CN110420335A - A kind of preparation and application of the nano immune preparation based on porous calcium carbonate - Google Patents
A kind of preparation and application of the nano immune preparation based on porous calcium carbonate Download PDFInfo
- Publication number
- CN110420335A CN110420335A CN201810890136.2A CN201810890136A CN110420335A CN 110420335 A CN110420335 A CN 110420335A CN 201810890136 A CN201810890136 A CN 201810890136A CN 110420335 A CN110420335 A CN 110420335A
- Authority
- CN
- China
- Prior art keywords
- idoi
- preparation
- calcium carbonate
- nano
- caco
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 title claims abstract description 64
- 238000002360 preparation method Methods 0.000 title claims abstract description 42
- 229910000019 calcium carbonate Inorganic materials 0.000 title claims abstract description 32
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 44
- 239000002105 nanoparticle Substances 0.000 claims abstract description 24
- 206010006187 Breast cancer Diseases 0.000 claims abstract description 10
- 208000026310 Breast neoplasm Diseases 0.000 claims abstract description 10
- 230000000694 effects Effects 0.000 claims abstract description 9
- 229940046166 oligodeoxynucleotide Drugs 0.000 claims abstract description 7
- 239000002246 antineoplastic agent Substances 0.000 claims abstract description 6
- 229940041181 antineoplastic drug Drugs 0.000 claims abstract description 6
- 239000003112 inhibitor Substances 0.000 claims abstract description 6
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 6
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 6
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 6
- 230000000903 blocking effect Effects 0.000 claims abstract description 5
- 150000002632 lipids Chemical class 0.000 claims abstract 3
- 230000002519 immonomodulatory effect Effects 0.000 claims abstract 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 17
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 16
- 239000000243 solution Substances 0.000 claims description 16
- 239000002502 liposome Substances 0.000 claims description 13
- 229940046168 CpG oligodeoxynucleotide Drugs 0.000 claims description 12
- KILNVBDSWZSGLL-KXQOOQHDSA-N 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCC KILNVBDSWZSGLL-KXQOOQHDSA-N 0.000 claims description 8
- 239000007864 aqueous solution Substances 0.000 claims description 8
- 235000012000 cholesterol Nutrition 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 6
- WTDRDQBEARUVNC-UHFFFAOYSA-N L-Dopa Natural products OC(=O)C(N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-UHFFFAOYSA-N 0.000 claims description 5
- MHUWZNTUIIFHAS-CLFAGFIQSA-N dioleoyl phosphatidic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(COP(O)(O)=O)OC(=O)CCCCCCC\C=C/CCCCCCCC MHUWZNTUIIFHAS-CLFAGFIQSA-N 0.000 claims description 5
- 229960004502 levodopa Drugs 0.000 claims description 5
- 238000001291 vacuum drying Methods 0.000 claims description 5
- 238000012986 modification Methods 0.000 claims description 4
- 230000004048 modification Effects 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 238000009792 diffusion process Methods 0.000 claims description 3
- 229940079593 drug Drugs 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 3
- 230000004957 immunoregulator effect Effects 0.000 claims description 3
- 239000011148 porous material Substances 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 2
- 239000003960 organic solvent Substances 0.000 claims description 2
- 238000005728 strengthening Methods 0.000 claims description 2
- 229960004756 ethanol Drugs 0.000 claims 2
- 239000003560 cancer drug Substances 0.000 claims 1
- 229960000935 dehydrated alcohol Drugs 0.000 claims 1
- 230000003053 immunization Effects 0.000 claims 1
- 238000002649 immunization Methods 0.000 claims 1
- 201000011510 cancer Diseases 0.000 abstract description 9
- 238000011282 treatment Methods 0.000 abstract description 9
- 239000000825 pharmaceutical preparation Substances 0.000 abstract description 2
- 235000010216 calcium carbonate Nutrition 0.000 description 17
- 241000699670 Mus sp. Species 0.000 description 10
- 238000000034 method Methods 0.000 description 7
- 230000028993 immune response Effects 0.000 description 5
- 230000003902 lesion Effects 0.000 description 5
- 102100031415 Hepatic triacylglycerol lipase Human genes 0.000 description 4
- 101000941289 Homo sapiens Hepatic triacylglycerol lipase Proteins 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000009169 immunotherapy Methods 0.000 description 3
- 238000001356 surgical procedure Methods 0.000 description 3
- 229960005486 vaccine Drugs 0.000 description 3
- 208000007660 Residual Neoplasm Diseases 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 238000002619 cancer immunotherapy Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 230000001506 immunosuppresive effect Effects 0.000 description 2
- 231100000053 low toxicity Toxicity 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000003446 memory effect Effects 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 230000002980 postoperative effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000001308 synthesis method Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 230000004614 tumor growth Effects 0.000 description 2
- 208000031648 Body Weight Changes Diseases 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000005784 autoimmunity Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000001815 biotherapy Methods 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000004579 body weight change Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000005934 immune activation Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011503 in vivo imaging Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 230000007787 long-term memory Effects 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 231100000057 systemic toxicity Toxicity 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 230000002100 tumorsuppressive effect Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
- A61K33/10—Carbonates; Bicarbonates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- 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
- A61K47/6905—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 colloid or an emulsion
- A61K47/6911—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 colloid or an emulsion the form being a liposome
- A61K47/6915—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 colloid or an emulsion the form being a liposome the form being a liposome with polymerisable or polymerized bilayer-forming substances, e.g. polymersomes
-
- 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
- A61K47/6949—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 inclusion complexes, e.g. clathrates, cavitates or fullerenes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Inorganic Chemistry (AREA)
- Dispersion Chemistry (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
技术领域technical field
本发明属于药物制剂领域，具体涉及一种纳米免疫制剂，以多孔CaCO3纳米颗粒为载体，并在其孔道内部装载免疫阻断抑制剂IDOi，表面携带免疫调节核酸CpG ODNs，其合成方法及其在强化乳腺癌疗效并防止其复发领域的应用。The invention belongs to the field of pharmaceutical preparations, and specifically relates to a nano - immune preparation, which uses porous CaCO3 nanoparticles as a carrier, and loads the immune blocking inhibitor IDOi inside the pores, and carries immune-regulating nucleic acid CpG ODNs on the surface, its synthesis method and its Application in the field of strengthening the curative effect of breast cancer and preventing its recurrence.
背景技术Background technique
癌症是严重威胁人类健康的恶性肿瘤，原发性肿瘤治疗后的复发是大多数癌症病人死亡的最主要原因。现阶段肿瘤的临床常规的治疗方法主要是手术、放疗和化疗，但是利用这些治疗方法治疗后机体仍会残留癌细胞(即微小病灶)，而这些微小病灶是癌症复发的最主要原因。因此，发展低毒性的、高效的治疗方法来治疗癌症，同时消灭微小病灶并防止癌症的复发是亟待解决的问题。Cancer is a malignant tumor that seriously threatens human health. The recurrence of primary tumor after treatment is the main cause of death for most cancer patients. At present, the conventional clinical treatment methods for tumors are mainly surgery, radiotherapy and chemotherapy, but after using these treatment methods, there will still be residual cancer cells in the body (ie, small lesions), and these small lesions are the main reason for cancer recurrence. Therefore, it is an urgent problem to develop low-toxicity and high-efficiency therapeutic methods to treat cancer, eliminate microscopic lesions and prevent cancer recurrence.
肿瘤的免疫治疗作为新一代的肿瘤治疗策略迅速发展并在实际临床应用中表现出巨大潜力。通过调整或刺激机体自身的免疫系统可以治疗肿瘤，并且利用机体自身免疫系统长时间的记忆效应，可以有效预防肿瘤的复发。近年来，已经发展了多种肿瘤疫苗通过激活病人的自身免疫来抑制肿瘤。研究表明现在常用的免疫疫苗法存在以下问题：首先，免疫疫苗引起的免疫反应效果有限，一般需要加入免疫佐剂来加强免疫反应，但是免疫佐剂(如氢氧化铝)的毒性较大。其次，当肿瘤发展到恶性的时候，大部分的肿瘤相关抗原通常都被清除了，这就是所谓的肿瘤免疫逃脱机制。并且肿瘤组织处的免疫抑制微环境通常很难被打破，这很容易导致肿瘤免疫治疗的失败。因此，发展一种安全有效的方法引发机体自身强烈的免疫响应，打破肿瘤的免疫抑制微环境，提高癌症免疫治疗的效率，并利用其记忆效应进一步防止癌症的复发是非常必要的，也是具有挑战性的研究工作。As a new generation of tumor treatment strategy, tumor immunotherapy has developed rapidly and has shown great potential in actual clinical application. Tumors can be treated by adjusting or stimulating the body's own immune system, and the long-term memory effect of the body's own immune system can be used to effectively prevent tumor recurrence. In recent years, a variety of tumor vaccines have been developed to suppress tumors by activating the patient's autoimmunity. Studies have shown that the commonly used immune vaccine method has the following problems: firstly, the immune response effect caused by the immune vaccine is limited, and it is generally necessary to add an immune adjuvant to strengthen the immune response, but the toxicity of the immune adjuvant (such as aluminum hydroxide) is relatively large. Secondly, when a tumor becomes malignant, most of the tumor-associated antigens are usually cleared, which is the so-called tumor immune escape mechanism. And the immunosuppressive microenvironment in tumor tissue is usually difficult to break, which can easily lead to the failure of tumor immunotherapy. Therefore, it is necessary and challenging to develop a safe and effective method to trigger the body's own strong immune response, break the tumor's immunosuppressive microenvironment, improve the efficiency of cancer immunotherapy, and use its memory effect to further prevent cancer recurrence sexual research work.
发明内容Contents of the invention
为克服临床常规癌症治疗方法的缺陷，本发明目的在于提供一种基于多孔碳酸钙的纳米免疫制剂，当纳米免疫制剂在血液中循环至肿瘤处时，CaCO3纳米材料会在弱酸性环境中特异性地溶解，在肿瘤部位释放出CpG ODNs、IDOi和Ca2+，三者原位协同作用，增强机体的免疫响应，确保肿瘤组织免疫反应的持续发生并保持记忆效应。这种新型的碳酸钙纳米制剂应用于生物体治疗中，具有强化癌症的免疫治疗的效率，并防止其复发的效果。In order to overcome the defects of conventional clinical cancer treatment methods, the purpose of the present invention is to provide a nano-immune preparation based on porous calcium carbonate. When the nano-immune preparation circulates in the blood to the tumor, the CaCO 3 nano-material will be specific in a weakly acidic environment. It dissolves permanently and releases CpG ODNs, IDOi and Ca 2+ at the tumor site. The three act synergistically in situ to enhance the body's immune response, ensure the continuous occurrence of tumor tissue immune response and maintain the memory effect. This new type of calcium carbonate nano-preparation is applied in the treatment of organisms, and has the effect of enhancing the efficiency of cancer immunotherapy and preventing its recurrence.
本发明的第一方面，提供一种基于多孔碳酸钙的纳米免疫制剂，该纳米制剂的组成为多孔碳酸钙纳米颗粒、免疫阻断抑制剂IDOi、脂质体层及免疫调节核酸CpG ODNs。The first aspect of the present invention provides a nano-immune preparation based on porous calcium carbonate. The nano-preparation is composed of porous calcium carbonate nanoparticles, immune blocking inhibitor IDOi, liposome layer and immunoregulatory nucleic acid CpG ODNs.
优选的，上述免疫制剂中，多孔碳酸钙纳米颗粒作为药物载体，颗粒的孔道内部装载免疫阻断抑制剂IDOi，装载免疫制剂的多孔碳酸钙纳米颗粒被脂质体层所包覆，脂质体层的表面具有免疫调节核酸CpG ODNs修饰。Preferably, in the above-mentioned immune preparation, the porous calcium carbonate nanoparticle is used as a drug carrier, and the inside of the pore channel of the particle is loaded with the immune blocking inhibitor IDOi, and the porous calcium carbonate nanoparticle loaded with the immune preparation is coated by a liposome layer, and the liposome The surface of the layer is modified with immunoregulatory nucleic acid CpG ODNs.
优选的，上述脂质体层是由DPPC、胆固醇和DSPE-PEG组成。Preferably, the liposome layer is composed of DPPC, cholesterol and DSPE-PEG.
本发明的第二方面，提供一种基于多孔碳酸钙的纳米免疫制剂的制备方法，步骤如下：A second aspect of the present invention provides a method for preparing a porous calcium carbonate-based nano-immune preparation, the steps are as follows:
(1)通过气体扩散反应合成多孔碳酸钙纳米颗粒，将制得的多孔碳酸钙纳米颗粒置于无水乙醇中装载IDOi，得到CaCO3@IDOi；(1) Synthesize porous calcium carbonate nanoparticles by gas diffusion reaction, place the prepared porous calcium carbonate nanoparticles in absolute ethanol to load IDOi, and obtain CaCO 3 @IDOi;
(2)采用脂质体层修饰步骤(1)中制得的CaCO3@IDOi得到CaCO3@IDOi@PEG@PEI；将得到的CaCO3@IDOi@PEG@PEI与CpG ODN在水溶液中搅拌，既得该纳米制剂。(2) Using the CaCO 3 @IDOi prepared in step (1) to modify the liposome layer to obtain CaCO 3 @IDOi@PEG@PEI; stir the obtained CaCO 3 @IDOi@PEG@PEI and CpG ODN in an aqueous solution, The nano preparation is obtained.
优选的，上述制备方法中的步骤如下：Preferably, the steps in the above-mentioned preparation method are as follows:
(1)将CaCl2·H2O和NH4HCO3同时置于真空干燥室中，将整个系统保持在真空环境中，最后通过离心分离制得的碳酸钙纳米颗粒；将分离得到的碳酸钙纳米颗粒置于无水乙醇溶液中搅拌过夜，装载IDOi，得到CaCO3@IDOi；(1) CaCl 2 ·H 2 O and NH 4 HCO 3 are placed in a vacuum drying chamber at the same time, the whole system is kept in a vacuum environment, and finally the calcium carbonate nanoparticles obtained by centrifugation are separated; The nanoparticles were placed in absolute ethanol solution and stirred overnight, and loaded with IDOi to obtain CaCO 3 @IDOi;
(2)将步骤(1)中制得的CaCO3@IDOi乙醇溶液与DOPA混合，然后将DPPC，胆固醇和DSPE-PEG加入上述溶液中，将溶液中的有机溶剂旋干，将制得的纳米颗粒溶于水溶液中，加入PEI后搅拌以制备CaCO3@IDOi@PEG@PEI；将得到的CaCO3@IDOi@PEG@PEI与CpG ODN在水溶液中搅拌，既得该纳米制剂。(2) Mix the CaCO 3 @IDOi ethanol solution prepared in step (1) with DOPA, then add DPPC, cholesterol and DSPE-PEG to the above solution, spin dry the organic solvent in the solution, and prepare the nano The particles were dissolved in an aqueous solution, and PEI was added and then stirred to prepare CaCO 3 @IDOi@PEG@PEI; the obtained CaCO 3 @IDOi@PEG@PEI and CpG ODN were stirred in an aqueous solution to obtain the nano-preparation.
进一步优选的，步骤(1)中将CaCl2·H2O和NH4HCO3同时置于真空干燥室中，设置温度为25-30℃，将整个系统保持在真空环境中20-30h后，通过10000-15000rpm离心分离制得碳酸钙纳米颗粒。Further preferably, in step (1), CaCl 2 ·H 2 O and NH 4 HCO 3 are placed in a vacuum drying chamber at the same time, the temperature is set at 25-30°C, and the whole system is kept in a vacuum environment for 20-30 hours, Calcium carbonate nanoparticles were prepared by centrifugal separation at 10000-15000rpm.
进一步优选的，步骤(1)中将分离得到的碳酸钙纳米颗粒置于无水乙醇溶液中装载IDOi，得到CaCO3@IDOi具体步骤为将碳酸钙与IDOi的乙醇溶液搅拌至少24小时。Further preferably, in step (1), place the separated calcium carbonate nanoparticles in absolute ethanol solution to load IDOi to obtain CaCO 3 @IDOi. The specific step is to stir the ethanol solution of calcium carbonate and IDOi for at least 24 hours.
进一步优选的，步骤(1)中CaCl2·H2O：NH4HCO3质量比为(0.5-2)：(30-50)，CaCO3和IDOi的质量比为(35-45)：(0.5-2.5)；更为优选的，CaCl2·H2O：NH4HCO3质量比为1:40，CaCO3和IDOi的质量比为40:1。Further preferably, in step (1), the mass ratio of CaCl 2 ·H 2 O:NH 4 HCO 3 is (0.5-2):(30-50), and the mass ratio of CaCO 3 and IDOi is (35-45):( 0.5-2.5); more preferably, the mass ratio of CaCl 2 ·H 2 O:NH 4 HCO 3 is 1:40, and the mass ratio of CaCO 3 and IDOi is 40:1.
进一步优选的，步骤(2)中CaCO3@IDOi：DOPA：DPPC：胆固醇：DSPE-PEG：PEI：CpGODN的质量比为(35-45)：(12-18)：(12-18)：(6-10)：(30-35)：(0.2-1)；更进一步的，为(38-42)：(14-16)：(14-16)：(7-9)：(31-33)：(0.3-0.5)；具体的，为(40-41)：16：16：8：32：0.48。Further preferably, the mass ratio of CaCO 3 @IDOi:DOPA:DPPC:cholesterol:DSPE-PEG:PEI:CpGODN in step (2) is (35-45):(12-18):(12-18):( 6-10): (30-35): (0.2-1); further, (38-42): (14-16): (14-16): (7-9): (31-33 ): (0.3-0.5); specifically, (40-41): 16: 16: 8: 32: 0.48.
本发明的第三方面，提供上述基于多孔碳酸钙的纳米免疫制剂在制备防治肿瘤药物领域的应用。The third aspect of the present invention provides the application of the above-mentioned porous calcium carbonate-based nano-immune preparation in the field of preparing anti-tumor drugs.
优选的，上述防治肿瘤药物为防治乳腺癌药物。Preferably, the above anti-tumor drug is a drug for preventing and treating breast cancer.
进一步优选的，防治肿瘤药物为强化乳腺癌疗效及防止肿瘤复发领域的药物。Further preferably, the anti-tumor drug is a drug in the field of enhancing the curative effect of breast cancer and preventing tumor recurrence.
本发明有益效果Beneficial effect of the present invention
1.本发明的多孔碳酸钙纳米免疫制剂具有良好的治疗效果，毒性低、免疫激活效率高，能够消除传统手术治疗方法所残存的微小病灶，提高乳腺癌的免疫治疗效率，有效防止乳腺癌的复发，能够用于生物体治疗。1. The porous calcium carbonate nano-immune preparation of the present invention has a good therapeutic effect, low toxicity and high immune activation efficiency, can eliminate the remaining tiny lesions in traditional surgical treatment methods, improve the immunotherapy efficiency of breast cancer, and effectively prevent the development of breast cancer. Recurrence can be used for biological therapy.
2.本发明中制剂的合成方法简单，不引入有毒试剂，制备的纳米制剂不存在有毒试剂残留问题，安全性高。制备过程中无需高温加热等复杂操作，适于规模化生产。2. The synthesis method of the preparation in the present invention is simple, does not introduce toxic reagents, and the prepared nano-preparation does not have the problem of residual toxic reagents, and has high safety. The preparation process does not require complex operations such as high-temperature heating, and is suitable for large-scale production.
附图说明Description of drawings
构成本申请的一部分的说明书附图用来提供对本申请的进一步理解，本申请的示意性实施例及其说明用于解释本申请，并不构成对本申请的不当限定。The accompanying drawings constituting a part of the present application are used to provide further understanding of the present application, and the schematic embodiments and descriptions of the present application are used to explain the present application, and do not constitute improper limitations to the present application.
图1小鼠肿瘤体积变化和体重变化曲线图；Figure 1 Curves of tumor volume change and body weight change in mice;
图2实施例2中防止肿瘤复发实验小鼠活体成像图。Fig. 2 In vivo imaging diagram of mice in the experiment for preventing tumor recurrence in Example 2.
具体实施方式Detailed ways
应该指出，以下详细说明都是例示性的，旨在对本申请提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。It should be pointed out that the following detailed description is exemplary and intended to provide further explanation to the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本申请的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。It should be noted that the terminology used here is only for describing specific implementations, and is not intended to limit the exemplary implementations according to the present application. As used herein, unless the context clearly dictates otherwise, the singular is intended to include the plural, and it should also be understood that when the terms "comprising" and/or "comprising" are used in this specification, they mean There are features, steps, operations, means, components and/or combinations thereof.
为了使得本领域技术人员能够更加清楚地了解本申请的技术方案，以下将结合具体的实施例与对比例详细说明本申请的技术方案。In order to enable those skilled in the art to understand the technical solution of the present application more clearly, the technical solution of the present application will be described in detail below in combination with specific examples and comparative examples.
实施例1基于多孔碳酸钙纳米制剂的制备Embodiment 1 is based on the preparation of porous calcium carbonate nano-preparation
(1)通过气体扩散反应合成多孔CaCO3纳米颗粒：将220毫克CaCl2·H2O和8克NH4HCO3同时置于真空干燥室中，控制温度为25℃，将整个系统保持在真空环境中24小时后，获得的CaCO3纳米颗粒通过在12000rpm离心分离。然后将在无水乙醇中CaCO3的孔道中装载IDOi，得到CaCO3@IDOi(缩写为CaI)。(1) Synthesis of porous CaCO 3 nanoparticles by gas diffusion reaction: 220 mg of CaCl 2 H 2 O and 8 g of NH 4 HCO 3 were placed in a vacuum drying chamber at the same time, the temperature was controlled at 25 °C, and the whole system was kept in vacuum After 24 h in ambient, the obtained CaCO 3 nanoparticles were separated by centrifugation at 12,000 rpm. IDOi was then loaded into the channels of CaCO 3 in absolute ethanol to obtain CaCO 3 @IDOi (abbreviated as CaI).
(2)用脂质体修饰上述纳米颗粒。首先，将20毫克CaI的乙醇溶液和2毫克DOPA混合。然后将DPPC，胆固醇和DSPE-PEG以4:4:2M比例加入上述溶液中，用旋转蒸发仪将溶剂旋干，将得到的纳米颗粒溶解在水溶液中以备进一步使用。然后，将获得的纳米颗粒与200毫克PEI一起搅拌以制备CaCO3@IDOi@PEG@PEI(缩写为CaIP)。(2) The above nanoparticles are modified with liposomes. First, 20 mg of CaI in ethanol and 2 mg of DOPA were mixed. Then DPPC, cholesterol and DSPE-PEG were added to the above solution at a ratio of 4:4:2M, the solvent was spin-dried by a rotary evaporator, and the obtained nanoparticles were dissolved in an aqueous solution for further use. Then, the obtained nanoparticles were stirred together with 200 mg of PEI to prepare CaCO 3 @IDOi@PEG@PEI (abbreviated as CaIP).
(3)将上述CaIP与12OD CpG ODN在水溶液中搅拌，得到最终产物，缩写为CaIPC。(3) The above CaIP and 12OD CpG ODN were stirred in an aqueous solution to obtain the final product, abbreviated as CaIPC.
对比例1Comparative example 1
作为对照，本发明合成了一种只以脂质体为载体的纳米免疫制剂(LIPC)，将IDOi装载入脂质体中，DPPC，胆固醇和DSPE-PEG以4:4:2M比例混合加入有机溶液中搅拌后旋干，后修饰12OD CpG ODN，将制备的纳米免疫制剂在8000rpm下离心10分钟并分散于溶解在生理盐水，配制成LIPC储备液储存待用。As a contrast, the present invention synthesized a nano-immune preparation (LIPC) that only uses liposomes as a carrier, and IDOi is loaded into liposomes, and DPPC, cholesterol and DSPE-PEG are mixed and added in a ratio of 4:4:2M Stir in the organic solution and spin dry, post-modify 12OD CpG ODN, centrifuge the prepared nano-immune preparation at 8000rpm for 10 minutes and disperse it in physiological saline, prepare LIPC stock solution and store it for later use.
实施例2小鼠造模实验Embodiment 2 mouse modeling experiment
2.1体内试验2.1 In vivo test
选取雌性Balb/c小鼠作为研究对象，通过皮下注射小鼠乳腺癌细胞制作荷瘤小鼠模型，待肿瘤生长至50mm3开始给药。给药组分别为CaIPC(实验组)、LIPC、生理盐水。通过静脉方式对荷瘤小鼠给药，实验周期中，隔天测量肿瘤体积及称重。实验结果表明，实施例1中CaIPC处理的小鼠肿瘤生长速度最慢，表明本发明可有效抑制小鼠模型中的肿瘤生长。相反，与生理盐水组相比，LIPC组显示出并不是很优异的肿瘤抑制效果，该结果表明，只有本发明处理的组表现出最优的肿瘤治疗效果。体重是评估材料对身体的全身毒性的重要参数，所有组的小鼠体重随着时间延长没有明显变化，这意味着利用本发明进行治疗没有对小鼠产生明显的副作用，适用于活体水平的肿瘤治疗(图1)。Female Balb/c mice were selected as research objects, and tumor-bearing mouse models were made by subcutaneously injecting mouse breast cancer cells, and the administration began when the tumor grew to 50 mm 3 . The administration groups were CaIPC (experimental group), LIPC, and normal saline. The tumor-bearing mice were administered intravenously, and the tumor volume and weight were measured every other day during the experimental period. The experimental results show that the CaIPC-treated mice in Example 1 have the slowest tumor growth rate, indicating that the present invention can effectively inhibit tumor growth in the mouse model. On the contrary, the LIPC group showed not very excellent tumor suppressive effect compared with the normal saline group, and this result indicated that only the group treated in the present invention showed the optimal tumor therapeutic effect. Body weight is an important parameter to evaluate the systemic toxicity of materials to the body. The body weight of mice in all groups has no obvious change over time, which means that the treatment of the present invention has no obvious side effects on mice, and is suitable for tumors at the living level Treatment (Figure 1).
2.2防止肿瘤复发实验2.2 Prevention of tumor recurrence experiment
为了研究本发明在实际的手术治疗后防止肿瘤复发的效果，本发明建立了肿瘤术后复发模型。首先，将小鼠乳腺癌细胞接种到雌性Balb/c小鼠腋下，对小鼠肿瘤进行手术切除并留下残留肿瘤病灶。手术后静脉注射和瘤旁注射本发明材料进行术后进一步治疗，观察小鼠的肿瘤复发情况。实验结果表明，生理盐水组的小鼠肿瘤出现了复发。而CaIPC实验组静脉或瘤旁注射均没有肿瘤复发的情况发生，表明该发明有明显地抑制肿瘤复发的能力(图2)。In order to study the effect of the present invention on preventing tumor recurrence after actual surgical treatment, the present invention established a postoperative tumor recurrence model. First, mouse breast cancer cells were inoculated into the armpit of female Balb/c mice, and the mouse tumors were surgically removed to leave residual tumor lesions. After the operation, the material of the present invention was injected intravenously and next to the tumor for further postoperative treatment, and the tumor recurrence in mice was observed. The experimental results showed that the tumors in the mice in the saline group recurred. In the CaIPC experimental group, no tumor recurrence occurred after intravenous or paratumoral injection, indicating that the invention has the ability to significantly inhibit tumor recurrence ( FIG. 2 ).
以上所述仅为本申请的优选实施例而已，并不用于限制本申请，对于本领域的技术人员来说，本申请可以有各种更改和变化。凡在本申请的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本申请的保护范围之内。The above descriptions are only preferred embodiments of the present application, and are not intended to limit the present application. For those skilled in the art, there may be various modifications and changes in the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of this application shall be included within the protection scope of this application.
Claims (10)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810890136.2A CN110420335B (en) | 2018-08-07 | 2018-08-07 | Preparation and application of nano immune preparation based on porous calcium carbonate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810890136.2A CN110420335B (en) | 2018-08-07 | 2018-08-07 | Preparation and application of nano immune preparation based on porous calcium carbonate |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110420335A true CN110420335A (en) | 2019-11-08 |
| CN110420335B CN110420335B (en) | 2022-05-27 |
Family
ID=68407246
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810890136.2A Expired - Fee Related CN110420335B (en) | 2018-08-07 | 2018-08-07 | Preparation and application of nano immune preparation based on porous calcium carbonate |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110420335B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023010776A1 (en) * | 2021-08-03 | 2023-02-09 | 苏州大学 | Nano-drug having tumor immunity microenvironment regulation function, preparation method therefor, and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101816631A (en) * | 2010-04-27 | 2010-09-01 | 上海交通大学 | Liposome preparation for tumor therapeutic vaccine and preparation method thereof |
-
2018
- 2018-08-07 CN CN201810890136.2A patent/CN110420335B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101816631A (en) * | 2010-04-27 | 2010-09-01 | 上海交通大学 | Liposome preparation for tumor therapeutic vaccine and preparation method thereof |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023010776A1 (en) * | 2021-08-03 | 2023-02-09 | 苏州大学 | Nano-drug having tumor immunity microenvironment regulation function, preparation method therefor, and application thereof |
| CN115702889A (en) * | 2021-08-03 | 2023-02-17 | 苏州大学 | Nano-medicine with tumor immune microenvironment regulation function and its preparation method and application |
| CN115702889B (en) * | 2021-08-03 | 2024-06-21 | 苏州大学 | Nanomedicine with tumor immune microenvironment regulation function and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110420335B (en) | 2022-05-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhu et al. | Responsive hydrogels based on triggered click reactions for liver cancer | |
| EP4233891A1 (en) | Targeting delivery system loaded with whole-cell components and use thereof | |
| CN110755613A (en) | Preparation and application of light-triggered erythrocyte membrane-wrapped NO nano-biomimetic donor material | |
| CN109568570B (en) | Anti-tumor vaccine compound, preparation method, injection and application | |
| CN106039326A (en) | Method for preparing nano anticancer probe of zirconium-porphyrin metal organic framework material | |
| CN104623646A (en) | Bi-function ligand targeting dendritic cell tumor vaccine and preparation method thereof | |
| Ao et al. | STING agonist-based hydrogel enhances immune activation in synergy with radiofrequency ablation for hepatocellular carcinoma treatment | |
| CN116763725B (en) | An intelligent responsive injectable hydrogel and its preparation method and application | |
| CN104940945A (en) | Hollow mesoporous copper sulfide compound modified by hyaluronic acid and preparation method and application thereof | |
| CN112546062B (en) | Perfluorocarbon silicon plastid and preparation method and application thereof | |
| WO2023040127A1 (en) | Use of cancer vaccine system based on whole cell components in preparation of drugs for cross-prevention or treatment of heterogeneous cancers | |
| CN115645371A (en) | Self-generated active oxygen nanoparticles for precise non-invasive colon cancer dynamic immunotherapy and preparation method and application thereof | |
| CN109364253B (en) | A kind of nanoparticle for improving the permeability of tumor tissue and its preparation method and application | |
| CN112535735B (en) | Combined medicine capable of simultaneously amplifying immunogenic cell death and enhancing anti-tumor effect | |
| CN110368501B (en) | RGD peptide modified boron drug-loading system and preparation and application thereof | |
| CN110420335B (en) | Preparation and application of nano immune preparation based on porous calcium carbonate | |
| CN113041216B (en) | Multifunctional liver extracellular matrix composite hydrogel for liver cancer treatment and repair integration and preparation method thereof | |
| CN114010799A (en) | Photo-thermal synergistic chemotherapy targeted degradable nano-drug carrier and preparation method thereof | |
| CN113827724A (en) | Drug-loaded Prussian blue @ manganese fibrin composite gel, and preparation method and application thereof | |
| CN110951072B (en) | Compound with cell calcification inducing capability, preparation method and application thereof | |
| CN114796492B (en) | An ultrasonically driven nano-acoustic vaccine and its preparation and application | |
| CN117100872A (en) | Nano delivery carrier for targeted tumor drug delivery and application thereof | |
| KR102473102B1 (en) | Core-shell Structured Nanoparticles Conjugated with Immune Adjuvants and use thereof | |
| CN112315940B (en) | Nanoparticle for promoting tumor coagulation and enzyme/ATP dual-responsive drug release, and preparation method and application thereof | |
| CN108273066A (en) | A kind of preparation method for the high targeted nano pharmaceutical carrier and antitumor drug being oriented to structure based on hydroxyl |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20220527 |