CN114767850B - Cell membrane targeting nano probe and preparation thereof and application thereof in regulating and controlling neuron calcium ion flow through light response - Google Patents
Cell membrane targeting nano probe and preparation thereof and application thereof in regulating and controlling neuron calcium ion flow through light response Download PDFInfo
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Abstract
Description
技术领域Technical field
本发明属于医用材料技术领域，具体涉及一种细胞膜靶向纳米探针及其制备和在光响应调控神经元钙离子流动上的应用。The invention belongs to the technical field of medical materials, and specifically relates to a cell membrane-targeted nanoprobe, its preparation and its application in light-responsive regulation of calcium ion flow in neurons.
背景技术Background technique
随着纳米技术的快速发展，纳米探针的出现为调控细胞活动提供了一种更为精准的干预策略。其中，由于无创、无损、非入侵式等特点，将纳米探针结合光刺激来调控细胞生命活动受到了生命医学领域研究人员的关注。通过调节光的波长、刺激时间、刺激时长、刺激位置等参数，研究人员可以实现时空特异性干预细胞的生命活动，从而进一步调控体内生物化学变化、辅助疾病治疗。With the rapid development of nanotechnology, the emergence of nanoprobes provides a more precise intervention strategy for regulating cellular activities. Among them, due to the characteristics of non-invasive, non-destructive and non-invasive, combining nanoprobes with light stimulation to regulate cell life activities has attracted the attention of researchers in the field of life medicine. By adjusting parameters such as the wavelength of light, stimulation time, stimulation duration, and stimulation location, researchers can achieve spatiotemporal specific intervention in the life activities of cells, thereby further regulating biochemical changes in the body and assisting in disease treatment.
钙离子是人体的重要组成元素，在各项生理活动中都发挥着特殊作用，尤其是在脑活动中扮演了传递者的角色。当钙离子通过细胞膜内流或者外溢后，神经元便产生了神经冲动，即神经电信号。电信号的产生促使神经元分泌神经递质，并传给下一个神经元。当下一个神经元接受到递质后，又进一步产生钙离子流动，然后将电信号逐级传递。通过离子流动、信号传递的周而反复，人们才能够形成意识、情感并学会认知、学习、记忆等高级技能。当神经元钙离子流动出现异常时，就有可能出现认知困难、反应迟钝，甚至导致老年痴呆、癫痫等疾病发生。因此，在钙离子活动异常后，有必要对其流动进行人为干预，以达到预防或是治疗疾病的目的。Calcium ions are an important component of the human body and play a special role in various physiological activities, especially as a transmitter in brain activities. When calcium ions flow in or out through the cell membrane, neurons generate nerve impulses, that is, nerve electrical signals. The generation of electrical signals prompts neurons to secrete neurotransmitters, which are passed on to the next neuron. When the next neuron receives the transmitter, it further generates a flow of calcium ions, and then transmits the electrical signal step by step. Through repeated cycles of ion flow and signal transmission, people can form consciousness, emotions, and learn advanced skills such as cognition, learning, and memory. When the flow of calcium ions in neurons is abnormal, cognitive difficulties, slow response, and even Alzheimer's disease, epilepsy and other diseases may occur. Therefore, after abnormal calcium ion activity, it is necessary to artificially intervene in its flow to prevent or treat diseases.
目前，可以实现调控神经元钙离子流动的方案主要有两类，分别是药物治疗和电极刺激两种。其中，药物治疗是指服用或注射钙离子受体拮抗剂或是激动剂，以抑制钙离子的内流或是外溢，这种仅适用于广泛区域的钙离子流动异常，却无法在特定空间(脑区)进行干预。另一种电极刺激的方案虽然精准，但却需要破坏性地植入脑内进行刺激，这种入侵式的干预策略必然不适用于长期调控。基于上述缺陷，亟需一种安全、精准调控钙离子流动的干预策略，对于基础研究和临床应用均具有重要意义。Currently, there are two main types of solutions that can regulate the flow of calcium ions in neurons, namely drug treatment and electrode stimulation. Among them, drug treatment refers to taking or injecting calcium ion receptor antagonists or agonists to inhibit the inflow or outflow of calcium ions. This kind of abnormal calcium ion flow is only applicable to a wide area, but cannot be treated in a specific space ( brain area) to intervene. Another electrode stimulation solution, although accurate, requires destructive implantation into the brain for stimulation. This invasive intervention strategy is certainly not suitable for long-term control. Based on the above defects, an intervention strategy for safe and precise regulation of calcium ion flow is urgently needed, which is of great significance for both basic research and clinical application.
发明内容Contents of the invention
本发明的第一个目的在于针对现有钙离子流动调控技术上无法同时实现非破坏式调控、可逆调控、响应时间不足的缺陷，提供一种神经元细胞膜靶向纳米探针的制备方法。该纳米探针可响应组织深度高，灵敏度好，单分散性好，合成路径清晰，可重复性强，调控钙离子时操作简便，适用于不同种类神经元钙离子流动的调控及相关疾病的防治研究。The first object of the present invention is to provide a preparation method for neuron cell membrane-targeted nanoprobes in view of the shortcomings of existing calcium ion flow control technology that cannot simultaneously achieve non-destructive control, reversible control, and insufficient response time. The nanoprobe can respond to tissue depth, has good sensitivity, good monodispersity, clear synthesis path, strong repeatability, and is easy to operate when regulating calcium ions. It is suitable for regulating the flow of calcium ions in different types of neurons and preventing and treating related diseases. Research.
本发明涉及的纳米探针是采用分步法逐级制备。The nanoprobes involved in the present invention are prepared step by step using a step-by-step method.
步骤(1)：长度为30-70nm、直径为5-10nm、长径比不小于5的金纳米棒制备；Step (1): Preparation of gold nanorods with a length of 30-70nm, a diameter of 5-10nm, and an aspect ratio of not less than 5;
作为优选，步骤(1)具体是将十六烷基苯磺酸钠、氯酸金混合后加入硝酸银搅拌一段时间，然后调节pH至1-2后再加入还原剂氢醌水溶液，制备得到金纳米种子(goldnanoseeds)；将金纳米种子置于一定温度(4-60℃)水浴中反应15分钟以上，快速加入硼氢化钠溶液作为老化剂，促使金纳米种子纵向生长形成金纳米棒。Preferably, step (1) specifically involves mixing sodium cetylbenzene sulfonate and gold chlorate, adding silver nitrate and stirring for a period of time, then adjusting the pH to 1-2 and then adding the reducing agent hydroquinone aqueous solution to prepare gold. Nanoseeds (goldnanoseeds): Place the gold nanoseeds in a water bath at a certain temperature (4-60°C) to react for more than 15 minutes, and quickly add sodium borohydride solution as an aging agent to promote the longitudinal growth of the gold nanoseeds to form gold nanorods.
更为优选，步骤(1)具体是配制10mL浓度为0.01-0.2M十六烷基苯磺酸钠溶液作为稳定剂，加入500μL浓度为1-20mM氯酸金溶液快速搅拌，然后再加入20μL浓度为0.1-2M的硝酸银溶液搅拌，最后加入10-30μL浓度为1M的盐酸调节pH后加入500μL浓度为0.1-1M的氢醌水溶液作为还原剂，制备得到金纳米种子；将上述金纳米种子在一定温度(4-60℃)水浴中反应15分钟以上，快速加入硼氢化钠溶液作为老化剂，促使金纳米种子纵向生长形成金纳米棒。More preferably, step (1) is to prepare 10 mL of sodium cetylbenzene sulfonate solution with a concentration of 0.01-0.2M as a stabilizer, add 500 μL of a gold chlorate solution with a concentration of 1-20 mM, stir quickly, and then add 20 μL of a concentration of 1-20 mM gold chlorate solution. Stir a 0.1-2M silver nitrate solution, finally add 10-30 μL of 1M hydrochloric acid to adjust the pH, and then add 500 μL of a 0.1-1M hydroquinone aqueous solution as a reducing agent to prepare gold nanoseeds; add the above gold nanoseeds in React in a water bath at a certain temperature (4-60°C) for more than 15 minutes, and quickly add sodium borohydride solution as an aging agent to promote the longitudinal growth of gold nanoseeds to form gold nanorods.
步骤(2)：巯基聚乙二醇-抗辣椒素受体抗体(HS-PEG-Anti-TRPV-1)二聚体的制备。Step (2): Preparation of thiol polyethylene glycol-anti-capsaicin receptor antibody (HS-PEG-Anti-TRPV-1) dimer.
在弱碱性环境中，在巯基聚乙二醇琥珀酰胺酯(HS-PEG-NHS)溶液中加入Anti-TRPV-1，低温(<25℃)下反应2小时以上，透析袋除去未反应物，将剩余溶液冻干，得到HS-PEG-Anti-TRPV-1二聚体。In a weakly alkaline environment, add Anti-TRPV-1 to the mercaptopolyethylene glycol succinamide ester (HS-PEG-NHS) solution, react at low temperature (<25°C) for more than 2 hours, and remove unreacted matter in a dialysis bag. , the remaining solution was freeze-dried to obtain HS-PEG-Anti-TRPV-1 dimer.
作为优选，pH值在5-9；Preferably, the pH value is between 5-9;
作为优选，Anti-TRPV-1与HS-PEG-NHS的摩尔比为1:10-10:1；Preferably, the molar ratio of Anti-TRPV-1 to HS-PEG-NHS is 1:10-10:1;
步骤(3)：HS-PEG-Anti-TRPV-1二聚体的修饰。Step (3): Modification of HS-PEG-Anti-TRPV-1 dimer.
在水相环境体系中，将金纳米棒和HS-PEG-Anti-TRPV-1二聚体混匀，利用具有高度亲和性Au-S键，得到HS-PEG-Anti-TRPV-1二聚体修饰的金纳米棒，即纳米探针的粗产品。通过离心水洗后，可除去未反应物，得到提纯后的纳米探针。In an aqueous environment system, the gold nanorods and HS-PEG-Anti-TRPV-1 dimer are mixed, and the high affinity Au-S bond is used to obtain the HS-PEG-Anti-TRPV-1 dimer. Body-modified gold nanorods, the crude product of nanoprobes. After centrifugation and water washing, unreacted substances can be removed to obtain the purified nanoprobe.
作为优选，金纳米棒和HS-PEG-Anti-TRPV-1二聚体的摩尔比为1:10-10:1。Preferably, the molar ratio of gold nanorods and HS-PEG-Anti-TRPV-1 dimer is 1:10-10:1.
本发明的第二个目的是提供一种神经元细胞膜靶向纳米探针，采用以上方法制备得到。The second object of the present invention is to provide a neuron cell membrane-targeted nanoprobe prepared by the above method.
本发明的第三个目的是提供一种神经元细胞膜靶向纳米探针在光响应调控神经元钙离子流动上的应用。The third object of the present invention is to provide an application of a neuron cell membrane-targeted nanoprobe in light-responsive regulation of calcium ion flow in neurons.
本发明的第四个目的是一种神经元细胞膜靶向纳米探针在用于干预或治疗与钙离子流动失衡相关的疾病试剂盒上的应用。The fourth object of the present invention is the application of a neuron cell membrane-targeted nanoprobe in a kit for intervening or treating diseases related to calcium ion flow imbalance.
本发明的第五个目的是一种用于干预或治疗与钙离子流动失衡相关的疾病试剂盒，包括上述神经元细胞膜靶向纳米探针。The fifth object of the present invention is a kit for intervening or treating diseases related to calcium ion flow imbalance, including the above-mentioned neuron cell membrane-targeted nanoprobe.
本发明的有益效果具体是：The beneficial effects of the present invention are specifically:
1)本发明采用以小尺寸、高长径比的金纳米棒为主体结构，以HS-PEG-NHS和Anti-TRPV-1抗体为靶向修饰材料，组装后得到的探针可实现非入侵、非破坏式的远程式安全探针。1) The present invention uses gold nanorods with small size and high aspect ratio as the main structure, and uses HS-PEG-NHS and Anti-TRPV-1 antibodies as targeted modification materials. The probe obtained after assembly can achieve non-invasiveness , non-destructive remote security probe.
2)本发明提出以小尺寸、高长径比的金纳米棒为主体结构，并引入Anti-TRPV-1抗体的修饰提高了纳米探针的细胞膜靶向特性，可将纳米探针结合到神经元膜表面的TRPV-1抗体上。TRPV-1对热刺激敏感的特征与金纳米棒对近红外光刺激的光热转化不谋而合，起到了协同作用，在金纳米棒的热转化下，可快速刺激TRPV-1通道的开关，进而实现了可逆调控神经元钙离子运动。2) The present invention proposes to use small-sized, high-aspect-ratio gold nanorods as the main structure, and introduce modifications with Anti-TRPV-1 antibodies to improve the cell membrane targeting properties of the nanoprobes, and can bind the nanoprobes to nerves. TRPV-1 antibodies on the surface of the membrane. The sensitivity of TRPV-1 to thermal stimulation coincides with the photothermal conversion of gold nanorods to near-infrared light stimulation, which plays a synergistic effect. The thermal conversion of gold nanorods can quickly stimulate the switching of TRPV-1 channels. , thereby achieving reversible regulation of neuronal calcium ion movement.
3)本发明采用Anti-TRPV-1将纳米材料精准靶向至TRPV-1附近，实现原位近距离调控神经元，具有时空特异性，反应灵敏，响应快。3) The present invention uses Anti-TRPV-1 to accurately target nanomaterials near TRPV-1 to achieve in-situ close-range regulation of neurons, with spatiotemporal specificity, sensitive response, and fast response.
4)本发明采用合适尺寸和长径比的金纳米棒，进而实现纳米探针的合适吸收光谱，从而直接影响纳米探针在生物脑内可响应的组织深度。4) The present invention uses gold nanorods of appropriate size and aspect ratio to achieve a suitable absorption spectrum of the nanoprobe, thereby directly affecting the tissue depth to which the nanoprobe can respond in the biological brain.
5)本发明采用的HS-PEG-NHS聚合物中PEG基团的引入则进一步提高了纳米探针的单分散性，防治纳米探针产生聚集，导致钙离子调控失败。5) The introduction of PEG groups into the HS-PEG-NHS polymer used in the present invention further improves the monodispersity of the nanoprobes and prevents the aggregation of the nanoprobes, resulting in failure of calcium ion regulation.
综上，本发明采用特制纳米探针实现光调控神经元钙离子流动，操作简单，思路明确，所得纳米探针结构组分明确、功能性质清晰，三者材料之间充分发挥了协同作用。通过进一步在神经元细胞和离体脑片上的研究验证，本发明提供了一种用于远程光响应调控钙离子流动的高灵敏度、时空特异性和可重复性的纳米探针。In summary, the present invention uses a special nanoprobe to achieve light regulation of calcium ion flow in neurons. The operation is simple and the idea is clear. The resulting nanoprobe has clear structural components and clear functional properties, and the synergy between the three materials is fully exerted. Through further research and verification on neuronal cells and isolated brain slices, the present invention provides a highly sensitive, spatiotemporally specific and reproducible nanoprobe for regulating calcium ion flow with long-range light response.
附图说明Description of drawings
图1.a)不同长径比的小尺寸金纳米棒溶液照片及其对应最大吸收波长。b)不同长径比的小尺寸金纳米棒的可见-近红外吸收光谱。Figure 1.a) Photos of solutions of small-sized gold nanorods with different aspect ratios and their corresponding maximum absorption wavelengths. b) Visible-near infrared absorption spectra of small-sized gold nanorods with different aspect ratios.
图2.不同长径比纳米探针溶液的透射电子显微镜照片。Figure 2. Transmission electron microscope images of nanoprobe solutions with different aspect ratios.
图3.金纳米棒(sAR)、PEG修饰的金纳米棒(sAR-PEG)、纳米探针(sAR-PEG-AA)的表面电荷分析。Figure 3. Surface charge analysis of gold nanorods (sAR), PEG-modified gold nanorods (sAR-PEG), and nanoprobes (sAR-PEG-AA).
图4.纳米探针的细胞膜靶向作用研究(预先将纳米探针进行红色荧光标记)。比例尺：20μm。Figure 4. Study on the cell membrane targeting effect of nanoprobes (the nanoprobes are labeled with red fluorescence in advance). Scale bar: 20 μm.
图5.近红外光刺激对金纳米棒(未经靶向修饰)孵育的神经细胞(同一区域)钙离子流动情况的影响(绿色荧光为钙离子)。比例尺：150μm。Figure 5. Effect of near-infrared light stimulation on calcium ion flow in nerve cells (same area) incubated with gold nanorods (without targeted modification) (green fluorescence is calcium ions). Scale bar: 150 μm.
图6.近红外光刺激对纳米探针孵育的神经细胞(同一区域)钙离子流动情况的影响(绿色荧光为钙离子)。比例尺：150μm。Figure 6. Effect of near-infrared light stimulation on calcium ion flow in nerve cells (same area) incubated with nanoprobes (green fluorescence is calcium ions). Scale bar: 150 μm.
图7.近红外光刺激对纳米探针孵育的离体脑片放电情况影响。红色线区域为给光刺激时间段。Figure 7. Effect of near-infrared light stimulation on the discharge of isolated brain slices incubated with nanoprobes. The red line area is the time period of light stimulation.
具体实施方式Detailed ways
如前所述，鉴于现有技术的不足，本案发明人经长期研究和大量实践，提出了本发明的技术方案，其主要是依据至少包括：1)本发明以小尺寸、高长径比的金纳米棒为光响应材料，结合抗辣椒素受体抗体，利用巯基聚乙二醇琥珀酰胺酯进行生物偶联，设计一种用于调控神经元钙离子流动的纳米探针。选取合适尺寸和长径比的金纳米棒实现本发明所需的脑内递送需求和更为深部的光热转化，对于脑内调控至关重要。2)Anti-TRPV-1可与神经元表面辣椒素受体(TRPV-1)精准结合，将纳米材料精准靶向至TRPV-1附近。当纳米材料产生特定刺激时，例如局部升温、机械力干预，TRVP-1作为通道蛋白则会打开，引导钙离子快速内流，其内流速度与刺激强度直接相关。3)HS-PEG-NHS作为一种功能聚合物，在本发明中不仅起到连接的作用，同时可以与生命环境中的水分子形成氢键，防止纳米材料的聚集，以延长调控时间窗。4)通过将金纳米棒、Anti-TRPV-1和HS-PEG-NHS逐级反应，得到的纳米探针对近红外光具有高响应性，具有精准干预神经元钙离子的作用，充分发挥了不同材料单元之间的协同作用。本发明所制的纳米探针在近红外二区附近具有极强的吸收，可以实现深部组织下的调控；尺寸小，有利于穿透血脑屏障；单分散性好，有助于快速、精准靶向。通过本发明所制的纳米探针，可用于安全、精准调控神经元钙离子流动，从而实现特定生化反应过程的干预与钙离子流动失衡相关的疾病治疗。As mentioned above, in view of the shortcomings of the existing technology, the inventor of the present case proposed the technical solution of the present invention after long-term research and extensive practice, which is mainly based on at least: 1) the present invention uses small size and high aspect ratio. Gold nanorods are light-responsive materials, combined with anti-capsaicin receptor antibodies, and bioconjugated using mercapto polyethylene glycol succinamide ester to design a nanoprobe for regulating the flow of calcium ions in neurons. Selecting gold nanorods of appropriate size and aspect ratio to achieve the intracerebral delivery requirements and deeper photothermal conversion required by the present invention is crucial for intracerebral regulation. 2) Anti-TRPV-1 can accurately bind to the capsaicin receptor (TRPV-1) on the surface of neurons and accurately target nanomaterials to the vicinity of TRPV-1. When nanomaterials produce specific stimuli, such as local heating or mechanical intervention, TRVP-1, as a channel protein, will open and guide the rapid influx of calcium ions. Its inflow speed is directly related to the intensity of the stimulus. 3) As a functional polymer, HS-PEG-NHS not only plays a connecting role in the present invention, but also can form hydrogen bonds with water molecules in the living environment to prevent the aggregation of nanomaterials and extend the control time window. 4) By reacting gold nanorods, Anti-TRPV-1 and HS-PEG-NHS step by step, the nanoprobe obtained is highly responsive to near-infrared light and has the function of accurately interfering with neuronal calcium ions, giving full play to its Synergy between different material units. The nanoprobe prepared by the present invention has extremely strong absorption near the second near-infrared region and can realize regulation under deep tissues; it is small in size and is conducive to penetrating the blood-brain barrier; and it has good monodispersity and is conducive to rapid and accurate operation. Targeting. The nanoprobe prepared by the present invention can be used to safely and accurately regulate the flow of calcium ions in neurons, thereby achieving intervention in specific biochemical reaction processes and treatment of diseases related to calcium ion flow imbalance.
实施例1：Example 1:
1)长70nm、直径9nm、长径比为8.3、最大吸收波长为1129nm金纳米棒的制备。1) Preparation of gold nanorods with a length of 70nm, a diameter of 9nm, an aspect ratio of 8.3, and a maximum absorption wavelength of 1129nm.
配制10mL浓度为0.1M十六烷基苯磺酸钠溶液作为稳定剂，加入新制备的500μL浓度为10mM氯酸金溶液作为原料快速搅拌，加入20μL浓度为0.1M的硝酸银溶液搅拌，然后加入20μL浓度为1M的盐酸调节pH，最后加入500μL浓度为0.1M的氢醌水溶液作为还原剂，制备金纳米种子(goldnanoseeds)。在30℃水浴中反应15分钟，快速加入10μL浓度为10mM硼氢化钠溶液作为老化剂，促使金纳米种子纵向生长形成金纳米棒，所得金纳米棒长70nm、宽9nm、长径比为8.3，最大吸收波长为1129nm的近红外二区。Prepare 10 mL of 0.1 M sodium cetylbenzene sulfonate solution as a stabilizer, add 500 μL of newly prepared 10 mM gold chlorate solution as raw material, stir quickly, add 20 μL of 0.1 M silver nitrate solution, stir, and then add 20 μL of hydrochloric acid with a concentration of 1 M was used to adjust the pH, and finally 500 μL of a hydroquinone aqueous solution with a concentration of 0.1 M was added as a reducing agent to prepare gold nanoseeds. React in a 30°C water bath for 15 minutes, and quickly add 10 μL of 10mM sodium borohydride solution as an aging agent to promote the longitudinal growth of gold nanoseeds to form gold nanorods. The resulting gold nanorods are 70nm long, 9nm wide, and have an aspect ratio of 8.3. The maximum absorption wavelength is the near-infrared second region of 1129nm.
2)HS-PEG-Anti-TRPV-1二聚体的制备。2) Preparation of HS-PEG-Anti-TRPV-1 dimer.
在弱碱性环境中，配制5mg/mL的HS-PEG-NHS溶液，其中HS-PEG-NHS分子量约为5000Da，加入与HS-PEG-NHS等摩尔量的Anti-TRPV-1，4℃反应12小时，透析袋除去未反应物，将剩余溶液冻干，得到HS-PEG-Anti-TRPV-1二聚体。In a weakly alkaline environment, prepare a 5 mg/mL HS-PEG-NHS solution, in which the molecular weight of HS-PEG-NHS is about 5000 Da. Add an equal molar amount of Anti-TRPV-1 to HS-PEG-NHS and react at 4°C. After 12 hours, unreacted materials were removed from the dialysis bag, and the remaining solution was freeze-dried to obtain HS-PEG-Anti-TRPV-1 dimer.
3)HS-PEG-Anti-TRPV-1二聚体的修饰。3) Modification of HS-PEG-Anti-TRPV-1 dimer.
在水相环境体系中，将金纳米棒和HS-PEG-Anti-TRPV-1二聚体以一定摩尔比(1:1)混匀，搅拌反应12小时，利用具有高度亲和性Au-S键，得到HS-PEG-Anti-TRPV-1二聚体修饰的金纳米棒，即纳米探针的粗产品。通过12000rpm超高速离心并用超纯水水洗2次后，可除去未反应物，得到提纯后的纳米探针。In an aqueous environment system, the gold nanorods and HS-PEG-Anti-TRPV-1 dimer were mixed at a certain molar ratio (1:1), stirred and reacted for 12 hours, using the highly affinity Au-S bond to obtain HS-PEG-Anti-TRPV-1 dimer-modified gold nanorods, which is the crude product of the nanoprobe. After ultra-high-speed centrifugation at 12,000 rpm and washing twice with ultrapure water, unreacted substances can be removed to obtain the purified nanoprobe.
4)纳米探针调控神经细胞钙离子内流。4) Nanoprobes regulate calcium ion influx in nerve cells.
将纳米探针以0.1mg/mL的浓度与ND7/23神经细胞共孵育，2小时即可快速靶向富集至神经细胞膜表面。对神经细胞给予近红外二区光刺激，刺激时长为5分钟，刺激强度为0.3W/cm2，通过共聚焦显微镜观察，确认神经细胞钙离子快速内流，实现光响应调控钙离子流动。The nanoprobes were incubated with ND7/23 nerve cells at a concentration of 0.1 mg/mL, and they could be rapidly targeted and enriched on the surface of nerve cell membranes in 2 hours. Nerve cells were given near-infrared second-zone light stimulation, with a stimulation duration of 5 minutes and a stimulation intensity of 0.3W/cm 2 . Through confocal microscopy observation, it was confirmed that calcium ions in the nerve cells rapidly flowed in and the calcium ion flow was regulated by light response.
如图1所示，本发明所提出的方案可制备900-1200nm近红外二区吸收的金纳米棒，所得金纳米棒溶液的颜色与其吸收光谱直接相关。As shown in Figure 1, the solution proposed by the present invention can prepare gold nanorods with near-infrared second-band absorption of 900-1200 nm. The color of the obtained gold nanorod solution is directly related to its absorption spectrum.
如图2所示，本发明所提出的方案可制备不同长径比的纳米探针。As shown in Figure 2, the solution proposed by the present invention can prepare nanoprobes with different aspect ratios.
如图3所示，本发明所制备金纳米棒(sAR)、PEG修饰的金纳米棒(sAR-PEG)、Anti-TRPV-1-PEG修饰的金纳米棒(sAR-PEG-AA)具有不同表明电荷，证明了纳米探针的制备。As shown in Figure 3, the gold nanorods (sAR), PEG-modified gold nanorods (sAR-PEG), and Anti-TRPV-1-PEG-modified gold nanorods (sAR-PEG-AA) prepared by the present invention have different properties. Indicates the charge, demonstrating the preparation of the nanoprobe.
如图4所示，本发明所制备的纳米探针可以快速富集到神经细胞表面，未经靶向修饰的金纳米棒不具有细胞膜靶向作用。As shown in Figure 4, the nanoprobes prepared in the present invention can be quickly enriched on the surface of nerve cells, and gold nanorods without targeted modification have no cell membrane targeting effect.
如图5、图6所示，在相同光刺激下，未经靶向修饰的金纳米棒(sAR)无法调控钙离子流动，而纳米探针(sAR-PEG-AA)可以促进神经细胞钙离子内流，且刺激强度越高，内流越明显。As shown in Figure 5 and Figure 6, under the same light stimulation, gold nanorods (sAR) without target modification cannot regulate the flow of calcium ions, while the nanoprobe (sAR-PEG-AA) can promote calcium ions in nerve cells. Inward flow, and the higher the stimulation intensity, the more obvious the inward flow.
实施例2：Example 2:
1)长35nm、宽7nm、长径比为5、最大吸收波长为953nm金纳米棒的制备。1) Preparation of gold nanorods with a length of 35 nm, a width of 7 nm, an aspect ratio of 5, and a maximum absorption wavelength of 953 nm.
配制10mL浓度为0.1M十六烷基苯磺酸钠溶液作为稳定剂，加入新制备的500μL浓度为10mM氯酸金溶液作为原料快速搅拌，加入20μL浓度为0.1M的硝酸银溶液搅拌，然后加入10μL浓度为1M的盐酸调节pH，最后加入500μL浓度为0.1M的氢醌水溶液作为还原剂，制备金纳米种子。在30℃水浴中反应15分钟，快速加入10μL浓度为10mM硼氢化钠溶液作为老化剂，促使金纳米种子纵向生长形成金纳米棒，所得金纳米棒长35nm、宽7nm、长径比为5，最大吸收波长为953nm。Prepare 10 mL of 0.1 M sodium cetylbenzene sulfonate solution as a stabilizer, add 500 μL of newly prepared 10 mM gold chlorate solution as raw material, stir quickly, add 20 μL of 0.1 M silver nitrate solution, stir, and then add 10 μL of hydrochloric acid with a concentration of 1 M was used to adjust the pH, and finally 500 μL of a hydroquinone aqueous solution with a concentration of 0.1 M was added as a reducing agent to prepare gold nanoseeds. React in a 30°C water bath for 15 minutes, and quickly add 10 μL of 10mM sodium borohydride solution as an aging agent to promote the longitudinal growth of gold nanoseeds to form gold nanorods. The resulting gold nanorods are 35nm long, 7nm wide, and have an aspect ratio of 5. The maximum absorption wavelength is 953nm.
2)HS-PEG-Anti-TRPV-1二聚体的制备。2) Preparation of HS-PEG-Anti-TRPV-1 dimer.
在弱碱性环境中，配制0.1-10mg/mL的HS-PEG-NHS溶液，其中HS-PEG-NHS分子量约为2000Da，加入一定质量的Anti-TRPV-1(与HS-PEG-NHS的摩尔比为1:2)，4℃反应12小时，透析袋除去未反应物，将剩余溶液冻干，得到HS-PEG-Anti-TRPV-1二聚体。In a weakly alkaline environment, prepare a 0.1-10mg/mL HS-PEG-NHS solution, in which the molecular weight of HS-PEG-NHS is about 2000Da, and add a certain mass of Anti-TRPV-1 (the molar ratio of HS-PEG-NHS Ratio is 1:2), react at 4°C for 12 hours, remove unreacted materials in a dialysis bag, and freeze-dry the remaining solution to obtain HS-PEG-Anti-TRPV-1 dimer.
3)HS-PEG-Anti-TRPV-1二聚体的修饰。3) Modification of HS-PEG-Anti-TRPV-1 dimer.
在水相环境体系中，将金纳米棒和HS-PEG-Anti-TRPV-1二聚体以一定摩尔比(1:1)混匀，搅拌反应12小时，利用具有高度亲和性Au-S键，得到HS-PEG-Anti-TRPV-1二聚体修饰的金纳米棒，即纳米探针的粗产品。通过12000rpm超高速离心并用超纯水水洗2次后，可除去未反应物，得到提纯后的纳米探针。In an aqueous environment system, the gold nanorods and HS-PEG-Anti-TRPV-1 dimer were mixed at a certain molar ratio (1:1), stirred and reacted for 12 hours, using the highly affinity Au-S bond to obtain HS-PEG-Anti-TRPV-1 dimer-modified gold nanorods, which is the crude product of the nanoprobe. After ultra-high-speed centrifugation at 12,000 rpm and washing twice with ultrapure water, unreacted substances can be removed to obtain the purified nanoprobe.
4)纳米探针调控神经元释放电信号。4) Nanoprobes regulate the release of electrical signals from neurons.
将纳米探针以0.1mg/mL的浓度与ICR幼鼠脑片共孵育，对脑片海马区域给予近红外光刺激，刺激强度为0.4W/cm2，利用电生理技术实时记录神经元放电情况。研究确认在光刺激下，纳米探针可以实时响应激活脑片，促使神经元放电。The nanoprobe was incubated with the brain slices of ICR young mice at a concentration of 0.1mg/mL, and near-infrared light stimulation was given to the hippocampus area of the brain slices with a stimulation intensity of 0.4W/cm 2 , and the neuronal discharge was recorded in real time using electrophysiological technology. . Research has confirmed that under light stimulation, nanoprobes can activate brain slices in real-time and prompt neurons to discharge.
如图7所示，给予近红外光刺激后，经纳米探针处理的神经元可以快速(20秒内)响应，释放电信号，而未处理的神经元对光刺激不响应。As shown in Figure 7, after being stimulated by near-infrared light, the neurons treated with the nanoprobe can respond quickly (within 20 seconds) and release electrical signals, while the untreated neurons do not respond to light stimulation.
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| WO2013029025A1 (en) * | 2011-08-24 | 2013-02-28 | The Rockefeller University | Compositions and methods to modulate cell activity |
| CN112620646A (en) * | 2020-12-17 | 2021-04-09 | 上海纳米技术及应用国家工程研究中心有限公司 | Preparation method of large length-diameter ratio gold nanorod with platinum particles growing at two ends and product thereof |
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