这项工作是部分由一线学术研究经费由新加坡教育部（RG十二分之六十四到CX）和纳米医学台大研究所西北支持。

1.准备<ol><li>干净的100毫升园使用王水（浓硝酸1份（HNO 3）和3份浓盐酸（HCl）的）中，用DI水冲洗烧瓶底部的烧瓶中。高压灭菌的烧瓶中并在100℃干燥它们在热空气烘箱中15分钟。换并存储无菌瓶中直至使用。 </li><li>消毒用70％的乙醇手持微型挤出集。 </li><li>打开旋转蒸发器和分别在37℃和4℃设置热水浴中冷却塔的温度。 </li><li>由374毫克的钙黄绿素溶解在0.1毫10毫升磷酸盐缓冲盐水（PBS）（pH 7.4）中制备60mM的钙黄绿素储备溶液。调整使用1M氢氧化钠（NaOH）溶液的pH至7.4。 </li></ol> 2.脂质体的合成<ol><li>除去脂质（1,2- Dipalmitoyl- SN -glycero -3-磷酸胆碱（DPPC），1-棕榈酰-2-羟基- SN -glycero -3- phosphocho线（MPPC）和1，2- distearoyl- SN -glycero -3- phosphoethanol-胺-N - [羧基（聚乙二醇）-2000]（铵盐）（DSPE-PEG2000））从冷冻机（-20℃）和它们解冻至室温。 </li><li>称重15.9毫克DPPC，1.3毫克MPPC，和2.8毫克DSPE-PEG2000。在2ml氯仿溶解在一起。 </li><li>转移氯仿溶液至无菌圆底烧瓶中，并用减压旋转蒸发器，以形成一个薄的，干的脂质层蒸发溶剂。 </li><li>水合在45℃用2ml水溶液脂质层含有1.95毫升60毫钙黄绿素在步骤1.4和50微升金纳米粒子（3.36×10 16个粒子/ ml）中制备30分钟。 </li><li>预加热加热块至45℃。放置过滤器支撑件之间的200纳米的聚碳酸酯膜过滤器，并装配微型挤出集。检查用DI水的潜在泄漏。 </li><li>填用1ml脂质体溶液的注射器中的一个来自步骤2.4和extru德通过将溶液的注射器在装配微型挤出机的另一端的样品。重复11次。 </li><li>通过将PD-10脱盐柱运行合成脂质体以除去游离的金纳米粒子，脂质和钙黄绿素使用PBS作为根据制造商的协议洗脱剂。 </li><li>存储在无菌试管中的样品在4℃和在2天内使用。 </li></ol> 3.从黄绿素脂质体释放暖气<ol><li>通过在步骤2.2相加DPPC，MPPC，和DSPE-PEG2000的摩尔浓度计算原液的脂质摩尔浓度。稀释脂质储备溶液用0.1毫的PBS缓冲液（pH 7.4）的5mM脂质浓度。转移样品到离心管（2毫升）中。 </li><li>放置在管中的热水浴中，逐步提高温度从25到70℃。提高温度以1℃/分钟此处的速率。 </li><li>在不同的温度点（27，32，37，39，41等分收集（10微升），43，45，52，57，62，67和70°C）。 </li><li>添加10微升的脂质体溶液2％的Triton X-100至2ml等分试样的消化脂质体在室温下10分钟，以实现钙黄绿素的完全释放。 </li><li>转移200微升脂质体溶液，以每孔在96孔微量并测量使用荧光酶标仪所收集样品的荧光强度。钙黄绿素的激发和发射波长分别为480和515纳米。 </li><li>服用的Triton X-100处理的样品为100％的释放的荧光强度，计算在每个时间点所释放的钙黄绿素的百分比，利用下式: <img alt="公式1" src="/files/ftp_upload/53619/53619formula1.jpg" /> Ft是在给定时间点的溶液的荧光强度。 的 F i和F x分别溶液分别归一化的初始和最终的荧光强度。 </li></ol> 4.重新钙黄绿从脂质体与脉冲激光租赁<ol><li>转移100微升脂质体溶液到石英比色皿，并将其放置在一个试管支架。使用脉冲Nd:YAG激光在波长532nm作为光源的6毫微秒的脉冲持续时间。使用下面的参数激光 - 重复率:1赫兹;激光的能量密度:1毫焦/厘米2;光束直径:0.5毫米。 </li><li>通过试管引导准直激光束，使得光穿过脂质体溶液和各种脉冲之后收集等分试样。 </li><li>添加10微升的脂质体溶液2％的Triton X-100至2ml等分试样的消化脂质体在室温下10分钟，以实现钙黄绿素的完全释放。 </li><li>考虑钙黄绿素是对光敏感并且激光实验期间可能被漂白，预先测量脉冲激光对钙黄绿素溶液的漂白效果，从脂质体释放正常化的数据。 <ol><li>具体来说，暴露钙黄绿素溶液（60毫米），脉冲激光无线1Hz的个频率用于改变脉冲数（0，25，50和100）。测量分别为480和515纳米的激发和发射波长钙黄绿素，前，激光照射后的荧光强度。 </li><li>计算漂白的量（具体脉冲数后的钙黄绿素的激光曝光/荧光强度前的钙黄绿素的荧光强度）。使用该因子通过与因子的脂质体的荧光强度乘以正常化从脂质体的样品得到的值。 </li></ol></li><li>测量分别利用在激发和发射波长的荧光酶标仪在480和515纳米的所收集样品的荧光强度。 </li><li>服用的Triton X-100处理的样品为100％的释放的荧光强度，计算每个脉冲数所释放的钙黄绿素的百分比，利用下式: <img alt="公式1" src="/files/ftp_upload/53619/53619formula1.jpg" /> 的 F i和F x分别溶液分别归一化的初始和最终的荧光强度。 </li></ol> 5.压力脉冲测量<ol><li>放置在显微镜载玻片100微升样品，并设置激光的焦点到样品。 </li><li>浸入针水听器（直径为1毫米和450纳伏/帕灵敏度）到溶液中。 注意:水听器必须不被激光光照射，以避免损坏。 </li><li>照射与脉冲激光具有不同的脉冲数（0-100）和脉冲能量（20-160μJ/脉冲）的样品。 </li><li>记录用数字示波器中的压力的​​信号。 </li></ol>

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没有利益冲突的声明。

薄膜水化是用于制备脂质体的常规方法。有机溶剂（在这种情况下，氯仿）首先用于溶解脂质，然后取出在旋转蒸发器于37℃，以产生在烧瓶的脂质薄膜。此脂质膜用含有60mM的钙黄绿素和5纳米金纳米粒子水溶液水合。在水合过程中，将温度维持在大约50℃，将该烧瓶不断受到烧瓶旋转搅拌。该步骤中的关键是在其中的蒸发和水合分别进行温度的选择。 DPPC，脂质体的主要成分，所述的相变温度（T m）为 41℃。在脂质的形成认为薄膜时，温度应低于41℃，以防止干燥脂类聚集团块的形成。在水化，被选择更高的温度比Tm值来驱动磷脂自组装成球形多层结构。虽然薄膜水化方法容易进行，它是通过在水溶液中的钙黄绿素和金纳米粒子的包封率差（&lt;1％）的限制。在未来，封装可能通过冻融得到改善。 随后大小挤出用手持小型挤出机，置于加热块上完成的。最多1毫升溶液可使用这种设置，这是理想的，在实验室小规模制备挤出。在这个步骤中的关键仍然是温度，这应高于脂质体的相变温度。通过在50℃下递挤出，脂质体的大小通过膜成为挤压的10次循环后均匀的200纳米的孔。在这个步骤中的运动应该是缓慢和轻柔的挤压机内的压力大，而该膜是脆弱的。 一个经常被忽视，但重要的STE含药脂质体的合成过程中，p是纯化或除去未包封的药物（ 即，钙黄绿素和金纳米这里）的。这是由凝胶过滤色谱法进行。这是至关重要的研究人员注意到，在这个研究中使用的PD-10柱只适合处理&lt;2 ml溶液。具有较大体积的样品可以通过使用多个列进行处理。 从脂质体的钙黄绿素释放的研究依赖于在高浓度的钙黄绿素和重新荧光的自猝灭作为稀释。当将样品分别暴露于热或光热或激光处理，样品的等分试样在不同的温度点，或脉冲数被不断地取出，并转移至单独的小瓶中。小瓶应当立即置于冰浴中，以防止钙黄绿素的潜力进一步释放，为精确测量。要注意的另一件事是，钙黄绿素对光敏感因此可以在激光实验期间漂白。为了克服这一点，在钙荧光素溶液的脉冲激光的漂白效果应该测量和从脂质体释放的数据相应地归一化。 这里的协议描述为对光敏感脂质体浅显的制备方法。热剥离首先通过利用响应磷脂的温度证实。激光设置说明和光触发释放脉冲激光来实现。的光的机制触发释放也探索和被发现是由于通过微泡空化对膜的破坏。 从所有报告的协议的不同，这个协议选择已在临床试验中常用的和在市场上广泛使用的材料（即，脂质和金纳米粒子）。使用容易获得的材料允许任何人通过自己编写这样的感光系统，而不NEED寻求技术帮助。此外，该协议是利用薄膜水化，制备脂质体，这是既具有成本效益和易于扩展。 这个简单但功能强大的协议将有利于研究人员和临床医生谁有兴趣获得药物控释浅表组织像皮肤一样。一个潜在的应用将是防晒霜中纳入该系统，可以提供防日晒剂。

可能使用外部的刺激是一个有吸引力的方式为客户提供药物以最大的特异性和最小的不利影响spatial-，temporal-和剂量控制的时装来触发药物释放。之间广泛的外源刺激响应系统（光，磁场，超声波，微波辐射），光触发的平台是有吸引力的，因为它们的非侵入性，简便性和适应性的诊所。在过去的十年1广泛的研究已经提供了各种平台技术，例如近红外光负责金的涂有智能聚合物（Au）的纳米笼，用药物，3和自组装porphysome纳米囊泡共轭2对光不稳定的，聚合物纳米颗粒（纳米颗粒）。4但是这些技术仍处于发展的临床前阶段，要求参与发起和续的工艺参数清醒的认识和优化滚动药物的释放。 一种用于制备这种系统的最简单和方便的方法是对金纳米粒子与热敏感脂质体5,6，这两者都是在市场上广泛使用，并已在临床前和甚至临床试验被广泛地研究集成。尽管金纳米粒子的深层组织活化的在其电浆波长的限制，相对于近红外活化的Au纳米结构（例如，纳米笼），在小动物或用于人类局部递送时使用该系统仍然保持很大的希望。 7中有金纳米粒子与脂质体相结合的用于光触发释放一些早期的努力。8-11虽然大多集中在材料的新颖性，需要解决可访问性和可扩展性的问题。此外，在使用这些纳米载体释放机构的报告仍然有限。 这里，在制造光响应脂质体，同时装载有药物和亲水性金纳米粒子已经描述。钙黄绿素被用作模型化合物以评价包封效率和系统的释放曲线。另外，在这个系统中，由金纳米粒子吸收的光消散至以热的形式的周围的微环境，从而增加在局部温度。空气微泡是在激光加热过程中产生的，并引起的脂质体（图1）的机械破坏。微泡空化的机制是由水听器测量证实。

<table><tbody><tr><td>1,2-Dipalmitoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphocholine (DPPC)</td><td>Avanti Polar Lipids (Alabama, US)</td><td>850355P</td><td>Powder, Store at -20 &amp;deg;C</td></tr><tr><td>1-palmitoyl-2-hydroxy-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphocholine (MPPC)</td><td>Avanti Polar Lipids (Alabama, US)</td><td>855675P</td><td>Powder, Store at -20 &amp;deg;C</td></tr><tr><td>1,2-distearoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphoethanol-amine-&lt;em&gt;N&lt;/em&gt;-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG2000)</td><td>Avanti Polar Lipids (Alabama, US)</td><td>880120P</td><td>Powder, Store at -20 &amp;deg;C</td></tr><tr><td>Gold Nanoparticles</td><td>Sigma Aldrich</td><td>752568-100mL</td><td>5&amp;nbsp;nm particles, stabilized at 0.1&amp;nbsp;mM PBS</td></tr><tr><td>Calcein</td><td>Sigma Aldrich</td><td>C0875-10g</td><td>60&amp;nbsp;mM, pH 7.4 (adjusted using NaOH)</td></tr><tr><td>phosphate buffered saline (PBS)</td><td>Sigma Aldrich</td><td>P5493</td><td>0.1 mM, pH 7.4</td></tr><tr><td>Double distilled water</td><td>Millipore Milli-DI water purification system</td><td></td><td></td></tr><tr><td>Triton X100&amp;nbsp;&amp;nbsp;</td><td>Sigma, Life Sciences</td><td>X-100</td><td>To disrupt the liposomes to calculate total encapsulation</td></tr><tr><td>Rotavapor&amp;nbsp;&amp;nbsp;</td><td>Buchi (Switzerland)</td><td>R 210</td><td>Used for Lipososme preparation</td></tr><tr><td>Heating bath</td><td>Buchi (Switzerland)</td><td>B 491</td><td>Used for Lipososme preparation</td></tr><tr><td>Vacuum Controller&amp;nbsp;&amp;nbsp;</td><td>Buchi (Switzerland)</td><td>V-850</td><td>Used for Lipososme preparation</td></tr><tr><td>Vacuum Pump</td><td>Buchi (Switzerland)</td><td>V-700</td><td>Used for Lipososme preparation</td></tr><tr><td>Recirculation bath with temperature controller</td><td>Polyscience</td><td></td><td>Used for Lipososme preparation</td></tr><tr><td>Mini-extruder assembly with heating block&amp;nbsp;</td><td>Avanti Polar Lipids (Alabama, US)</td><td>610000</td><td>Used for extrusion of liposomes</td></tr><tr><td>Syringes, 1,000 &amp;#956;l</td><td>Avanti Polar Lipids (Alabama, US)</td><td>610017</td><td>Used for extrusion of liposomes</td></tr><tr><td>Polycarbonate filter membrane, 200&amp;nbsp;nm</td><td>Whatmann</td><td>800281</td><td>Used for extrusion of liposomes</td></tr><tr><td>Filter Support</td><td>Avanti Polar Lipids (Alabama, US)</td><td>610014</td><td>Used for extrusion of liposomes</td></tr><tr><td>PD 10 Desalting coulumns, Sephadex G-25 medium</td><td>GE Healthcare, Life sciences</td><td>17-0851-01</td><td>Used to purify the liposomes</td></tr><tr><td>Centrifuge&amp;nbsp;&amp;nbsp;</td><td>Sigma Laboratory Centrifuges</td><td>3K30</td><td>Used to concentrate the liposomal solution&amp;nbsp;</td></tr><tr><td>Rotor</td><td>Sigma</td><td>19777-H</td><td>Used to concentrate the liposomal solution&amp;nbsp;</td></tr><tr><td>Zetasizer&amp;nbsp;&amp;nbsp;</td><td>Nano ZS Malvern</td><td></td><td>Used for the determination of liposome size and zetapotential</td></tr><tr><td>UV-Visible Spectrophotometer</td><td>Shimadzu</td><td>UV-2450</td><td>Used to measure the absorbance of the samples</td></tr><tr><td>Fluorescent Spectrofluorometer&amp;nbsp;&amp;nbsp;</td><td>Molecular Devices</td><td>SpectraMax M5</td><td>Used to measure the fluorescence emission of the samples</td></tr><tr><td>Nd:YAG Laser</td><td>NewWave Research</td><td></td><td>532 nm; Maximum power: 17&amp;nbsp;mJ; Width: 406&amp;nbsp;nsec; Used for sample irradiation</td></tr><tr><td>HNR Hydrophone</td><td>ONDA</td><td>HNR-1000</td><td>1 mm diameter and 450 nV/Pa sensitivity, Proper working frequency range: 0.25-10&amp;nbsp;MHz; Calibration: 50&amp;nbsp;mV/Bar; Used to measure the acoustic signals</td></tr><tr><td>Digital Osciloscope</td><td>LECORY - Wave Runner 64Xi-A</td><td></td><td>Frequency: 600 MHz; Max sample rate: 10&amp;nbsp;Gs/sec (at two channel); Used to record the measured acoustic signals</td></tr></tbody></table>

synthesis of gold nanoparticle integrated photo-responsive liposomes and measurement of their microbubble cavitation upon pulse laser excitation

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control over the drug release. However, most of the relevant technologies are still in the development process and are unprocurable by clinics. Here, we describe a facile fabrication of these photo-responsive NPs with commercially available gold NPs and thermo-responsive liposomes. Calcein is used as a model drug to evaluate the encapsulation efficiency and the release kinetic profile upon heat/light stimulation. Finally, we show that this photo-triggered release is due to the membrane disruption caused by microbubble cavitation, which can be measured with hydrophone.

10:4:4或7.95:使用具有DPPC，MPPC和DSPE-PEG2000的常规薄膜水化技术中的86摩尔比制备的脂质体0.65:1.39毫克/毫升12金纳米粒子的大小是至关重要的，以确定所述光下面激光激发实验期间加热的转换效率。金纳米粒子的尺寸小，高为13因此为5nm金纳米粒子，从供应商的最小样本，被选择为包封传感效率。在合成过程中，加入含有金纳米粒子和水合介质钙黄绿素生成多层状囊泡中，将其受到大小挤出和凝胶过滤层析以除去游离的金纳米粒子和钙黄绿素。 钙黄绿素是亲水性荧光染料在60毫浓度的脂质体的水性核心内包封。在这样高的浓度，C的荧光alcein自淬灭。然而，因为它是从脂质体释放，从而导致荧光重新增益钙黄绿素被稀释，指示触发药物释放。14依托钙黄绿素的脂质体的这种特性中稀释至5mM和脂质体系统的对温度变化的响应是观察到的。 如图 2A中所示 ，无论金纳米粒子的存在，所述脂质体在生理温度（即，37℃）具有小于10％的泄漏百分比几乎完好无损。然而，当温度升高到42℃（稍微高于脂质的转变温度），60％的包封的钙黄绿素-80％是从脂质体释放的2分钟内。在释放百分比没有显著差与和不金纳米粒子的脂质体，当释放利用热触发内观察到。这是由于该系统的相变温度。此外，快速和有效的释放是由于prese的NCE 10％MPPC，这增强了脂质体双层的渗透性在转变温度。 接着，从在激光照射的脂质体的钙黄绿素释放进行了检查。脂质体溶液溶解在其中放置在试管架的石英比色皿。 532纳米的Nd:YAG脉冲的6纳秒的脉冲持续时间和166.67千瓦/ cm 2的瞬时功率密度的激光被聚焦到脂质体溶液。样品的等分试样在不同的脉冲数（0，25，50，和100），其中脉冲频率固定在1赫兹收集。将样品立即转移至微量离心管中，并置于冰浴中。这是为了防止钙黄绿素的任何进一步释放。同时收集样品的激光器被关闭。 如图 2B所示，用Au纳米颗粒的脂质体释放激发时包封的钙黄绿素，其中释放的钙黄绿素的量随着脉冲民BER的增加。 最后一步是研究从水听器的脂质体的钙黄绿素释放的机构;图3A是记录金纳米粒子（红色），用Au纳米颗粒（黑色）和脂质体而不金纳米粒子（蓝色）的脂质体的声信号。虽然金纳米粒子和脂质体与金纳米粒子表现出特有的压力波信号，从没有金纳米粒子脂质体观察到没有信号。该压力脉冲包括正面和负面的阶段（ 图 3A插入图示）。它表明，微气泡形成（正相）和破坏（负相）中的溶液中。的最大值和在含NP-凹脂质体所记录的峰的最小值分别为0.00093和-0.00074诉最后，将压力脉冲作为增加激光能量的函数的平均值的最大值和最小值计算。正如预期的，声学信号幅度逐渐日益增加的激光增大能量（图3B）。这应该是由于归因于随后的热弹性膨胀和收缩颗粒的振荡的强度增加。15,16 <img alt="图1" src="/files/ftp_upload/53619/53619fig1.jpg" /> 图1. 光响应脂质体的提议的药物释放机制:金纳米（红点）的照射时，产生破坏脂质体膜，从而触发钙黄绿素释放钙黄绿素，自猝灭脂质体中（如图所示），发荧光的微泡。作为被释放到周围。 <a href="https://www.jove.com/files/ftp_upload/53619/53619fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53619/53619fig2.jpg" /> 图2. &lt;强&gt;热和脉冲激光触发从脂质体的钙黄绿素释放（A）中从具有或不具有金纳米粒子，它被放置在水浴各种温度下进行2分钟的脂质体释放的钙黄绿素的百分比。从与金纳米粒子，将其用脉冲激光处理的脂质体释放的钙黄绿素（B）的百分比（脉冲持续时间= 6纳秒;功率密度〜12毫瓦/厘米2）。误差棒代表平均值±一式三份进行的三个独立实验的标准差（SD）。 <a href="https://www.jove.com/files/ftp_upload/53619/53619fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53619/53619fig3.jpg" /> 图3.（A）声信号，使用水听系统的金纳米粒子（红色），与金纳米粒子（黑色）和脂质体脂质体没有经过测量ü纳米粒（蓝色）。虽然金纳米粒子和脂质体与金纳米粒子表现出特有的压力波信号，从没有金纳米粒子脂质体观察到没有信号。插图显示光响应脂质体的声信号的放大图。 （B）中的最大和压力脉冲作为增加激光功率的函数的最小峰值。声学信号幅度与增加激光能量逐渐增大。误差棒代表平均值±在10次运行进行三个独立实验的标准差。 <a href="https://www.jove.com/files/ftp_upload/53619/53619fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation at JoVE.com

Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

这个协议描述了金纳米颗粒集成光响应的脂质体与市售材料的简单制备方法。这也说明了如何在脉冲激光治疗测量合成脂质体的微泡空化进程。

在脉冲激光激发金纳米粒子集成光电响应脂质体及其微泡空化的测量的合成

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control ...

synthesis-gold-nanoparticle-integrated-photo-responsive-liposomes

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.1K 次观看.  Nanyang Technological University. 这个协议描述了金纳米颗粒集成光响应的脂质体与市售材料的简单制备方法。这也说明了如何在脉冲激光治疗测量合成脂质体的微泡空化进程。 

视频: Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

Advanced Compositional Analysis of Nanoparticle-polymer Composites Using Direct Fluorescence Imaging

The fabrication of polymer-nanoparticle composites is extremely important in the development of many functional materials. Identifying the precise composition of these materials is essential, especially in the design of surface catalysts, where the surface concentration of the active component determines the activity of the material. Antimicrobial materials which utilize nanoparticles are a particular focus of this technology. Recently swell encapsulation has emerged as a technique for inserting antimicrobial nanoparticles into a host polymer matrix. Swell encapsulation provides the advantage of localizing the incorporation to the external surfaces of materials, which act as the active sites of these materials. However, quantification of this nanoparticle uptake is challenging. Previous studies explore the link between antimicrobial activity and surface concentration of the active component, but this is not directly visualized. Here we show a reliable method to monitor the incorporation of nanoparticles into a polymer host matrix via swell encapsulation. We show that the surface concentration of CdSe/ZnS nanoparticles can be accurately visualized through cross-sectional fluorescence imaging. Using this method, we can quantify the uptake of nanoparticles via swell encapsulation and measure the surface concentration of encapsulated particles, which is key in optimizing the activity of functional materials.

Here we present a reliable method to monitor the incorporation of nanoparticles into a polymer host matrix via swell encapsulation. We show that the surface concentration of cadmium selenide quantum dots can be accurately visualized through cross-sectional fluorescence imaging.

纳米粒子 - 聚合物复合材料使用直接荧光成像的先进成分分析

The fabrication of polymer-nanoparticle composites is extremely important in the development of many functional materials. Identifying the precise ...

科研

工程学

Controllable Nucleation of Cavitation from Plasmonic Gold Nanoparticles for Enhancing High Intensity Focused Ultrasound Applications

In this study, plasmonic gold nanoparticles were simultaneously exposed to pulsed near-infrared laser light and high intensity focused ultrasound (HIFU) for the controllable nucleation of cavitation in tissue-mimicking gel phantoms. This in vitro protocol was developed to demonstrate the feasibility of this approach, for both enhancement of imaging and therapeutic applications for cancer. The same apparatus can be used for both imaging and therapeutic applications by varying the exposure duration of the HIFU system. For short duration exposures (10 &#181;s), broadband acoustic emissions were generated through the controlled nucleation of inertial cavitation around the gold nanoparticles. These emissions provide direct localization of nanoparticles. For future applications, these particles may be functionalized with molecular-targeting antibodies (e.g. anti-HER2 for breast cancer) and can provide precise localization of cancerous regions, complementing routine diagnostic ultrasound imaging. For continuous wave (CW) exposures, the cavitation activity was used to increase the localized heating from the HIFU exposures resulting in larger thermal damage in the gel phantoms. The acoustic emissions generated from inertial cavitation activity during these CW exposures was monitored using a passive cavitation detection (PCD) system to provide feedback of cavitation activity. Increased localized heating was only achieved through the unique combination of nanoparticles, laser light and HIFU. Further validation of this technique in pre-clinical models of cancer is necessary.

This protocol demonstrates the controllable nucleation of cavitation in gel phantoms, through simultaneous exposure to both near-infrared pulsed laser light and high intensity focused ultrasound (HIFU). The cavitation activity can then be used for enhancing imaging and/or therapeutic uses of HIFU.

等离子金纳米粒子空化的可控核增强高强度聚焦超声应用

In this study, plasmonic gold nanoparticles were simultaneously exposed to pulsed near-infrared laser light and high intensity focused ultrasound (HIFU) ...

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

含有金纳米棒蜂窝车辆的制备及光声分析

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

生物工程

Nanoparticle Tracking Analysis of Gold Nanoparticles in Aqueous Media through an Inter-Laboratory Comparison

In the field of nanotechnology, analytical characterization plays a vital role in understanding the behavior and toxicity of nanomaterials (NMs). Characterization needs to be thorough and the technique chosen should be well-suited to the property to be determined, the material being analyzed and the medium in which it is present. Furthermore, the instrument operation and methodology need to be well-developed and clearly understood by the user to avoid data collection errors. Any discrepancies in the applied method or procedure can lead to differences and poor reproducibility of obtained data. This paper aims to clarify the method to measure the hydrodynamic diameter of gold nanoparticles by means of Nanoparticle Tracking Analysis (NTA). This study was carried out as an inter-laboratory comparison (ILC) amongst seven different laboratories to validate the standard operating procedure&#8217;s performance and reproducibility. The results obtained from this ILC study reveal the importance and benefits of detailed standard operating procedures (SOPs), best practice updates, user knowledge, and measurement automation.

The protocol described here aims to measure the hydrodynamic diameter of spherical nanoparticles, more specifically gold nanoparticles, in aqueous media by means of Nanoparticle Tracking Analysis (NTA). The latter involves tracking the movement of particles due to Brownian motion and implementing the Stokes-Einstein equation to obtain the hydrodynamic diameter.

通过实验室间比较，对液态介质中的金纳米粒子进行纳米粒子跟踪分析

In the field of nanotechnology, analytical characterization plays a vital role in understanding the behavior and toxicity of nanomaterials (NMs). ...

化学

Gold Nanoparticle Synthesis

Gold nanoparticles (Au nanoparticles) that are ~12 nm in diameter were synthesized by rapidly injecting a solution of 150 mg (0.15 mmol) of tetrachloroauric acid in 3.0 g (3.7 mmol, 3.6 mL) of oleylamine (technical grade) and 3.0 mL of toluene into a boiling solution of 5.1 g (6.4 mmol, 8.7 mL) of oleylamine in 147 mL of toluene. While boiling and mixing the reaction solution for 2 hours, the color of the reaction mixture changed from clear, to light yellow, to light pink, and then slowly to dark red. The heat was then turned off, and the solution was allowed to gradually cool down to room temperature for 1 hour. The gold nanoparticles were then collected and separated from the solution using a centrifuge and washed three times; by vortexing and dispersing the gold nanoparticles in 10 mL portions of toluene, and then precipitating the gold nanoparticles by adding 40 mL portions of methanol and spinning them in a centrifuge. The solution was then decanted to remove any remaining byproducts and unreacted starting materials. Drying the gold nanoparticles in a vacuum environment produced a solid black pellet; which could be stored for long periods of time (up to one year) for later use, and then redissolved in organic solvents such as toluene.

A protocol for synthesizing ~12 nm diameter gold nanoparticles (Au nanoparticles) in an organic solvent is presented. The gold nanoparticles are capped with oleylamine ligands to prevent agglomeration. The gold nanoparticles are soluble in organic solvents such as toluene.

金纳米粒子合成

Gold nanoparticles (Au nanoparticles) that are ~12 nm in diameter were synthesized by rapidly injecting a solution of 150 mg (0.15 mmol) of ...

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)&#8211;Cell Interaction and the Resultant Bioeffects at the Single-cell Level

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass substrate, that precisely controls the generation of tandem bubbles and individual cells patterned nearby with well-defined locations and shapes. We then demonstrate the generation of tandem bubbles by using two pulsed lasers illuminating a pair of gold dots with a few-microsecond time delay. We visualize the bubble-bubble interaction and jet formation by high-speed imaging and characterize the resultant flow field using particle image velocimetry (PIV). Finally, we present some applications of this technique for single cell analysis, including cell membrane poration with macromolecule uptake, localized membrane deformation determined by the displacements of attached integrin-binding beads, and intracellular calcium response from ratiometric imaging. Our results show that a fast and directional jetting flow is produced by the tandem bubble interaction, which can impose a highly localized shear stress on the surface of a cell grown in close proximity. Furthermore, different bioeffects can be induced by altering the strength of the jetting flow by adjusting the standoff distance from the cell to the tandem bubbles.

A microfluidic chip was fabricated to produce pairs of gold dots for tandem bubble generation and fibronectin-coated islands for single-cell patterning nearby. The resultant flow field was characterized by particle image velocimetry and was employed to study various bioeffects, including cell membrane poration, membrane deformation, and intracellular calcium response.

用的表面图案化微流体系统用于在单细胞水平调查空泡（S） - 细胞的相互作用及产生的生物效应

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass ...

通过激光烧蚀合成纳米颗粒和纳米结构

Ultrafast Laser-Ablated Nanoparticles and Nanostructures for Surface-Enhanced Raman Scattering-Based Sensing Applications

The technique of ultrafast laser ablation in liquids has evolved and matured over the past decade, with several impending applications in various fields such as sensing, catalysis, and medicine. The exceptional feature of this technique is the formation of nanoparticles (colloids) and nanostructures (solids) in a single experiment with ultrashort laser pulses. We have been working on this technique for the past few years, investigating its potential using the surface-enhanced Raman scattering (SERS) technique in hazardous materials sensing applications. Ultrafast laser-ablated substrates (solids and colloids) could detect several analyte molecules at the trace levels/mixture form, including dyes, explosives, pesticides, and biomolecules. Here, we present some of the results achieved using the targets of Ag, Au, Ag-Au, and Si. We have optimized the nanostructures (NSs) and nanoparticles (NPs) obtained (in liquids and air) using different pulse durations, wavelengths, energies, pulse shapes, and writing geometries. Thus, various NSs and NPs were tested for their efficiency in sensing numerous analyte molecules using a simple, portable Raman spectrometer. This methodology, once optimized, paves the way for on-field sensing applications. We discuss the protocols in (a) synthesizing the NPs/NSs via laser ablation, (b) characterization of NPs/NSs, and (c) their utilization in the SERS-based sensing studies.

作者聚焦:通过液体中的激光烧蚀进行纳米颗粒合成的进展和应用 

Ultrafast laser ablation in liquid is a precise and versatile technique for synthesizing nanomaterials (nanoparticles [NPs] and nanostructures [NSs]) in liquid/air environments. The laser-ablated nanomaterials can be functionalized with Raman-active molecules to enhance the Raman signal of analytes placed on or near the NSs/NPs.

超快激光烧蚀纳米颗粒和纳米结构在基于表面增强拉曼散射的传感应用中的应用

The technique of ultrafast laser ablation in liquids has evolved and matured over the past decade, with several impending applications in various fields ...

Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been devoted to their development. In this protocol, a recently developed, facile method for preparation of water soluble, biocompatible, and colloidally stable near-infrared emitting AuNCs have been described in detail. This room-temperature, bottom-up chemical synthesis provides easily functionalizable AuNCs capped with thioctic acid and thiol-modified polyethylene glycol in aqueous solution. The synthetic approach requires neither organic solvents or additional ligand exchange nor extensive knowledge of synthetic chemistry to reproduce. The resulting AuNCs offer free surface carboxylic acids, which can be functionalized with various biological molecules bearing a free amine group without adversely affecting the photoluminescent properties of the AuNCs. A quick, reliable procedure for flow cytometric quantification and confocal microscopic imaging of AuNC uptake by HeLa cells also been described. Due to the large Stokes shift, proper setting of filters in flow cytometry and confocal microscopy is necessary for efficient detection of near-infrared photoluminescence of AuNCs.

A reliable and easily reproducible method for preparation of functionalizable, near-infrared emitting photoluminescent gold nanoclusters and their direct detection inside HeLa cells by flow cytometry and confocal laser scanning microscopy is described.

生物应用近红外发光金纳米簇的合成

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been ...

在脉冲激光激发金纳米粒子集成光电响应脂质体及其微泡空化的测量的合成

摘要

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金纳米粒子合成

用的表面图案化微流体系统用于在单细胞水平调查空泡（S） - 细胞的相互作用及产生的生物效应

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超快激光烧蚀纳米颗粒和纳米结构在基于表面增强拉曼散射的传感应用中的应用

超快激光烧蚀纳米颗粒和纳米结构在基于表面增强拉曼散射的传感应用中的应用

生物应用近红外发光金纳米簇的合成