WO2021127814A1 - 用于癌症治疗的光纳米疫苗及其制备方法和应用 - Google Patents

用于癌症治疗的光纳米疫苗及其制备方法和应用 Download PDF

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WO2021127814A1
WO2021127814A1 PCT/CN2019/127330 CN2019127330W WO2021127814A1 WO 2021127814 A1 WO2021127814 A1 WO 2021127814A1 CN 2019127330 W CN2019127330 W CN 2019127330W WO 2021127814 A1 WO2021127814 A1 WO 2021127814A1
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exosomes
cancer
tumor
hex
quantum dots
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French (fr)
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刘利平
刘权
严巧婷
王斗
葛晨晨
张玉华
鲍世韵
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深圳市人民医院
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Priority to PCT/CN2019/127330 priority Critical patent/WO2021127814A1/zh
Priority to CN201980003493.5A priority patent/CN111344015B/zh
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
    • A61K9/5068—Cell membranes or bacterial membranes enclosing drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention belongs to the technical field of immunology, and specifically relates to a photonano vaccine and a preparation method and application thereof, in particular to a photonano vaccine for cancer treatment and a preparation method and application thereof.
  • Nanovaccine is an emerging field with varying degrees of success in animal and clinical cancer models.
  • An effective nano-vaccine usually consists of antigens (tumor-specific peptides, proteins or nucleic acids), adjuvants, and nanoparticles that may serve as carriers.
  • the purpose of the nano-vaccine is to achieve efficient co-transfusion of antigen and adjuvant to draining lymph nodes (dLNs), trigger antigen-specific adaptive immunity and long-term immune memory effect on cancer cells, thereby avoiding immune tolerance and tumor recurrence.
  • DNLNs draining lymph nodes
  • nano-vaccine may show significantly different therapeutic effects on established tumors and different cancer patients.
  • Personalized cancer immunotherapy vaccines have become a new promising cancer treatment strategy.
  • innovations that continue to emerge in the digital age promote the development of vaccination, vaccinating patients with a single tumor mutation may become the first truly personalized cancer treatment. Therefore, we urgently need a safe and effective immunogenic nano-vaccine suitable for personalized treatment of patient-specific neoantigens.
  • Cancer photothermal therapy is a promising alternative to traditional treatment methods due to its high efficiency and minimal invasiveness.
  • gold nanoparticles, graphene oxide, indocyanine green (ICG) and other near-infrared (NIR) responsive nano-preparations have been proven to effectively convert light energy into heat energy and improve the efficacy of photothermal ablation therapy.
  • ICG indocyanine green
  • NIR near-infrared
  • black phosphorus nanosheets have unique physical structure, good optical properties, high surface area and drug loading, and can be used as drug delivery systems such as tumor synergistic photodynamic/photothermal therapy, tumor hyperthermia, and other biological Medical applications.
  • BPQDs BP quantum dots
  • BPQDs BP quantum dots
  • studies have shown that BPQDs are almost non-toxic and have good biocompatibility, degradability, treatment and penetration potential. Its main element, phosphorus, is one of the basic elements of living organisms. Under acidic conditions, the degradation of BP increases, thereby accelerating the release of anti-tumor drugs and tumor-associated antigens in the acidic tumor microenvironment.
  • ultra-small size ( ⁇ 10nm) BPQDs have a long half-life and are not easily swallowed by macrophages.
  • BPQDs are expected to become excellent photothermal agents for the treatment of tumors.
  • ablative tumor treatments have been reported to induce strong tumor-specific immune responses by releasing tumor-specific antigens, promoting DC maturation and T cell activation. Local fever and hyperthermia can increase blood flow, vascular permeability and interstitial pressure, and promote T cells to fight cancer cells to enter the tumor.
  • Exosomes are a type of 50-150nm lipid bilayer membrane vesicles secreted by cells. They are intercellular messengers of protein, messenger RNA and microRNA. They have biology in intercellular communication, maintaining normal physiological processes, and disease pathology. effect. In recent years, exosomes have been widely studied for diagnosis and as drug delivery vehicles. However, exosomes with multiple functions in photonanoparticle delivery vehicles and tumor immunotherapy have not been reported yet.
  • the purpose of the present invention is to provide a photonano vaccine and a preparation method and application thereof, in particular to provide a photonano vaccine for cancer treatment and a preparation method and application thereof.
  • the present invention provides a photonanovaccine for cancer treatment.
  • the photonanovaccine includes black phosphorous quantum dots and exosomes, and the black phosphorous quantum dots are encapsulated in the exosomes.
  • the present invention creatively combines nanoparticle-based photothermal therapy with immunotherapy, and establishes a new and effective personalized photonano vaccine, that is, using exosomes as the carrier of black phosphorus quantum dots and tumor-specific antigens. Effective carrier.
  • the photonanovaccine has a long blood circulation effect, and the exosomes can protect the black phosphorous quantum dots and avoid their rapid biodegradation;
  • the immunogenicity is induced, and the exosomes of tumor cells contain a A series of tumor antigens can be used as antigen carriers to act on dendritic cells (DCs) and major cytotoxic T lymphocytes, and produce immune responses to tumors;
  • Targeted selection, exosomes contain cell membranes, which can pass through A series of surface adhesion proteins and carrier ligands (such as integrins, CD11b and CD18 receptors) attach to target cells and deliver payloads to target cells.
  • Exosomes can carry cell type-specific proteins found in the mother cell membrane, such as myelin proteins from oligodendrocytes and specific antigens from tumor cells in exosomes, with unique homing selectivity.
  • the photonano vaccine of the present invention has better long-term stability in vivo, photothermal conversion and tumor targeting; in addition, it has a significant therapeutic effect on tumors and promotes
  • the infiltration of T lymphocytes in tumor tissues is an innovative photonano vaccine for the treatment of cancer.
  • the mass ratio of the black phosphorous quantum dots to the exosomes is 1:(8-12), such as 1:8, 1:9, 1:10, 1:11 or 1:12.
  • the mass ratio of the black phosphorous quantum dots to the exosomes is specifically selected to be a mass ratio of 1:(8-12), because the above-mentioned beneficial effects can be optimized under this condition.
  • the cancer includes lung cancer, liver cancer, breast cancer, bladder cancer, pancreatic cancer, lymphoma, esophageal cancer, stomach cancer or colon cancer.
  • the photonanovaccine involved in the present invention can be used for the prevention and treatment of the above-mentioned but not limited to the above-mentioned tumor types.
  • the exosomes are exosomes of tumor cells.
  • the exosomes are exosomes of tumor cells after ablation.
  • the exosomes of tumor cells carry more kinds of tumor antigens, which can act as antigen carriers on dendritic cells (DCs) and main cytotoxic T lymphocytes, and produce immune responses to tumors.
  • DCs dendritic cells
  • main cytotoxic T lymphocytes main cytotoxic T lymphocytes
  • the present invention provides a method for preparing a photonanovaccine for cancer treatment as described above, and the preparation method includes the following steps:
  • step (2) Load the black phosphorous quantum dots prepared in step (1) into exosomes to obtain the photonano vaccine for cancer treatment.
  • the preparation method of the photonano vaccine involved in the present invention is simple and easy to operate, and is convenient for industrial production.
  • the method for separating exosomes in step (1) is: performing photothermal treatment on the tumor patient, taking plasma and separating the exosomes from the plasma.
  • the tumor area is irradiated with a 808 nm laser (1 W/cm 2 ) for 10 minutes, and the above operation is performed again on the next day, the plasma is separated after 24 hours, and the exosomes in the plasma are separated by ultracentrifugation.
  • the plasma was centrifuged at 1000g for 10min, and then centrifuged at 10000g for 30min; the supernatant was collected, filtered with a 0.22 ⁇ m filter membrane, and then ultracentrifuged at 100,000g for 1h; the exosomes were in phosphate buffered saline (PBS Washed in) and recovered by centrifugation at 100,000 g for 1 h; the part rich in exosomes was washed twice with PBS and stored at -80°C.
  • PBS Washed in phosphate buffered saline
  • the method of photothermal treatment is: injecting black phosphorous nanoparticles into the tumor, and irradiating the tumor area with a near-infrared laser.
  • the separation of exosomes from plasma adopts an ultracentrifugation method.
  • the method for synthesizing black phosphorous quantum dots in step (1) is a liquid phase exfoliation method.
  • the liquid phase exfoliation method includes: mixing black phosphorus crystals with isopropanol and then performing bath ultrasound, centrifuging, and concentrating and drying the supernatant.
  • the ratio of the black phosphorus crystals to isopropanol is (1.5-2.5) mg/mL, such as 1.5 mg/mL, 1.8 mg/mL, 1.9 mg/mL, 2.0 mg/mL, 2.2 mg/mL, 2.3mg/mL or 2.5mg/mL, etc.
  • the bath ultrasound time is 45-50h, such as 45h, 46h, 47h, 48h, 49h or 50h.
  • the power of the bath ultrasound is 340-380W, such as 340W, 345W, 350W, 355W, 360W, 365W, 370W or 380W.
  • the centrifugation is centrifugation at (8000-10000) rpm (for example, 8000rpm, 8500rpm, 8800rpm, 9000rpm, 9200rpm, 9500rpm, 9800rpm or 10000rpm, etc.) for 20-40min (20min, 25min, 30min, 35min, 38min or 40min) Wait).
  • the invention adopts a simple liquid phase exfoliating method to prepare black phosphorous quantum dots.
  • isopropanol is used as a solvent, which is easy to volatilize and remove.
  • bath ultrasound is used instead of probe ultrasound.
  • 100 mg of black phosphorus crystals and 50 mL of isopropanol were mixed in a 60 mL brown bottle, and after 48h bath ultrasound (360W), centrifuged at 9000 rpm for 30 min; the supernatant was a dispersion of black phosphorus quantum dots, Collect and concentrate at 18000 rpm. Then, the dispersion was dried in a vacuum oven overnight.
  • the prepared black phosphorous quantum dots are stored in an argon glove box without light.
  • the method for loading black phosphorous quantum dots into exosomes in step (2) includes: mixing black phosphorous quantum dots, exosomes and PBS solution, sonicating, and cooling.
  • the ultrasound time is 2.5-3.5min, such as 2.5min, 2.6min, 2.8min, 2.9min, 3.0min, 3.2min or 3.5min.
  • the cooling time is 1.5-2.5min, such as 1.5min, 1.7min, 1.8min, 2.0min, 2.2min, 2.3min, 2.4min or 2.5min.
  • the preparation method of the photonanovaccine for cancer treatment includes the following steps:
  • the present invention provides an application of the photonanovaccine for cancer treatment as described above in the preparation of a medicine for cancer treatment or cancer prevention.
  • the dosage form of the drug includes any pharmacologically acceptable dosage form, such as tablets, capsules, suspensions, suppositories, sprays, injections or drops.
  • any pharmacologically acceptable dosage form such as tablets, capsules, suspensions, suppositories, sprays, injections or drops.
  • the drug further includes any one or a combination of at least two of pharmaceutically acceptable pharmaceutical excipients
  • the pharmaceutical excipients include excipients, carriers, solubilizers, solubilizers, stabilizers, Sustained release agent, etc.
  • the carrier is, for example, liposomes, micelles, dendrimers, microspheres, microcapsules and the like.
  • the drugs also include direct anti-tumor drugs, auxiliary anti-tumor drugs or molecular targeted drugs.
  • the direct anti-cancer drugs include chemotherapeutic drugs, such as fluorouracil, cyclophosphamide and other drugs; in addition, they also include anti-tumor antibiotics, such as doxorubicin, paclitaxel, taxanes, and the like.
  • the auxiliary anticancer drugs are mostly endocrine therapy drugs that regulate the internal environment, such as anastrozole, aromatase inhibitors and the like. In most cases, the molecular targeted drugs can destroy the blood vessels of tumor cells, make tumor cells lack blood supply, and starve cancer cells; in addition, some molecular targeted drugs block the growth signal on the surface of cancer cells, so that the tumor cells cannot start to grow. .
  • the present invention has the following beneficial effects:
  • the present invention creatively combines nanoparticle-based photothermal therapy with immunotherapy, and establishes a new and effective personalized photonano vaccine, that is, using exosomes as the carrier of black phosphorus quantum dots and tumor-specific antigens. Effective carrier.
  • the photonanovaccine has a long blood circulation effect, and exosomes can protect black phosphorus quantum dots and avoid their rapid biodegradation;
  • immunogenicity is induced, tumor cell exosomes carry a variety of Tumor antigens can act as antigen carriers on dendritic cells (DCs) and main cytotoxic T lymphocytes, and produce immune responses to tumors;
  • Targeted selection, exosomes contain cell membranes, which can pass a series of Surface adhesion proteins and carrier ligands (such as integrins, CD11b and CD18 receptors) attach to target cells and deliver the payload to the target cells.
  • DCs dendritic cells
  • carrier ligands such as integrins, CD11b and CD18 receptors
  • Exosomes can carry cell type-specific proteins found in the mother cell membrane, such as myelin proteins from oligodendrocytes and specific antigens from tumor cells in exosomes, with unique homing selectivity.
  • the photonano vaccine of the present invention has better long-term stability in vivo, photothermal conversion and tumor targeting; in addition, it has a significant therapeutic effect on tumors and promotes
  • the infiltration of T lymphocytes in tumor tissues is an innovative photonano vaccine for the treatment of cancer.
  • Figure 1 is a graph showing the expression results of human lung cancer antigen MUC1 and epithelial cell adhesion molecule EpCAM in the experimental group and the control group in Example 1;
  • FIG. 2 is a graph showing the expression results of glypican-3 (GPC3) in the experimental group and the control group in Example 1;
  • Figure 3 is a particle size distribution diagram of exosomes separated in Example 1;
  • Figure 4 is a TEM image of the exosomes isolated in Example 1.
  • Figure 5 is a diagram showing the results of the uptake of exosomes by DC cells in Example 1;
  • Figure 6 is a graph showing the proportion of exosomes induced to form CD8 ⁇ +CD11c+;
  • Figure 7 is a diagram showing the results of exosomes inducing the maturation of DC cells
  • Figure 8 is a TEM image of black phosphorous quantum dots
  • Figure 9 is a high-resolution TEM image of black phosphorous quantum dots
  • Figure 10 is a morphological information diagram of black phosphorous quantum dots
  • Figure 11 is a graph of thickness information of black phosphorous quantum dots
  • Figure 12 is a Raman spectrum of black phosphorous quantum dots
  • Figure 13 is a transmission electron microscope image of hEX@BP involved in the present invention.
  • Figure 14 is a transmission electron micrograph of hEX@BP involved in the present invention.
  • Figure 15 is a particle size distribution diagram of hEX@BP involved in the present invention.
  • Figure 16 is a diagram showing the relationship between loading efficiency and dose of BPQD
  • Figure 17 is a graph showing the uptake of hEX@BP by tumor cells
  • Figure 18 is a graph showing the cytotoxicity results of hEX@BP involved in the present invention on U251 (human glioma cells), LLC (mouse lung cancer cells) and 4T1 (breast cancer cells);
  • Figure 19 is a graph showing the cytotoxicity of hEX@BP to LLC (mouse lung cancer cells) involved in the present invention.
  • Figure 20 is a concentration-dependent temperature rise curve diagram of hEX@BP under 808nm laser irradiation
  • Figure 21 is a characterization diagram of hEX@BP photothermal stability performance
  • Figure 22 is a fluorescence imaging diagram of hEX@BP in tumor mice in Example 5.
  • Figure 23 is a fluorescence imaging diagram of hEX@BP in various tissues in tumor mice in Example 5;
  • Figure 24 is a fluorescence image of a tumor section in Example 5.
  • Figure 25 is a graph showing the long-term PTT efficacy evaluation results of hEX@BP after injection
  • Figure 26 is a fluorescence imaging image of mice injected with hEX-DiR or hEX@BP-DiR on the left foot of the mouse;
  • Figure 27 is a graph showing the trend of changes in tumor volume over time in the therapeutic vaccine treatment program of hEX@BP involved in the present invention.
  • Fig. 28 is a graph showing the change trend of mouse body weight over time in the therapeutic vaccine treatment program of hEX@BP involved in the present invention.
  • Figure 29 is a graph showing the trend of mouse survival rate over time in the hEX@BP therapeutic vaccine treatment program involved in the present invention.
  • Figure 30 is a graph showing the trend of changes in tumor volume over time in the prophylactic vaccine treatment program of hEX@BP involved in the present invention.
  • Figure 31 is a graph showing the trend of changes in the body weight of mice over time in the preventive vaccine treatment program of hEX@BP involved in the present invention.
  • Figure 32 is a graph showing the effect of hEX@BP on the secretion level of IFN- ⁇ involved in the present invention.
  • Figure 33 is a graph showing the effect of hEX@BP on the secretion level of IL-2 involved in the present invention.
  • Figure 34 is a graph showing the effect of hEX@BP on the secretion level of IL-6 involved in the present invention.
  • Figure 35 is a graph showing the effect of hEX@BP on the secretion level of TNF- ⁇ involved in the present invention.
  • Figure 36 is a graph showing the effect of hEX@BP on the secretion level of IL-10 involved in the present invention.
  • Fig. 37 is a graph showing the effect of hEX@BP on the proportion of CD4+ T cells involved in the present invention.
  • Figure 38 is a graph showing the effect of hEX@BP on the proportion of CD8+ T cells involved in the present invention.
  • liver tumor cells Hepa1-6 cells
  • LCC cells 5 ⁇ 10 5 lung tumor cells
  • H1975 cells human lung tumor cells
  • BP black phosphorus
  • the mouse whole blood was separated, centrifuged at 3000g for 15 minutes, cells and cell debris were collected, and plasma was collected. Ultracentrifugation was used to separate exosomes in plasma.
  • the ultracentrifugation method is specifically as follows: the plasma is first centrifuged at 1000g for 10min, and then centrifuged at 10000g for 30min. The supernatant was collected, filtered with a 0.22 ⁇ m filter (SLGP033RS, Merck Millipore, Billerica, MA, USA), and then subjected to ultracentrifugation at 100,000 g for 1 hour.
  • exosomes were washed in phosphate buffered saline (PBS) and recovered by centrifugation at 100,000 g for 1 h. The part rich in exosomes was washed twice with PBS and stored at -80°C.
  • PBS phosphate buffered saline
  • the total protein was extracted from exosomes using RIPA lysis buffer (P0013B, Byotime). The concentration was detected with BCA protein quantification kit (P0010, Byotime). Then perform SDS-PAGE and western blotting. The sample (containing 10 ⁇ g total protein and 1 ⁇ loading buffer) was separated by standard SDS-PAGE, and then stained with Coomassie brilliant blue or the sample was transferred to a polyvinylidene difluoride membrane (Millipore).
  • MUC1 16564S; 1:1000, Cell Signalling Technology, CST
  • EpCAM 14452S; 1:1000, CST
  • GPC3 ab95363; 1:1000, Abcam
  • CD63 ab217345, ab134045; 1:1000, Abcam
  • CD9 ab92726; 1:1000, Abcam
  • HRP horseradish peroxidase
  • the results of the lung cancer (H1975) mouse model are shown in Figure 1: Compared with the control mice, the experimental group can detect more abundant human lung cancer antigen MUC1 and epithelial cell adhesion molecule EpCAM expression.
  • liver cancer (Hepa1-6) mouse model The results of the liver cancer (Hepa1-6) mouse model are shown in Figure 2: Compared with the control mice, the experimental group can detect more abundant glypican-3 (GPC3) expression. It shows that the secretion body isolated from the tumor mice after hyperthermia carries more tumor-specific antigens.
  • GPC3 glypican-3
  • DC Dendritic cells
  • LLC hyperthermia lung cancer
  • the fluorescence imaging image is shown in Figure 5 (the nucleus is stained with Hoechst (blue), and the exosomes are stained with DiR (red)): the internalized DiR-labeled exosomes are distributed around the nucleus and are punctate in appearance .
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • a simple liquid phase exfoliation method is used to prepare black phosphorus quantum dots.
  • 100 mg of black phosphorus crystals and 50 mL of isopropanol are mixed in a 60 mL brown bottle. After sonication in the bath for 48h (360W), it was centrifuged at 9000rpm for 30min. The supernatant was collected and concentrated by centrifugation at 18000 rpm. Then, the dispersion was dried in a vacuum oven for 24 hours.
  • the prepared black phosphorous quantum dots are stored in an argon glove box without light.
  • BPQDs were measured by TEM (FEI Tecnai G2 spirit, 80kv), as shown in Figure 8:
  • the black phosphorous quantum dots are spherical in appearance, uniform in morphology, and 2.8 ⁇ 1.6nm in particle size.
  • the high-resolution TEM image is shown in Figure 9: showing the 0.224nm lattice fringes on the (041) plane of the BP crystal.
  • the photonanovaccine was prepared by ultrasonic method (represented by hEX@BP): 100 ⁇ g/mL black phosphorous quantum dots and the secretion obtained from the LCC lung cancer mouse model in Example 1 were stirred in PBS (the mass ratio of hEX and BPQDs was 10 :1). Then, the mixture was sonicated for 6 cycles, each cycle was 30 seconds, a total of 3 minutes, and a cooling time of 2 minutes. Finally, the BPQD was loaded into the exosomes (hEX@BP).
  • the prepared hEX@BP was characterized by transmission electron microscopy (TEM), as shown in Figure 13 and Figure 14: hEX@BP exhibits a good spherical shape and uniform size, and it is clear that the black phosphorous quantum dots are covered by exosomes. .
  • the particle size of hEX@BP was measured by DLS, as shown in Figure 15: the average hydrodynamic size is about 100nm.
  • the loading efficiency of BPQDs in hEX The results are shown in Figure 16: The loading efficiency of BPQD is related to the dosage. When the feeding ratio reaches 1:5, the weight of coated BPQDs is more than 8 times that of hEX.
  • cell viability (%) (average cell viability of the treatment group/average cell viability of the control group) ⁇ 100%.
  • Figure 18 at a concentration of 100 ⁇ g/mL, the cytotoxicity of hEX@BP is very small and almost negligible (the bars in the figure are U251, LLC, and 4T1 groups from left to right).
  • AO green fluorescence indicates live cells
  • PI propidium iodide
  • hEX@BP dispersion experienced 10min of 808nm laser illumination and natural cooling in each cycle , The attenuation of the temperature rise can be ignored, indicating that hEX@BP has good photothermal stability, and the photothermal conversion efficiency of hEX@BP can be calculated to be 23.2%.
  • the photonano vaccine hEX@BP involved in the present invention has significant in vitro biocompatibility and photothermal effect, and can promote phototriggered ablation of cancer cells.
  • the biodistribution of LLC-induced lung cancer in Balb/c nude mice was detected by intravenous injection of exosomes (hEX-DiR and hEX@BP-DiR) labeled with the commonly used near-infrared fluorescent dye DiR, and fluorescence imaging was performed.
  • the specific operation is: 5 ⁇ 10 5 LLC tumor cells are injected into the right side of the back of Balb/c nude mice.
  • the tumor-bearing BALB/c mice are divided into three groups, and intravenous injection of normal saline and hEX-DiR (0.5 mg/ kg hEX) and hEX@BP-DiR (5mg/kg BP, 0.5mg/kg hEX).
  • the Xenogen IVIS-Spectrum imaging system was used for whole-body imaging. As shown in Figure 22, 24h after the injection, considerable fluorescence can be observed in the tumor, which can be clearly seen from other tissues.
  • the subcutaneous tumor is outlined in the middle; 48 hours after injection, the fluorescence intensity within the tumor gradually increases, indicating that exosomes can continue to accumulate at the tumor site.
  • the tumor-bearing mice were euthanized, the main organs (heart, liver, spleen, lung, kidney, tumor) were imaged in vitro, and the fluorescence intensity per gram of the tumor and the main organs was counted (3 parallel tests).
  • hEX-DiR or hEX@BP-DiR was injected into the left foot of the mouse, and 6 hours after the injection, the migration of the mouse was observed by in vivo fluorescence imaging technology.
  • the results are shown in Figure 26: hEX-Dir or hEX@BP-Dir can effectively migrate to lymph nodes and interact with antigen presenting cells (APCs), which has an important impact on the induced immune response.
  • APCs antigen presenting cells
  • Therapeutic vaccine treatment procedure 9 days after tumor inoculation (5 ⁇ 10 5 LLC cells/mouse), C57BL/6 mice were randomly divided into three groups (6 mice in each group), and 100 ⁇ L of different vaccine formulas were injected intravenously: PBS (control group); hEX@BP (5mg/kg BP and 0.5mg/kg hEX); hEX@BP+NIR (each mouse’s hEX@BP is 5mg/kg BP and 0.5mg/kg hEX, using the nearest Infrared illumination, 1W/cm 2 , 8min), two PTT treatments were given on the 9th and 16th days after tumor cell inoculation, and the tumor size was measured every other day after treatment.
  • hEX@BP nano vaccine can effectively inhibit the early growth of tumors, and the tumors of the other two groups (treated with PBS alone or treated with hEX alone) then grow rapidly, resulting in all of the LLC cells within 15-25 days after inoculation.
  • the mouse died; the weight change curve with time after different treatments is shown in Figure 28; the survival of the mouse is shown in Figure 29: 80% of the mice have tumors disappeared after the combined treatment, and the 40th after tumor cell inoculation Days to survive. It shows that our developed exosome-coated BPQD nano-vaccine combined with PTT therapy may be a very attractive cancer immune combination therapy strategy.
  • Preventive vaccine treatment procedure C57BL/6 mice were randomly divided into four groups (5 in each group), and intravenously injected three times (one week apart). The different vaccine formulas were injected intravenously: 100 ⁇ L PBS (control group); PBS Naked BPQDs in solution (5 mg/kg BP per mouse); hEX@BP (5 mg/kg BP and 0.5 mg/kg hEX per mouse); hEX@BP (5 mg/kg BP and 0.5 mg per mouse /kg hEX). Seven days after the last vaccination, 5 ⁇ 10 5 LLC cells were injected subcutaneously into the right side of each mouse.
  • mice in the hEX@BP+NIR group were simultaneously vaccinated After hEX@BP vaccine and BP-based PTT vaccine, tumor growth was significantly delayed, and there was no abnormal weight change (as shown in Figure 31), which may be due to the immune stimulation induced by hEX@BP and near-infrared tumor ablation.
  • cytokines in the supernatant uses mouse interferon- ⁇ (IFN- ⁇ , 430807), interleukin-2 (IL-2, 431007), interleukin-6 (IL-6, 431307), and interleukin -10 (IL-10, 431417) and tumor necrosis factor ⁇ (TNF- ⁇ ) enzyme-linked immunosorbent assay (ELISA) kit (Biolegend).
  • IFN- ⁇ interferon- ⁇
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • IL-10 interleukin -10
  • TNF- ⁇ tumor necrosis factor ⁇
  • ELISA tumor necrosis factor ⁇
  • FACS Fluorescence activated cell sorting
  • the present invention uses the above-mentioned examples to illustrate a photonanovaccine for cancer treatment of the present invention and its preparation method and application, but the present invention is not limited to the above-mentioned examples, which does not mean that the present invention must It can be implemented only by relying on the above-mentioned embodiments.
  • Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

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Abstract

本发明公开了一种用于癌症治疗的光纳米疫苗及其制备方法和应用。所述光纳米疫苗包括黑磷量子点和外泌体，所述黑磷量子点包裹于所述外泌体中。

Description

[根据细则37.2由ISA制定的发明名称]　用于癌症治疗的光纳米疫苗及其制备方法和应用 技术领域
本发明属于免疫学技术领域，具体涉及一种光纳米疫苗及其制备方法和应用，尤其涉及一种用于癌症治疗的光纳米疫苗及其制备方法和应用。
背景技术
目前的癌症免疫疗法，如癌症疫苗、检查点阻断疗法和嵌合抗原受体T细胞(CAR-T)，被证明是一种有效的治疗多种癌症的方法。纳米疫苗是一个新兴领域，在动物和临床癌症模型中取得了不同程度的成功。一种有效的纳米疫苗通常由抗原(肿瘤特异性肽、蛋白质或核酸)、佐剂以及可能作为载体的纳米颗粒组成。纳米疫苗的目的是实现抗原和佐剂高效共输至引流淋巴结(dLNs)，触发抗原特异性适应性免疫和对癌细胞的长期免疫记忆效应，从而避免免疫耐受和肿瘤复发。由于肿瘤抗原表达谱具有较大的异质性，纳米疫苗对已建立的肿瘤和不同的癌症患者可能表现出显著不同的疗效。个性化的癌症免疫治疗疫苗已经成为一种新的有前途的癌症治疗策略。随着数字时代不断涌现的创新推动了疫苗接种的发展，为患者接种带有单个肿瘤突变的疫苗可能成为第一个真正个性化的癌症治疗方法。因此，我们迫切需要一种安全而有效的免疫原性纳米疫苗，以适合于患者特异性新抗原的个性化治疗。
癌症光热治疗(PTT)因其高效率和最小的侵袭性，是传统治疗方法的一个有前途的替代方法。近十年来，金纳米颗粒、氧化石墨烯、吲哚菁绿(ICG)等近红外(NIR)响应纳米制剂已被证明可以有效地将光能转化为热能，提高光热消融治疗的疗效。近年来研究表明，黑磷纳米片具有独特的物理结构、良好的光学性能、较高的表面积和载药量，可作为肿瘤协同光动力/光热治疗、肿瘤热疗等药物传递系统和其他生物医学应用。事实上，在光热/光动力协同抗肿瘤治疗中，BP量子点(BPQDs)作为传递纳米颗粒的平台表现出良好的组织穿透性。重要的是，研究表明，BPQDs几乎是无毒的，具有良好的生物相容性、降解性、治疗和渗透潜力。它的主要元素磷是生物体生命活动的基本元素之一。在酸性条件下，BP降解增加，从而加速抗肿瘤药物和肿瘤相关抗原在酸性肿瘤微环境中的释放。此外，超小尺寸(<10nm)的BPQDs半衰期较长，不易被巨噬细胞吞噬。因此，它们更可能通过增强的通透性和保留(EPR)活性而分布于肿瘤组织中，使负载的抗肿瘤药物能够在肿瘤部位积聚。这些优势使得基于BP纳米材料的无毒高效治疗成为可能。根据上述特点，BPQDs有望成为治疗肿瘤的优良光热剂。最后，许多消融性肿瘤治疗，被报道可通过释放肿瘤特异性抗原、促进DC成熟和T细胞活化来诱导强肿瘤特异性免疫反应。局部发热和热疗可增加血流量、血管通透性和间质压力， 促进T细胞对抗癌细胞进入肿瘤。
外泌体是一类由细胞分泌的50-150nm脂质双层膜囊泡，是蛋白质、信使RNA和microRNA的细胞间信使，在细胞间通讯、维持正常生理过程和疾病病理等方面具有生物学作用。近年来，外泌体被广泛研究用于诊断和作为药物传递载体。然而，在光纳米颗粒传递载体和肿瘤免疫治疗中具有多功能作用的外泌体尚未见报道。
发明内容
针对现有技术的不足，本发明的目的在于提供一种光纳米疫苗及其制备方法和应用，尤其提供一种用于癌症治疗的光纳米疫苗及其制备方法和应用。
为达到此发明目的，本发明采用以下技术方案：
一方面，本发明提供一种用于癌症治疗的光纳米疫苗，所述光纳米疫苗包括黑磷量子点和外泌体，所述黑磷量子点包裹于所述外泌体中。
本发明创造性地将纳米颗粒为基础的光热治疗与免疫治疗结合起来，建立了一种新的有效的个性化光纳米疫苗，即使用外泌体作为携带黑磷量子点和肿瘤特异性抗原的有效载体。其优势包括：(1)该光纳米疫苗具有长血循环效应，且外泌体可以保护黑磷量子点，避免其快速生物降解；(2)免疫原性诱导，肿瘤细胞的外泌体中含有一系列肿瘤抗原，可作为抗原载体作用于树突状细胞(DCs)和主要的细胞毒性T淋巴细胞，并对肿瘤产生免疫应答；(3)靶向性选择，外泌体含有细胞膜，可通过一系列表面粘附蛋白和载体配体(如整合素、CD11b和CD18受体)附着于靶细胞，并将有效载荷传递到靶细胞。外泌体可携带在母细胞膜中发现的细胞类型特异性蛋白，如来自少突胶质细胞的髓鞘蛋白和来自外泌体中肿瘤细胞的特异性抗原，具有独特的归巢选择性。与单纯的黑磷量子点相比，本发明所涉及的光纳米疫苗具有更好的体内长期稳定性、光热转换性和肿瘤靶向性；此外，其对肿瘤具有的显著治疗效果，并促进了肿瘤组织T淋巴细胞的浸润，是一种创新的用于治疗癌症的光纳米疫苗。
优选地，所述黑磷量子点与外泌体的质量比为1:(8-12)，例如1:8、1:9、1:10、1:11或1:12等。
所述黑磷量子点与外泌体的质量比特定选择为1:(8-12)的质量配比，是因为在此条件下才能使上述有益效果达到最佳。
优选地，所述癌症包括肺癌、肝癌、乳腺癌、膀胱癌、胰腺癌、淋巴癌、食管癌、胃癌或结肠癌。本发明所涉及的光纳米疫苗可以用于上述但不仅限于上述肿瘤类型的预防和治疗。
优选地，所述外泌体为肿瘤细胞的外泌体。
优选地，所述外泌体为经消融后的肿瘤细胞的外泌体。肿瘤细胞的外泌体经消融后携带 更多种的肿瘤抗原，可作为抗原载体作用于树突状细胞(DCs)和主要的细胞毒性T淋巴细胞，并对肿瘤产生免疫应答。
另一方面，本发明提供一种如上所述的用于癌症治疗的光纳米疫苗的制备方法，所述制备方法包括如下步骤：
(1)分离外泌体和合成黑磷量子点；
(2)将步骤(1)制得的黑磷量子点加载到外泌体中，得到所述用于癌症治疗的光纳米疫苗。
本发明所涉及的光纳米疫苗的制备方法简单易操作，便于工业化生产。
优选地，步骤(1)所述分离外泌体的方法为：对肿瘤患者进行光热治疗，取血浆并从血浆中分离出外泌体。
示例性地，肿瘤区域使用808nm的激光进行辐照(1W/cm 2)10min，并于第二天再次进行上述操作，24h后分离血浆，采用超离心法分离血浆中的外泌体。简而言之，血浆先以1000g离心10min，然后以10000g离心30min；收集上层清液，用0.22μm过滤膜过滤，然后在10万g超离心作用1h；外泌体在磷酸盐缓冲盐水(PBS)中洗涤，10万g离心1h回收；富含外泌体的部分用PBS洗涤两次，并在-80℃保存。
优选地，所述进行光热治疗的方式为：在肿瘤内注射黑磷纳米颗粒，使用近红外激光辐照肿瘤区域。
优选地，所述从血浆中分离出外泌体采用超离心法。
优选地，步骤(1)所述合成黑磷量子点的方法为液相去角质法。
优选地，所述液相去角质法包括：将黑磷晶体与异丙醇混合后进行浴超声，离心，上清液浓缩并干燥。
优选地，所述黑磷晶体与异丙醇的比例为(1.5-2.5)mg/mL，例如1.5mg/mL、1.8mg/mL、1.9mg/mL、2.0mg/mL、2.2mg/mL、2.3mg/mL或2.5mg/mL等。
优选地，所述浴超声的时间为45-50h，例如45h、46h、47h、48h、49h或50h等。
优选地，所述浴超声的功率为340-380W，例如340W、345W、350W、355W、360W、365W、370W或380W等。
优选地，所述离心为在(8000-10000)rpm(例如8000rpm、8500rpm、8800rpm、9000rpm、9200rpm、9500rpm、9800rpm或10000rpm等)下离心20-40min(20min、25min、30min、35min、38min或40min等)。
本发明采用简单的液相去角质法制备了黑磷量子点。为了提高黑磷量子点的生物相容性， 采用异丙醇作为溶剂，易于挥发和除去。此外，为了避免超声探头可能带来的杂质，使用浴式超声而不是探针式超声。具体地，将100mg的黑磷晶体和50mL的异丙醇混合在一个60mL的棕色瓶子里，在48h浴超声(360W)后，以9000rpm离心30min；上清液是黑磷量子点的分散物，收集并以18000rpm浓缩。然后，将分散物在真空炉中干燥一夜。制备好的黑磷量子点保存在氩气手套箱中，不需要光照。
优选地，步骤(2)所述将黑磷量子点加载到外泌体中的方法包括：将黑磷量子点、外泌体和PBS液混合搅拌，超声，冷却。
优选地，所述超声的时间为2.5-3.5min，例如2.5min、2.6min、2.8min、2.9min、3.0min、3.2min或3.5min等。
优选地，所述冷却的时间为1.5-2.5min，例如1.5min、1.7min、1.8min、2.0min、2.2min、2.3min、2.4min或2.5min等。
作为本发明的优选技术方案，所述用于癌症治疗的光纳米疫苗的制备方法包括如下步骤：
(1)分离外泌体：肿瘤区域使用808nm的激光进行辐照(1W/cm 2)后分离血浆，采用超离心法分离血浆中的外泌体，并在-80℃保存；
(2)合成黑磷量子点：将黑磷晶体和异丙醇混合避光，浴超声(360W)48h后，以9000rpm离心30min；收集上清液并以18000rpm离心浓缩、干燥；
(3)将黑磷量子点、外泌体和PBS液混合搅拌，超声2.5-3.5min，冷却1.5-2.5min，将黑磷量子点加载于外泌体中。
再一方面，本发明提供一种如上所述的用于癌症治疗的光纳米疫苗在制备用于癌症治疗或癌症预防的药物中的应用。
优选地，所述药物的剂型包括药剂学上的可接受的任意一种剂型，例如片剂、胶囊剂、混悬剂、栓剂、喷剂、注射剂或滴剂等。
优选地，所述药物还包括药剂学上可接受的药用辅料中的任意一种或至少两种的组合，所述药用辅料包括赋形剂、载体、增溶剂、助溶剂、稳定剂、缓释剂等。所述载体例如脂质体、胶束、树枝状大分子、微球、微囊等。
优选地，所述药物还包括直接抗肿瘤药物、辅助抗肿瘤药物或分子靶向药物。
所述直接抗癌药物包括化疗药物，例如氟尿嘧啶、环磷酰胺等药物；另外还包括抗肿瘤抗生素，例如阿霉素、紫杉醇类、紫杉烷类等。所述辅助抗癌药物多为调节内环境的内分泌治疗药物，如阿那曲唑、芳香化酶抑制剂等。所述分子靶向药物大部分情况可破坏肿瘤细胞的血管，使肿瘤细胞缺乏供血，饿死癌细胞；另外部分分子靶向药物是将癌细胞表面生长信 号封闭，达到肿瘤细胞无法启动生长的目的。
相对于现有技术，本发明具有以下有益效果：
本发明创造性地将纳米颗粒为基础的光热治疗与免疫治疗结合起来，建立了一种新的有效的个性化光纳米疫苗，即使用外泌体作为携带黑磷量子点和肿瘤特异性抗原的有效载体。其优势包括：(1)该光纳米疫苗具有长血循环效应，且外泌体可以保护黑磷量子点，避免其快速生物降解；(2)免疫原性诱导，肿瘤细胞的外泌体携带多种肿瘤抗原，可作为抗原载体作用于树突状细胞(DCs)和主要的细胞毒性T淋巴细胞，并对肿瘤产生免疫应答；(3)靶向性选择，外泌体含有细胞膜，可通过一系列表面粘附蛋白和载体配体(如整合素、CD11b和CD18受体)附着于靶细胞，并将有效载荷传递到靶细胞。外泌体可携带在母细胞膜中发现的细胞类型特异性蛋白，如来自少突胶质细胞的髓鞘蛋白和来自外泌体中肿瘤细胞的特异性抗原，具有独特的归巢选择性。与单纯的黑磷量子点相比，本发明所涉及的光纳米疫苗具有更好的体内长期稳定性、光热转换性和肿瘤靶向性；此外，其对肿瘤具有的显著治疗效果，并促进了肿瘤组织T淋巴细胞的浸润，是一种创新的用于治疗癌症的光纳米疫苗。
附图说明
图1是实施例1中实验组和对照组的人肺癌抗原MUC1和上皮细胞粘附分子EpCAM表达结果图；
图2是实施例1中实验组和对照组的glypican-3(GPC3)表达结果图；
图3是实施例1中分离得到的外泌体的粒径分布图；
图4是实施例1中分离得到的外泌体的TEM图；
图5是实施例1中外泌体被DC细胞的摄取结果图；
图6是外泌体诱导形成CD8α+CD11c+的比例结果图；
图7是外泌体诱导DC细胞成熟的结果图；
图8是黑磷量子点的TEM图；
图9是黑磷量子点的高分辨TEM图；
图10是黑磷量子点的形态信息图；
图11是黑磷量子点的厚度信息图；
图12是黑磷量子点的拉曼光谱图；
图13是本发明所涉及的hEX@BP的透射电镜图；
图14是本发明所涉及的hEX@BP的透射电镜图；
图15是本发明所涉及的hEX@BP的粒径分布图；
图16是BPQD的装载效率与剂量关系图；
图17是肿瘤细胞对hEX@BP的摄取结果图；
图18是本发明所涉及的hEX@BP对U251(人神经胶质瘤细胞)、LLC(小鼠肺癌细胞)和4T1(乳腺癌细胞)的细胞毒性结果图；
图19是本发明所涉及的hEX@BP对LLC(小鼠肺癌细胞)的细胞毒性结果图；
图20是hEX@BP在808nm激光照射下的浓度依赖性温升曲线图；
图21是hEX@BP光热稳定性能表征图；
图22是实施例5中hEX@BP在肿瘤小鼠体内的荧光成像图；
图23是实施例5中hEX@BP在肿瘤小鼠中各组织中的荧光成像图；
图24是实施例5中肿瘤切片的荧光图；
图25是hEX@BP在注射后长期的PTT功效评价结果图；
图26是小鼠左侧足底注射hEX-DiR或hEX@BP-DiR后的荧光成像图；
图27是本发明所涉及的hEX@BP在治疗性疫苗治疗程序中肿瘤体积随时间的变化趋势图；
图28是本发明所涉及的hEX@BP在治疗性疫苗治疗程序中小鼠体重随时间的变化趋势图；
图29是本发明所涉及的hEX@BP在治疗性疫苗治疗程序中小鼠生存率随时间的变化趋势图；
图30是本发明所涉及的hEX@BP在预防性疫苗治疗程序中肿瘤体积随时间的变化趋势图；
图31是本发明所涉及的hEX@BP在预防性疫苗治疗程序中小鼠体重随时间的变化趋势图；
图32是本发明所涉及的hEX@BP对IFN-γ的分泌水平影响结果图；
图33是本发明所涉及的hEX@BP对IL-2的分泌水平影响结果图；
图34是本发明所涉及的hEX@BP对IL-6的分泌水平影响结果图；
图35是本发明所涉及的hEX@BP对TNF-α的分泌水平影响结果图；
图36是本发明所涉及的hEX@BP对IL-10的分泌水平影响结果图；
图37是本发明所涉及的hEX@BP对CD4+T细胞比例影响结果图；
图38是本发明所涉及的hEX@BP对CD8+T细胞比例影响结果图。
具体实施方式
下面通过具体实施方式来进一步说明本发明的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本发明，不应视为对本发明的具体限制。
以下实施例中涉及到的动物实验得到伦理委员会批准(暨南大学)，根据动物福利委员会批准的标准指导方针进行的动物实验(暨南大学，中国)。
除另有说明外，以下实施例的所有数据均以平均值±标准差表示。治疗组和对照组之间的统计学差异通过使用SPSS 22.0版软件(SPSS，Inc.，Chicago，IL，USA)进行单因素或双因素方差分析和Tukey事后检验(多重比较)。生存资料采用log-rank检验。*表示差异有统计学意义，p<0.05；**，p<0.01；和***，p<0.001。
实施例1
分离外泌体及相关评价
(1)建立肺癌小鼠模型和肝癌小鼠模型
分别在雌性C57BL/6小鼠皮下(s.c.)注射5×10 5肝肿瘤细胞(Hepa1-6细胞)，在雌性C57BL/6小鼠皮下(s.c.)注射5×10 5肺肿瘤细胞(LCC细胞)，在雌性BALB/C裸鼠皮下(s.c.)注射5×10 6人肺肿瘤细胞(H1975细胞)，10天后，肿瘤体积达到200-300mm 3，LCC肺癌小鼠模型、H1975肺癌小鼠模型和Hepa1-6肝癌小鼠模型成功建立。
(2)光热治疗
在Hepa1-6肿瘤模型和H1975肿瘤模型的小鼠瘤内注射黑磷(BP)纳米颗粒，1h后，小鼠被麻醉，肿瘤区域使用808nm的激光辐照(1W/cm 2)10min，第二天重新进行第二次肿瘤光疗，此为实验组(n＝20)，以没有经过光热治疗的疾病模型小鼠为对照组(n＝20)。
(3)分离外泌体(用EX或hEX表示，前者为未热疗分离的，后者为热疗后分离的)
24h后分离小鼠全血，3000g离心15min，取细胞及细胞碎片，取血浆。采用超离心法分离血浆中的外泌体。超离心法具体为：血浆先以1000g离心10min，然后以10000g离心30min。收集上层清液，0.22μm滤膜过滤(SLGP033RS，Merck Millipore，Billerica，MA，USA)，然后在10万g超离心作用1h。外泌体在磷酸盐缓冲盐水(PBS)中洗涤，10万g离心1h回收。富含外泌体的部分用PBS洗涤两次，并在-80℃保存。
(4)蛋白样品制备，SDS-PAGE和western blotting
利用RIPA裂解缓冲液(P0013B，Byotime)从外泌体中提取总蛋白。用BCA蛋白定量试剂盒(P0010，Byotime)检测浓度。然后进行SDS-PAGE和western blotting。样本(包含10μg总蛋白和1×加样缓冲液)被标准的SDS-PAGE分离，其后用考马斯亮蓝染色或样本转 到聚乙二烯二氟化膜(Millipore)。这些膜与MUC1(16564S；1:1000，Cell Signalling Technology，CST)、EpCAM(14452S；1:1000，CST)、GPC3(ab95363；1:1000，Abcam)、CD63(ab217345，ab134045；1:1000，Abcam)、CD9(ab92726；1:1000，Abcam)抗体4℃过夜。接下来，用适当的辣根过氧化物酶(HRP)标记的抗兔IgG或HRP标记的抗小鼠二抗室温孵育膜1小时，用增强型化学发光试剂(Applygen)检测反应条带。
肺癌(H1975)小鼠模型的结果如图1所示：与对照组小鼠相比，实验组可检测到更加丰富的人肺癌抗原MUC1和上皮细胞粘附分子EpCAM表达。
肝癌(Hepa1-6)小鼠模型的结果如图2所示：与对照组小鼠相比，实验组可检测到更加丰富的glypican-3(GPC3)表达。说明从经过热疗的肿瘤小鼠体内分离到的分泌体携带有更多的肿瘤特异性抗原。
分别用DLS和TEM(透射电镜)分析肺癌模型组分离的外泌体的大小和形态分布，由图3和图4所示：外泌体的大小(中位径100nm)均匀，形态相似。
树突状细胞(DC)是一组异质化细胞，研究表明CD8α+DC有能力参与Th1细胞的分化和CD8+T细胞抗原提呈，在激活免疫反应中这是很重要的策略。为了测试外泌体是否能被DCs吸收，使用热疗肺癌(LLC)小鼠模型来源的外泌体与DCs共孵育，并在共孵育24h后检测细胞摄取情况。荧光成像图如图5所示(细胞核用Hoechst(蓝色)染色，外泌体用DiR(红色)染色)：内化的DiR标记的外泌体在外观上是分布于核周并呈点状。
为了分析外泌体对未被DiR染色的DC细胞分化和成熟的影响，我们以GM-CSF(粒细胞-巨噬细胞集落刺激因子)培养的DC细胞为阳性对照组，以单纯的DC细胞对空白对照组，以未经过热疗分离出的外泌体培养的DC细胞为阴性对照组，以经过热疗分离出的外泌体培养的DC细胞为实验组，利用细胞流式术探究各条件下DC细胞分化和成熟情况，采用Flow-jo软件进行统计和分析。由图6可知：与各对照组比较，光热治疗肿瘤小鼠内的外泌体诱导CD8α+CD11c+的比例增加；由图7可知：与各对照组比较，光热治疗肿瘤小鼠内的外泌体最能诱导DC成熟，表现出最高的CD8α以及CD80和CD86的表达，这表明经热疗后肿瘤小鼠中分离的分泌体能够激活DCs，具有高的免疫原性。
实施例2
黑磷量子点(用BPQDs表示)的合成及相关评价
采用简单的液相去角质法制备黑磷量子点，将100mg的黑磷晶体和50mL的异丙醇混合在一个60mL的棕色瓶子里。在浴超声48h(360W)后，以9000rpm离心30min。收集上清 液并18000rpm下离心浓缩。然后，将分散物在真空炉中干燥24h。制备好的黑磷量子点保存在氩气手套箱中，不需要光照。
用TEM(FEI Tecnai G2 spirit，80kv)测定BPQDs的形态和尺寸，如图8所示：黑磷量子点外观呈球形，形态分布均匀，粒径2.8±1.6nm。高分辨率TEM图像如图9所示：显示BP晶体(041)平面的0.224nm的晶格条纹。将BPQDs分散在IPA中，滴在Si/SiO 2衬底上，通过AFM测量BPQDs，获得形态信息(如图10所示)和厚度信息(如图11所示，此图为图10所示线的高度剖面图，图中line1和line2分别为图10中线性标记区域)。采用拉曼光谱法(InVia反射共焦拉曼显微镜)测定了BPQDs的拉曼光谱，如图12所示。
实施例3
制备光纳米疫苗及相关评价
采用超声法制备光纳米疫苗(用hEX@BP表示)：将100μg/mL的黑磷量子点和实施例1LCC肺癌小鼠模型分离得到的分泌体在PBS中搅拌(hEX与BPQDs的质量比为10:1)。然后，将混合物超声6个周期，每个周期30s，共3min，2min的冷却时间，最终使BPQD加载到外泌体中(hEX@BP)。
对制得的hEX@BP进行透射电镜(TEM)表征，如图13和图14所示：hEX@BP表现出良好的球形和均匀的尺寸，且清晰可见黑磷量子点被外泌体包覆。用DLS测量hEX@BP的粒径，如图15所示：平均水动力尺寸为100nm左右。此外，我们还考察了BPQDs在hEX中的装载效率，结果如图16所示：BPQD的装载效率与剂量有关，当投料比达到1:5时，包覆BPQDs的重量是hEX的8倍以上。
实施例4
光纳米疫苗光热效应和体外生物相容性评价
用DiR和Calcein-AM分别标记外泌体和细胞质，将LLC和Hepa1-6细胞与hEX@BP(LCC肺癌小鼠模型和Hepa1-6肝癌小鼠模型)孵育4h后，用Hoechst、Calcein染细胞，DiR标记hEX@BP，结果如图17所示：显示肿瘤细胞可以有效吸收hEX@BP(图中Merge表示重叠图，标尺为25μm)。
将U251(人神经胶质瘤细胞)、LLC(小鼠肺癌细胞)和4T1(乳腺癌细胞)种在96孔板(2×10 4细胞)中，在完全培养基中培养12h后，细胞培养基替换为100mL含有不同浓度的实施例3中hEX@BP的H-DMEM(内部BPQDs浓度为0、5、10、25、50和100μg/mL)，重复四个样本，24h后用CCK-8法测定细胞活力，不经任何处理将细胞活力归一化至对照组， 计算细胞生长抑制率：细胞活力(％)＝(处理组细胞活力平均值/对照组细胞活力平均值)×100％。结果如图18所示：在100μg/mL浓度下hEX@BP的细胞毒性很小，几乎可以忽略不计(图中柱形从左至右依次为U251、LLC和4T1组)。
将LLC细胞与上述hEX@BP在不同浓度(0、5、10、25、50、100μg/mL)孵化4h，然后暴露在波长808nm的激光中(1.0W/cm2，10min)，然后用吖啶橙(AO，绿色荧光指示活细胞)和碘化丙啶(PI，红色荧光指示死细胞)对LLC细胞进行荧光染色，以测定辐照后细胞的活力。结果如图19所示：随着hEX@BP浓度的增加，大部分LLC细胞被光热消融逐渐杀死，用红色荧光标记的死亡细胞数量也随之增加(图中标尺为25μm)。
我们还探究了hEX@BP(1mL，分散在PBS中)在808nm激光照射(1W/cm 2)下的浓度依赖性温升曲线，如图20所示：在808nm激光照射下，hEX@BP色散可在10min内上升到近50℃，表现出良好的光热性能。此外，光稳定性对于光热剂来说也是至关重要的，所以我们设置了6个循环，如图21所示：hEX@BP色散在每个循环中经历了10min的808nm激光照明和自然冷却，温度升高的衰减可以忽略不计，说明hEX@BP具有良好的光热稳定性，可以计算出hEX@BP的光热转换效率为23.2％。
综上，本发明所涉及的光纳米疫苗hEX@BP具有显著的体外生物相容性和光热效应，可以促进癌细胞的光触发消融。
实施例5
光纳米疫苗对肿瘤和淋巴结的靶向性评价
通过静脉注射常用的近红外荧光染料DiR标记的外泌体(hEX-DiR和hEX@BP-DiR)，检测Balb/c裸小鼠的LLC诱导肺癌的生物分布，并进行荧光成像检测。具体操作为：Balb/c裸小鼠背部右侧注入5×10 5LLC肿瘤细胞，第10天将荷瘤BALB/c小鼠分三组，分别静脉注射生理盐水、hEX-DiR(0.5mg/kg hEX)和hEX@BP-DiR(5mg/kg BP，0.5mg/kg hEX)。在注射后预定的时间点，使用荧光(Xenogen IVIS-Spectrum)成像系统进行全身成像，如图22所示，在注射后24h，从肿瘤中可以观察到相当大的荧光，可以明确地从其他组织中勾画出皮下肿瘤；注射后48h，肿瘤内的荧光强度逐渐增强，说明外泌体可以在肿瘤部位持续积累。48h后，将荷瘤小鼠安乐死，主要器官(心、肝、脾、肺、肾、肿瘤)进行体外成像，并对肿瘤及主要器官每克组织的荧光强度(平行测试3次)进行统计，如图23所示(图中从左到右，从上到下依次为心、肝、脾、肺、肾、肿瘤)：得到与上述相同结论。肿瘤被临时冻结并切成10μm切片，切片用10％中性缓冲福尔马林固定，石蜡包埋，整个肿瘤切片使用DAPI 标记所有肿瘤细胞的细胞核。肿瘤切片的荧光图像在Leica DM4000B荧光显微镜上观察，如图24所示：显示细胞核(DAPI染色，蓝色显示)和外泌体(红色显示)明显共域化，证实了外泌体(hEX和hEX@BP)在肿瘤细胞内的有效穿透。
此外，我们进行了动物实验，以评价hEX@BP在注射后长期的PTT功效。静脉注射hEX@BP和裸BPQDs(5mg/kg)，ΔT被热监控。1天之后，ΔT在生理盐水对照组仅有9℃，而BPQD和hEX@BP治疗组分别为17.8和30.5℃(如图25所示)。再7天后，测量ΔT，结果表明：即使在7天之后，ΔT在BPQD组和hEX@BP组分别达到10℃和20℃，表明hEX@BP在注射后长期的PTT功效。
同时，在小鼠左侧足底注射hEX-DiR或hEX@BP-DiR，注射后6h，用体内荧光成像技术观察小鼠的迁移情况。结果如图26所示：hEX-Dir或hEX@BP-Dir均能有效迁移至淋巴结，与抗原呈递细胞(APCs)相互作用，这对诱导的免疫应答有重要影响。
实施例6
光纳米疫苗对肿瘤的治疗效果评价
治疗性疫苗治疗程序：肿瘤接种后9天(5×10 5LLC细胞/小鼠)，C57BL/6小鼠被随机分为三组(每组6只)，静脉注射接种100μL不同的疫苗配方：PBS(对照组)；hEX@BP(5mg/kg BP和0.5mg/kg hEX)；hEX@BP+NIR(每只小鼠的hEX@BP为5mg/kg BP和0.5mg/kg hEX，采用近红外照明，1W/cm 2，8min)，在接种肿瘤细胞后第9天和第16天分别给予两次PTT治疗，治疗后每隔1天测量肿瘤大小，计算公式为：体积＝(宽) 2×长×0.5。
肿瘤体积曲线图如图27所示：hEX@BP纳米疫苗能有效抑制肿瘤早期生长，另外两组(PBS单独处理或hEX单独处理)的肿瘤随后迅速生长，导致LLC细胞接种后15-25天内所有小鼠死亡；不同治疗后随时间体重变化曲线图如图28所示；小鼠的生存情况如图29所示：80％的小鼠在联合治疗后肿瘤消失，并在肿瘤细胞接种后第40天存活。表明我们开发的外泌体包被的BPQD纳米疫苗联合PTT治疗可能是一种很有吸引力的癌症免疫联合治疗策略。
预防性疫苗治疗程序：C57BL/6小鼠随机分为四组(每组5只)，静脉注射接种三次(间隔一个星期)，静脉注射不同的疫苗配方分别是：100μL PBS(对照组)；PBS溶液中裸BPQDs(每只小鼠5mg/kg BP)；hEX@BP(每只小鼠5mg/kg BP和0.5mg/kg hEX)；hEX@BP(每只小鼠5mg/kg BP和0.5mg/kg hEX)。在最后一次接种后7天，将5×10 5LLC细胞皮下注射到每只小鼠的右侧。接种肿瘤9天后，第4组小鼠接受近红外照射(1W/cm 2，8min)，每隔1天测量肿瘤大小，计算公式为：体积＝(宽) 2×长×0.5。当肿瘤体积达到1500mm 3时处死小鼠。
如图30所示：与对照组(PBS)相比，免疫组和基于BP的PTT治疗组仅能部分抑制肿瘤的进展，与其他三组相比，hEX@BP+NIR组小鼠同时接种了hEX@BP疫苗和基于BP的PTT疫苗后，肿瘤生长明显延迟，且无体重异常变化(如图31所示)，这可能是由于hEX@BP诱导的免疫刺激作用和近红外肿瘤消融所致。
实施例7
基于hEX@BP的光热消融诱导免疫系统的激活评价
本发明所涉及的光纳米疫苗hEX@BP的高负载量、高效的细胞摄取和良好的抗肿瘤效果促使我们在体内进一步研究其潜在的免疫佐剂性能。用实施例6的治疗性疫苗程序处理C57BL/6小鼠，在最后一次治疗7天后，所有小鼠均被处死，并切除脾脏。
(1)脾捣碎，经过70μm细胞过滤器，并使用10mL的红细胞溶解液裂解红细胞30min。PBS洗两次，在1200rpm离心2min，以1×10 7细胞的密度接种于6孔板中，2mL 1640完全培养基，培养3天。上清液中细胞因子的检测使用鼠interferon-γ(IFN-γ，430807)，白细胞介素-2(IL-2，431007)，白细胞介素-6(IL-6，431307)，白细胞介素-10(IL-10，431417)和肿瘤坏死因子α(TNF-α)酶联免疫吸附试验(ELISA)试剂盒(Biolegend)。结果如图32-图36所示：hEX@BP治疗组在808nm近红外光谱辐照相对于其他群体使得IFN-γ、IL-2、IL-6、TNF-α的分泌水平大大提高，对分泌的IL-10水平并没有显著变化。与此同时，我们发现hEX@BP免疫没有近红外光谱辐照也引发了如IL-2/6和TNF-α细胞因子的分泌。
(2)通过荧光激活细胞分选(FACS)分析，进一步检测脾脏中CD4+T细胞(CD3e+CD4+作为标记)和CD8+T细胞(CD3e+CD8+作为标记)的频率。用抗小鼠CD3e-APC抗体、抗小鼠CD4-FITC抗体、抗小鼠CD8-PE抗体(均购自美国eBioscience公司)对分离的细胞进行染色，流式细胞术分析，并使用FlowJo软件分析，定量分析各组脾脏CD3e、CD4+、CD8+T细胞比例(n＝3)，结果如图37-38所示：在808nm NIR照射下免疫hEX@BP的小鼠CD4+和CD8+T细胞的频率高于其他各组，同时，与对照组相比，未近红外照射的hEX@BP组脾脏CD8+T细胞比例明显升高，说明hEX@BP单独在体内激活免疫系统是有效的。
综上结果表明，PTT可以增加炎症细胞的浸润，如CD4+T细胞和CD8+T细胞。此外，hEX@BP介导的PTT与免疫应答的协同作用较单独的hEX@BP介导的免疫应答有更好的协同免疫增强作用。综上所述，这些结果表明hEX@BP介导的PTT联合疫苗增强了对肿瘤的反应。
申请人声明，本发明通过上述实施例来说明本发明的一种用于癌症治疗的光纳米疫苗及 其制备方法和应用，但本发明并不局限于上述实施例，即不意味着本发明必须依赖上述实施例才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。

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  1. 一种用于癌症治疗的光纳米疫苗，其特征在于，所述光纳米疫苗包括黑磷量子点和外泌体，所述黑磷量子点包裹于所述外泌体中。
  2. 如权利要求1所述的用于癌症治疗的光纳米疫苗，其特征在于，所述黑磷量子点与外泌体的质量比为1:(8-12)。
  3. 如权利要求1或2所述的用于癌症治疗的光纳米疫苗，其特征在于，所述癌症包括肺癌、肝癌、乳腺癌、膀胱癌、胰腺癌、淋巴癌、食管癌、胃癌或结肠癌。
  4. 如权利要求1-3中任一项所述的用于癌症治疗的光纳米疫苗，其特征在于，所述外泌体为肿瘤细胞的外泌体；
    优选地，所述外泌体为经消融后的肿瘤细胞的外泌体。
  5. 如权利要求1-4中任一项所述的用于癌症治疗的光纳米疫苗的制备方法，其特征在于，所述制备方法包括如下步骤：
    (1)分离外泌体和合成黑磷量子点；
    (2)将步骤(1)制得的黑磷量子点加载到外泌体中，得到所述用于癌症治疗的光纳米疫苗。
  6. 如权利要求5所述的用于癌症治疗的光纳米疫苗的制备方法，其特征在于，步骤(1)所述分离外泌体的方法为：对肿瘤患者进行光热治疗，取血浆并从血浆中分离出外泌体；
    优选地，所述进行光热治疗的方式为：在肿瘤内注射黑磷纳米颗粒，使用近红外激光辐照肿瘤区域；
    优选地，所述从血浆中分离出外泌体采用超离心法。
  7. 如权利要求5或6所述的用于癌症治疗的光纳米疫苗的制备方法，其特 征在于，步骤(1)所述合成黑磷量子点的方法为液相去角质法；
    优选地，所述液相去角质法包括：将黑磷晶体与异丙醇混合后进行浴超声，离心，上清液浓缩并干燥；
    优选地，所述黑磷晶体与异丙醇的比例为(1.5-2.5)mg/mL；
    优选地，所述浴超声的时间为45-50h；
    优选地，所述浴超声的功率为340-380W；
    优选地，所述离心为在(8000-10000)rpm下离心20-40min。
  8. 如权利要求5-7中任一项所述的用于癌症治疗的光纳米疫苗的制备方法，其特征在于，步骤(2)所述将黑磷量子点加载到外泌体中的方法包括：将黑磷量子点、外泌体和PBS液混合搅拌，超声，冷却；
    优选地，所述超声的时间为2.5-3.5min；
    优选地，所述冷却的时间为1.5-2.5min。
  9. 如权利要求1-4中任一项所述的用于癌症治疗的光纳米疫苗在制备用于癌症治疗或癌症预防的药物中的应用。
  10. 如权利要求9所述的应用，其特征在于，所述药物的剂型包括药剂学上的可接受的任意一种剂型；
    优选地，所述药物还包括药剂学上可接受的药用辅料中的任意一种或至少两种的组合；
    优选地，所述药物还包括直接抗肿瘤药物、辅助抗肿瘤药物或分子靶向药物。
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