技术领域technical field
本发明涉及一种金纳米星材料的制备，具体涉及一种交联具有pH响应的穿膜小肽pHLIP的金纳米星材料的制备及其应用。The invention relates to the preparation of a gold nano-star material, in particular to the preparation and application of a gold nano-star material cross-linked with a pH-responsive membrane-penetrating small peptide pHLIP.
背景技术Background technique
肿瘤的治疗研究具有重大的医学和社会意义，是提高我国居民健康的重大迫切需求。传统的抗肿瘤治疗主要包括化疗或物理治疗，但均是有创性损伤，如某些化疗药物阿霉素、顺铂、丝裂霉素等本身具有极大的毒性，副作用大，通过静脉全身给药会严重影响正常细胞的机能，破坏免疫功能；大多数化疗药物体内循环时间较短，很难靶向肿瘤部位发挥药效，并且长时间的给药会使某些肿瘤细胞产生耐药性。Tumor treatment research has great medical and social significance, and it is a major and urgent need to improve the health of Chinese residents. Traditional anti-tumor treatment mainly includes chemotherapy or physical therapy, but all of them are invasive injuries. For example, certain chemotherapy drugs such as doxorubicin, cisplatin, and mitomycin have great toxicity and side effects. Drug administration will seriously affect the function of normal cells and destroy immune function; most chemotherapy drugs have a short circulation time in the body, it is difficult to target the tumor site to exert their drug effect, and long-term administration will cause some tumor cells to develop drug resistance .
纳米医学的发展为解决上述问题提供了新的方向，基于纳米载体治疗方式的探索为改善传统治疗方式，提高治疗疗效带来了新的契机。近红外(Near infraredfluorescence，NIR)激光技术以其特殊的波长范围(650nm～1000nm)，较强的光吸收和高效的光热转化率、低毒且非侵袭性，较好的生物相容性，深穿透能力等优点受到纳米医学研究领域的青睐。2015年，Shell等在Accounts of Chemical Research(if：22.323，2015，48：2866-2874)高度评价近红外光热技术作为一种安全特异的刺激源在临床载药及治疗中的应用。其中，金纳米材料以其良好的生物相容性、低毒性、简单的合成步骤，在近红外区有强吸收并能转化成热能等优点受到广泛研究，本发明通过简单的合成方法制备出具有特殊形貌及理化性质的金纳米星材料，调整使其具有特定的近红外区吸收峰，并能高效的转换成热能，短时间内迅速提高材料富集区的温度，利用热效应杀死肿瘤细胞，避免引起全身毒性。The development of nanomedicine provides a new direction to solve the above problems, and the exploration of nanocarrier-based treatment methods brings new opportunities to improve traditional treatment methods and improve therapeutic efficacy. Near infrared (NIR) laser technology has a special wavelength range (650nm ~ 1000nm), strong light absorption and high photothermal conversion rate, low toxicity and non-invasiveness, and good biocompatibility. Advantages such as deep penetration ability are favored by the field of nanomedicine research. In 2015, Shell et al. in Accounts of Chemical Research (if: 22.323, 2015, 48: 2866-2874) highly evaluated the application of near-infrared photothermal technology as a safe and specific stimulus in clinical drug loading and treatment. Among them, gold nanomaterials have been widely studied due to their good biocompatibility, low toxicity, simple synthesis steps, strong absorption in the near-infrared region and the ability to convert them into heat energy. Gold nanostar materials with special morphology and physical and chemical properties are adjusted to have specific absorption peaks in the near-infrared region, and can be efficiently converted into heat energy, rapidly increasing the temperature of the material-enriched area in a short period of time, and killing tumor cells by thermal effect , to avoid systemic toxicity.
同时，纳米材料在肿瘤部位的高选择通透性和滞留效应，即增强渗透滞留效应(enhanced permeability and retention effect，EPR)，使得纳米材料本身具有被动靶向肿瘤的作用。但EPR效率容易受到肿瘤微环境如致密间质、突变细胞外基质、间质液压力，肿瘤病理生理异质性等的影响；另外，虽然EPR效应能够提高纳米载体在肿瘤组织的积聚，但细胞内化效率低及细胞内药物释放不足也干扰了抗癌药物的利用率。若达到较理想的靶向肿瘤治疗作用，需通过化学连接方法结合某些具有肿瘤靶向识别作用的生物分子，但这些生物分子在正常细胞中也会有表达，会导致边缘效应的发生。At the same time, the high selective permeability and retention effect of nanomaterials in tumor sites, that is, the enhanced permeability and retention effect (EPR), enables nanomaterials to passively target tumors. However, the EPR efficiency is easily affected by the tumor microenvironment such as dense stroma, mutant extracellular matrix, interstitial fluid pressure, tumor pathophysiological heterogeneity, etc. In addition, although the EPR effect can increase the accumulation of nanocarriers in tumor tissues, the cells Low internalization efficiency and insufficient intracellular drug release also interfere with the availability of anticancer drugs. In order to achieve a more ideal targeted tumor therapy, it is necessary to combine certain biomolecules with tumor targeting recognition through chemical linkage, but these biomolecules are also expressed in normal cells, which will lead to edge effects.
利用肿瘤特征性的微酸性环境来实现纳米材料的肿瘤靶向运输的策略越来越受到关注。新型穿膜小肽pHLIP可响应肿瘤微环境，在肿瘤pH酸性环境下迅速折叠并插入肿瘤细胞细胞膜形成稳定的跨膜α螺旋，并协助交联的材料进入细胞，提高细胞对材料的吞噬量，进而使材料更好的发挥作用。The strategy of using the characteristic slightly acidic environment of tumors to achieve tumor-targeted delivery of nanomaterials has attracted more and more attention. The new membrane-penetrating small peptide pHLIP can respond to the tumor microenvironment, rapidly fold and insert into the tumor cell membrane to form a stable transmembrane α helix in the acidic environment of the tumor pH, and assist the cross-linked material to enter the cell, increasing the phagocytosis of the material by the cell, And then make the material play a better role.
迄今为止尚无交联pH响应的跨膜小肽的金纳米星材料的制备及光热治疗应用的相关文献报道。So far, there is no relevant literature report on the preparation of gold nanostar materials cross-linked with pH-responsive transmembrane small peptides and the application of photothermal therapy.
发明内容Contents of the invention
为了克服现有技术中存在的不足，本发明提出一种交联pH响应跨膜小肽pHLIP的金纳米星材料的制备与应用。In order to overcome the deficiencies in the prior art, the present invention proposes the preparation and application of a gold nanostar material cross-linked with pH-responsive transmembrane small peptide pHLIP.
技术方案：为解决上述技术问题，本发明的一种交联pH响应跨膜小肽的金纳米星材料通过如下方法制备得到：Technical solution: In order to solve the above technical problems, a gold nanostar material cross-linked pH-responsive transmembrane small peptide of the present invention is prepared by the following method:
(1)用水分别配制2mM马来酰亚胺-聚乙二醇修饰的小肽pHLIP(所述多肽是序列为ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG的pHLIP肽)溶液和2mM氨基-聚乙二醇-巯基溶液，将两者按照摩尔比1∶0.8～1混合均匀，得到混合液。(1) Prepare 2mM maleimide-polyethylene glycol-modified small peptide pHLIP (the polypeptide is a pHLIP peptide whose sequence is ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG) solution and 2mM amino-polyethylene glycol-sulfhydryl solution respectively with water, mix the two Mix evenly according to the molar ratio of 1:0.8~1 to obtain a mixed liquid.
(2)2.5mL 0.01M的氯金酸加入到97.5mL的水中，加热搅拌5min～10min，随后加入3mL 1％的柠檬酸三钠，溶液颜色由无色变成暗红色，停止加热和搅拌，制得柠檬酸钠金溶液。(2) Add 2.5mL of 0.01M chloroauric acid into 97.5mL of water, heat and stir for 5min to 10min, then add 3mL of 1% trisodium citrate, the color of the solution changes from colorless to dark red, stop heating and stirring, A sodium citrate gold solution was prepared.
(3)0.25mL 0.01M的氯金酸溶液溶解在10mL水中，取0.1mL上述(2)的柠檬酸钠金溶液加入氯金酸溶液，在室温搅拌下，向该混合液中同时加入0.05mL 0.1M硝酸银和0.04mL0.01M抗坏血酸，继续搅拌使溶液颜色由浅粉变成深蓝色，反应5～10min后加入0.02mL步骤(1)得到的混合物，然后将得到的产物离心、洗涤3次、弃上清，并分散至1mL水中，形成交联pH响应跨膜小肽的金纳米星材料储备液。(3) Dissolve 0.25mL of 0.01M chloroauric acid solution in 10mL of water, take 0.1mL of the sodium citrate gold solution of (2) above and add the chloroauric acid solution, stir at room temperature, and add 0.05mL of 0.1M silver nitrate and 0.04mL0.01M ascorbic acid, continue to stir to make the solution color change from light pink to dark blue, add 0.02mL of the mixture obtained in step (1) after reacting for 5-10min, then centrifuge the obtained product, wash 3 times, The supernatant was discarded and dispersed into 1 mL of water to form a gold nanostar material stock solution of cross-linked pH-responsive transmembrane small peptides.
优选地，所述步骤(2)中搅拌条件为800～1000转/分钟。Preferably, the stirring condition in the step (2) is 800-1000 rpm.
步骤(2)中，离心条件为：相对离心力为7500～8000，离心10～15分钟。In step (2), the centrifugation condition is: the relative centrifugal force is 7500-8000, centrifuged for 10-15 minutes.
经过表征结果，利用本发明方法制备得到的金纳米星材料形状为多支状星型，比表面积大，尺寸小，直径为50～80nm。According to the characterization results, the shape of the gold nano-star material prepared by the method of the invention is a multi-branched star shape, the specific surface area is large, the size is small, and the diameter is 50-80 nm.
本发明进一步提出了上述交联pH响应跨膜小肽的金纳米星材料在制备用于肿瘤光热治疗药物中的应用。The present invention further proposes the application of the above-mentioned gold nanostar material cross-linked with pH-responsive transmembrane small peptides in the preparation of drugs for photothermal therapy of tumors.
本发明中金纳米星采用改良的湿化学法合成，氨基-聚乙二醇-巯基首先与巯基化pHLIP交联，后修饰到金纳米星表面。交联小肽的金纳米星具有良好的生物相容性、水溶性、低毒性，且尺寸小，在生物环境中能保持较好的分散性，借助小肽在酸性环境下稳定插入细胞膜的性质，能提高肿瘤细胞对金纳米星的吞噬率，利用金纳米星良好的近红外光吸收和热转换的功能，杀死肿瘤细胞，提高治疗疗效。In the present invention, the gold nanostar is synthesized by an improved wet chemical method. The amino-polyethylene glycol-mercapto group is firstly cross-linked with the thiolated pHLIP, and then modified onto the surface of the gold nanostar. Gold nanostars cross-linked with small peptides have good biocompatibility, water solubility, low toxicity, and small size, and can maintain good dispersion in biological environments. With the help of small peptides, they can stably insert into cell membranes in acidic environments. , can increase the phagocytosis rate of tumor cells on gold nanostars, and use the good near-infrared light absorption and heat conversion functions of gold nanostars to kill tumor cells and improve the therapeutic effect.
本发明制备得到的新型pH响应的小肽pHLIP在肿瘤pH酸性环境下能快速折叠，并能迅速插入到肿瘤细胞膜磷脂双分子层中，形成稳定的跨膜α螺旋；同时协助交联的材料快速被肿瘤细胞识别产生胞吞作用，提高细胞对纳米材料的吞噬量。此小肽pHLIP安全、生物相容性好、性质稳定，与易于表面修饰的金纳米星交联，提高金纳米星的细胞吞噬量；金纳米星高效的光热治疗作用可以迅速杀死富集材料的肿瘤细胞，显著提高治疗疗效，实现靶向肿瘤微酸性环境的光热治疗。因pHLIP靶向作用受到肿瘤酸性环境的调节，因此，此携带小肽的金纳米星体系可以广泛用于其他临床肿瘤的光热治疗，具有重要的应用前景。The novel pH-responsive small peptide pHLIP prepared by the present invention can be rapidly folded in the acidic pH environment of the tumor, and can be quickly inserted into the phospholipid bilayer of the tumor cell membrane to form a stable transmembrane α-helix; It is recognized by tumor cells to produce endocytosis, which increases the phagocytosis of nanomaterials by cells. This small peptide pHLIP is safe, has good biocompatibility, and stable properties. It can be cross-linked with gold nanostars that are easy to modify the surface and increase the cell phagocytosis of gold nanostars; the efficient photothermal treatment of gold nanostars can quickly kill and enrich The tumor cells of the material can significantly improve the therapeutic efficacy and realize the photothermal therapy targeting the slightly acidic environment of the tumor. Because the targeting effect of pHLIP is regulated by the acidic environment of the tumor, this gold nanostar system carrying small peptides can be widely used in the photothermal therapy of other clinical tumors, and has important application prospects.
有益效果：本发明的交联具有pH响应的小肽的金纳米星材料，与现有技术相比，具有以下优点：Beneficial effects: Compared with the prior art, the cross-linked gold nanostar material with pH-responsive small peptides of the present invention has the following advantages:
1、该金纳米星材料在pH小于或等于6.4时可被细胞大量吞噬，在pH大于或等于7.4或不交联小肽时不易被细胞吞噬，这种性质可用于识别肿瘤特征性微酸环境；1. The gold nanostar material can be phagocytized by cells when the pH is less than or equal to 6.4, and it is not easy to be phagocytized by cells when the pH is greater than or equal to 7.4 or when there is no cross-linked small peptide. This property can be used to identify the characteristic slightly acidic environment of tumors ;
2、该金纳米星材料具有多支的星形结构，均一的尺寸，在酸性、中性及含蛋白的水溶液中均具有良好的生物相容性和分散性。2. The gold nanostar material has a multi-branched star structure and uniform size, and has good biocompatibility and dispersibility in acidic, neutral and protein-containing aqueous solutions.
3、该金纳米星材料具有近红外的吸收峰，其吸收光谱峰值在750～850nm之间，因此具备良好的近红外光热治疗作用。在近红外激光的照射下，能将光能转化成热能，迅速提高材料富集区的温度，杀死肿瘤细胞，降低材料未富集区的毒性，在肿瘤光热治疗方面具有重要的潜力。3. The gold nanostar material has a near-infrared absorption peak, and its absorption spectrum peak is between 750-850 nm, so it has a good near-infrared photothermal treatment effect. Under the irradiation of near-infrared laser, it can convert light energy into heat energy, rapidly increase the temperature of material-enriched area, kill tumor cells, and reduce the toxicity of material-unenriched area, which has important potential in tumor photothermal therapy.
附图说明Description of drawings
图1本发明在实施例1的工艺条件下制得的未交联小肽(左图)和交联小肽的金纳米星材料(右图)电子显微镜照片；Fig. 1 electron micrograph of the gold nanostar material (right figure) of uncrosslinked small peptide (left figure) and crosslinked small peptide prepared under the processing conditions of embodiment 1 of the present invention;
图2本发明在实施例1的工艺条件下制得的交联小肽及未交联小肽的金纳米星材料紫外-可见光吸收光谱；Fig. 2 the ultraviolet-visible light absorption spectrum of cross-linked small peptide and gold nanostar material of uncross-linked small peptide prepared under the processing conditions of embodiment 1 of the present invention;
图3本发明在实施例1的工艺条件下制得的交联小肽的具有pH响应的金纳米星材料在1W/cm2的808nm近红外激光照射下温度变化曲线；Fig. 3 is the cross-linked small peptide prepared under the process conditions of Example 1. The pH-responsive gold nanostar material has a temperature change curve under 1W/cm2 808nm near-infrared laser irradiation;
图4本发明在实施例1的工艺条件下制得的交联小肽的具有pH响应的金纳米星材料在pH 6.4和pH 7.4条件下的细胞吞噬定量分析；Fig. 4 Quantitative analysis of cell phagocytosis of the pH-responsive gold nanostar material of the cross-linked small peptide prepared under the process conditions of Example 1 under the conditions of pH 6.4 and pH 7.4;
图5在实施例1的工艺条件下制得的交联小肽的具有pH响应的金纳米星在pH 6.4条件下对细胞生长抑制作用；Fig. 5 inhibits cell growth under the conditions of pH 6.4 of the cross-linked small peptide prepared under the process conditions of Example 1 with pH-responsive gold nanostars;
图6在实施例1的工艺条件下制得的交联小肽的具有pH响应的金纳米星在pH 7.4条件下对细胞生长抑制作用。Fig. 6 The cross-linked small peptide prepared under the process conditions of Example 1 has a pH-responsive gold nanostar on cell growth inhibition at pH 7.4.
具体实施方式detailed description
本发明公开了一种交联pH响应跨膜小肽的金纳米星材料的制备方法，通过将柠檬酸钠金溶液加入氯金酸溶液，在室温搅拌下，向该混合液中同时加入硝酸银和抗坏血酸，充分搅拌后加入pHLIP和聚乙二醇的混合物。相对于金纳米球颗粒未附有银离子的部位，表面附有银离子的部位金还原的速度更快，因此这些部位生长的速度更快，最终形成分支状的金纳米星结构。之后加入聚乙二醇交联的pHLIP混合物，通过巯基-巯基，氨基-巯基的相互作用，形成稳定的纳米结构。聚乙二醇的作用既能增强小肽与金纳米星之间的连接，又能提高整体纳米材料的生物相容性。在pH酸性环境下，pHLIP小肽迅速插入细胞膜并形成牢固的跨膜α螺旋结构，所交联的材料能较多的分布在肿瘤细胞膜周围，促使细胞大量吞噬。进入到细胞内的金纳米星材料具有稳定的分散性，并保持良好的近红外光热转化效率，在近红外激光的刺激下，能迅速提高材料富集区的表面温度，杀死肿瘤细胞。最终获得具有pH响应的靶向肿瘤进行光热治疗的金纳米星材料。由于pHLIP在肿瘤酸性环境下能广泛插入到细胞膜，所以本发明的金纳米星材料还能应用于其他肿瘤的光热治疗，提高肿瘤的治疗疗效。The invention discloses a method for preparing a cross-linked pH-responsive transmembrane small peptide gold nanostar material. Adding sodium citrate gold solution to chloroauric acid solution and stirring at room temperature, silver nitrate is added to the mixed solution at the same time. and ascorbic acid, after stirring well, add the mixture of pHLIP and polyethylene glycol. Compared with the parts without silver ions attached to the gold nanosphere particles, the gold reduction speed of the parts with silver ions attached to the surface is faster, so these parts grow faster, and finally form a branched gold nanostar structure. Afterwards, the polyethylene glycol-crosslinked pHLIP mixture is added to form a stable nanostructure through the interaction of mercapto-mercapto and amino-mercapto groups. The role of polyethylene glycol can not only enhance the connection between small peptides and gold nanostars, but also improve the biocompatibility of the overall nanomaterial. In an acidic pH environment, the small pHLIP peptide quickly inserts into the cell membrane and forms a firm transmembrane α-helix structure, and the cross-linked material can be more distributed around the tumor cell membrane, prompting a large number of cells to phagocytize. The gold nanostar material entering the cell has stable dispersion and maintains good near-infrared photothermal conversion efficiency. Under the stimulation of near-infrared laser, it can rapidly increase the surface temperature of the material-enriched area and kill tumor cells. Finally, a pH-responsive gold nanostar material targeting tumors for photothermal therapy was obtained. Since the pHLIP can be widely inserted into the cell membrane in the acidic environment of the tumor, the gold nanostar material of the present invention can also be applied to the photothermal therapy of other tumors to improve the curative effect of the tumor.
下面通过具体的实施例并结合附图对本发明作更进一步的说明。The present invention will be further described below through specific embodiments in conjunction with the accompanying drawings.
在下述实施例中，马来酰亚胺-聚乙二醇修饰的小肽pHLIP由南京莱昂生物科技有限公司合成；氨基-聚乙二醇-巯基溶液由上海西宝生物科技有限公司合成；In the following examples, the maleimide-polyethylene glycol modified small peptide pHLIP was synthesized by Nanjing Leon Biotechnology Co., Ltd.; the amino-polyethylene glycol-sulfhydryl solution was synthesized by Shanghai Xibao Biotechnology Co., Ltd.;
实施例1：粒径为60nm左右的交联小肽的具有pH响应的金纳米星制备和鉴定。Example 1: Preparation and identification of pH-responsive gold nanostars with cross-linked small peptides with a particle size of about 60 nm.
(1)用水分别配0.02mL、2mM的马来酰亚胺-聚乙二醇修饰的小肽pHLIP，及0.02mL、2mM氨基-聚乙二醇-巯基，并将两化合物充分混合，摇匀。(1) Prepare 0.02mL, 2mM maleimide-polyethylene glycol-modified small peptide pHLIP, and 0.02mL, 2mM amino-polyethylene glycol-sulfhydryl with water, mix the two compounds well, and shake well .
(2)2.5mL 0.01M的氯金酸加入到97.5mL的水中，加热搅拌5min～10min，随后加入3mL 1％的柠檬酸三钠，溶液颜色由无色变成暗红色，停止加热和搅拌，制得柠檬酸钠金溶液。(2) Add 2.5mL of 0.01M chloroauric acid into 97.5mL of water, heat and stir for 5min to 10min, then add 3mL of 1% trisodium citrate, the color of the solution changes from colorless to dark red, stop heating and stirring, A sodium citrate gold solution was prepared.
(3)氯金酸(0.25mL，0.01M)加入到10mL水中，混匀后加入0.1mL(2)中制备的柠檬酸钠金溶液，然后同时加入0.05mL 0.1M还原剂抗坏血酸及0.04mL 0.01M硝酸银，溶液颜色由浅粉变成深蓝色，初步制备金纳米星材料。(3) Add chloroauric acid (0.25mL, 0.01M) to 10mL water, mix well, add 0.1mL sodium citrate gold solution prepared in (2), then add 0.05mL 0.1M reducing agent ascorbic acid and 0.04mL 0.01 M silver nitrate, the color of the solution changed from light pink to dark blue, and the gold nanostar material was preliminarily prepared.
(3)反应5分钟后，加入0.02mL步骤(1)所得的产物，室温搅拌1h，初步制备交联小肽的金纳米星材料。(3) After reacting for 5 minutes, add 0.02 mL of the product obtained in step (1) and stir at room temperature for 1 h to preliminarily prepare a cross-linked small peptide gold nanostar material.
(4)将步骤(2)(3)所得产物分别离心(相对离心力为7500～8000，离心10～15分钟)、洗涤3次、弃上清，将材料分散至1mL水中，形成未交联和交联pH响应跨膜小肽的金纳米星材料，如图1。(4) Centrifuge the products obtained in steps (2) and (3) respectively (relative centrifugal force is 7500-8000, and centrifuge for 10-15 minutes), wash 3 times, discard the supernatant, and disperse the material into 1 mL of water to form uncrosslinked and A gold nanostar material cross-linked with a pH-responsive transmembrane small peptide, as shown in Figure 1.
(5)测定步骤(4)所得的金纳米星材料的紫外-可见光吸收光谱，如图2所示。(5) Determination of the ultraviolet-visible light absorption spectrum of the gold nanostar material obtained in step (4), as shown in FIG. 2 .
(6)将步骤(4)所得的交联pH响应跨膜小肽的金纳米星材料置于1W/cm2的波长为808nm的近红外激光照射下，记录温度变化如图3所示。(6) The cross-linked pH-responsive transmembrane small peptide gold nanostar material obtained in step (4) was irradiated by a near-infrared laser with a wavelength of 808 nm at 1 W/cm2 , and the temperature change was recorded as shown in Figure 3 .
本实施例中得到的未交联和交联pH响应跨膜小肽的金纳米星材料，均具有多枝状星形分支，如图1透射电镜扫描分析所示其直径约50～70nm，如图2所示紫外-可见光吸收光谱峰值均在750～850nm左右，如图3所示在近红外激光照射下，交联pH响应跨膜小肽的金纳米星材料温度迅速升高。The uncrosslinked and crosslinked gold nanostar materials with pH-responsive transmembrane small peptides obtained in this example all have multi-branched star-shaped branches, as shown in Figure 1. The diameter is about 50-70nm as shown in the scanning analysis of the transmission electron microscope. The peaks of the ultraviolet-visible light absorption spectra shown in Figure 2 are all around 750-850 nm. As shown in Figure 3, under the near-infrared laser irradiation, the temperature of the cross-linked pH-responsive transmembrane small peptide gold nanostar material increases rapidly.
实施例2：pH 6.4及pH 7.4条件下的细胞吞噬定量分析Example 2: Quantitative analysis of cell phagocytosis under pH 6.4 and pH 7.4 conditions
(1)无菌条件下，配置pH 6.4的DMEM培养基，将950mL的DMEM水溶液培养基中先后加入4.93mL 7.5％NaHCO3溶液和44.37mL 5.2％NaCl溶液。之后加入10mL的青霉素/链霉素抗生素混合液，加入100mL的新鲜肽牛血清，经测定，获得pH 6.4的DMEM培养基。而pH 7.4的DMEM培养基，无需额外加入盐溶液，只加入10mL的青霉素/链霉素抗生素混合液和100mL的新鲜肽牛血清即可获得。(1) Under sterile conditions, prepare DMEM medium with pH 6.4, add 4.93mL 7.5% NaHCO3 solution and 44.37mL 5.2% NaCl solution to 950mL DMEM aqueous medium successively. Afterwards, 10 mL of penicillin/streptomycin antibiotic mixture was added, and 100 mL of fresh peptide bovine serum was added to obtain a DMEM medium with a pH of 6.4. The DMEM medium with pH 7.4 can be obtained by adding only 10 mL of penicillin/streptomycin antibiotic mixture and 100 mL of fresh peptide bovine serum without adding additional salt solution.
(2)细胞培养：乳腺癌细胞系MCF-7于pH 7.4的DMEM培养基中分裂生长，并放置在37℃，5％的CO2的细胞培养箱中培养。待细胞长到70％～80％的汇合度，分别更换新鲜pH6.4及pH 7.4的培养基，并将制得的未交联和交联pH响应跨膜小肽的金纳米星材料溶于培养基中，使得材料终浓度为2mg/L。(2) Cell culture: the breast cancer cell line MCF-7 was divided and grown in DMEM medium with pH 7.4, and cultured in a cell culture incubator at 37° C. and 5% CO2 . When the cells grow to a confluence of 70% to 80%, replace the medium with fresh pH 6.4 and pH 7.4 respectively, and dissolve the prepared uncrosslinked and crosslinked gold nanostar materials with pH-responsive transmembrane small peptides in In the culture medium, the final concentration of the material was 2 mg/L.
(3)待细胞与材料混合培养24h后，测定金纳米材料的细胞吞噬量：PBS缓冲液洗涤细胞，胰酶消化细胞后离心收集细胞，最终分散在3mL PBS中。取0.4mL的细胞PBS悬液，以每分钟950转速离心细胞5min，细胞沉淀中加入200μL的细胞裂解液，超声破碎、每分钟13000转速离心5min后，取上清测定蛋白浓度。剩余2.6mL细胞PBS悬液，再次离心后，向细胞沉淀中加入2mL的王水消化，在100℃的平台上蒸干，最终加入4mL 2％的稀硝酸定容，用ICP测定金浓度。金纳米星细胞吞噬量＝总金浓度/总蛋白浓度。如图4，测量得pH 6.4条件下，MCF-7细胞交联小肽的金纳米星吞噬量为0.67±0.12，未交联小肽的金纳米星的吞噬量为0.19±0.01；pH 7.4条件下，MCF-7细胞交联小肽的金纳米星吞噬量为0.38±0.09，未交联小肽的金纳米星的吞噬量为0.15±0.05。(3) After the cells and the material were mixed and cultured for 24 hours, the phagocytosis of the gold nanomaterials was measured: the cells were washed with PBS buffer, the cells were digested with trypsin and centrifuged to collect the cells, and finally dispersed in 3 mL of PBS. Take 0.4 mL of cell suspension in PBS, centrifuge the cells at 950 rpm for 5 min, add 200 μL of cell lysate to the cell pellet, ultrasonically break, and centrifuge at 13,000 rpm for 5 min, then take the supernatant to determine the protein concentration. The remaining 2.6 mL of cell PBS suspension was centrifuged again, and 2 mL of aqua regia was added to the cell pellet for digestion, evaporated to dryness on a platform at 100°C, and finally 4 mL of 2% dilute nitric acid was added to make up to volume, and the gold concentration was determined by ICP. Phagocytosis of gold nanostar cells = total gold concentration/total protein concentration. As shown in Figure 4, under the condition of pH 6.4, the phagocytosis of gold nanostars cross-linked with small peptides in MCF-7 cells was 0.67±0.12, and the phagocytosis of gold nanostars without cross-linked small peptides was 0.19±0.01; pH 7.4 conditions Under the same conditions, the phagocytosis of gold nanostars cross-linked with small peptides by MCF-7 cells was 0.38±0.09, and the phagocytosis of gold nanostars without cross-linked small peptides was 0.15±0.05.
实施例3：交联小肽的具有pH响应的金纳米星在pH 6.4条件下对细胞生长抑制作用鉴定。Example 3: Identification of pH-responsive gold nanostars cross-linked with small peptides on cell growth inhibition at pH 6.4.
将对数生长期的MCF-7细胞接种于96孔培养板，待细胞汇合度为80％左右后，每孔加入浓度为0mg/L、0.5mg/L、1mg/L、2mg/L及4mg/L的溶于pH 6.4的DMEM培养基中的交联小肽的金纳米星颗粒，终体积为100μL，设五个复孔，孵育4h后，弃上清，PBS洗涤3遍后，加入100μL新鲜培养基，用1W/cm2的808nm近红外激光照射6min，另设对照组不照射激光，之后每孔加入四甲基偶氮唑蓝(5mg/mL)20μL，继续培养4h，小心吸弃上清液，加二甲基亚砜(200μL/孔)，震荡10min后，检测A570光密度值。肿瘤细胞生存率＝(实验组或对照组平均光密度值/阴性对照组平均光密度值)*100％。MCF-7 cells in the logarithmic growth phase were inoculated in a 96-well culture plate. After the confluence of the cells was about 80%, the concentrations of 0mg/L, 0.5mg/L, 1mg/L, 2mg/L and 4mg were added to each well. /L cross-linked small peptide gold nanostar particles dissolved in DMEM medium with pH 6.4, the final volume is 100 μL, set up five duplicate wells, after incubation for 4 hours, discard the supernatant, wash with PBS 3 times, add 100 μL Fresh culture medium was irradiated with 1W/cm2 808nm near-infrared laser for 6min, another control group was not irradiated with laser, and then 20μL of tetramethylazolazolium blue (5mg/mL) was added to each well, continued to culture for 4h, carefully aspirate and discard Add dimethyl sulfoxide (200 μL/well) to the supernatant, shake for 10 min, and detect the A570 optical density value. Tumor cell survival rate=(average optical density value of experimental group or control group/average optical density value of negative control group)*100%.
本实施例中在pH 6.4条件下，交联pH响应小肽的金纳米星在没有近红外光照情况下，不引起细胞死亡；在酸性条件下，随着细胞吞噬材料增多，其光热作用增强，近红外激光照射下，肿瘤生长显著受到抑制。结果如图5所示。In this example, under the condition of pH 6.4, the cross-linked pH-responsive small peptide gold nanostars did not cause cell death in the absence of near-infrared light; under acidic conditions, as the cells phagocytized more materials, the photothermal effect was enhanced , tumor growth was significantly inhibited under near-infrared laser irradiation. The result is shown in Figure 5.
实施例4：交联小肽的具有pH响应的金纳米星在pH 7.4条件下对细胞生长抑制作用鉴定。Example 4: Identification of pH-responsive gold nanostars cross-linked with small peptides on cell growth inhibition at pH 7.4.
将对数生长期的MCF-7细胞接种于96孔培养板，待细胞汇合度为80％左右后，每孔加入浓度为0mg/L、0.5mg/L、1mg/L、2mg/L及4mg/L的溶于pH 7.4的DMEM培养基中的交联小肽的金纳米星颗粒，终体积为100μL，设五个复孔，孵育4h后，弃上清，PBS洗涤3遍后，加入100μL新鲜培养基，用1W/cm2的808nm近红外激光照射6min，另设对照组不照射激光，之后每孔加入四甲基偶氮唑蓝(5mg/mL)20μL，继续培养4h，小心吸弃上清液，加二甲基亚砜(200μL/孔)，震荡10min后，检测A570光密度值。肿瘤细胞生存率＝(实验组或对照组平均光密度值/阴性对照组平均光密度值)*100％。MCF-7 cells in the logarithmic growth phase were inoculated in a 96-well culture plate. After the confluence of the cells was about 80%, the concentrations of 0mg/L, 0.5mg/L, 1mg/L, 2mg/L and 4mg were added to each well. /L cross-linked small peptide gold nanostar particles dissolved in DMEM medium with pH 7.4, the final volume is 100 μL, set up five duplicate wells, after incubation for 4 hours, discard the supernatant, wash with PBS three times, add 100 μL Fresh culture medium was irradiated with 1W/cm2 808nm near-infrared laser for 6min, another control group was not irradiated with laser, and then 20μL of tetramethylazolazolium blue (5mg/mL) was added to each well, continued to culture for 4h, carefully aspirate and discard Add dimethyl sulfoxide (200 μL/well) to the supernatant, shake for 10 min, and detect the A570 optical density value. Tumor cell survival rate=(average optical density value of experimental group or control group/average optical density value of negative control group)*100%.
本实施例中在pH 7.4条件下，交联pH响应小肽的金纳米星在没有近红外光照情况下，不引起细胞死亡；在pH 7.4条件下，由于小肽的靶向肿瘤酸性环境的作用较弱，细胞吞噬材料相对较少，随着材料浓度的增加，近红外激光照射下，肿瘤生长会受到抑制，但治疗结果显著弱于在酸性条件下的治疗结果。结果如图6所示。In this example, under the condition of pH 7.4, the gold nanostars cross-linked with pH-responsive small peptides did not cause cell death in the absence of near-infrared light; Weaker, the cells phagocytized relatively less material, and with the increase of material concentration, tumor growth would be inhibited under near-infrared laser irradiation, but the treatment results were significantly weaker than those under acidic conditions. The result is shown in Figure 6.
本发明在室温条件下，利用简单的步骤合成了稳定、无毒、分散性好的交联pHLIP的金纳米星材料。pHLIP可以响应肿瘤的酸性环境，稳定插入到肿瘤细胞膜磷脂双分子层中，并协助金纳米星被肿瘤细胞识别，提高细胞吞噬量。金纳米星本身具有近红外光热治疗效应，可以利用安全高效的光热治疗手段，杀死肿瘤细胞，最终实现靶向肿瘤微酸性环境的光热治疗的应用。The present invention synthesizes a stable, non-toxic and well-dispersed cross-linked pHLIP gold nano-star material through simple steps at room temperature. pHLIP can respond to the acidic environment of the tumor, stably insert into the phospholipid bilayer of the tumor cell membrane, and assist gold nanostars to be recognized by tumor cells to increase cell phagocytosis. Gold nanostars themselves have near-infrared photothermal therapy effects, and can use safe and efficient photothermal therapy methods to kill tumor cells, and finally realize the application of photothermal therapy targeting the slightly acidic environment of tumors.
在本发明中，交联pH响应的跨膜小肽的金纳米星在紫外-可见光吸收光谱峰值在750～850nm之间，在近红外激光照射下能迅速提高材料摄取部位的温度。In the present invention, the cross-linked pH-responsive transmembrane small peptide gold nanostar has an ultraviolet-visible light absorption spectrum peak between 750 and 850 nm, and can rapidly increase the temperature of the material uptake site under near-infrared laser irradiation.
交联pH响应的跨膜小肽的金纳米星，在pH小于或等于6.4的情况下，小肽pHLIP构型发生改变，插入肿瘤细胞膜，可被肿瘤细胞大量吞噬，在近红外激光的照射下，迅速产生高热并显著抑制肿瘤细胞生长；在pH大于或等于7.4或没有交联小肽的情况下，不易被细胞吞噬或吞噬量较少，在近红外激光照射下，不会显著抑制肿瘤细胞的生长。Cross-linked pH-responsive transmembrane small peptide gold nanostars, when the pH is less than or equal to 6.4, the configuration of the small peptide pHLIP changes, inserts into the tumor cell membrane, and can be phagocytized by tumor cells in large quantities. Under the irradiation of near-infrared laser , quickly generate high heat and significantly inhibit the growth of tumor cells; when the pH is greater than or equal to 7.4 or without cross-linked small peptides, it is not easy to be phagocytized by cells or the amount of phagocytosis is small, and it will not significantly inhibit tumor cells under near-infrared laser irradiation growth.
以上所述仅是本发明的优选实施方式，应当指出：对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications are also possible. It should be regarded as the protection scope of the present invention.
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