WO2023155861A1 - 一种铝纳米晶复合免疫药物及其制备方法和应用 - Google Patents

一种铝纳米晶复合免疫药物及其制备方法和应用 Download PDF

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WO2023155861A1
WO2023155861A1 PCT/CN2023/076660 CN2023076660W WO2023155861A1 WO 2023155861 A1 WO2023155861 A1 WO 2023155861A1 CN 2023076660 W CN2023076660 W CN 2023076660W WO 2023155861 A1 WO2023155861 A1 WO 2023155861A1
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aluminum
immunomedicine
nanocrystal
nanocrystal composite
tumor
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French (fr)
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王亚玲
陈春英
赵宇亮
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广东粤港澳大湾区国家纳米科技创新研究院
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Publication of WO2023155861A1 publication Critical patent/WO2023155861A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/52—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an inorganic compound, e.g. an inorganic ion that is complexed with the active ingredient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/208—IL-12
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • 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 relates to the fields of biomedical technology and vaccine technology, in particular to an aluminum nanocrystal composite immunomedicine and its preparation method and application.
  • tumor is still a disease that is difficult to cure and has a high lethality rate.
  • cancer cancer
  • Surgical therapy directly removes the diseased part through surgical operation, which is better for early-stage tumors or benign tumors with low spreading ability, but for cancer patients that have metastasized, its curative effect and prognosis are poor. Due to the lack of specificity, chemotherapy can also cause great damage to the patient's own immune system and stem cells while treating tumors.
  • Immunotherapy is a general term for a large class of treatments that activate the body's immune system to treat cancer.
  • the immune system in the human body has always acted as a "police officer", expelling foreign invaders like the body.
  • Cancer immunotherapy works in three ways: (1) designing monoclonal antibodies to boost the immune response to destroy cancer cells; (2) using immune checkpoint inhibitors to help the immune system recognize and attack cancer cells; (3) Synthesize cancer vaccines to elicit immune responses to treat and prevent cancer.
  • immunotherapy primarily gives the body the ability to attack tumor cells by activating the immune system, a process that may last long after initial treatment due to the memory function of the body's immune system.
  • the immune system also has the ability to distinguish cancer cells from normal cells, and can selectively attack cancer cells by activating the immune response And avoid damaging normal cells.
  • Cytokines are one of the major players in the regulation of immune responses, and cytokine-based approaches offer an alternative strategy for cancer immunotherapy. Over the past few decades, cytokines and cytokine receptors have been extensively studied as targets for cancer therapy. Cytokines are expressed by immune cells (such as monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.) It is a kind of small molecular protein secreted with a wide range of biological activities, and can regulate various functions such as innate immunity and adaptive immunity, hematopoiesis, cell growth, APSC pluripotent cells and damaged tissue repair by binding to corresponding receptors.
  • immune cells such as monocytes, macrophages, T cells, B cells, NK cells, etc.
  • non-immune cells endothelial cells, epidermal cells, fibroblasts, etc.
  • Cytokines can be divided into interleukins, interferons, some members of the tumor necrosis factor superfamily, colony-stimulating factors, chemokines, growth factors, etc.
  • cytokines are key mediators of cellular communication. Dysregulation of cytokine production by malignant cells, immune cells, and stromal cells is involved in all stages of tumorigenesis and progression. Certain cytokines are closely associated with tumor development, progression, and metastasis, and abnormal production of inflammatory cytokines is a common downstream consequence of oncogenic changes in nonmalignant cells. Therefore, the treatment of tumor tissue can be effectively achieved by utilizing the immunostimulatory effect of cytokines and the neutralization of cytokines when they are out of balance.
  • systemic administration of cytokines has certain systemic toxicity due to lack of targeting, and has potential adverse effects on embryonic tissues. Therefore, local administration of cytokine drugs is a relatively effective and safe way.
  • the present invention provides an aluminum nanocrystal composite immune drug, its preparation method and application, and functional polypeptide sequences and cytokines linked to the surface of the aluminum nanocrystal based on the aluminum nanocrystal composite immune drug.
  • the aluminum nanocrystals provided by the present invention can effectively combine functional polypeptide sequences and cytokines through polyethylene glycol molecules to construct compound immune drugs, effectively improve the binding ability of compound immune drugs to tumor cells at tumor sites through functional polypeptide sequences, and increase The accumulation time of compound immune drugs in the tumor site; at the same time, it can enhance the stability of cytokines and play an immune role in the tumor site.
  • the time window for immune regulation ability can effectively improve the body's immune response to tumor tissue and inhibit the growth of tumor tissue.
  • the invention provides an aluminum nanocrystal compound immunomedicine, which is constructed based on the aluminum nanocrystal through the combination of polyethylene glycol molecules with functional polypeptide sequences and cytokine molecules.
  • the functional polypeptide sequence includes a polypeptide sequence that can specifically bind to proteins highly expressed on the surface of tumor cells.
  • the aluminum nanocrystal compound immune drug, the functional polypeptide sequence is one or both of the circular RGD sequence and the TAT sequence that can assist the immune drug to enter cells.
  • the cytokine molecules are selected from interleukin factors, and the interleukin factors include IL-2, IL-7, IL-12, IL-15, IL -18, IL-21, interferon gamma IFN- ⁇ or tumor necrosis factor TNF ⁇ one or more.
  • the construction method is that the amino group of the M-PEG-NH 2 on the surface of the aluminum nanocrystal forms a covalent amide bond with the carboxyl group on the cytokine molecule.
  • the aluminum nanocrystals are prepared through the following steps:
  • the solute of the aluminum salt solution is one or more of aluminum chloride, aluminum nitrate, aluminum sulfate and aluminum acetate;
  • the solvent used in the aluminum salt solution is pure water or sodium acetate with a concentration of 0.01mol/L solution;
  • polyethylene glycol is a mixture of phosphoserine-modified polyethylene glycol (PS-PEG) and polyethylene glycol (M-PEG-NH 2 ) with maleimide groups and terminal carboxylic acids reagent.
  • NaOH aqueous solution is added to adjust the pH to 5.0-8.0.
  • the present invention also provides a method for preparing the above-mentioned aluminum nanocrystal compound immunomedicine, comprising the following steps:
  • Cytokine molecules are added to the MES buffer, and the carboxyl groups on the EDC/NHS cytokine activation molecules are added, and then the aluminum nanocrystal solution is added to make the ammonia of M-PEG-NH 2
  • the base forms a covalent amide bond with the carboxyl group on the cytokine molecule to obtain the aluminum nanocrystal composite immune drug modified by the cytokine molecule.
  • the solvent is 0.1M phosphate buffer or 0.1M ammonium acetate buffer.
  • the functional polypeptide sequence includes a polypeptide sequence that can specifically bind to proteins highly expressed on the surface of tumor cells.
  • the functional polypeptide sequence is one or both of a circular RGD sequence and a TAT sequence that can assist immune drugs to enter cells.
  • the cytokine molecule is selected from interleukin factors, which include IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, gamma One or more of interferon IFN- ⁇ , tumor necrosis factor TNF ⁇ .
  • the present invention also provides the application of the aluminum nanocrystal composite immunomedicine or the aluminum nanocrystal composite immunomedicine prepared by the preparation method in the immunotherapy of tumor models.
  • the application is intravenous injection, mucosal administration, subcutaneous injection, intradermal injection, peritumoral injection or intratumoral injection.
  • a method for immunotherapy of tumors using the aluminum nanocrystal composite immunomedicine or the aluminum nanocrystal composite immunomedicine prepared by the preparation method.
  • the method of administration in the method includes: intravenous injection, mucosal administration, subcutaneous injection, intradermal injection, peritumoral injection or intratumoral injection.
  • the polyethylene glycol-assisted aluminum nanocrystals provided by the present invention can effectively combine functional polypeptide sequences and cytokines, effectively enhance the stability of cytokines and the time window for exerting immune regulation ability at tumor sites, and improve the body's ability to respond to tumor tissues.
  • the immune response inhibits the growth of tumor tissue.
  • Fig. 1 shows the effect analysis of the inhibition of tumor growth of the aluminum nanocrystal composite immunomedicine constructed in Examples 6 and 7 of the present invention
  • Fig. 2 shows the effect analysis of the inhibition of tumor growth of the aluminum nanocrystal composite immune drug constructed in Examples 8-13 of the present invention
  • Fig. 3 shows the analysis of the accumulation and stability of the aluminum nanocrystal composite immune drug in the mouse tumor model of the present invention
  • Fig. 4 shows that the aluminum nanocrystal composite immune drug constructed in Examples 6 and 7 of the present invention regulates the proliferation of CD8 T cells in tumor tissue in a mouse tumor model;
  • Fig. 5 shows the effect analysis of inhibiting the growth of remote tumor tissue by the aluminum nanocrystal composite immune drug constructed in Examples 6 and 7 of the present invention
  • Fig. 6 shows a schematic diagram of the construction process of the aluminum nanocrystal composite immune drug of the present invention.
  • the invention discloses an aluminum nanocrystal composite immune drug and its preparation method, as well as functional polypeptide sequences and cytokines linked to the surface of aluminum nanocrystals in the aluminum nanocrystal composite immune drug.
  • Those skilled in the art can refer to the content of this article and make appropriate improvements. Process parameters are realized.
  • all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.
  • the method and application of the present invention have been described through preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the method and application described herein without departing from the content, spirit and scope of the present invention to realize and Apply the technology of the present invention.
  • the raw materials and reagents used in Examples 1 to 19 provided by the present invention can be purchased from the market.
  • Embodiment 1 Aluminum nanocrystal (Al-PEG-1) preparation
  • Al-PEG-1 Dissolve AlCl 3 6H 2 O in 0.01mol/L NaAc to prepare an aluminum salt solution with an aluminum ion content of 0.05mol/L; add PS-PEG and M with a total molecular concentration of 0.05mol/L to the obtained aluminum salt solution -PEG-NH 2 molar ratio is a mixed solution of 1:1, wherein the molar ratio between Al 3+ and phosphoserine is 1:2; add NaOH aqueous solution to adjust the pH value to 7.6, after the reaction is completed, let it stand for 7 hours, Wash after centrifugation, and finally sterilize by high pressure steam or filter membrane to obtain aluminum nanocrystals, which are named Al-PEG-1.
  • Al(NO 3 ) 3 Dissolve Al(NO 3 ) 3 in pure water to prepare an aluminum salt solution with an aluminum ion content of 0.05 mol/L; add PS-PEG and M-PEG with a total molecular concentration of 0.05 mol/L to the obtained aluminum salt solution -NH 2 mixed solution with a molar ratio of 1:2, in which the molar ratio between Al 3+ and phosphoserine is 1:1; add NaOH aqueous solution to adjust the pH value to 5.0, after the reaction is completed, let it stand for 10 hours, and centrifuge Washing, and finally autoclaving or sterilizing through a filter membrane to obtain aluminum nanocrystals, named Al-PEG-2.
  • This embodiment prepares a kind of aluminum nanocrystal (Al-PEG-3), and the preparation method is basically the same as that of Example 1, the difference is that the aluminum salt used is aluminum acetate, and the molar ratio of PS-PEG and M-PEG-NH is 2:1, add NaOH aqueous solution to adjust the pH value to 8.0, and let it stand for 3 hours after the reaction is completed.
  • This example prepares an aluminum nanocrystal (Al-PEG-4), the preparation method is basically the same as that of Example 1, the difference is that the aluminum salt used is aluminum sulfate, NaOH aqueous solution is added to adjust the pH value to 6.5, and the static Set time is 8 hours.
  • the concentration of the aluminum nanocrystals was diluted to 10 ⁇ g/mL, and the particle size of the aluminum nanocrystals was tested with a nanoparticle size analyzer.
  • cytokine IL-12 is added to 50mM MES buffer, and the carboxyl group on the EDC/NHS activation molecule is added, and then the solution of the aluminum nanocrystal obtained in step (1) is added, wherein the aluminum nanocrystal surface M-PEG -
  • the ratio of NH 2 to cytokine molecules is 1.5:1, and the aluminum nanocrystal composite immune drug further modified by cytokine molecules is obtained, which is named Al-RGD-IL12.
  • Example 7 Aluminum nanocrystal composite immune drug Al-TAT-TNF ⁇
  • Example 8 Aluminum nanocrystal composite immune drug Al-TAT-IL2
  • cytokine IL2 The cytokine IL2 molecule is added to 50mM MES buffer, and the carboxyl group on the EDC/NHS activation molecule is added, and then the solution of aluminum nanocrystals obtained in step (1) is added, wherein the surface of aluminum nanocrystals is M-PEG-NH
  • the ratio of 2 to cytokine molecules was 1.2:1, and the aluminum nanocrystal composite immune drug further modified by cytokine molecules was obtained, which was named Al-TAT-IL2.
  • Example 9 Aluminum nanocrystal composite immune drug Al-RGD-IL15
  • cytokine IL15 The cytokine IL15 molecule is added to 50mM MES buffer, and the carboxyl group on the EDC/NHS activation molecule is added, and then the solution of the aluminum nanocrystal obtained in step (1) is added, wherein the surface of the aluminum nanocrystal is M-PEG-NH
  • the ratio of 2 to cytokine molecules was 1.3:1, and the aluminum nanocrystal composite immune drug further modified by cytokine molecules was obtained, which was named Al-RGD-IL15.
  • Example 10 Aluminum nanocrystal composite immune drug Al-TAT-IL18
  • Cytokine IL18 molecules were added to 50mM MES buffer, and added The carboxyl group on the EDC/NHS activation molecule is then added to the solution of the aluminum nanocrystals obtained in step (1), wherein the M-PEG-NH on the surface of the aluminum nanocrystals is 1.4: 1 with the cytokine molecule ratio, and the cytokine molecule is further obtained.
  • the modified aluminum nanocrystal composite immune drug is named Al-TAT-IL18.
  • Example 11 Aluminum nanocrystal composite immune drug Al-RGD-IL21
  • cytokine IL21 The cytokine IL21 molecule is added to 50mM MES buffer, and the carboxyl group on the EDC/NHS activation molecule is added, and then the solution of the aluminum nanocrystal obtained in step (1) is added, wherein the surface of the aluminum nanocrystal is M-PEG-NH
  • the ratio of 2 to cytokine molecules was 1.3:1, and the aluminum nanocrystal composite immune drug further modified by cytokine molecules was obtained, which was named Al-RGD-IL21.
  • Example 12 Aluminum nanocrystal composite immune drug Al-RT-IFN- ⁇
  • Example 13 Aluminum nanocrystal composite immune drug Al-RT-IL7
  • Example 1 The aluminum nanocrystals prepared in Example 1 (the product obtained in Example 1 is only used as a representative of subsequent experiments, does not mean that only the aluminum nanocrystals obtained in Example 1 can be used in subsequent experiments) and c(RGDfC) and TAT peptide (1.5:1 molar ratio) add 0.1M phosphate buffer or 0.1M In the ammonium acetate buffer solution, the ratio of the peptide sequence to the maleimide contained in the PEG molecule on the surface of the aluminum nanocrystal is 1.5:1, stirred and reacted for 24 hours, and centrifugally washed and purified to obtain the aluminum nanocrystal modified by the RGD peptide sequence ;
  • cytokine IL7 The cytokine IL7 molecule is added to 50mM MES buffer, and the carboxyl group on the EDC/NHS activation molecule is added, and then the solution of the aluminum nanocrystal obtained in step (1) is added, wherein the surface of the aluminum nanocrystal is M-PEG-NH
  • the ratio of 2 to cytokine molecules was 1.3:1, and the aluminum nanocrystal composite immune drug further modified by cytokine molecules was obtained, which was named Al-RT-IL7.
  • mice under the premise of following the national animal health protocol, BALB/c mice aged 6-8 weeks were selected for subcutaneous tumor implantation on the posterior side.
  • the tumor model was B16F10 mouse melanoma cells with high metastases, and the tumor tissue grown to 100mm
  • the mice were randomly divided into 5 groups, and 50 ⁇ L of the following drugs were injected into the tumor: 1, injection of saline (Ctrl); 2, commercial aluminum adjuvant Alum and a simple mixed sample containing 0.25 ⁇ g IL-12 (Alum -IL12); 3, the Al-RGD-IL12 compound immunomedicine containing 0.25 ⁇ g IL-12 constructed in Example 6; 4, commercial aluminum adjuvant Alum combined with A simple mixed sample (Alum-TNF ⁇ ) with 0.25 ⁇ g TNF ⁇ ; 5, the Al-TAT-TNF ⁇ composite immune drug containing 0.25 ⁇ g IL-12 constructed in Example 7. And the change of tumor volume in mice was recorded.
  • Figure 1 is an analysis of the effect of the constructed aluminum nanocrystal composite immune drug on inhibiting tumor growth.
  • the average size of the tumors in the control group was 1942 mm 3 ; the average size of the tumor volume in the Alum-IL12 group was 1364 mm 3 ; and the average size of the tumor volume in the Al-RGD-IL12 group was 183 mm 3 ;
  • the average tumor volume of the Alum-TNF ⁇ group was 1540mm 3 ; while the average tumor volume of the Al-TAT-TNF ⁇ group was 204mm 3 ; the constructed aluminum nanocrystal composite immune drug loaded with cytokines can effectively inhibit the growth of tumors.
  • mice under the premise of following the national animal health protocol, BALB/c mice aged 6-8 weeks were selected for subcutaneous tumor implantation on the posterior side.
  • the tumor model was B16F10 mouse melanoma cells with high metastases, and the tumor tissue grown to 100mm At about 3 , the mice were randomly divided into 5 groups, and 50 ⁇ L of the aluminum nanocrystal composite immune drug constructed in Examples 8-13 containing 0.25 ⁇ g of cytokines or gamma interferon was injected into the tumor. And the change of tumor volume in mice was recorded.
  • Figure 2 is an analysis of the effect of the aluminum nanocrystal composite immune drugs constructed in Examples 8-13 on inhibiting tumor growth.
  • the average tumor volume of the mice treated with the aluminum nanocrystal compound immune drugs constructed in Examples 8-13 was 181mm 3 -254mm 3 , which were significantly lower than those of the control group tumors shown in Example 15.
  • the ability to suppress tumors is the most direct indicator for verifying delivery efficiency.
  • the aluminum nanocrystal composite immune drugs obtained in Examples 6-13 of the present invention can significantly inhibit the growth of melanoma. Since their components are similar and their mechanisms of action are basically similar, only The Al-RGD-IL12 compound immune drug constructed in Example 5 and the Al-TAT-TNF ⁇ compound immune drug constructed in Example 6 were selected for subsequent further mechanism verification.
  • Example 15 For each group of mice in Example 15, the accumulation and accumulation of cytokines in the tumor tissues were analyzed. After 24 hours and 48 hours after the intratumoral injection of the drug, respectively, in 2, 3, 4 of Example 15 , 5 One mouse was randomly selected from each of the four groups, and its tumor tissue was dissected after humane sacrifice, and the operation was performed according to the instructions of the ELISA kit for interleukin 12 (IL-12) or tumor necrosis factor (TNF ⁇ ) The storage levels of IL-12 or TNF ⁇ in tumor tissues were analyzed.
  • IL-12 interleukin 12
  • TNF ⁇ tumor necrosis factor
  • FIG. 3 is an analysis of cytokine accumulation in the mouse tumor model in Example 15.
  • the stock level of interleukin 12 in the tumor tissue was: Alum-IL12 experimental group, 24.1 pg/mL; Al-RGD-IL12 experimental group, 60.9 pg/mL.
  • the stock level of interleukin-12 in tumor tissue was: Alum-IL12 experimental group, 11.4pg/mL; Al-RGD-IL12 experimental group, 52.3pg/mL.
  • the storage level of tumor necrosis factor in tumor tissue was: Alum-TNF ⁇ experimental group, 18.2pg/mL; Al-TAT-TNF ⁇ experimental group, 46.7pg/mL.
  • the storage level of tumor necrosis factor in tumor tissue was: Alum-TNF ⁇ experimental group, 8.4pg/mL; Al-TAT-TNF ⁇ experimental group, 41.5pg/mL. From the data, it can be seen that the constructed aluminum nanocrystal composite immune drug can significantly improve the stability and accumulation time of cytokines in the tumor environment.
  • FIG. 4 shows the proliferation of CD8 T cells in the tumor tissue in the mouse tumor model in Example 15.
  • the proportion of CD8T cells in tumor tissue was: Alum-IL12 experimental group, 1.5%; Al-RGD-IL12 experimental group, 7.1%; Alum-TNF ⁇ experimental group, 1.7%; Al -TAT-TNF ⁇ experimental group, 6.6%.
  • the proportion of CD8T cells in tumor tissue was: Alum-IL12 experimental group, 2.4%; Al-RGD-IL12 experimental group, 9.0%; Alum-TNF ⁇ experimental group, 2.3%; Al-TAT-TNF ⁇ experimental group , 8.4%. From the data, it can be seen that the constructed aluminum nanocrystal composite immune drug can significantly improve CD8T cell Cell proliferation ability, effectively regulate the immune response of tumor tissue.
  • Aluminum nanocrystal compound immune drug inhibits distant tumor growth.
  • B16F10 mouse melanoma cells were inoculated subcutaneously into the right hind leg of Balb/c mice, which was recorded as tumor in situ, and B16F10 mouse melanoma cells with half the number of cells were inoculated subcutaneously into the left hind leg of Balb/c mice, which was recorded as Distant tumors.
  • the average volume of the orthotopic tumor grows to 100mm 3
  • the average volume of the distal tumor is about 50mm 3
  • the mice are randomly divided into 5 groups, and 50 ⁇ L of the following drugs are injected into the orthotopic tumor: 1.
  • Figure 5 is an analysis of the effect of the constructed aluminum nanocrystal composite immune drug on inhibiting the growth of distant tumor tissue. It can be seen from Figure 5 that at 20 days, the average size of the distal tumors in the control group was 1286mm 3 ; the average size of the distal tumors in the Alum-IL12 group was 984mm 3 ; and the average size of the distal tumors in the Al-RGD-IL12 group was 253mm 3 ; the average volume of distal tumors in the Alum-TAT-TNF ⁇ group was 880mm 3 ; while the average volume of distal tumors in the Al-TAT-TNF ⁇ group was 382mm 3 ; Drugs can effectively inhibit the growth of distant tumors. It shows that the constructed aluminum nanocrystal composite immunomedicine can not only produce obvious inhibitory effect on the growth of existing tumors, but also have obvious inhibitory effect on the growth of new tumors.

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Abstract

本发明涉及生物医药技术与疫苗技术领域，尤其涉及一种铝纳米晶复合免疫药物及其制备方法和应用。其中铝纳米晶作为载体，在铝纳米晶表面覆盖有聚乙二醇，并通过聚乙二醇端位基团链接功能多肽序列和细胞因子分子，形成类病毒颗粒免疫药物，提升细胞因子稳定性以及在肿瘤组织停留时间，有效提升细胞因子辅助抗肿瘤效果。

Description

一种铝纳米晶复合免疫药物及其制备方法和应用
本申请要求于2022年02月18日提交中国专利局、申请号为202210152866.9、发明名称为“一种铝纳米晶复合免疫药物及其制备方法和应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物医药技术与疫苗技术领域，特别涉及一种铝纳米晶复合免疫药物及其制备方法和应用。
背景技术
截止目前，虽然临床医学上理论研究以及相关技术已经有了较大的进步，但是肿瘤(癌症)仍然是难以治愈和高致死率的疾病。临床上，肿瘤主要有手术、化学和放射三种传统治疗方案。手术疗法通过外科手术直接切除患病部位，对于早期肿瘤或扩散能力较低的良性肿瘤治疗效果较好，但是对于已经发生转移的癌症患者，其疗效和预后生活质量较差。化学疗法由于缺乏特异性，在治疗肿瘤的同时，对于患者自身免疫系统和干细胞也有较大伤害。近些年来，随着对肿瘤生长发展过程不断深入的研究，提出了免疫疗法这一创新性治疗方案，极大促进了癌症治疗的发展，并于2013年被Science杂志评为当年的十大科技突破之首，是近些年来癌症临床治疗的最重要的研究方向之一。
免疫疗法是一大类通过激活人体的免疫系统达到治疗癌症目的的治疗手段的统称。人体内的免疫系统一直充当着“警务人员”的角色，驱除像体内的外来入侵者。肿瘤免疫疗法可通过以下三种方式起作用：(1)设计单克隆抗体来增强免疫反应以破坏癌细胞；(2)使用免疫检查点抑制剂来帮助免疫系统识别和攻击癌细胞；(3)合成癌症疫苗以激发免疫应答以治疗和预防癌症。与其他治疗癌症手段不同，免疫疗法主要是通过激活免疫系统来赋予人体攻击肿瘤细胞的能力，由于人体免疫系统的记忆功能，其过程在初始治疗后可能会持续很长时间。此外，免疫系统还具备区分癌细胞与正常细胞的能力，并可通过激活免疫反应，从而选择性攻击癌细胞 并避免损坏正常细胞。
细胞因子是调节免疫反应的主要参与者之一，基于细胞因子的方法为癌症免疫治疗提供了另一种策略。在过去的几十年中，细胞因子和细胞因子受体作为癌症治疗的靶点已被广泛研究。细胞因子是由免疫细胞(如单核、巨噬细胞、T细胞、B细胞、NK细胞等)和某些特定的非免疫细胞(内皮细胞、表皮细胞、纤维母细胞等)经刺激而表达、分泌的一类具有广泛生物学活性的小分子蛋白质，并可通过结合相应受体调节固有免疫和适应性免疫、血细胞生成、细胞生长、APSC多能细胞以及损伤组织修复等多种功能。细胞因子可被分为白细胞介素、干扰素、肿瘤坏死因子超家族的部分成员、集落刺激因子、趋化因子、生长因子等。在肿瘤微环境中，细胞因子是细胞通讯的关键介质。恶性细胞、免疫细胞和基质细胞的细胞因子产生失调参与肿瘤发生和发展的所有阶段。某些细胞因子与肿瘤的发展、进展和转移密切相关，并且炎性细胞因子的异常产生是非恶性细胞中致癌变化的常见下游结果。因此，利用细胞因子的免疫刺激作用和细胞因子失调时中和它们的作用，能够有效实现肿瘤组织的治疗。然而，全身给予细胞因子由于缺乏靶向性，具有一定的全身毒性，并对胚胎组织也有潜在的不良作用，因此细胞因子药物进行局部给药是一种比较有效和安全的方式。
因此，研制一种基于铝纳米晶的复合免疫药物，解决细胞因子机体稳定性以及局部蓄积时间，提升细胞因子在肿瘤组织内与细胞作用时间，充分发挥调节免疫的作用，将极具实用价值。
发明内容
有鉴于此，本发明提供一种铝纳米晶复合免疫药物及其制备方法和应用，以及基于铝纳米晶复合免疫药物中的铝纳米晶表面链接的功能多肽序列和细胞因子。本发明所提供的铝纳米晶能够通过聚乙二醇分子有效结合功能多肽序列和细胞因子，构建复合免疫药物，通过功能多肽序列有效提升复合免疫药物在肿瘤部位与肿瘤细胞的结合能力，并增加复合免疫药物在肿瘤部位的蓄积时间；同时增强细胞因子稳定性和在肿瘤部位发挥免 疫调节能力的时间窗口，有效改善机体对肿瘤组织的免疫反应，抑制肿瘤组织的增长。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种铝纳米晶复合免疫药物，基于铝纳米晶通过聚乙二醇分子结合功能多肽序列和细胞因子分子构建而成。
进一步的，所述铝纳米晶复合免疫药物中，所述功能多肽序列包括可特异性结合肿瘤细胞表面高表达蛋白的多肽序列。
进一步的，所述铝纳米晶复合免疫药物，所述功能多肽序列为环形RGD序列、可协助免疫药物进入细胞的TAT序列中的一种或两种。
进一步的，所述铝纳米晶复合免疫药物，所述细胞因子分子选自白细胞介素类因子，所述白细胞介素类因子包括IL-2、IL-7、IL-12、IL-15、IL-18、IL-21、伽马干扰素IFN-γ或肿瘤坏死因子TNFα中的一种或多种。
进一步的，所述构建方式为铝纳米晶表面M-PEG-NH2的氨基与细胞因子分子上的羧基形成共价酰胺键。
进一步地，所述铝纳米晶通过以下步骤制备：
将铝盐溶液与聚乙二醇溶液搅拌充分混合，并加入NaOH水溶液调节pH后静置得沉淀，离心洗涤纯化后得聚乙二醇修饰的铝纳米晶。
进一步地，所述铝盐溶液的溶质为氯化铝、硝酸铝、硫酸铝和醋酸铝中的一种及以上；所述铝盐溶液所用溶剂为纯水或浓度为0.01mol/L的醋酸钠溶液；
进一步地，所述聚乙二醇为磷酸丝氨酸修饰的聚乙二醇(PS-PEG)与马来酰亚胺基团和末端羧酸的聚乙二醇(M-PEG-NH2)的混合试剂。
进一步地，加入NaOH水溶液调节所述pH为5.0～8.0。
本发明还提供了上述铝纳米晶复合免疫药物的制备方法，包括如下步骤：
(1)将铝纳米晶和功能多肽序列加入溶剂中，搅拌反应，并离心洗涤提纯，重悬至超纯水得到功能多肽序列修饰的铝纳米晶溶液；
(2)将细胞因子分子加入MES缓冲液中，并加入EDC/NHS细胞因子活化分子上的羧基，之后加入所述铝纳米晶溶液，使M-PEG-NH2的氨 基与细胞因子分子上的羧基形成共价酰胺键，得到细胞因子分子修饰的铝纳米晶复合免疫药物。
进一步地，所述溶剂为0.1M磷酸盐缓冲液或0.1M醋酸铵缓冲液。
进一步地，所述功能多肽序列包括可特异性结合肿瘤细胞表面高表达蛋白的多肽序列。
更进一步地，所述功能多肽序列为环形RGD序列、可协助免疫药物进入细胞的TAT序列中的一种或两种。
进一步地，所述细胞因子分子选自白细胞介素类因子，所述白细胞介素类因子包括IL-2、IL-7、IL-12、IL-15、IL-18、IL-21、伽马干扰素IFN-γ、肿瘤坏死因子TNFα中的一种或多种。
本发明还提供了上述铝纳米晶复合免疫药物或所述制备方法制得的铝纳米晶复合免疫药物在肿瘤模型免疫治疗中的应用。
所述应用为静脉注射使用、粘膜施用、皮下注射使用、皮内注射使用、瘤周注射使用或瘤内注射使用。
免疫治疗肿瘤的方法，施用所述的铝纳米晶复合免疫药物或所述制备方法制得的铝纳米晶复合免疫药物。
进一步地，所述方法中所述施用的方式包括：静脉注射使用、粘膜施用、皮下注射使用、皮内注射使用、瘤周注射使用或瘤内注射使用。
本发明具有的技术效果为：
本发明所提供的聚乙二醇辅助制备的铝纳米晶能够有效结合功能多肽序列和细胞因子，有效增强了细胞因子稳定性和在肿瘤部位发挥免疫调节能力的时间窗口，改善了机体对肿瘤组织的免疫反应，抑制了肿瘤组织的增长。
附图说明
图1示本发明实施例6、7所构建的铝纳米晶复合免疫药物抑制肿瘤生长的效果分析；
图2示本发明实施例8～13所构建的铝纳米晶复合免疫药物抑制肿瘤生长的效果分析；
图3示本发明实施例6、7所构建的铝纳米晶复合免疫药物在小鼠肿瘤模型中细胞因子蓄积和稳定性情况分析；
图4示本发明实施例6、7所构建的铝纳米晶复合免疫药物在小鼠肿瘤模型中调节肿瘤组织中CD8T细胞增殖情况；
图5示本发明实施例6、7所构建的铝纳米晶复合免疫药物抑制远端肿瘤组织生长的效果分析；
图6示本发明铝纳米晶复合免疫药物构建过程示意图。
具体实施方式
本发明公开了一种铝纳米晶复合免疫药物及其制备方法以及基于铝纳米晶复合免疫药物中的铝纳米晶表面链接的功能多肽序列和细胞因子，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明提供的实施例1～实施例19中所用原料及试剂均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1铝纳米晶(Al-PEG-1)制备
将AlCl3·6H2O溶于0.01mol/LNaAc中，配制成铝离子含量0.05mol/L的铝盐溶液；向得到的铝盐溶液加入总分子浓度为0.05mol/L的PS-PEG与M-PEG-NH2摩尔比例为1:1的混合溶液，其中Al3+与磷酸丝氨酸之间的摩尔比为1:2；加入NaOH水溶液调节pH值为7.6，反应完成后，静置7小时，离心后洗涤，最后高压蒸汽灭菌或经过滤膜除菌，制得铝纳米晶，命名为Al-PEG-1。
实施例2铝纳米晶(Al-PEG-2)制备
将Al(NO3)3溶于纯水中，配制成铝离子含量0.05mol/L的铝盐溶液；向得到的铝盐溶液加入总分子浓度为0.05mol/L的PS-PEG与M-PEG-NH2摩尔比例为1:2的混合溶液，其中Al3+与磷酸丝氨酸之间的摩尔比为1：1；加入NaOH水溶液调节pH值为5.0，反应完成后，静置10小时，离心后洗涤，最后高压蒸汽灭菌或经过滤膜除菌，制得铝纳米晶，命名为Al-PEG-2。
实施例3铝纳米晶(Al-PEG-3)制备
本实施例制备一种铝纳米晶(Al-PEG-3)，制备方法与实施例1基本相同，区别在于，使用的铝盐为醋酸铝，PS-PEG与M-PEG-NH2摩尔比例为2:1，加入NaOH水溶液调节pH值为8.0，反应完成后静置时间为3小时。
实施例4铝纳米晶(Al-PEG-4)制备
本实施例制备一种铝纳米晶(Al-PEG-4)，制备方法与实施例1基本相同，区别在于，使用的铝盐为硫酸铝，加入NaOH水溶液调节pH值为6.5，反应完成后静置时间为8小时。
实施例5
对实施例1～实施例4获得的铝纳米晶或铝纳米晶复合免疫药物进行粒径测试。
25℃条件下，将铝纳米晶的浓度稀释至10μg/mL，用纳米粒度仪测试铝纳米晶的粒径。
其中，实施例1、2、3、4制备的铝纳米晶的理化性质如下表1所示。
表1

由表1可知，本发明实施例1、2、3、4制备实施例均得到纳米尺寸的铝纳米晶，且粒径大小相近，由于使用的主要反应底物类似，因此得到的铝纳米晶性质相似。
实施例6铝纳米晶复合免疫药物Al-RGD-IL12制备
(1)将实施例1制备的铝纳米晶和可特异性结合肿瘤细胞表面高表达的功能蛋白整合素的多肽序列c(RGDfC)加入0.1M磷酸盐缓冲液或0.1M醋酸铵缓冲液中，其中多肽序列与铝纳米晶表面PEG分子中含有的马来酰亚胺的比例为1.2：1，搅拌反应24小时，并离心洗涤提纯，得到RGD肽序列修饰的铝纳米晶；
(2)将细胞因子IL-12分子加入50mM MES缓冲液中，并加入EDC/NHS活化分子上的羧基，之后加入步骤(1)得到的铝纳米晶的溶液，其中铝纳米晶表面M-PEG-NH2的与细胞因子分子比例为1.5:1，得到细胞因子分子进一步修饰的铝纳米晶复合免疫药物，命名为Al-RGD-IL12。
实施例7铝纳米晶复合免疫药物Al-TAT-TNFα
(1)将实施例1制备的铝纳米晶和可有效载带纳米颗粒进入细胞内部的穿膜肽序列TAT加入0.1M磷酸盐缓冲液或0.1M醋酸铵缓冲液中，其中多肽序列与铝纳米晶表面PEG分子中含有的马来酰亚胺的比例为1.5:1，搅拌反应24小时，并离心洗涤提纯，得到TAT肽序列修饰的铝纳米晶；
(2)将肿瘤坏死因子TNFα分子加入50mM MES缓冲液中，并加入EDC/NHS活化分子上的羧基，之后加入步骤(1)得到的铝纳米晶的溶液，其中铝纳米晶表面M-PEG-NH2的与细胞因子分子比例为1.3:1，得到细胞因子分子进一步修饰的铝纳米晶复合免疫药物，命名为Al-TAT-TNFα。
实施例8铝纳米晶复合免疫药物Al-TAT-IL2
(1)将实施例2制备的铝纳米晶和可有效载带纳米颗粒进入细胞内部的穿膜肽序列TAT加入0.1M磷酸盐缓冲液或0.1M醋酸铵缓冲液中，其中多肽序列与铝纳米晶表面PEG分子中含有的马来酰亚胺的比例为1.1:1，搅拌反应15小时，并离心洗涤提纯，得到TAT肽序列修饰的铝纳米晶；
(2)将细胞因子IL2分子加入50mM MES缓冲液中，并加入EDC/NHS活化分子上的羧基，之后加入步骤(1)得到的铝纳米晶的溶液，其中铝纳米晶表面M-PEG-NH2的与细胞因子分子比例为1.2:1，得到细胞因子分子进一步修饰的铝纳米晶复合免疫药物，命名为Al-TAT-IL2。
实施例9铝纳米晶复合免疫药物Al-RGD-IL15
(1)将实施例4制备的铝纳米晶和c(RGDfC)加入0.1M磷酸盐缓冲液或0.1M醋酸铵缓冲液中，其中多肽序列与铝纳米晶表面PEG分子中含有的马来酰亚胺的比例为1.5:1，搅拌反应24小时，并离心洗涤提纯，得到RGD肽序列修饰的铝纳米晶；
(2)将细胞因子IL15分子加入50mM MES缓冲液中，并加入EDC/NHS活化分子上的羧基，之后加入步骤(1)得到的铝纳米晶的溶液，其中铝纳米晶表面M-PEG-NH2的与细胞因子分子比例为1.3:1，得到细胞因子分子进一步修饰的铝纳米晶复合免疫药物，命名为Al-RGD-IL15。
实施例10铝纳米晶复合免疫药物Al-TAT-IL18
(1)将实施例3制备的铝纳米晶和可有效载带纳米颗粒进入细胞内部的穿膜肽序列TAT加入0.1M磷酸盐缓冲液或0.1M醋酸铵缓冲液中，其中多肽序列与铝纳米晶表面PEG分子中含有的马来酰亚胺的比例为1.3:1，搅拌反应20小时，并离心洗涤提纯，得到TAT肽序列修饰的铝纳米晶；
(2)将细胞因子IL18分子加入50mM MES缓冲液中，并加入 EDC/NHS活化分子上的羧基，之后加入步骤(1)得到的铝纳米晶的溶液，其中铝纳米晶表面M-PEG-NH2的与细胞因子分子比例为1.4:1，得到细胞因子分子进一步修饰的铝纳米晶复合免疫药物，命名为Al-TAT-IL18。
实施例11铝纳米晶复合免疫药物Al-RGD-IL21
(1)将实施例2制备的铝纳米晶和c(RGDfC)加入0.1M磷酸盐缓冲液或0.1M醋酸铵缓冲液中，其中多肽序列与铝纳米晶表面PEG分子中含有的马来酰亚胺的比例为1.5:1，搅拌反应24小时，并离心洗涤提纯，得到RGD肽序列修饰的铝纳米晶；
(2)将细胞因子IL21分子加入50mM MES缓冲液中，并加入EDC/NHS活化分子上的羧基，之后加入步骤(1)得到的铝纳米晶的溶液，其中铝纳米晶表面M-PEG-NH2的与细胞因子分子比例为1.3:1，得到细胞因子分子进一步修饰的铝纳米晶复合免疫药物，命名为Al-RGD-IL21。
实施例12铝纳米晶复合免疫药物Al-RT-IFN-γ
(1)将实施例4制备的铝纳米晶和c(RGDfC)与TAT肽(摩尔比为1:1)加入0.1M磷酸盐缓冲液或0.1M醋酸铵缓冲液中，其中多肽序列与铝纳米晶表面PEG分子中含有的马来酰亚胺的比例为1.3:1，搅拌反应24小时，并离心洗涤提纯，得到RGD肽序列修饰的铝纳米晶；
(2)将伽马干扰素IFN-γ分子加入50mM MES缓冲液中，并加入EDC/NHS活化分子上的羧基，之后加入步骤(1)得到的铝纳米晶的溶液，其中铝纳米晶表面M-PEG-NH2的与细胞因子分子比例为1.5:1，得到细胞因子分子进一步修饰的铝纳米晶复合免疫药物，命名为Al-RT-IFN-γ。
实施例13铝纳米晶复合免疫药物Al-RT-IL7
(1)将实施例1制备的铝纳米晶(实施例1所得产品仅作为后继实验的代表物，不表示仅实施例1得到的铝纳米晶可用于后继实验)和c(RGDfC)与TAT肽(摩尔比为1.5:1)加入0.1M磷酸盐缓冲液或0.1M 醋酸铵缓冲液中，其中多肽序列与铝纳米晶表面PEG分子中含有的马来酰亚胺的比例为1.5:1，搅拌反应24小时，并离心洗涤提纯，得到RGD肽序列修饰的铝纳米晶；
(2)将细胞因子IL7分子加入50mM MES缓冲液中，并加入EDC/NHS活化分子上的羧基，之后加入步骤(1)得到的铝纳米晶的溶液，其中铝纳米晶表面M-PEG-NH2的与细胞因子分子比例为1.3:1，得到细胞因子分子进一步修饰的铝纳米晶复合免疫药物，命名为Al-RT-IL7。
实施例14
经过与实施例5类似的水合粒径检测，实施例6～13所得到的产物理化性质如下表2所示。由表中数据可知，不同细胞因子或干扰素等制得的铝纳米晶复合免疫药物粒径均明显增大。
表2
实施例15
将上述对所构建的铝纳米晶复合免疫药物(以实施例6构建的Al-RGD-IL12复合免疫药物和实施例7构建的Al-TAT-TNFα复合免疫药物为代表)应用于肿瘤模型免疫治疗中，进行肿瘤生长抑制评估。
在遵循国家动物保健协议的前提下，选取6-8周龄的BALB/c小鼠进行后侧皮下肿瘤种植，肿瘤模型选取B16F10小鼠黑色素瘤高转移细胞，待所种植的肿瘤组织生长至100mm3左右时，将小鼠随机分为5组，并于瘤内注射50μL如下药物：①、注射盐水(Ctrl)；②、商业铝佐剂Alum与含有0.25μg IL-12的简单混合样(Alum-IL12)；③、实施例6构建的含有0.25μg IL-12的Al-RGD-IL12复合免疫药物；④、商业铝佐剂Alum与含 有0.25μg TNFα的简单混合样(Alum-TNFα)；⑤、实施例7构建的含有0.25μg IL-12的Al-TAT-TNFα复合免疫药物。并记录小鼠肿瘤体积变化情况。
结果如图1所示，图1为所构建的铝纳米晶复合免疫药物抑制肿瘤生长的效果分析。
由图1可知，20天时，对照组(Ctrl组)小鼠肿瘤平均大小为1942mm3；Alum-IL12组肿瘤体积平均大小为1364mm3；而Al-RGD-IL12组肿瘤体积平均大小为183mm3；Alum-TNFα组肿瘤体积平均大小为1540mm3；而Al-TAT-TNFα组肿瘤体积平均大小为204mm3；可得所构建的载带细胞因子的铝纳米晶复合免疫药物能够有效抑制肿瘤的生长。
实施例16
在遵循国家动物保健协议的前提下，选取6-8周龄的BALB/c小鼠进行后侧皮下肿瘤种植，肿瘤模型选取B16F10小鼠黑色素瘤高转移细胞，待所种植的肿瘤组织生长至100mm3左右时，将小鼠随机分为5组，并于瘤内注射50μL实施例8～13所构建的含有0.25μg细胞因子或伽马干扰素的铝纳米晶复合免疫药物。并记录小鼠肿瘤体积变化情况。
结果如图2所示，图2为实施例8～13所构建的铝纳米晶复合免疫药物抑制肿瘤生长的效果分析。
由图2可知，20天时，实施例8～13所构建的铝纳米晶复合免疫药物处理的小鼠肿瘤体积平均大小为181mm3～254mm3，均显著低于实施例15所示对照组别肿瘤平均体积；可得实施例8～13所构建的铝纳米晶复合免疫药物也能够有效抑制肿瘤的生长。
肿瘤抑制能力是验证递送效率最直接的指标，本发明实施例6～13所获得的铝纳米晶复合免疫药物都能够显著抑制黑色素瘤生长，由于其组成成分类似，其作用机制基本类似，故仅选取以实施例5构建的Al-RGD-IL12复合免疫药物和实施例6构建的Al-TAT-TNFα复合免疫药物进行后续更进一步的机制验证。
实施例17
针对实施例15中的各组小鼠，分析其肿瘤组织中细胞因子的积累和蓄积情况，分别在瘤内注射药物后的24小时和48小时后，分别于实施例15的②、③、④、⑤四个组别内随机各取出一只小鼠，人道主义处死后解剖获取其肿瘤组织，并按照白细胞介素12(IL-12)或肿瘤坏死因子(TNFα)的ELISA试剂盒说明书的操作对肿瘤组织中的IL-12或TNFα的存量水平进行分析。
结果如图3所示，图3为实施例15中小鼠肿瘤模型中细胞因子蓄积情况分析。由图3所示，24小时时，肿瘤组织中白细胞介素12存量水平为：Alum-IL12实验组，24.1pg/mL；Al-RGD-IL12实验组，60.9pg/mL。48小时时，肿瘤组织中白细胞介素12存量水平为：Alum-IL12实验组，11.4pg/mL；Al-RGD-IL12实验组，52.3pg/mL。24小时时，肿瘤组织中肿瘤坏死因子存量水平为：Alum-TNFα实验组，18.2pg/mL；Al-TAT-TNFα实验组，46.7pg/mL。48小时时，肿瘤组织中肿瘤坏死因子存量水平为：Alum-TNFα实验组，8.4pg/mL；Al-TAT-TNFα实验组，41.5pg/mL。由此数据可知，所构建的铝纳米晶复合免疫药物能够显著提升细胞因子在肿瘤环境中的稳定性和蓄积时间。
实施例18
对实施例15提取的注射药物后小鼠的肿瘤组织，进行CD8+T细胞增值情况研究。将部分肿瘤组织称重并研磨，然后通过100μm细胞过滤器过滤。用抗CD8对细胞悬液进行表面标记染色，然后在流式细胞仪上进行分析。结果如图4所示，图4为实施例15中小鼠肿瘤模型中，肿瘤组织中CD8T细胞增殖情况。由图4所示，24小时时，肿瘤组织中CD8T细胞占比水平为：Alum-IL12实验组，1.5％；Al-RGD-IL12实验组，7.1％；Alum-TNFα实验组，1.7％；Al-TAT-TNFα实验组，6.6％。48小时时，肿瘤组织中CD8T细胞占比水平为：Alum-IL12实验组，2.4％；Al-RGD-IL12实验组，9.0％；Alum-TNFα实验组，2.3％；Al-TAT-TNFα实验组，8.4％。由此数据可知，所构建的铝纳米晶复合免疫药物能够显著提升CD8T细 胞的增殖能力，有效调节肿瘤组织的免疫反应。
实施例19
铝纳米晶复合免疫药物抑制远端肿瘤生长。
将B16F10小鼠黑色素瘤细胞接种到Balb/c小鼠右后腿皮下，记为原位肿瘤，将一半细胞数目的B16F10小鼠黑色素瘤细胞接种到Balb/c小鼠左后腿皮下，记为远端肿瘤。待原位肿瘤平均体积长到100mm3时，远端肿瘤体积平均大小约50mm3，然后将小鼠随机分为5组，并于原位瘤内注射50μL如下药物：①、注射盐水(Ctrl)；②、商业铝佐剂Alum与含有0.25μg IL-12的简单混合样(Alum-IL12)；③、实施例6构建的含有0.25μg IL-12的Al-RGD-IL12复合免疫药物；④、商业铝佐剂Alum与含有0.25μg TNFα的简单混合样(Alum-TNFα)；⑤、实施例7构建的含有0.25μg IL-12的Al-TAT-TNFα复合免疫药物。每3天治疗一次，并记录小鼠远端肿瘤体积变化情况。
结果如图5所示，图5为所构建的铝纳米晶复合免疫药物抑制远端肿瘤组织生长的效果分析。由图5可知，20天时，对照组小鼠远端肿瘤平均大小为1286mm3；Alum-IL12组远端肿瘤体积平均大小为984mm3；而Al-RGD-IL12组远端肿瘤体积平均大小为253mm3；Alum-TAT-TNFα组远端肿瘤体积平均大小为880mm3；而Al-TAT-TNFα组远端肿瘤体积平均大小为382mm3；可得所构建的载带细胞因子的铝纳米晶复合免疫药物能够有效抑制远端肿瘤的生长。说明所构建的铝纳米晶复合免疫药物除了可以对已有肿瘤的生长产生明显抑制效果，对于新生肿瘤的生长也有明显的抑制作用。
以上对本发明所提供的一种铝纳米晶复合免疫药物及其制备方法和应用进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (17)

  1. 一种铝纳米晶复合免疫药物，其特征在于，由铝纳米晶通过聚乙二醇分子结合功能多肽序列和细胞因子分子构建而成。
  2. 根据权利要求1所述的铝纳米晶复合免疫药物，其特征在于，所述功能多肽序列包括可特异性结合肿瘤细胞表面高表达蛋白的多肽序列。
  3. 根据权利要求2所述的铝纳米晶复合免疫药物，其特征在于，所述功能多肽序列为环形RGD序列、可协助免疫药物进入细胞的TAT序列中的一种或两种。
  4. 根据权利要求1所述的铝纳米晶复合免疫药物，其特征在于，所述细胞因子分子选自白细胞介素类因子，所述白细胞介素类因子包括IL-2、IL-7、IL-12、IL-15、IL-18、IL-21、伽马干扰素IFN-γ或肿瘤坏死因子TNFα中的一种或多种。
  5. 根据权利要求1所述的铝纳米晶复合免疫药物，其特征在于，所述铝纳米晶通过以下步骤制备：
    将铝盐溶液与聚乙二醇溶液搅拌混合，并加入NaOH水溶液调节pH后静置得沉淀，离心洗涤纯化后得聚乙二醇修饰的铝纳米晶。
  6. 根据权利要求5所述的铝纳米晶复合免疫药物，其特征在于，所述铝盐溶液的溶质为氯化铝、硝酸铝、硫酸铝和醋酸铝中的一种或多种；所述铝盐溶液所用溶剂为纯水或浓度为0.01mol/L的醋酸钠溶液。
  7. 根据权利要求5所述的铝纳米晶复合免疫药物，其特征在于，所述聚乙二醇溶液为磷酸丝氨酸修饰的聚乙二醇与马来酰亚胺基团和末端羧酸的聚乙二醇的混合试剂。
  8. 根据权利要求5所述的铝纳米晶复合免疫药物，其特征在于，加入NaOH水溶液调节所述pH为5.0～8.0。
  9. 根据权利要求1至8任一项所述的一种铝纳米晶复合免疫药物的制备方法，其特征在于，包括如下步骤：
    (1)将铝纳米晶和功能多肽序列加入溶剂中，搅拌反应，并离心洗涤提纯，重悬至超纯水得到功能多肽序列修饰的铝纳米晶溶液；
    (2)将细胞因子分子加入MES缓冲液中，并加入EDC/NHS活化细 胞因子分子上的羧基，之后加入所述铝纳米晶溶液，使M-PEG-NH2的氨基与细胞因子分子上的羧基形成共价酰胺键，得到细胞因子分子修饰的铝纳米晶复合免疫药物。
  10. 根据权利要求9所述的铝纳米晶复合免疫药物的制备方法，其特征在于，所述溶剂为0.1M磷酸盐缓冲液或0.1M醋酸铵缓冲液。
  11. 根据权利要求9所述的铝纳米晶复合免疫药物的制备方法，其特征在于，所述功能多肽序列包括可特异性结合肿瘤细胞表面高表达蛋白的多肽序列。
  12. 根据权利要求9所述的铝纳米晶复合免疫药物的制备方法，其特征在于，所述细胞因子分子选自白细胞介素类因子，所述白细胞介素类因子包括IL-2、IL-7、IL-12、IL-15、IL-18、IL-21、伽马干扰素IFN-γ或肿瘤坏死因子TNFα中的一种或多种。
  13. 根据权利要求9所述的铝纳米晶复合免疫药物的制备方法，其特征在于，所述功能多肽序列为环形RGD序列、可协助免疫药物进入细胞的TAT序列中的一种或两种。
  14. 权利要求1～8任意一项铝纳米晶复合免疫药物或权利要求9～13任意一项所述制备方法制得的铝纳米晶复合免疫药物在肿瘤模型免疫治疗中的应用。
  15. 权利要求14所述的应用，其特征在于，所述应用为静脉注射使用、粘膜施用、皮下注射使用、皮内注射使用、瘤周注射使用或瘤内注射使用。
  16. 免疫治疗肿瘤的方法，其特征在于，施用权利要求1～8任一项所述的铝纳米晶复合免疫药物或权利要求9～13任一项所述制备方法制得的铝纳米晶复合免疫药物。
  17. 根据权利要求15所述的方法，其特征在于，所述施用的方式包括：静脉注射使用、粘膜施用、皮下注射使用、皮内注射使用、瘤周注射使用或瘤内注射使用。
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