CN116421727A - Filiform silk fibroin/calcium carbonate hybrid particles, and preparation method and application thereof - Google Patents
Filiform silk fibroin/calcium carbonate hybrid particles, and preparation method and application thereof Download PDFInfo
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- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 title claims abstract description 158
- 239000002245 particle Substances 0.000 title claims abstract description 87
- 229910000019 calcium carbonate Inorganic materials 0.000 title claims abstract description 79
- 108010022355 Fibroins Proteins 0.000 title claims abstract description 39
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 31
- 229960004657 indocyanine green Drugs 0.000 claims abstract description 27
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 13
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- 229910000029 sodium carbonate Inorganic materials 0.000 claims abstract description 8
- 238000006243 chemical reaction Methods 0.000 claims abstract description 4
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- BHTJEPVNHUUIPV-UHFFFAOYSA-N pentanedial;hydrate Chemical compound O.O=CCCCC=O BHTJEPVNHUUIPV-UHFFFAOYSA-N 0.000 claims 2
- 230000001225 therapeutic effect Effects 0.000 claims 1
- MOFVSTNWEDAEEK-UHFFFAOYSA-M indocyanine green Chemical compound [Na+].[O-]S(=O)(=O)CCCCN1C2=CC=C3C=CC=CC3=C2C(C)(C)C1=CC=CC=CC=CC1=[N+](CCCCS([O-])(=O)=O)C2=CC=C(C=CC=C3)C3=C2C1(C)C MOFVSTNWEDAEEK-UHFFFAOYSA-M 0.000 abstract description 26
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
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- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 4
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- ZQYZRCRCINWUKA-UHFFFAOYSA-L calcium ethane-1,2-diol dichloride Chemical compound C(CO)O.[Cl-].[Ca+2].[Cl-] ZQYZRCRCINWUKA-UHFFFAOYSA-L 0.000 description 3
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- CVOFKRWYWCSDMA-UHFFFAOYSA-N 2-chloro-n-(2,6-diethylphenyl)-n-(methoxymethyl)acetamide;2,6-dinitro-n,n-dipropyl-4-(trifluoromethyl)aniline Chemical compound CCC1=CC=CC(CC)=C1N(COC)C(=O)CCl.CCCN(CCC)C1=C([N+]([O-])=O)C=C(C(F)(F)F)C=C1[N+]([O-])=O CVOFKRWYWCSDMA-UHFFFAOYSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000005809 anti-tumor immunity Effects 0.000 description 1
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Abstract
本发明属于生物医药领域，涉及一种花状丝素蛋白/碳酸钙杂化微粒及其制备方法和应用，包括以碳酸钠溶液与氯化钙溶液为原料、并采用沉淀反应法制备的碳酸钙微粒；修饰增强碳酸钙微粒稳定性的丝素蛋白；以及负载使其拥有光热疗能力的吲哚菁绿，还包括带正电的鱼精蛋白。本发明能够发挥良好的光热治疗效果，与球形微粒相比能够更多的吸附肿瘤细胞免疫原性死亡产生的肿瘤相关抗原，激活机体免疫，对远端肿瘤的生长产生明显抑制作用，并显著延长荷瘤小鼠的生存时间；本发明的方法易于控制尺寸、形状，操作简单，制备工艺普适性强，适合于规模化生产，所用材料具有良好的生物相容性、生物可降解性；本发明适合在多功能肿瘤治疗中应用。
The invention belongs to the field of biomedicine, and relates to a flower-like silk fibroin/calcium carbonate hybrid particle and its preparation method and application, including calcium carbonate particle prepared by precipitation reaction method using sodium carbonate solution and calcium chloride solution as raw materials ; silk fibroin, which is modified to enhance the stability of calcium carbonate particles; and indocyanine green, which is loaded with photothermotherapy ability, including positively charged protamine. Compared with spherical particles, the present invention can exert a good effect of photothermal therapy. Compared with spherical particles, it can adsorb more tumor-associated antigens produced by immunogenic death of tumor cells, activate the body's immunity, and have a significant inhibitory effect on the growth of distant tumors, and significantly Prolong the survival time of tumor-bearing mice; the method of the invention is easy to control the size and shape, simple to operate, the preparation process is universal, suitable for large-scale production, and the materials used have good biocompatibility and biodegradability; The invention is suitable for application in multifunctional tumor treatment.
Description
技术领域technical field
本发明属于生物医药领域，涉及一种花状丝素蛋白/碳酸钙杂化微粒及其制备方法和应用。The invention belongs to the field of biomedicine, and relates to a flower-like silk fibroin/calcium carbonate hybrid particle and a preparation method and application thereof.
背景技术Background technique
恶性肿瘤是世界范围内的主要公共卫生问题之一，是造成人类死亡及影响人类寿命延长的重要原因。除了传统的手术、放疗、化疗等疗法，近年，光疗、免疫治疗以及基因治疗等新的治疗方法飞速发展，丰富了人类抗击恶性肿瘤的方法及手段。然而诸多研究和临床试验表明，肿瘤单一治疗模式难以获得预期效果，易发生复发、转移，联合治疗已成为肿瘤治疗的必然趋势。Malignant tumor is one of the major public health problems worldwide, and it is an important cause of human death and prolongation of human life span. In addition to traditional surgery, radiotherapy, chemotherapy and other treatments, in recent years, new treatment methods such as phototherapy, immunotherapy and gene therapy have developed rapidly, enriching the methods and means for human beings to fight malignant tumors. However, many studies and clinical trials have shown that it is difficult to obtain the expected effect in the single treatment mode of tumors, and recurrence and metastasis are prone to occur. Combination therapy has become an inevitable trend in tumor treatment.
光疗是一种非侵入性或微创性的治疗策略，主要包括光热疗和光动力治疗等。光热疗(PTT)利用光热转换剂的光热效应，吸收光产生热量从而提高周围环境的温度，引发肿瘤细胞死亡，可以高效消除多种类型的肿瘤。使用外部激光，照射剂量可调，可以精确地照射肿瘤部位，从而将对周围健康组织的损害降至最低。此外，光热疗也可以诱发免疫原性细胞死亡，诱导一定的抗肿瘤免疫。然而，光疗诱导的免疫反应相对较弱且持续时间较短。因此，需要一定的办法提高光热疗后的免疫效应。佐剂可以提高疫苗的免疫原性，增强非特异性机体免疫应答，颗粒型佐剂特别是微米级粒子具有与病原微生物相似的尺寸，能更好地刺激免疫系统做出应答反应。Phototherapy is a non-invasive or minimally invasive treatment strategy, mainly including photothermal therapy and photodynamic therapy. Photothermotherapy (PTT) uses the photothermal effect of photothermal conversion agents to absorb light to generate heat to increase the temperature of the surrounding environment, trigger tumor cell death, and effectively eliminate various types of tumors. Using an external laser, the dose can be adjusted to precisely irradiate the tumor site, minimizing damage to surrounding healthy tissue. In addition, photothermal therapy can also induce immunogenic cell death and induce certain antitumor immunity. However, phototherapy-induced immune responses were relatively weak and short-lived. Therefore, certain methods are needed to improve the immune effect after photothermia. Adjuvants can improve the immunogenicity of vaccines and enhance the non-specific immune response of the body. Particle-type adjuvants, especially micron-sized particles, have a size similar to that of pathogenic microorganisms, which can better stimulate the immune system to respond.
碳酸钙(CaCO3)微粒制备简单，生物相容性好，生物可降解，比表面积大，能够负载多种药物或者生物活性分子，同时制备过程“生物友好”。碳酸钙具有pH响应性，一方面被抗原呈递细胞摄取后，在酸性的溶酶体中分解，可以帮助递送的抗原逃逸到胞浆中进行随后的交叉呈递，从而实现更好的免疫激活作用。另一方面，未被摄取的碳酸钙可以作为质子清除剂来调节肿瘤环境的酸度，一定程度地抑制肿瘤。碳酸钙在生物医药领域具有广泛的应用前景。目前就已经有将碳酸钙应用于药物载体的研究先例，为药物递送载体提供了新的平台。除此之外，碳酸钙可以作为模板吸附聚合物壳层，研究者已经开发出了多种以碳酸钙为模板的载体，包括使用碳酸钙作为功能核或将核移除的中空微囊。Calcium carbonate (CaCO 3 ) particles are easy to prepare, have good biocompatibility, are biodegradable, have a large specific surface area, and can be loaded with various drugs or bioactive molecules, and the preparation process is "bio-friendly". Calcium carbonate is pH-responsive. On the one hand, after being taken up by antigen-presenting cells, it is decomposed in acidic lysosomes, which can help the delivered antigen escape into the cytoplasm for subsequent cross-presentation, thereby achieving better immune activation. On the other hand, uningested calcium carbonate can act as a proton scavenger to regulate the acidity of the tumor environment and inhibit the tumor to a certain extent. Calcium carbonate has broad application prospects in the field of biomedicine. At present, there have been research precedents of applying calcium carbonate to drug carriers, which provides a new platform for drug delivery carriers. In addition, calcium carbonate can be used as a template to adsorb polymer shells. Researchers have developed a variety of calcium carbonate-templated carriers, including hollow microcapsules using calcium carbonate as a functional core or removing the core.
碳酸钙微粒可以非特异性的吸附抗原类分子，与球形碳酸钙微粒相比，花状碳酸钙微粒有着更强的吸附能力，将碳酸钙粒子特别是花状碳酸钙粒子作为佐剂具有良好的应用前景。然而，碳酸钙结晶的重现性较低(大小、形状等)，所制备碳酸钙微粒分散性和稳定性较差。所以，寻找一种简单易行且重复性好的碳酸钙制备方法，且提高所制备碳酸钙微粒的稳定性显得尤为重要。本发明在溶剂中加入乙二醇改善碳酸钙微粒的分散性，利用丝素蛋白修饰提高了碳酸钙微粒的稳定性，并优选出有更强抗原吸附能力的花状碳酸钙微粒，将花状丝素蛋白/碳酸钙微粒作为肿瘤疫苗并结合光热疗的研究还未见报道。Calcium carbonate particles can non-specifically adsorb antigen molecules. Compared with spherical calcium carbonate particles, flower-shaped calcium carbonate particles have stronger adsorption capacity. Calcium carbonate particles, especially flower-shaped calcium carbonate particles, have good application as adjuvants prospect. However, the reproducibility of calcium carbonate crystals is low (size, shape, etc.), and the dispersibility and stability of the prepared calcium carbonate particles are poor. Therefore, it is particularly important to find a simple and reproducible method for preparing calcium carbonate, and to improve the stability of the prepared calcium carbonate particles. In the present invention, ethylene glycol is added to the solvent to improve the dispersibility of calcium carbonate particles, the stability of calcium carbonate particles is improved by silk fibroin modification, and the flower-shaped calcium carbonate particles with stronger antigen adsorption capacity are optimized. The study of silk fibroin/calcium carbonate microparticles as a tumor vaccine combined with photothermia has not been reported yet.
发明内容Contents of the invention
鉴于此，本发明的目的在于，提供一种工艺简单、重现性好、形状可控、稳定性强，且具有良好肿瘤治疗效果的花状丝素蛋白/碳酸钙微粒；In view of this, the purpose of the present invention is to provide a flower-like silk fibroin/calcium carbonate particle with simple process, good reproducibility, controllable shape, strong stability, and good tumor treatment effect;
本发明的第二个目的是提供一种花状丝素蛋白/碳酸钙微粒的制备方法；The second object of the present invention is to provide a preparation method of floral fibroin/calcium carbonate particles;
本发明的第三个目的是提供一种花状丝素蛋白/碳酸钙微粒在肿瘤联合治疗中的应用。The third object of the present invention is to provide an application of fibroin/calcium carbonate particles in combined treatment of tumors.
为了达到上述目的，进而采取的技术方案如下：In order to achieve the above object, the technical scheme adopted is as follows:
一种花状丝素蛋白/碳酸钙杂化微粒，包括以碳酸钠溶液与氯化钙溶液为原料、并采用沉淀反应法制备的碳酸钙微粒；A flower-like silk fibroin/calcium carbonate hybrid particle, including calcium carbonate particle prepared by precipitation reaction method using sodium carbonate solution and calcium chloride solution as raw materials;
修饰增强碳酸钙微粒稳定性的丝素蛋白；Modification of silk fibroin that enhances the stability of calcium carbonate particles;
以及负载使其拥有光热疗能力的吲哚菁绿。And indocyanine green loaded with photothermotherapy ability.
优选地，还包括带正电的鱼精蛋白。Preferably, positively charged protamine is also included.
优选地，所述碳酸钙微粒大小为1～10μm；Preferably, the size of the calcium carbonate particles is 1-10 μm;
所述碳酸钙微粒是指微粒表面有分支结构。The calcium carbonate particle refers to a branched structure on the surface of the particle.
一种花状丝素蛋白/碳酸钙杂化微粒在肿瘤联合治疗中的应用。Application of a fibroin/calcium carbonate hybrid particle in combined therapy of tumors.
一种花状丝素蛋白/碳酸钙杂化微粒的制备方法，包括以下步骤：A preparation method of floral silk fibroin/calcium carbonate hybrid particles, comprising the following steps:
步骤一：将5mL浓度为0.5mol/L～1mol/L的碳酸钠溶液与2.5mL～5mL的乙二醇混合，得混合液一；Step 1: Mix 5 mL of sodium carbonate solution with a concentration of 0.5 mol/L to 1 mol/L and 2.5 mL to 5 mL of ethylene glycol to obtain a mixed solution 1;
将5mL浓度为0.05mol/L～0.5mol/L的氯化钙溶液与2.5mL～5mL的乙二醇混合，得混合液二；Mix 5 mL of calcium chloride solution with a concentration of 0.05 mol/L to 0.5 mol/L and 2.5 mL to 5 mL of ethylene glycol to obtain mixed
将混合液二在磁力搅拌下注入混合液一中，得到混合液三；Inject mixed
将混合液三离心，水洗，得到第一种微粒；Centrifuge the mixed solution three times and wash with water to obtain the first particle;
其中，乙二醇溶液中的水和乙二醇体积比为1:2～1:0.5；Wherein, the volume ratio of water and ethylene glycol in the ethylene glycol solution is 1:2～1:0.5;
步骤二:将浓度为2mg/mL的丝素蛋白溶液和第一种微粒混合，得混合液四，旋转混合60min，离心，除去上清液，清洗，离心，得到第二种微粒；Step 2: Mix the silk fibroin solution with a concentration of 2 mg/mL and the first particle to obtain the mixed solution 4, rotate and mix for 60 minutes, centrifuge, remove the supernatant, wash, and centrifuge to obtain the second particle;
将第二种微粒与甲醇溶液混合，得混合液五，旋转混合30min，离心，除去上清液，清洗，离心，得到第三种微粒；Mix the second particle with the methanol solution to obtain the
步骤三：重复步骤二2～4正整数次；Step 3: Repeat
步骤四：向步骤三获得的微粒中加入戊二醛水溶液进行交联，离心，水洗，得到第四种微粒；Step 4: adding an aqueous solution of glutaraldehyde to the microparticles obtained in step 3 for cross-linking, centrifuging, and washing with water to obtain the fourth microparticles;
步骤五：向步骤四获得微粒中加入吲哚菁绿(ICG)溶液，旋转混合60min，离心，除去上清液，清洗，离心，得到最终微粒。Step 5: Add indocyanine green (ICG) solution to the microparticles obtained in step 4, rotate and mix for 60 minutes, centrifuge, remove the supernatant, wash, and centrifuge to obtain final microparticles.
一种花状丝素蛋白/碳酸钙杂化微粒的制备方法，向步骤三获得微粒中加入带正电的鱼精蛋白溶液，旋转混合60min，离心，除去上清液，清洗，离心，得到第五种微粒；A preparation method of floral fibroin/calcium carbonate hybrid microparticles, adding positively charged protamine solution to the microparticles obtained in step 3, rotating and mixing for 60 minutes, centrifuging, removing the supernatant, washing, and centrifuging to obtain the fifth kind of particle;
戊二醛水溶液加入第五种微粒中进行交联，离心，水洗，得到第六种微粒；Aqueous glutaraldehyde solution is added to the fifth particle for cross-linking, centrifuged and washed with water to obtain the sixth particle;
最终得到经过鱼精蛋白修饰的丝素蛋白/碳酸钙杂化材料。Finally, the protamine-modified silk fibroin/calcium carbonate hybrid material was obtained.
本发明的有益效果是：The beneficial effects of the present invention are:
本发明的花状丝素蛋白/碳酸钙微粒能够发挥良好的光热治疗效果，与球形微粒相比能够更多的吸附肿瘤细胞免疫原性死亡产生的肿瘤相关抗原，激活机体免疫，对远端肿瘤的生长产生明显抑制作用，并显著延长荷瘤小鼠的生存时间；The floral fibroin/calcium carbonate microparticles of the present invention can exert a good photothermal treatment effect, and compared with spherical microparticles, can adsorb more tumor-associated antigens produced by immunogenic death of tumor cells, activate the body's immunity, and The growth of the tumor was significantly inhibited, and the survival time of the tumor-bearing mice was significantly prolonged;
本发明的方法易于控制尺寸、形状，操作简单，制备工艺普适性强，适合于规模化生产，所用材料具有良好的生物相容性、生物可降解性；The method of the invention is easy to control the size and shape, simple to operate, and the preparation process is highly universal, suitable for large-scale production, and the materials used have good biocompatibility and biodegradability;
本发明适合在多功能肿瘤治疗中应用。The invention is suitable for application in multifunctional tumor treatment.
附图说明Description of drawings
构成本申请的一部分的附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：The accompanying drawings constituting a part of this application are used to provide further understanding of the present invention, and the schematic embodiments and descriptions of the present invention are used to explain the present invention, and do not constitute an improper limitation of the present invention. In the attached picture:
图1为实施例一所制备花状碳酸钙微粒的扫描电镜照片；Fig. 1 is the scanning electron micrograph of the prepared flower-shaped calcium carbonate particle of embodiment one;
图2为实施例二所制备花状碳酸钙微粒的扫描电镜照片；Fig. 2 is the scanning electron micrograph of the prepared flower-shaped calcium carbonate particle of embodiment two;
图3为实施例三所制备花状碳酸钙微粒的扫描电镜照片；Fig. 3 is the scanning electron micrograph of the prepared flower-shaped calcium carbonate particle of embodiment three;
图4为实施例四所制备的花状丝素蛋白/碳酸钙微粒(a)Flo@SC/ICG和(b)Flo@PSC/ICG；Fig. 4 is the floral silk fibroin/calcium carbonate particle (a) Flo@SC/ICG and (b) Flo@PSC/ICG prepared by embodiment four;
图5为实施例五中球状和花状丝素蛋白/碳酸钙微粒对(a)蛋白质和(b)DNA的吸附情况；Fig. 5 is the adsorption situation of spherical and flower-like silk fibroin/calcium carbonate particles to (a) protein and (b) DNA in embodiment five;
图6为实施例六中丝素蛋白/碳酸钙微粒的肿瘤治疗，(a-c)分别为Control、Flo@SC/ICG、Flo@PSC/ICG组治疗后原位肿瘤的生长曲线；Figure 6 is the tumor treatment of silk fibroin/calcium carbonate microparticles in Example 6, (a-c) are the growth curves of tumors in situ after treatment in the Control, Flo@SC/ICG, and Flo@PSC/ICG groups respectively;
(d-f)分别为Control、Flo@SC/ICG、Flo@PSC/ICG组治疗后对侧肿瘤的生长曲线；(d-f) are the growth curves of contralateral tumors after treatment in Control, Flo@SC/ICG, and Flo@PSC/ICG groups, respectively;
图7为实施例六中注射丝素蛋白/碳酸钙微粒的肿瘤治疗下，Control、Flo@SC/ICG、Flo@PSC/ICG组治疗后小鼠的存活时间曲线。Fig. 7 is the survival time curve of mice treated with Control, Flo@SC/ICG, and Flo@PSC/ICG groups under the tumor treatment injected with silk fibroin/calcium carbonate microparticles in Example 6.
具体实施方式Detailed ways
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本发明。It should be noted that, in the case of no conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and examples.
为了使本技术领域的人员更好地理解本申请方案，下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本申请一部分的实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都应当属于本申请保护的范围。In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiment of the application will be clearly and completely described below in conjunction with the accompanying drawings in the embodiment of the application. Obviously, the described embodiment is only It is an embodiment of a part of the application, but not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by persons of ordinary skill in the art without creative efforts shall fall within the scope of protection of this application.
实施例一Embodiment one
花状碳酸钙微粒的制备：Preparation of flower-like calcium carbonate particles:
取两个100mL容器，均加入2.5mL乙二醇溶液，然后分别加入5mL 1M的碳酸钠溶液与5mL 0.1M的氯化钙溶液，磁力搅拌使其混合均匀。Take two 100mL containers, add 2.5mL ethylene glycol solution to both, then add 5mL 1M sodium carbonate solution and 5mL 0.1M calcium chloride solution respectively, stir magnetically to make them evenly mixed.
将氯化钙-乙二醇溶液缓缓注入碳酸钠-乙二醇溶液中，磁力搅拌，4000rpm离心，乙醇/水混合溶液(V乙醇∶V水＝1∶1)清洗沉淀一次，之后用超纯水洗继续洗涤两次得到花状碳酸钙微粒悬液。The calcium chloride-ethylene glycol solution is slowly injected into the sodium carbonate-ethylene glycol solution, magnetically stirred, centrifuged at 4000rpm, and the ethanol/water mixed solution (V ethanol : V water =1: 1) washes the precipitate once, and then uses an ultrasonic Wash with pure water and continue to wash twice to obtain flower-shaped calcium carbonate particle suspension.
将上述碳酸钙微粒悬液滴在硅片上自然干燥，扫描电镜下拍照，见图1。The above-mentioned suspension of calcium carbonate particles was dropped on a silicon wafer to dry naturally, and photographed under a scanning electron microscope, as shown in FIG. 1 .
实施例二Embodiment two
花状碳酸钙微粒的制备：Preparation of flower-like calcium carbonate particles:
取两个100mL容器，均加入4mL乙二醇溶液，然后分别加入4mL 1M的碳酸钠溶液与4mL 0.1M的氯化钙溶液，磁力搅拌使其混合均匀。Take two 100mL containers, add 4mL ethylene glycol solution to both, then add 4mL 1M sodium carbonate solution and 4mL 0.1M calcium chloride solution respectively, stir magnetically to make them evenly mixed.
将氯化钙-乙二醇溶液缓缓注入碳酸钠-乙二醇溶液中，磁力搅拌，4000rpm离心，乙醇/水混合溶液(V乙醇∶V水＝1∶1)清洗沉淀一次，之后用超纯水洗继续洗涤两次得到花状碳酸钙微粒悬液。The calcium chloride-ethylene glycol solution is slowly injected into the sodium carbonate-ethylene glycol solution, magnetically stirred, centrifuged at 4000rpm, and the ethanol/water mixed solution (V ethanol : V water =1: 1) washes the precipitate once, and then uses an ultrasonic Wash with pure water and continue to wash twice to obtain flower-shaped calcium carbonate particle suspension.
将上述碳酸钙微粒悬液滴在硅片上自然干燥，扫描电镜下拍照，见图2。Drop the above-mentioned suspension of calcium carbonate particles on a silicon wafer to dry naturally, and take pictures under a scanning electron microscope, as shown in FIG. 2 .
实施例三Embodiment three
花状碳酸钙微粒的制备：Preparation of flower-like calcium carbonate particles:
取两个100mL容器，均加入2.5mL乙二醇溶液，然后分别加入5mL 0.5M的碳酸钠溶液与5mL 0.05M的氯化钙溶液，磁力搅拌使其混合均匀。Take two 100mL containers, add 2.5mL ethylene glycol solution to both, then add 5mL 0.5M sodium carbonate solution and 5mL 0.05M calcium chloride solution respectively, stir magnetically to make them evenly mixed.
将氯化钙-乙二醇溶液缓缓注入碳酸钠-乙二醇溶液中，磁力搅拌，4000rpm离心，乙醇/水混合溶液(V乙醇∶V水＝1∶1)清洗沉淀一次，之后用超纯水洗继续洗涤两次得到花状碳酸钙微粒悬液。The calcium chloride-ethylene glycol solution is slowly injected into the sodium carbonate-ethylene glycol solution, magnetically stirred, centrifuged at 4000rpm, and the ethanol/water mixed solution (V ethanol : V water =1: 1) washes the precipitate once, and then uses an ultrasonic Wash with pure water and continue to wash twice to obtain flower-shaped calcium carbonate particle suspension.
将上述碳酸钙微粒悬液滴在硅片上自然干燥，扫描电镜下拍照，见图3。Drop the suspension of calcium carbonate particles above on a silicon wafer to dry naturally, and take pictures under a scanning electron microscope, as shown in FIG. 3 .
实施例四Embodiment Four
花状丝素蛋白/碳酸钙微粒的制备方法，包括如下步骤：The preparation method of floral silk fibroin/calcium carbonate particle, comprises the steps:
(1)将浓度为2mg/mL的丝素蛋白溶液和花状碳酸钙微粒混合，得混合液四，旋转混合60min，离心，除去上清液，清洗，离心，得到第二种微粒；将第二种微粒与甲醇溶液混合，得混合液五，旋转混合30min，离心，除去上清液，清洗，离心，得到第三种微粒；(1) Mix the silk fibroin solution with a concentration of 2 mg/mL and flower-like calcium carbonate particles to obtain the mixed solution 4, rotate and mix for 60 minutes, centrifuge, remove the supernatant, wash, and centrifuge to obtain the second particle; Mix the two kinds of microparticles with methanol solution to obtain a mixed solution five, rotate and mix for 30 minutes, centrifuge, remove the supernatant, wash, and centrifuge to obtain the third kind of microparticles;
(2)重复步骤(1)2～4正整数次；(2) Repeat step (1) 2 to 4 positive integer times;
(3)向步骤(2)获得的微粒中加入戊二醛水溶液进行交联，离心，水洗，得到第四种微粒；(3) adding an aqueous solution of glutaraldehyde to the microparticles obtained in step (2) for cross-linking, centrifuging, and washing with water to obtain the fourth microparticles;
(4)向步骤(3)获得微粒中加入吲哚菁绿(ICG)溶液，旋转混合60min，离心，除去上清液，清洗，离心，得到最终微粒。用Flo@SC/ICG表示，简称(a)。(4) Add indocyanine green (ICG) solution to the microparticles obtained in step (3), rotate and mix for 60 min, centrifuge, remove supernatant, wash, and centrifuge to obtain final microparticles. Represented by Flo@SC/ICG, referred to as (a).
花状鱼精蛋白修饰的丝素蛋白/碳酸钙微粒的制备方法，包括如下步骤：The preparation method of the silk fibroin/calcium carbonate particle modified by flower-like protamine comprises the steps:
(1)将浓度为2mg/mL的丝素蛋白溶液和花状碳酸钙微粒混合，得混合液四，旋转混合60min，离心，除去上清液，清洗，离心，得到第二种微粒；将第二种微粒与甲醇溶液混合，得混合液五，旋转混合30min，离心，除去上清液，清洗，离心，得到第三种微粒；(1) Mix the silk fibroin solution with a concentration of 2 mg/mL and flower-like calcium carbonate particles to obtain the mixed solution 4, rotate and mix for 60 minutes, centrifuge, remove the supernatant, wash, and centrifuge to obtain the second particle; Mix the two kinds of microparticles with methanol solution to obtain a mixed solution five, rotate and mix for 30 minutes, centrifuge, remove the supernatant, wash, and centrifuge to obtain the third kind of microparticles;
(2)重复步骤(1)2～4正整数次；(2) Repeat step (1) 2 to 4 positive integer times;
(3)向步骤(2)获得微粒中加入硫酸鱼精蛋白溶液，旋转混合60min，离心，除去上清液，清洗，离心，得到第五种微粒；(3) Add protamine sulfate solution to the particles obtained in step (2), rotate and mix for 60 minutes, centrifuge, remove the supernatant, wash, and centrifuge to obtain the fifth particle;
(4)向步骤(3)获得的微粒中加入戊二醛水溶液进行交联，离心，水洗，得到第六种微粒；(4) adding an aqueous solution of glutaraldehyde to the microparticles obtained in step (3) for cross-linking, centrifuging, and washing with water to obtain the sixth microparticles;
(5)向步骤(4)获得微粒中加入ICG溶液，旋转混合60min，离心，除去上清液，清洗，离心，得到最终微粒。用Flo@PSC/ICG表示，简称(b)。(5) Add ICG solution to the microparticles obtained in step (4), rotate and mix for 60 min, centrifuge, remove supernatant, wash, and centrifuge to obtain final microparticles. Represented by Flo@PSC/ICG, referred to as (b).
上述花状丝素蛋白/碳酸钙微粒滴在硅片上自然干燥，扫描电镜下拍照，见图4。The above-mentioned flower-like silk fibroin/calcium carbonate particles were dropped on a silicon wafer and dried naturally, and photographed under a scanning electron microscope, as shown in FIG. 4 .
实施例五Embodiment five
丝素蛋白/碳酸钙微粒对肿瘤光热疗后释放蛋白和DNA的吸附作用，包括如下步骤：The adsorption of silk fibroin/calcium carbonate microparticles to released protein and DNA after tumor photothermia includes the following steps:
(1)取状态良好的4T1细胞，在12孔板中每孔接种2×105个，在37℃细胞培养箱中孵育过夜；(1) Take 4T1 cells in good condition, inoculate 2× 105 cells in each well of a 12-well plate, and incubate overnight in a cell culture incubator at 37°C;
(2)细胞贴壁后弃去原有培养基，用PBS清洗三遍，加入1mL含ICG浓度为10μg/mL的PBS，孵育2h；(2) Discard the original medium after the cells attach to the wall, wash with PBS three times, add 1 mL of PBS containing ICG with a concentration of 10 μg/mL, and incubate for 2 h;
(3)用1w/cm2的707nm激光照射10min，光照完成后，弃去含ICG的PBS，并加入500μLPBS溶液，37℃孵育24h使细胞释放蛋白和DNA；(3) Irradiate with 1w/cm 2 707nm laser for 10min. After the illumination is completed, discard the PBS containing ICG, add 500μL PBS solution, and incubate at 37°C for 24h to release the protein and DNA from the cells;
(4)将各孔中PBS收集到15mL离心管中，离心，上清液收集备用；(4) Collect the PBS in each well into a 15mL centrifuge tube, centrifuge, and collect the supernatant for later use;
(5)取尺寸匹配的球状和花状的丝素蛋白/碳酸钙微粒(Sph@SC/ICG和Flo@SC/ICG)，以及球状和花状的鱼精蛋白修饰的丝素蛋白/碳酸钙微粒(Sph@PSC/ICG和Flo@PSC/ICG)的混悬液于1.5mLEP管中，用PBS稀释到100μL，使溶液中ICG的浓度为50μg/mL，并以不加碳酸钙微粒的PBS作为对照组；(5) Take size-matched spherical and flower-like silk fibroin/calcium carbonate particles (Sph@SC/ICG and Flo@SC/ICG), and spherical and flower-like protamine-modified silk fibroin/calcium carbonate The suspension of microparticles (Sph@PSC/ICG and Flo@PSC/ICG) was placed in a 1.5mLEP tube, diluted to 100μL with PBS, so that the concentration of ICG in the solution was 50μg/mL, and the solution was diluted with PBS without calcium carbonate microparticles. as a control group;
(6)分别加入400μLPBS上清，孵育2h之后离心，用BCA试剂盒测定上清液中蛋白含量，用DNA浓度测定试剂盒测定上清液中DNA的含量，计算各组蛋白和DNA吸附比例，见图5。(6) Add 400 μL PBS supernatant respectively, centrifuge after incubation for 2 hours, measure the protein content in the supernatant with a BCA kit, measure the DNA content in the supernatant with a DNA concentration determination kit, and calculate the adsorption ratio of each histone protein and DNA, See Figure 5.
结果表明，丝素蛋白/碳酸钙微粒能够吸附光热后释放的蛋白和DNA，而且花状微粒比球状微粒的吸附性更强，鱼精蛋白修饰有利于增强蛋白和DNA的吸附，推测该类碳酸钙微粒可以吸附抗原并起到佐剂的作用。The results show that silk fibroin/calcium carbonate particles can adsorb proteins and DNA released after light and heat, and the adsorption of flower-shaped particles is stronger than that of spherical particles. The modification of protamine is beneficial to enhance the adsorption of proteins and DNA. Calcium carbonate particles can adsorb antigens and act as an adjuvant.
实施例六Embodiment six
丝素蛋白/碳酸钙微粒对肿瘤联合治疗作用，包括如下步骤：Silk fibroin/calcium carbonate microparticles have the effect of combined therapy on tumors, including the following steps:
将带有乳腺癌皮下肿瘤模型的小鼠分为三组，其中包括：Mice bearing subcutaneous tumor models of breast cancer were divided into three groups consisting of:
第一组，对照组(仅注射生理盐水)；The first group, the control group (only inject normal saline);
第二组，注射Flo@SC/ICG并光照；The second group, injected with Flo@SC/ICG and illuminated;
第三组，注射Flo@PSC/ICG并光照。In the third group, Flo@PSC/ICG was injected and illuminated.
对小鼠进行相应的治疗后，在对侧皮下种植乳腺癌细胞，测量其原位肿瘤和对侧肿瘤的生长，结果见于图6和图7。结果表明，相比较对照组，第二组和第三组的肿瘤生长得到了有效的抑制。表明花状丝素蛋白/碳酸钙制剂能够实现对肿瘤的光热治疗，并抑制远端肿瘤的生长。After the mice were treated accordingly, breast cancer cells were subcutaneously planted on the contralateral side, and the growth of the orthotopic tumor and the contralateral tumor were measured, and the results are shown in Fig. 6 and Fig. 7 . The results showed that, compared with the control group, the tumor growth of the second group and the third group was effectively inhibited. It shows that the fibroin/calcium carbonate preparation can achieve photothermal treatment of tumors and inhibit the growth of distant tumors.
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.
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