotherapy is mainly divided into active immunotherapy and passive immunotherapy, and compared with passive immunization represented by CAR-T, antibody and cytokine therapy, active immunization represented by tumor vaccine and the like not only has better curative effect in a plurality of tumors, but also has the advantages of long-term immunity and low adverse reaction.
At present, the combination of the physical therapy and the in-situ tumor vaccine is the front of discipline, wherein the combination of the antigen generated by the cryoablation and the in-situ tumor vaccine is still a blank field, and the combination of the nano in-situ antigen vaccine and the cryoablation and the generated antigen thereof is expected to become a new tumor treatment mode. Although the tumor fragments generated in the tumor area after cryoablation cannot effectively activate immune response due to insufficient immunosuppressive environment of the tumor area and insufficient activation of antigen presenting cells, the tumor fragments retain more complete antigen information because the biological properties are not changed by freezing, and the tumor fragments are hopeful to provide antigen fragments with stronger immunogenicity for nano tumor vaccines. While other tumor physical therapies such as radiotherapy, photothermal therapy, radiofrequency ablation, photodynamic therapy and the like can cause the change of the tumor fragment characters, for example, the photothermal therapy and the radiofrequency ablation can cause protein denaturation, and the radiotherapy and the photodynamic therapy can cause the change of genetic materials. Cryoablation may be the optimal tumor physiotherapy with in situ tumor vaccines. On one hand, tumor fragments generated by cryoablation retain more antigen information, and an antigen presentation system can be activated more efficiently; on one hand, the surface loading capacity of the nano particles is limited, and after the task of manufacturing the tumor antigen is replaced by cryoablation, the nano particles can carry more pattern recognition receptor agonists or medicines, so that the antigen presenting cell targeting and activating capacity is enhanced.
In view of the above, the disclosure provides a stimuli-responsive nanomaterial and an antigen loaded by the nanomaterial generated by cryoablation to form a nano in-situ tumor vaccine, including a related preparation method and an application thereof.
Disclosure of Invention
The disclosure provides a stimulus response type nano material and an antigen generated by loading cryoablation thereof to form a nano in-situ tumor vaccine so as to realize the functions of lymph node targeting, in-vivo tumor fragment adsorption, targeted activation of antigen presenting cells, PH response drug controlled release, drug loading and the like, and provides a new method and a new technology for tumor immunotherapy.
Another object of the present disclosure is to provide a method for preparing the stimuli-responsive nanomaterial.
It is yet another object of the present disclosure to propose the use of said stimuli-responsive nanomaterial to form a nano in situ tumor vaccine therapy by loading antigens generated by cryoablation.
The present disclosure provides a stimulus-responsive nanomaterial, wherein the stimulus-responsive nanomaterial comprises a stimulus-responsive carrier, an antigen-adsorbing functional group and a pattern-recognition receptor agonist or drug, wherein the stimulus-responsive carrier is capable of responding to environmental changes, the antigen-adsorbing functional group is capable of adsorbing tumor antigens, and the pattern-recognition receptor agonist or drug is capable of enhancing targeting of antigen-presenting cells and activating the antigen-presenting ability thereof.
The present disclosure provides a stimulus-responsive nanomaterial, wherein the stimulus-responsive nanomaterial is in a spherical shell shape, the spherical shell is formed by a stimulus-responsive carrier, the surface of the stimulus-responsive carrier is linked with an antigen-adsorbing functional group, and a pattern recognition receptor agonist or a drug is encapsulated in the spherical shell formed by the stimulus-responsive carrier.
The present disclosure provides a stimulus-responsive nanomaterial, wherein the stimulus-responsive carrier is selected from one or more of polyethylene glycol (PEG), propylene oxide-ethylene oxide copolymer (Poloxamer), polylactic-co-glycolic acid (PLGA), polyglycolic acid (PGA), chitosan, or derivatives thereof; preferably, the stimulus responsive carrier is selected from one or more of Pluronic F127 and/or chitosan.
The present disclosure provides the stimulus-responsive nanomaterial of any one of the above, wherein the antigen-adsorbing functional group is selected from maleimide and derivatives thereof.
In one aspect of the present disclosure, wherein, preferably, the antigen adsorbing functional group is selected from one or more of maleimide, N-methylmaleimide, N-cyclohexylmaleimide, maleimidobutyric acid, maleimidopolyethylene glycol maleimide, phospholipid polyethylene glycol maleimide.
The present disclosure provides any one of the above stimuli-responsive nanomaterials, wherein the pattern recognition receptor agonist or drug is selected from one or more of interferon inducers, immune activators, and anti-tumor drugs.
In one aspect of the present disclosure, wherein, preferably, the pattern recognition receptor agonist or drug is selected from one or more of polynucleotide, polyinosinic acid, polyinosinic-polycytidylic acid, ginseng polysaccharide, astragalus polysaccharide, hemagglutinin, canavalin, and placental polysaccharide.
In another aspect of the present disclosure, wherein, preferably, the pattern recognition receptor agonist or drug is astragalus polysaccharides and/or polyinosinic-polycytidylic acid.
The present disclosure provides any one of the above stimuli-responsive nanomaterials, wherein the stimuli-responsive carrier, the antigen-adsorbing functional group, the pattern recognition receptor agonist or the drug in the stimuli-responsive nanomaterials are in a mass ratio of (5-18): (1-3): (1-3), preferably (8-15): (1-2): (1-2), more preferably 10: 1: 1.
the present disclosure provides a method for preparing any one of the above stimuli-responsive nanomaterials, which is characterized in that the method comprises the following steps:
(1) preparing a stimulus-responsive polymer carrier: preparing a stimulus-responsive polymer carrier by using a nano-particle preparation method by using a biocompatible polymer material as a raw material;
(2) surface functional modification of stimulus response type polymer carrier: crosslinking an antigen adsorption functional group with the stimulus response type high polymer material to finish surface functional modification;
(3) packaging: mixing the shock response type nanometer material with a pattern recognition receptor agonist or medicine to be loaded, and completing encapsulation by utilizing the expansion characteristic of the shock response type nanometer material under low-temperature stimulation;
preferably, the nanoparticle preparation method in step (1) comprises one or more of an ultrasonic method, a micro-emulsion method, a double-emulsion method and a reverse-emulsion method;
preferably, the antigen adsorption functional group is crosslinked with the prepared stimulus-responsive polymer material in step (2) by a chemical crosslinking method.
The present disclosure provides a pharmaceutical composition, wherein the pharmaceutical composition comprises any one of the above stimuli-responsive nanomaterials; preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers or excipients.
The present disclosure provides an application of any one of the above stimuli-responsive nanomaterials or the above pharmaceutical compositions in the preparation of a medicament for preventing or treating tumors.
In one aspect of the disclosure, the use of the stimuli-responsive nanomaterial according to any one of the above or the pharmaceutical composition as described above in the preparation of a medicament for preventing or treating a tumor, wherein the medicament for preventing or treating a tumor is a tumor vaccine; preferably, the medicament for preventing or treating the tumor is a stimulus-responsive nano in-situ tumor vaccine; preferably, the stimulus-responsive nano in situ tumor vaccine comprises any one of the stimulus-responsive nano materials and tumor antigen generated by cryoablation; preferably, the stimulus-responsive nano in situ tumor vaccine is prepared by loading tumor antigens generated by cryoablation on any one of the stimulus-responsive nano materials.
The technical scheme of the disclosure has the following advantages:
(1) the tumor vaccine disclosed by the invention can utilize the difference between the intracellular environment (PH is 5.6) and the extracellular environment (PH is 7.4) after endocytosis, not only can realize drug controlled release, but also can destroy the internal balance of an inner membrane/lysosome through PH response, thereby breaking the speed-limiting step of antigen presentation and improving the antigen presentation efficiency;
(2) ability to capture tumor fragments: the surface of the nano tumor vaccine is modified by maleimide, and has the capacity of capturing tumor fragments through in vitro experiment detection, wherein the antigen adsorption capacity is about 762 mu g/mg;
(3) lymph node targeting ability: as proved by researches, substances with the tumor part smaller than 30nm are metabolized through blood backflow, substances with the tumor part of 30nm to 400nm are mostly metabolized through lymphatic vessel backflow, so the size of nano particles is controlled within the range of 30nm to 400nm before and after antigen adsorption, CY5 fluorescent dye is used for marking nano tumor vaccine, a living body imaging system is utilized, 24 hours after nano tumor vaccine is injected into a mouse tumor, the living body imaging system is dissected, and the bilateral inguinal lymph nodes and axillary lymph nodes are taken out, and the fluorescence response is found;
(4) the biological safety of the main material: the main body of the nano-particle is constructed by Pluronic F127 and/or chitosan, so that the DC phagocytosis can be enhanced, and the nano-particle has high biological safety property;
(5) can stably carry the medicine: through absorbance detection, the nano in-situ tumor vaccine disclosed by the invention can effectively carry Astragalus Polysaccharide (APS) or Lipopolysaccharide (LPS);
(6) the APNPs and PNPs disclosed by the invention have the effects of breaking the immunosuppression environment of a tumor region and enhancing T cell recruitment after cryoablation, and have a synergistic effect.
(7) The PNPs and APNPs of the present disclosure, in combination with cryoablation-generated antigens, can inhibit distal tumor growth and reduce the recurrence rate of in situ tumor after ablation.
(8) The present disclosure not only has the above in vivo lymph node targeting capability, can activate systemic immunotherapy, and cryoablation can prolong the time of stasis of the APNPs and PNPs in the tumor region.
The stimulus-responsive nanomaterial provided by the disclosure can be combined with an antigen generated by cryoablation to form a nano in-situ tumor vaccine, can realize the functions of lymph node targeting, in-vivo tumor fragment adsorption, targeted activation of antigen presenting cells, PH response drug controlled release, drug loading and the like, and provides a new method and a new technology for tumor immunotherapy. On one hand, tumor fragments generated by cryoablation retain more antigen information, and an antigen presentation system can be activated more efficiently; on the other hand, the stimulus response type nano material can effectively adsorb tumor fragments and can carry a mode recognition receptor agonist or a drug, so that the capability of targeting and activating antigen presenting cells is enhanced. The antigen generated by the cryoablation and the stimuli-responsive nano in-situ tumor vaccine disclosed by the invention make up the defect of the cryoablation on killing the far-end focus and free tumor cells, and are expected to induce the whole-body immunotherapy while killing the local focus.
Drawings
Fig. 1 is a schematic diagram of a stimulus-responsive nanomaterial of the present disclosure.
Fig. 2 is a schematic diagram of the combination of nanomaterials of the present disclosure with cryoablation and antigens produced thereby to form a nano in situ tumor vaccine.
Fig. 3 is a graph showing a change in particle size of the nanomaterial in response to pH in effect example 1 of the present disclosure.
Fig. 4 is a graph showing a change in particle size of the nanomaterial in effect example 1 of the present disclosure in response to temperature.
Fig. 5 is a graph of the co-localization analysis of antigen and lysosome of the nano in situ tumor vaccine of effect example 2 of the present disclosure.
Fig. 6 is a diagram of flow-based quantitative analysis of phagocytosis by nanomaterial Dendritic Cells (DCs) in example 3 of the present disclosure.
Fig. 7 is a graph showing the ability of the nanomaterial in effect example 4 of the present disclosure to adsorb tumor fragments.
Fig. 8 is a graph that measures the ability of nanomaterials to stimulate DC maturation in effect example 5 of the present disclosure.
Fig. 9 shows the nanomaterial cytotoxicity test in effect example 6 of the present disclosure.
Fig. 10 shows primary tumor growth curves for each group in effect example 7 of the present disclosure.
Fig. 11 shows the distal tumor growth inhibition curves for each group in effect example 7 of the present disclosure.
Fig. 12 shows lung metastasis in each group in effect example 7 of the present disclosure, and metastatic tumors are shown in the circular arc.
Fig. 13 shows survival of mice in effect example 7 of the present disclosure.
Fig. 14 shows a thermographic image of a mouse lymph node in effect example 8 of the present disclosure.
Fig. 15 shows the proportion of T cells in distal tumors of mice in effect example 9 of the present disclosure.
FIG. 16 shows the proportion of MDSC cells in distal tumors of mice in example 9 of the effect of the present disclosure.
Fig. 17 shows the proportion of DC cells in distal tumors of mice in effect example 9 of the present disclosure.
Fig. 18 shows the proportion of Treg cells in distal tumors of mice in effect example 9 of the present disclosure.
Fig. 19 shows the proportion of Treg cells in the spleen of mice in effect example 9 of the present disclosure.
Fig. 20 shows the proportion of DC cells in the spleen of a mouse in effect example 9 of the present disclosure.
Detailed Description
Based on the above disclosure, other modifications, substitutions and alterations can be made without departing from the basic technical concept of the present disclosure as it is known and customary in the art.
I. Definition of
Throughout the specification and claims, unless explicitly stated otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component.
The term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The term "treating" includes inhibiting, alleviating, preventing or eliminating one or more symptoms or side effects associated with the disease, disorder or condition being treated.
The terms "reduce", "inhibit", "reduce" or "decrease" are used relative to a control. One skilled in the art will readily determine the appropriate control for each experiment. For example, a decreased response in a subject or cell treated with a compound is compared to a response in a subject or cell not treated with a compound.
As used herein, the term "effective amount" or "therapeutically effective amount" refers to a dose sufficient to treat, inhibit or alleviate one or more symptoms of the disease state being treated or to otherwise provide a desired pharmacological and/or physiological effect. The precise dosage will vary depending on a variety of factors, such as the subject-dependent variables (e.g., age, immune system health, etc.), the disease or disorder, and the treatment being administered. The effective amount of the effect may be relative to a control. These controls are known in the art and discussed herein, and may be, for example, the condition of the subject prior to or without administration of the drug or drug combination, or in the case of a drug combination, the effect of the combination may be compared to the effect of administration of only one drug.
The term "pharmaceutical composition" means a composition comprising at least one pharmaceutically acceptable ingredient selected from the group consisting of, but not limited to, the following, depending on the mode of administration and the nature of the dosage form: carriers, diluents, adjuvants, excipients, preservatives, fillers, disintegrating agents, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, temperature sensitive materials, temperature regulating agents, adhesives, stabilizers, suspending agents, and the like.
The term "excipient" is used herein to include any other compound that is not a therapeutic or biologically active compound that may be contained in or on the microparticles. Thus, the excipient should be pharmaceutically or biologically acceptable or relevant, e.g., the excipient is generally non-toxic to the subject. "excipient" includes a single such compound, and is also intended to include multiple compounds.
The foregoing and other aspects of the present disclosure are achieved by the following detailed description of the embodiments. It should not be understood that the scope of the above-described subject matter of the present disclosure is limited to the following examples. All the technologies realized based on the above contents of the present disclosure belong to the scope of the present disclosure.
Example II
The disclosure is further illustrated with reference to the following examples. The description of the specific exemplary embodiments of the present disclosure has been presented for purposes of illustration and description. It is not intended to limit the disclosure to the precise form disclosed, and obviously many modifications and variations are possible in light of the teaching of the present disclosure. The exemplary embodiments were chosen and described in order to explain certain principles of the disclosure and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the disclosure and various alternatives and modifications thereof.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1: temperature and PH stimulus response type nano material APNPs and preparation thereof
The embodiment is a preparation method of a temperature and PH stimulus responsive nano material, which specifically comprises the following steps:
(1) preparation of stimulus-responsive polymer carrier
Pluronic F127(300 mgmL) activated with 4-nitrophenyl chloroformate (4-NPC)-1500mL) was dissolved in methylene chloride, and added dropwise to an aqueous chitosan solution (15 mgmL) under sonication (30% ultrasonic power, 3 minutes)-15mL, pH 10), after removing dichloromethane by rotary evaporation, the resulting solution was dialyzed overnight in deionized water using a dialysis tube (50kDa), and then dialyzed in deionized water using a (1000kDa) dialysis tube for 3 hours, and the prepared stimulus-responsive polymeric carrier was lyophilized for further use;
(2) surface functional modification of stimulus response type polymer carrier
N- (methoxycarbonyl) maleimide (0.1mM) was added to the prepared aqueous stimulus-responsive polymer carrier solution (10 mgmL)-1) After the crosslinking is finished, dialyzing the mixture in ionized water for 8 hours by a (20kDa) dialysis tube to remove unreacted N- (methoxycarbonyl) maleimide and finish surface functional modification;
(3) package with a metal layer
Adding Astragalus polysaccharides (1mg) into shock response type nanometer material water solution (10 mgmL)-110mL), free drug can be encapsulated after swelling for 1h at 4 ℃, then the drug-encapsulated stimuli-responsive nanomaterial is dialyzed in ionized water for 8 hours to remove unencapsulated drug, and finally, the preparation (APNPs) is finished by freeze-drying.
The prepared temperature and PH stimulus responsive nano material can be combined with cryoablation and antigens generated by the cryoablation for stimulus responsive nano in-situ tumor vaccine therapy, and is expected to induce whole-body immunotherapy while killing local lesions.
The temperature and PH stimuli responsive nanomaterials of this example are schematically illustrated in fig. 1, and their stimuli responsive nano in situ tumor vaccines in combination with cryoablation and its generated antigens are schematically illustrated in fig. 2.
Example 2: temperature and PH stimulus response type nano material PNPs and preparation thereof
The same procedure as that for preparing APNPs in example 1 is carried out, and temperature and PH stimulus responsive nanomaterial PNPs are obtained only by adding no Astragalus Polysaccharide (APS) during packaging.
Example 3: other temperature and PH stimulus responsive nano material and preparation thereof
This example differs from example 1 only in that the methylene chloride in step (1) of example 1 is replaced with other organic solvents such as styrene, perchloroethylene, trichloroethylene, ethylene glycol ether and triethanolamine.
In this example, temperature and PH stimuli responsive nanomaterials encapsulating astragalus polysaccharides and polyinosinic-polycytidylic acid were prepared.
Example 4: other temperature and PH stimulus responsive nano material and preparation thereof
This example differs from example 1 only in that the concentration of Pluronic F127 in step (1) of example 1 was changed (100 to 500 mgmL)-1)；
In this example, temperature and PH stimuli responsive nanomaterials encapsulating astragalus polysaccharides and polyinosinic-polycytidylic acid were prepared.
Example 5: other temperature and PH stimulus responsive nano material and preparation thereof
This example differs from example 1 only in that the concentration of the aqueous chitosan solution in step (1) of example 1 was changed (10 to 100 mgmL)-1)。
In this example, temperature and PH stimuli responsive nanomaterials encapsulating astragalus polysaccharides and polyinosinic-polycytidylic acid were prepared.
Example 6: other temperature and PH stimulus responsive nano material and preparation thereof
The present embodiment is different from embodiment 1 only in that the ultrasonic treatment power and time in step (1) of embodiment 1 are changed (ultrasonic power is 10 to 70%, 3 to 20 minutes).
In this example, temperature and PH stimuli responsive nanomaterials encapsulating astragalus polysaccharides and polyinosinic-polycytidylic acid were prepared.
Example 7: other temperature and PH stimulus responsive nano material and preparation thereof
This example differs from example 1 only in that the N- (methoxycarbonyl) maleimide in step (2) of example 1 was replaced with other maleimide derivatives such as N-methylmaleimide, N-cyclohexylmaleimide, maleimidobutyric acid and the like.
In this example, temperature and PH stimuli responsive nanomaterials encapsulating astragalus polysaccharides and polyinosinic-polycytidylic acid were prepared.
Example 8: other temperature and PH stimulus responsive nano material and preparation thereof
This example is different from example 1 only in that the astragalus polysaccharide and polyinosinic-polycytidylic acid in step (3) of example 1 are replaced by one or more other drugs, such as other artificially synthesized double-stranded RNA (e.g. polynucleotide, polyinosinic acid, etc.), polyamine compounds, fungal polysaccharides, traditional Chinese medicine and other traditional Chinese medicine effective components (e.g. ginseng, astragalus, hemagglutinin, sword bean protein, placental polysaccharide, etc.).
In this embodiment, a temperature and PH stimulus responsive nanomaterial with an encapsulated mode for recognizing a receptor agonist or a drug is prepared.
Effect example 1: the disclosed nano material is characterized and functionally tested
The experimental method comprises the following steps: dissolving the nano-material APNPs prepared in example 1 in a PBS solution at a concentration of 1mg/ml, observing by a transmission electron microscope after ultrasonic stirring, detecting the particle size change of the nano-material at different pHs and temperatures by a nano-particle size and Zeta potential analyzer as shown in figures 3 and 4, detecting the APS drug entrapment rate of 10% by a Nanodrop spectrophotometer, and ultrasonically mixing the APNPs and FITC-OVA (Bioss company, cat # bs-0283P-FITC) according to a ratio of 1:1 to obtain an APNPs-FITC-OVA compound solution (APNPs-MAL @ OVA); PNPs-FITC-OVA Complex solution (PNPs-MAL @ OVA) was prepared in a similar manner using PNPs
The experimental results are as follows:
experiments prove that the nano material disclosed by the invention has the following characteristics: (1) temperature responsiveness and PH responsiveness, particle size changes can occur at different PHs and/or temperatures, which indicates that the nanomaterial disclosed in the present disclosure has responsiveness to changes in environmental temperature and PH (fig. 3 and fig. 4), and meanwhile, the nano tumor vaccine disclosed in the present disclosure can not only achieve controlled drug release by utilizing the difference between the intracellular environment (PH 5.6) and the extracellular environment (PH 7.4) after endocytosis, but also show that PH response can break the internal equilibrium of intima/lysosome, thereby breaking the rate-limiting speed for antigen presentation; (2) ability to capture tumor fragments: the surface of the nano tumor vaccine is modified by maleimide, and the nano tumor vaccine has the capacity of capturing tumor fragments through in vitro experiment detection; (3) lymph node targeting ability: as the research proves that substances with the tumor part being less than 30nm are metabolized through blood reflux, and substances with the tumor part being 30nm to 400nm are mostly metabolized through lymphatic vessel reflux, the size of the nano particles is controlled within the range of 30nm to 400nm before and after antigen adsorption; (4) the biological safety of the main material: the main body of the nano-particle is constructed by Pluronic F127 and/or chitosan, so that the DC phagocytosis can be enhanced, and the nano-particle has high biological safety property; (5) can stably carry the medicine: through absorbance detection, the nano in-situ tumor vaccine disclosed by the invention can be effectively loaded with Astragalus Polysaccharide (APS) or Lipopolysaccharide (LPS).
Effect example 2: the disclosed detection experiment for escape capacity of lysosome of nano material
Grouping experiments:
PNPs-MAL @ OVA experimental group: PNPs-FTIC-OVA Complex solution (prepared in reference Effect example 1)
APNPs-MAL @ OVA experimental group: APNPs-FTIC-OVA complex solution (prepared according to Effect example 1)
OVA control group: FITC-OVA solution (Bioss Co., Cat # bs-0283P-FITC)
The experimental method comprises the following steps:
1) preparation of mouse bone marrow-derived dendritic cells (BMDCs): killing the mouse by removing the neck, taking out the femur and the tibia through an operation, washing out bone marrow to a bacterial culture dish (adding 15mL of culture medium) with the diameter of 100mm, collecting bone marrow suspension, filtering by using a 200-mesh nylon net, centrifuging the filtrate at 1200r/min for 5min, discarding supernatant, adding 0.3mL of erythrocyte lysate into the filtrate, incubating at room temperature for 2 min, and then adding 1.5mL of culture medium to stop lysis; mouse bone marrow cells were obtained by resuspending the cells in RPMI 1640 medium with 10% FBS and 1% double antibody.
The cell count of the obtained mice was adjusted to 1×106Spreading onto culture plate, adding recombinant mouse GM-CSF and IL-4 at 37 deg.C and 5% CO2The culture medium is replaced every 2 days, the cell factors are replenished, and suspension cells and loose adherent growth cells are collected on the 6 th day. The cells obtained were plated in the same way into 100mm petri dishes or 6-well plates at 37 ℃ in 5% CO2The culture chamber was continued for 2 days to obtain mature BMDCs.
2) FITC-OVA solution, PNPs-FTIC-OVA complex solution, and APNPs-FTIC-OVA complex solution were prepared (see Effect example 1).
3) Flow cytometry detection: transferring the BMDC prepared in the step 1) into a 12-well plate, adding a blank solution, the FITC-OVA solution prepared in the step 2), the PNPs-FITC-OVA compound solution and the APNPs-FITC-OVA compound solution (the final concentration of FITC-OVA is 20 mu g/ml), transferring a culture dish into a cell incubator, and incubating for 12 hours; centrifuging at 1400rpm for 10min to collect cells, and discarding the supernatant; centrifuging at 1400rpm for 10min to collect cells, and discarding the supernatant; nuclear staining was performed by adding DAPI at a final concentration of 1 μ g/ml and the redundant OVA lysosomal escape was observed using flow cytometry.
And (4) experimental conclusion: as shown in FIG. 5, OVA is labeled by FITC, and after detection, the OVA and lysosome do not have co-localization relationship in the PNPs-FITC-OVA compound solution and APNPs-FITC-OVA compound solution groups, while the co-localization relationship of the FITC-OVA compound solution group is clear. The experimental result shows that the APNPs and PNPs disclosed by the invention have the lysosome escape function and can accelerate the presentation process of the carried antigens.
Effect example 3: flow quantification of phagocytosis of the disclosed nanomaterials by Dendritic Cells (DCs)
Grouping experiments:
1) PNPs-MAL @ OVA experimental group: PNPs-FTIC-OVA complex solution (prepared according to Effect example 1);
2) APNPs-MAL @ OVA experimental group: APNPs-FTIC-OVA complex solution (prepared according to Effect example 1);
3) blank control group: RPMI 1640 complete medium;
the experimental steps are as follows:
1) BMDC culture and extraction are the same as the effect example 2;
2) BMDCs were transferred to 12-well plates and controls, PNPs-FITC-OVA complex solution, APNPs-FTIC-OVA complex solution (FITC-OVA final concentration was 20. mu.g/ml) were added and the dishes were transferred to a cell incubator and incubated for 12 hours. (4 groups of 4 duplicate wells for 16 wells); centrifuging at 1400rpm for 10min, and collecting and discarding the supernatant; adding APC-CD11c antibody, and incubating at 37 ℃ for 30min to mark DC cells; centrifuging at 1400rpm for 10min, and collecting and discarding the supernatant; DAPI was added to a final concentration of 1. mu.g/ml and tested on the flow machine.
And (4) experimental conclusion: as shown in fig. 6, the most important antigen presenting cells, Dendritic Cells (DCs), can efficiently take up the APNPs and PNPs of the present disclosure, which is beneficial to the presentation of the loaded antigens of the APNPs and PNPs, the activation of DC cells and the acceleration of the antigen presenting process.
Effect example 4: BCA method for measuring OVA adsorption capacity of nano-materials of the disclosure
Grouping experiments:
1) PNPs-MAL @ OVA experimental group: PNPs-FTIC-OVA complex solution (prepared according to Effect example 1);
2) APNPs-MAL @ OVA experimental group: APNPs-FTIC-OVA complex solution (prepared according to Effect example 1);
3) group of PNPs: PNPs solution, taking 1.0mg/mL PNPs and 1mg/mL OVA solution, adding the OVA solution into the PNPs solution, whirling for 30s, and standing for 30 min;
4) APNPs: and (3) taking 1.0mg/mL PNPs and 1mg/mL OVA solution, adding the OVA solution into the APNPs solution, whirling for 30s, and standing for 30 min.
The experimental method comprises the following steps:
respectively adding the PNPs-FTIC-OVA compound solution, the APNPs-FTIC-OVA compound solution, the PNPs solution and the APNPs solution into an ultrafiltration centrifugal tube, centrifuging at 8000rpm for 10 minutes, detecting the protein content of the solution at the lower layer of the ultrafiltration centrifugal tube by using a BCA protein detection kit, and calculating the antigen capture amount of the PNPs by using a formula.
Antigen capture amount (μ g/mg) ═ M1-C V)/M2;
c is the protein concentration of the solution at the lower layer of the ultrafiltration centrifugal tube, V is the volume of the solution at the lower layer of the ultrafiltration centrifugal tube, M1 is the total mass of OVA, and M2 is the mass of PNPs or APNPs.
And (4) experimental conclusion: as shown in FIG. 7, the PNPs and APNPs of the present disclosure can efficiently adsorb protein fragments, and have excellent ability of adsorbing tumor antigen fragments, wherein the antigen adsorption ability is about 762 μ g/mg.
Effect example 5: ability of the disclosed nanomaterials to stimulate DC maturation
Grouping experiments: OVA was purchased from Solarbio, Inc., cat # A8041
1) Positive control: LPS + BMDC +4 antibodies (CD40, CD86, CD11c, MHC-II4, 100ng/ml, the same below)
2) Negative control: OVA (20. mu.g/mL) + BMDC +4 antibodies
3) Experimental group 1: APNPs-MAL @ OVA (150. mu.g/ml OVA-adsorbed PNPs) + OVA + BMDC +4 antibodies
4) Experimental group 2: PNPs-MAL @ OVA (150. mu.g/ml OVA-adsorbed PNPs) + OVA + BMDC +4 antibodies
5) Experimental group 3: APS (10. mu.g/ml) + OVA (20. mu.g/ml) + BMDC +4 antibodies
The experimental steps are as follows:
1) BMDC is added at 106Inoculating to 6-well plate at a concentration of one ml, and culturing in a specific manner as described in example 2
2) Detecting the activation level of DC cells, selecting CD40, CD86, CD11c and MHC-II4 staining monoclonal antibodies for detection, adding 1 μ g of FITC-CD40, 1 μ g of PE-CD86 and 0.25 μ g of APC-CD11 into 100 μ l of solution C1. mu.g of MHC-II.
3) Immature dendritic cells on day 8 in the bacterial culture dish were transferred to a 50mL centrifuge tube and centrifuged at 1200rpm for 5 minutes, the supernatant was discarded, and the RPMI-1640 complete culture resuspended cell pellet. Immature dendritic cells were seeded in 6-well plates at a controlled seeding density of 5X 106one/mL, then, different materials were added separately, grouped according to above, with 3 multiple wells per group. The 6-well plate was placed in a cell incubator and incubated for 24 hours. Subsequently, the cell suspension in each well was transferred to a 1.5mL centrifuge tube and centrifuged at 1200rpm for 5 minutes.
4) The cells were washed twice with PBS and left to stand at 37 ℃ for 4 min. Digestion was stopped by adding 400 μ l of complete medium and adherent cells were blown down and transferred to centrifuge tubes. 1000r/min, centrifuging for 5min, and discarding the supernatant. Resuspend the cells in 1ml PBS, repeat the centrifugation step, then use 100 u L diluted to the working concentration of fluorescent antibody heavy suspension cells, according to the requirements at 37 degrees C rest 15min, every 10min shake. The resuspension step was repeated twice.
5) After 4 hours of co-incubation, the cell suspension in each well was collected into a 1.5mL centrifuge tube and centrifuged at 1200rpm for 5 minutes. In one aspect, the obtained supernatant was transferred to a new 1.5mL centrifuge tube for analysis of the cytokine TNF-. alpha.in an ELISA assay. On the other hand, the cell pellet was centrifuged and washed with 1mL of 1% BSA solution, resuspended in 100. mu.L of 1% BSA solution, and then added with mouse anti-APC-CD 11c, mouse anti-CD 40-PE, and mouse anti-CD 86-FITC monoclonal antibody, and incubated at room temperature in the dark for 20 minutes. Finally, centrifugation washes were performed with 1mL of 1% BSA solution to remove unbound fluorescent monoclonal antibodies. Add 500. mu.l PBS to resuspend the cells, filter into a flow tube with cell screen, and place on ice. Detection was performed using a flow cytometer.
And (4) experimental conclusion: as shown in FIG. 8, the APNPs and PNPs of the present disclosure can efficiently stimulate DC cells (the most predominant antigen presenting cells) to mature, thereby activating anti-tumor immunity
Effect example 6: cytotoxicity detection of the nanomaterials of the disclosure
The DC cytotoxicity of APNPs and PNPs was evaluated by CCK-8 method.
The experimental steps are as follows: the well-grown DC2.4 cells were collected at 1X105Cell density per well, seeded in 96-well plates, 5% CO2And culturing at 37 ℃ for 12h, adding PNPs and APNPs solutions with different concentrations respectively, and setting 6 parallel groups for each concentration by taking cells which are not treated by the solution as blank control. And (4) continuing incubation for 24h, using a CCK-8 solution according to the instruction, and calculating the cell survival rate according to the following formula after detection by an enzyme-labeling instrument:
cell survival (%) ═ A1/A2X 100%
In the formula: a1 is absorbance of sample group, A2 is absorbance of blank control group
And (4) experimental conclusion: as shown in fig. 9, the results indicate that the APNPs and PNPs of the present disclosure have very good biosafety.
Effect example 7: in vivo efficacy experiments with nanomaterials of the disclosure
Grouping experiments:
1) blank group: physiological saline 100 mu L
2) Experimental group 1: saline solution of PNPs (200. mu.g)
3) Experimental group 2: APNPs (200. mu.g) saline solution
4) Experimental group 3: APS (80 μ g) saline solution
5) Experimental group 4: physiological saline solution 100 mu L after cryoablation
6) Experimental group 5: saline solution of PNPs (200. mu.g) after cryoablation
7) Experimental group 6: saline solution of APNPs (200 μ g) after cryoablation
8) Experimental group 7: APS (80 μ g) saline solution after cryoablation
The experimental steps are as follows:
1) LLC cells are collected in logarithmic growth phase and made up to 1X10 concentration using sterile physiological saline6Single cell suspension/ml, and subcutaneous injection containing 1X10 on day 0 in the left dorsal aspect of female C57BI/6J mice6LLC cells, day 3, were injected subcutaneously into the right dorsal side of mice containing 3X106LLC cells, and establishing a mouse double primary tumor model. And the round subcutaneous solid tumor with the diameter of about 0.5cm to 0.7cm is used as a qualified animal model for standby.
2) Cryoablation was performed according to experimental groups on day 15, and normal saline, APNPs, PNPs, APS were injected into the left dorsal tumor (primary lesion) of the mouse according to experimental groups on days 15, 17, 19, 21, respectively, and the primary and distal tumors were measured during the experiment using a vernier caliper to monitor the progression of the tumor, and the tumor volume calculation formula was as follows:
wherein W and L represent the minor and major diameters of the tumor, respectively.
3) On day 60, each group of mice was sacrificed, the tumor recurrence rate after cryoablation was counted for each group of mice, and lung tissues were dissected, and the metastatic tumor condition of the lung was observed and measured for each group of mice.
The results show that PNPs and APNPs of the present disclosure, alone or in combination with cryoablation-generated antigens, can inhibit primary tumor growth (fig. 10) as well as distal tumor growth (fig. 11), while reducing pulmonary tumor metastasis (fig. 12), reducing the recurrence rate after in situ tumor ablation (table 1 below), and extending the survival of mice (fig. 13).
TABLE 1 in situ tumor cryoablation postoperative recurrence Rate statistics
Effect example 8: lymph node targeting capability detection of nanomaterials of the present disclosure
And evaluating the living lymph node targeting capability of the APNPs by using a living body imaging technology.
The animal molding method was the same as in effect example 7.
Experiment grouping
1) Experimental group 1: APNPs @ CY5
2) Experimental group 2: PNPs @ CY5
3) Experimental group 3: APS group
4) Experimental group 4: cryoablation + APNPs @ CY5
5) Experimental group 5: cryoablation + PNPs @ CY5
Intratumoral injection was performed at the primary tumor left in each group of female C57BI/6J mice, and fluorescence images were taken at 0h, 4h, 8h, 12h, and 24h after injection on a small animal in vivo fluorescence imaging system, and at 24h after dissection of the lymph nodes of the mice for fluorescence imaging.
In the fluorescence image shown in FIG. 14, the upper part is a thermal image of the back of the mouse, the middle part is a thermal image of the abdomen of the mouse, and the lower part is a local thermal image. The APNPs and PNPs disclosed by the invention have living lymph node targeting capability, and the freeze ablation can prolong the stagnation time of the APNPs and PNPs in a tumor area, so that the tumor treatment effect is further improved.
Effect example 9: effect of the disclosed nanomaterials on the distal tumor immune microenvironment
Grouping experiments:
1) blank group: physiological saline 100. mu.l
2) Experimental group 1: cryoablation + APNPs (200 μ g) saline solution
3) Experimental group 2: APNPs (200. mu.g) saline solution
4) Experimental group 3: cryoablation
The mouse spleen and distal tumor were obtained by dissection one week after successful model creation and administration of the intervention drugs on alternate days in groups, as in effect example 7, and after labeling CD3, CD45, CD11c, FOXP3, CD11b, CD25, CD4, CD8, Gr-1, and MHC-II with FLOW antibody, they were analyzed by FLOW cytometry, and as a result, data was processed using FLOW J.
As shown in FIGS. 15-20, APNPs and PNPs of the present disclosure, combined with cryoablation and antigens generated thereby, have the effects of breaking the immunosuppressive environment of tumor regions and enhancing T cell recruitment. Among them, figure 15 demonstrates T cell recruitment in the tumor area, with CD8+ T being the most dominant cell for tumor killing (CD45+ CD3+ cells); FIG. 16 demonstrates the effect of reducing MDSC cells (CD45+ CD11b + Gr-1 cells) in the tumor area, which are one of the immunosuppressive cells in the tumor area; FIG. 17 demonstrates recruitment and activation of DC cells (CD11c + MHC-II + cells) that are upstream of T cell activation in the tumor region; fig. 18 demonstrates the effect of reducing Treg cells in the tumor region (CD25+ FOXP3+ cells), which are one of the tumor region immunosuppressive cells (CD45+ CD4+ cells); FIG. 19 demonstrates that Treg cells in the spleen, which are one of the immunosuppressive cells, are reduced; FIG. 20 demonstrates that there is recruitment activation of splenic DC cells, which are upstream cells of T cell activation.
The above embodiments are merely illustrative of the specific embodiments of the present disclosure, and are not restrictive of the scope of the present disclosure, and many modifications and variations may be made by those skilled in the art based on the prior art, and various changes and modifications may be made by those skilled in the art without departing from the spirit of the present disclosure, which is intended to be covered by the appended claims.
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