This research was supported by a Marie Curie FP7 Career Integration Grant 334134 within the seventh European Union Framework Program.

1.从家蚕蚕茧反向工程的丝溶液的制备注:此方法是基于其他地方12,27描述的协议。 <ol><li> 5切断克干蚕茧用剪刀分成5 x 5毫米件。卸下所有脏层。 </li><li>称出4.24克碳酸钠和小心添加此2升沸腾蒸馏水的。 注意:这产生了0.02 2M碳酸钠溶液。 </li><li>切件茧添加到沸腾的碳酸钠溶液煮沸60分钟，脱胶的蚕丝纤维。炒出的丝绸偶尔确保均匀样加工。 </li><li>除去脱胶的丝，用20分钟1升的蒸馏水洗净;重复洗涤步骤至少3次。 </li><li>取出水洗丝和挤压它很好地去除多余的液体，然后解开/用手拉丝。放置解开丝绸在通风橱中空气干燥过夜。这通常会产生3.6克脱胶的S之流纤维。 </li><li>第二天，称量满分5 G空气干燥的脱胶丝纤维和紧密包装丝到50ml烧杯的底部。 </li><li>准备一个新鲜的9.3中号溴化锂溶液。用一根1G的丝绸〜4毫升溴化锂比例溶于溴化锂丝纤维。用铝箔覆盖丝溴化锂样品以防止蒸发，并允许丝完全溶解在60℃。这一步需要长达4小时，并通过搅拌偶尔帮助。 </li><li>润湿在水中的透析盒（3,500道尔顿分子量截留）5分钟。注入15ml的所述丝溴化锂溶液进入15毫升透析盒和使用针和注射器，以除去任何气泡。 </li><li>透析针对1升蒸馏水，并在1,3和6小时（ 即 ，在第一天3的变化），并再次在第二天早晨和晚上（ 即 ，2的变化的第二天），并再次改变水第二天早上（ 即 1变第三天）。 </li><li>收集丝绸soluti从透析盒，离心20分钟，在5℃下以9500×g的溶液。恢复上清，两次重复此过程离心。 </li><li>确定空称量船（W1）的重量和加入1毫升丝溶液。再次（W2）记录重量，然后通过使在60ºC称量舟过夜干燥样品。接下来，确定的总干重（W 3）（干燥丝和称量船）。丝溶液的浓度（重量/体积）为:（％）=（W3-W1 / W2-W1）×100。 </li></ol>从反向工程丝溶液丝绸纳米2.准备工作<ol><li>添加5％（重量/体积）丝溶液滴加到丙酮，同时保持&gt; 75％（体积/体积）丙酮溶液。例如，添加9mL经5％（重量/体积）丝溶液滴加至34毫升丙酮（以50滴/分钟的速度10微升/滴）。 </li><li>离心沉淀物以48000×g离心在4℃下2小时。 </li><li>吸出上清液，重悬PELLE吨蒸馏水通过首先用刮刀松脱沉淀，然后加入20ml蒸馏水。用枪头从锅铲去除沉淀。使用的超声波探头，在30％的幅度，持续30秒，20秒，随后由两个超声处理周期涡旋，顶部向上离心管以用蒸馏水后容量。 </li><li>重复离心和再悬浮步骤至少两次。 </li><li>悬浮在蒸馏水中，如在4℃直至使用在2.3和存储详述6毫升沉淀。对于细胞培养研究，纳米丝绸个股可以伽马辐射17。 </li></ol> 3.丝绸纳米浓度测定<ol><li>离心机丝纳米颗粒以48,000 xg离心在4℃下2小时。 </li><li>收集所有纳米颗粒在3毫升的蒸馏水，然后由两个超声处理周期，在30％的幅度，持续30秒。 </li><li>除以3 ml储备丝纳米颗粒于2ml和1ml地段并转入公关电子称重2ml管。记录2 ml样品的总重量。存储1ml的很多在4℃直至使用;此1ml样品将被用于产生校准曲线。 </li><li>捕捉冻结，然后在一夜之间冻干2 mL萃取丝绸纳米很多冷冻干燥机。冷冻干燥后，重新称重2毫升管和计算蚕丝纳米颗粒（毫克），该最初2 ml样品中的存在量。 </li><li>稀释1ml的丝的纳米颗粒的库存用蒸馏水，以产生一个5点标准曲线（0.04-7毫克/毫升）。保证样品不超过最大吸光度。 </li><li>确定在600nm每标准稀释的吸光度。这是通过使用一个96孔板设置最佳完成。情节吸光度与浓度（mg / ml）的标准曲线。然后用这个标准曲线定期确定丝绸纳米粒子悬浮液中的浓度。 </li></ol> 4.负载阿霉素的丝绸纳米颗粒的制备<ol><li&gt;阿霉素溶液的制备<ol><li>溶解1.2毫克阿霉素HCl的蒸馏水加8毫升</li><li>补至10ml，用蒸馏水，得到的116微克/毫升（0.2微摩尔/毫升）的工作原液。 </li></ol></li><li>负载阿霉素的丝绸纳米颗粒的制备<ol><li>混合料2毫升0.2微摩尔/毫升阿霉素的溶液用200μl的10，30或50mg /在一个2毫升管毫升丝纳米颗粒。 </li><li>在室温下孵育（25℃）过夜在旋转混合器丝 - 阿霉素悬浮液。 </li><li>接下来，离心丝绸阿霉素在悬挂194,000 XG 30分钟。用蒸馏水洗涤负载阿霉素的纳米粒子的丝绸和重复此过程两次。 </li><li>池上清液并注意的总体积（该样本被用于确定包封效率）。 </li><li>悬浮负载阿霉素的纳米颗粒丝在蒸馏水中，从光并存储保护在4ºC未直到使用。 </li></ol></li><li>包封率和载药量的测定<ol><li>吸取200微升从步骤4.2.4上清变成了黑色微孔板。 </li><li>使用荧光酶标仪在一个固定的光电倍增管设置测量阿霉素相关的荧光。 </li><li>激发波长到485纳米，发射波长设置为590nm处并记录荧光值。 </li><li>产生阿霉素的校准曲线。保证测量获得具有相同仪器设置（ 即，具有固定的光电倍增管设置）。使用校正曲线，在合并上清液计算阿霉素浓度。在三个独立的实验重复这种测量。 </li><li>用公式（1）确定包封率: <img alt="式（1）" src="/files/ftp_upload/54669/54669equation1.jpg" /></li></ol></li></ol> 5.表征丝绸纳米粒子<ol><li>新鲜制备和贮存丝绸纳米粒子的尺寸和Zeta电位的评价。 <ol><li>在蒸馏水商店丝纳米颗粒在4℃和25℃。 </li><li>测量丝的纳米颗粒上使用动态光散射（DLS）天0，14和28的大小和ζ电位。设置折射率1.33蒸馏水和1.60蛋白17。计算粒径与所述用户界面的软件。 </li></ol></li><li>丝绸纳米粒子的形态评价用扫描电子显微镜（SEM）。 <ol><li>放置一个碳粘合剂光盘上的SEM存根并随后附加的硅晶片。 </li><li>稀释丝纳米至1mg / ml的浓度。移取10微升样品在硅晶片上的，冷冻在-80℃下的样品和冻干过夜使用冷冻干燥系统按照制造商的说明。 </li><li>涂层用金样品层达20纳米厚使用低真空溅射镀膜机。 注:仪器设置模式之间变化。这里使用的模型是完全自动化的并且仅由厚度进行操作。 </li><li>图像样品在5kV的扫描型电子显微镜和40,000倍的放大倍率。 </li></ol></li></ol> 6. 在控制的体外细胞毒性和负载阿霉素的纳米丝绸<ol><li>细胞活力暴露于纳米丝绸。 <ol><li>培养的MDA-MB-231细胞在RPMI 1640有10％v / v的FBS。组织培养板的细胞处理的聚苯乙烯，并在37℃下在湿润的5％的CO 2气氛中孵育。在80％汇合常规传代培养每2-3天。 </li><li>板的MDA-MB-231细胞在96孔板一个2×10 4个细胞/ cm 2的密度。使细胞恢复过夜。 </li><li>加（ⅰ）0.001微克自由扩散阿霉素，（ⅱ）0.001〜0.5毫克丝纳米颗粒和0.1mg丝纳米颗粒装载有0.001微克的阿霉素为96孔板（最终体积每孔100μl）。 </li><li>确定细胞存活力，并通过加入（3-（4,5-二甲基吡啶-2-基）-2,5-二苯基溴（MTT 5毫克/ PBS中）溶液在72小时的半最大抑制浓度（IC 50）。孵育5小时，小心地排出孔用吸管和溶解用100μl二甲基亚砜的甲，测量560nm处的吸光度。在三个独立的实验重复这种测量。 注:未处理的对照组的吸光度值作为100％细胞生存力的基准值。 </li></ol></li><li>暴露于纳米丝绸细胞SEM。 <ol><li>以2×10 4个细胞/ cm 2的密度种子的MDA-MB-231细胞在无菌盖玻片。使细胞恢复过夜。露出元到达所希望的处理条件为72小时。 </li><li>固定用PBS中2％的V / V戊二醛细胞30分钟，用二洗平息两次水，脱水用乙醇系列，以及临界点干燥的样品，其他地方28详述。 </li><li>溅射涂层用金样品层高达20nm厚使用低真空溅射镀膜机。 注:仪器设置模式之间变化。这里使用的模型是完全自动化的并且仅由厚度进行操作。 </li><li>图片使用5千伏和700倍的放大倍率的电子加速通过SEM样品。 </li></ol></li></ol>

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<li>Seib, F. P., Müller, K., Franke, M., Grimmer, M., Bornhäuser, M., Werner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineered+extracellular+matrices+modulate+the+expression+profile+and+feeder+properties+of+bone+marrow-derived+human+multipotent+mesenchymal+stromal+cells%5BTitle%5D)+AND+%22Tissue.+Eng.+Part+A%22%5BJournal%5D)">Engineered extracellular matrices modulate the expression profile and feeder properties of bone marrow-derived human multipotent mesenchymal stromal cells.</a> Tissue. Eng. Part A. 15 (10), 3161-3171 (2009).</li>
<li>Lai, P., Daear, W., Löbenberg, R., Prenner, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Overview+of+the+preparation+of+organic+polymeric+nanoparticles+for+drug+delivery+based+on+gelatine%2C+chitosan%2C+poly%28d%2Cl-lactide-co-glycolic+acid%29+and+polyalkylcyanoacrylate%5BTitle%5D)+AND+%22Colloids+Surf.%2C+B%2C+Biointerfaces%22%5BJournal%5D)">Overview of the preparation of organic polymeric nanoparticles for drug delivery based on gelatine, chitosan, poly(d,l-lactide-co-glycolic acid) and polyalkylcyanoacrylate.</a> Colloids Surf., B, Biointerfaces. 118, 154-163 (2014).</li>
<li>Subia, B., Kundu, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drug+loading+and+release+on+tumor+cells+using+silk+fibroin-albumin+nanoparticles+as+carriers%5BTitle%5D)+AND+%22Nanotechnology%22%5BJournal%5D)">Drug loading and release on tumor cells using silk fibroin-albumin nanoparticles as carriers.</a> Nanotechnology. 24 (3), 035103(2013).</li>
<li>Zhang, Y. Q., Shen, W. D., Xiang, R. L., Zhuge, L. J., Gao, W. J., Wang, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Formation+of+silk+fibroin+nanoparticles+in+water-miscible+organic+solvent+and+their+characterization%5BTitle%5D)+AND+%22J.+Nanopart.+Res%22%5BJournal%5D)">Formation of silk fibroin nanoparticles in water-miscible organic solvent and their characterization.</a> J. Nanopart. Res. 9 (5), 885-900 (2006).</li>
<li>Jin, H. J., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanism+of+silk+processing+in+insects+and+spiders%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Mechanism of silk processing in insects and spiders.</a> Nature. 424 (6952), 1057-1061 (2003).</li>
<li>Yhr Bae,, Park, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+drug+delivery+to+tumors%3A+myths%2C+reality+and+possibility%5BTitle%5D)+AND+%22J.+Control.+Release%22%5BJournal%5D)">Targeted drug delivery to tumors: myths, reality and possibility.</a> J. Control. Release. 153 (3), 198-205 (2011).</li>
<li>Lammel, A., Schwab, M., Hofer, M., Winter, G., Scheibel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recombinant+spider+silk+particles+as+drug+delivery+vehicles%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Recombinant spider silk particles as drug delivery vehicles.</a> Biomaterials. 32 (8), 2233-2240 (2011).</li>
<li>Holliday, D. L., Speirs, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Choosing+the+right+cell+line+for+breast+cancer+research%5BTitle%5D)+AND+%22Breast.+Cancer.+Res%22%5BJournal%5D)">Choosing the right cell line for breast cancer research.</a> Breast. Cancer. Res. 13, 215(2011).</li>
</ol>

The authors have nothing to disclose.

各种方法可用来生产丝纳米颗粒，包括聚乙烯醇共混20，喷雾干燥21，盐析22，毛细管网点印刷23，超临界CO 2的沉淀24和纳米沉淀16,25（参照26中综述）。然而，纳米沉淀，由于其整体简单，是用于产生丝纳米颗粒的最流行的技术。因此，本研究的目的是要应用纳米沉淀到反向工程丝来制造基于丝的纳米颗粒，可用于各种应用，包括溶...

纳米尺寸的药物递送系统通常用来控制药物释放和递送多样化的治疗有效载荷 - 例如，蛋白质，肽和小分子量药物 - 靶细胞和组织。这些治疗的有效载荷常常结合到各种大分子药物载体，例如脂质体，水溶性聚合物（包括树枝状聚合物），以及微颗粒和纳米颗粒1。纳米颗粒（典型地在1纳米至1000纳米的粒度范围）被广泛作为潜在的药物载体，特别是用于抗癌药物递送2。成功引入Abraxane的的（装载紫杉醇120nm的尺寸的基于白蛋白的纳米颗粒）放入常规临床实践3已经催化的领域中，以便用于药物递送更多的纳米颗粒正在进入临床试验4。实体瘤一般显示较差淋巴引流，并有漏水的血管，这意味着是n高达200纳米anoparticles将被动地定位到静脉给药这些肿瘤。这种被动靶向现象被称为增强的渗透性和保留（EPR）效果，并在1986年5首次报道该EPR效应可导致50到100倍的增加中的药物浓度在肿瘤微环境中对于给定的药物剂量时药物有效负荷使用大分子药物载体的方法，而不是无药无承运人交付。设计用于抗癌药物递送载药纳米颗粒具有到达肿瘤微环境和通常必须输入特定的胞内区室，通常通过内吞摄取，用于药物，以实现其所需的治疗效果3。设计用于细胞内药物递送的纳米颗粒利用内吞作为网关进入细胞，以及克服耐药机制的路由。从纳米粒子药物释放往往是专门邻CCur函数在溶酶体（ 即 ，溶酶体药物递送）6，其中该纳米颗粒载体的pH响应性（溶酶体pH值约4.5）可以用作触发药物释放或溶酶体酶从载体7释放有效载荷。 许多不同类型的材料可以用于产生纳米颗粒（ 例如，金属和许多有机和无机材料）。然而，生物聚合物不断涌现，因为他们已知的生物相容性，生物可降解性和低毒性8吸引力的材料。许多生物聚合物正在探索，其中包括白蛋白，海藻酸钠，壳聚糖和丝绸。其中，丝绸已成为一个有前途的竞争者发展注入药物输送系统9。各种类型的丝绸是由一些节肢动物，包括蜘蛛（ 例如 ， 络clavipes）和蚕（ 例如 ， 家蚕生产）。蚕丝被使用更为分配延长sively比蜘蛛丝，因为蚕驯化充分，因此，其丝绸代表重现的原料。蚕丝是食品和药物管理局（FDA）批准的材料供人类使用，特别是作为一个缝合材料;它在人类中健壮的安全记录，并已知在体内 10降解。丝的降解轮廓可以微调以从小时（低结晶丝），以12个月或以上（高结晶丝）的范围内。丝降解产物是无毒的并且在主体10中被代谢。丝绸结构赋予结合小分子量化合物和大分子蛋白药物11的能力，使其成为一个好材料药物控释。蛋白质药物（ 例如，抗体）易受变性，聚集，蛋白水解裂解和清除由免疫系统。然而，丝稳定的治疗性蛋白质，由于其纳米晶重的缓冲能力gions及其在纳米尺度11来调整水分的能力。这些独特的功能提供人身保护，并降低流动性的有效载荷11和通常不与其他（生物）聚合物看到。许多抗癌药物递送系统，例如基于丝的水凝胶12，薄膜13-15和纳米颗粒16,17，现在已经开发了利用这些特征（参考文献18,19中综述） 此处，丝纳米颗粒通过确定在延长的时间帧的大小和电荷特征。阿霉素，临床相关的抗癌药物，被用来作为在用载药丝纳米颗粒处理过的三阴性人类乳癌细胞对药物装载和细胞毒性的研究模型药物。

<table><tbody><tr><td>Acetone</td><td>VWR International, Radnor, PA, USA</td><td>20066.33</td><td></td></tr><tr><td>Automated Critical Point Dryer</td><td>Leica Microsystems, Wetzlar, Germany</td><td>EM CPD300</td><td></td></tr><tr><td>Balancing</td><td>Mettler Toledo, Greifensee, Switzerland</td><td>NewClassic MS</td><td></td></tr><tr><td>Black polystyrene microplate, 96 well</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>3991</td><td></td></tr><tr><td>Capillary cell (DTS 1070)</td><td>Malvern Instrument, Worcestershire, UK</td><td>DTS107</td><td></td></tr><tr><td>Carbon adhesive disc</td><td>Agar Scientific, Essex, UK</td><td>G3347N</td><td></td></tr><tr><td>Centrifuge&amp;nbsp;</td><td>Hermle Labortechnik, Wehingen, Germany</td><td>Z323K</td><td></td></tr><tr><td>Centrifuge&amp;nbsp;</td><td>Beckman Coulter, Brea, CA, USA</td><td>Avanti J-E, Rotor: J20</td><td></td></tr><tr><td>Centrifuge&amp;nbsp;</td><td>Beckman Coulter, Brea, CA, USA</td><td>Optima L-70K, Rotor: 50.2 Ti, Adaptor 303392</td><td></td></tr><tr><td>Coater, low vacuum</td><td>Leica Microsystems, Wetzlar, Germany</td><td>EM ACE200</td><td></td></tr><tr><td>Cuvettes, polystyrene, disposable</td><td>Fisher Scientific, Waltham, MA, USA</td><td>FB55147</td><td></td></tr><tr><td>Doxorubixin&amp;nbsp;</td><td>LC Laboratories, Boston, MA, USA</td><td>D4000</td><td></td></tr><tr><td>Electronic pipetting, Easypet&amp;nbsp;</td><td>Eppendorf, Hamburg, Germany</td><td>N/A</td><td></td></tr><tr><td>FE-SEM</td><td>Hitachi High-Technologies, Krefeld, Germany</td><td>SU6600</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Scientific, Waltham, MA, USA</td><td>16000-044</td><td></td></tr><tr><td>Freeze dryer</td><td>Martin Christ, Osterode, Germany</td><td>Epsilon 2-4</td><td></td></tr><tr><td>Heat inactivated Bombyx mori silk cocoons</td><td>Tajima Shoji, Kanagawa, Japan</td><td>N/A</td><td></td></tr><tr><td>Hotplate with Stirrer</td><td>Bibby Scientific, Stanffordshire, UK</td><td>US 152</td><td></td></tr><tr><td>Incubator</td><td>Memmert, Schwabach, Germany</td><td>INB 200</td><td></td></tr><tr><td>Insulin, human recombinant, zinc solution</td><td>Thermo Scientific, Waltham, MA, USA</td><td>12585-014</td><td></td></tr><tr><td>Lithium bromide</td><td>Acros Organics, Geel, Belgium</td><td>AC199870025</td><td></td></tr><tr><td>MDA-MB-231</td><td>ATCC, Manassas, VA, U.S.A</td><td>N/A</td><td></td></tr><tr><td>Micropipette and tips</td><td>Eppendorf, Hamburg, Germany</td><td>N/A</td><td></td></tr><tr><td>Microplate Reader</td><td>Molecular devices, Sunnyvale, CA, USA</td><td>SpectraMax M5</td><td></td></tr><tr><td>Oak Ridge High-Speed Centrifuge Tubes, 50 ml</td><td>Thermo Scientific, Waltham, MA, USA</td><td>N/A</td><td></td></tr><tr><td>Open-Top Thickwall Polycarbonate tube, 4 ml</td><td>Beckman Coulter, Brea, CA, USA</td><td>355645</td><td></td></tr><tr><td>Penicilin/streptomycin&amp;nbsp;</td><td>Thermo Scientific, Waltham, MA, USA</td><td>15140-122</td><td></td></tr><tr><td>RPMI medium</td><td>Thermo Scientific, Waltham, MA, USA</td><td>11875-093</td><td></td></tr><tr><td>Serological pipettes, 5 ml</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td></td><td></td></tr><tr><td>Silicon wafers</td><td>Agar Scientific, Essex, UK</td><td>G3391</td><td></td></tr><tr><td>Slide-A-Lyzer Dialysis cassettes, 3.5K MWCO, 15 ml</td><td>Thermo Scientific, Waltham, MA, USA</td><td>87724</td><td></td></tr><tr><td>Sodium carbonate anhydrous</td><td>Fisher Scientific, Waltham, MA, USA</td><td>S/2840/62</td><td></td></tr><tr><td>Specimen stubs for SEM</td><td>Agar Scientific, Essex, UK</td><td>G301</td><td></td></tr><tr><td>Ultrasonic homogenizer</td><td>Bandelin, Berlin, Germany</td><td>Sonoplus HD 2070</td><td></td></tr><tr><td>UV transparent microplate, 96 well</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>3635</td><td></td></tr><tr><td>Vortex</td><td>IKA, Staufen, Germany</td><td>Genius 3</td><td></td></tr><tr><td>Zetasizer</td><td>Malvern Instrument, Worcestershire, UK</td><td>Nano ZS</td><td></td></tr><tr><td>Zetasizer Software version 7.11</td><td></td><td>DLS software</td><td></td></tr><tr><td>Micro Modulyo</td><td>&amp;nbsp;Thermo Fisher</td><td>230</td><td>Freeze drying system&amp;nbsp;</td></tr></tbody></table>

manufacture and drug delivery applications of silk nanoparticles

丝是用于生物医学和制药应用的有前途的生物聚合物，由于其优异的机械特性，生物相容性和生物降解性，以及它的保护和随后释放其有效载荷响应于触发的能力。而丝可以配制成各种材料的格式，丝绸纳米颗粒正在成为有希望的药物递送系统。因此，本文涵盖了逆向工程丝茧的步骤以产生可用于产生稳定的丝纳米粒子的再生丝溶液。这些纳米颗粒随后被表征，药物加载并探索作为潜在的抗癌药物递送系统。简要地说，丝茧反向首先通过脱胶茧工程化，随后丝溶解和清理，得到水性丝溶液。接下来，再生丝溶液进行纳米沉淀产生的丝绸纳米粒子 - 一个简单而有效的方法产生均匀的纳米颗粒。根据它们的大小，ζ电位，形态和稳定在水介质中，以及其对截留化疗有效载荷和杀死人乳腺癌细胞的能力的丝的纳米颗粒的特征。总体而言，所描述的方法产生可以容易地探索应用无数均匀丝纳米颗粒，包括它们作为潜在的纳米使用。

进行统计学分析数据如以前17详细说明。用于随后的Bonferroni的多重比较事后检验对多个样品采样对和方差（ANOVA）的单向分析的学生t检验。一个星号表示统计显着性，如下所示:* P &lt;0.05和** P &lt;0.001。所有数据表示为平均值±标准差（SD）和在括号中的数字表示的独立实验的数目。 再生丝溶液的制备，随后滴加到丙酮?...

Watch this Scientific Journal Video about Manufacture and Drug Delivery Applications of Silk Nanoparticles at JoVE.com

Manufacture and Drug Delivery Applications of Silk Nanoparticles

纳米颗粒正在成为有希望的药物递送系统用于范围广泛适应症。在这里，我们将介绍使用逆向工程桑蚕丝制作蚕丝纳米颗粒的简单而有效的方法。这些丝的纳米颗粒可以容易地加载有治疗有效载荷，并随后探索用于药物递送应用。

纳米丝绸制造和药物输送应用

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and ...

manufacture-and-drug-delivery-applications-of-silk-nanoparticles

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

15.7K 次观看.  University of Strathclyde. 纳米颗粒正在成为有希望的药物递送系统用于范围广泛适应症。在这里，我们将介绍使用逆向工程桑蚕丝制作蚕丝纳米颗粒的简单而有效的方法。这些丝的纳米颗粒可以容易地加载有治疗有效载荷，并随后探索用于药物递送应用。 

视频: Manufacture and Drug Delivery Applications of Silk Nanoparticles

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications 3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications 9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are a novel class of biomaterials readily customizable for an array of biomedical applications. The presented silk film culture system is highly adaptable to a variety of in vitro analyses. This system represents a biomaterial design platform offering in vitro optimization before direct translation to in vivo models.

丝绸电影文化系统在体外分析和生物材料设计

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ...

科研

生物工程

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi) and domestic mulberry silk (Bombyx mori) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.

Blending is an efficient approach to generate biomaterials with a broad range of properties and combined features. By predicting the molecular interactions between different natural silk proteins, new silk-silk protein alloy platforms with tunable mechanical resiliency, electrical response, optical transparency, chemical processability, biodegradability, or thermal stability can be designed.

设计丝绸蚕丝蛋白合金生物医用材料

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

多孔硅微粒进行交付的siRNA治疗的

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Reactive Inkjet Printing and Propulsion Analysis of Silk-based Self-propelled Micro-stirrers

In this study, a protocol for using reactive inkjet printing to fabricate enzymatically propelled silk swimmers with well-defined shapes is reported. The resulting devices are an example of self-propelled objects capable of generating motion without external actuation and have potential applications in medicine and environmental sciences for a variety of purposes ranging from micro-stirring, targeted therapeutic delivery, to water remediation (e.g., cleaning oil spills). This method employs reactive inkjet printing to generate well-defined small-scale solid silk structures by converting water soluble regenerated silk fibroin (silk I) to insoluble silk fibroin (silk II). These structures are also selectively doped in specific regions with the enzyme catalase in order to produce motion via bubble generation and detachment. The number of layers printed determines the three-dimensional (3D) structure of the device, and so here the effect of this parameter on the propulsive trajectories is reported. The results demonstrate the ability to tune the motion by varying the dimensions of the printed structures.

This protocol demonstrates the ability to utilize reactive inkjet printing to print self-motile biocompatible and environmentally friendly micro-stirrers for use in biomedical and environmental applications.

基于丝绸的自行式微搅拌器的反应式喷墨打印及推进分析

In this study, a protocol for using reactive inkjet printing to fabricate enzymatically propelled silk swimmers with well-defined shapes is reported. The ...

化学

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

固体脂质纳米粒（前哨淋巴结）细胞内定位的应用

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device

The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based RNA delivery systems, namely, RNA-loaded LNPs have attracted attention for RNA therapy. In particular, mRNA-loaded LNP vaccines were approved to prevent COVID-19, thereby leading to the paradigm shift toward the development of next-generation nanomedicines. For the LNP-based nanomedicines, the LNP size is a significant factor in controlling the LNP biodistribution and LNP performance. Therefore, a precise LNP size control technique is indispensable for the LNP production process. Here, we report a protocol for size controlled LNP production using a microfluidic device, named iLiNP. siRNA loaded LNPs are also produced using the iLiNP device and evaluated by in vitro experiment. Representative results are shown for the LNP size, including siRNA-loaded LNPs, Z-potential, siRNA encapsulation efficiency, cytotoxicity, and target gene silencing activity.

Microfluidic-based lipid nanoparticle (LNP) production methods have attracted attention in drug delivery systems (DDSs), including RNA delivery. This protocol describes the fabrication, LNP (siRNA-loaded LNP) production, and LNP evaluation processes using our original microfluidic device named iLiNP.

使用微流体装置生产siRNA负载的脂质纳米颗粒

The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based ...

生物化学

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

The quest to understand how neural circuits process information in order to drive behavioral output has been greatly aided by recently-developed optical methods for manipulating and monitoring the activity of neurons in vivo. These types of experiments rely on two main components: 1) implantable devices that provide optical access to the brain, and 2) light-sensitive proteins that change neuronal excitability or provide a readout of neuronal activity. There are a number of ways to express light-sensitive proteins, but stereotaxic injection of viral vectors is currently the most flexible approach because expression can be controlled with genetic, anatomical, and temporal precision. Despite the great utility of viral vectors, delivering the virus to the site of optical implants poses numerous challenges. Stereotaxic virus injections are demanding surgeries that increase surgical time, increase the cost of studies, and pose a risk to the animal's health. The surrounding tissue can be physically damaged by the injection syringe, and by immunogenic inflammation caused by the abrupt delivery of a bolus of high-titer virus. Aligning injections with optical implants is especially difficult when targeting small regions deep in the brain. To overcome these challenges, we describe a method for coating multiple types of optical implants with films composed of silk fibroin and Adeno-associated viral (AAV) vectors. Fibroin, a polymer derived from the cocoon of Bombyx mori, can encapsulate and protect biomolecules and can be processed into forms ranging from soluble films to ceramics. When implanted into the brain, silk/AAV coatings release virus at the interface between optical elements and the surrounding brain, driving expression precisely where it is needed. This method is easily implemented and promises to greatly facilitate in vivo studies of neural circuit function.

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

Here, we present a method for delivering viral expression vectors into the brain using silk fibroin films. This method allows targeted delivery of expression vectors using silk/AAV coated optical fibers, tapered optical fibers, and cranial windows.

在 vivosilk/aav 薄膜在光基因表达中的靶向表达

The quest to understand how neural circuits process information in order to drive behavioral output has been greatly aided by recently-developed optical ...

神经科学

<meta charset="utf8"></head><body>FN-Silk 膜的形成和附着到细胞培养物的插入物上

Nanofibrillar Basement Membrane Mimic Made of Recombinant Functionalized Spider Silk in Custom-Made Tissue Culture Inserts

Replicating tissue barriers&#160;is critical for generating relevant in vitro models for evaluating novel therapeutics. Today, this is commonly done using tissue culture inserts with a plastic membrane, which generates an apical and a basal side. Besides providing support for the cells, these membranes come far from emulating their native counterpart, the basement membrane, which is a nanofibrillar, protein-based matrix. In this work, we show a simple way to considerably improve the biological relevance of the tissue culture inserts by replacing the plastic membrane with one made from a pure recombinant functionalized spider silk protein. The silk membrane forms through self-assembly and will spontaneously adhere to a membrane-free tissue culture insert, where it can provide support for cells. Custom-designed tissue culture inserts can be printed using a standard 3D printer, following the instructions provided in the protocol, or commercial ones can be purchased and used instead. This protocol shows how the culture system with silk membranes in inserts is set up and, subsequently, how the same cell culturing techniques that are used with traditional, commercially available inserts can be implemented.

<meta charset="utf8"></head><body>作者聚焦：使用重组功能化蜘蛛丝膜增强体外细胞培养模型

Tissue culture inserts with plastic membranes are the golden standard in cell culture labs as permeable supports to establish cell layers and models of barrier tissues. Herein, we present a simple method to replace the plastic membrane with a more biologically relevant membrane made from a recombinant functionalized spider silk protein.

纳米原纤维基底膜模拟物，由重组功能化蜘蛛丝制成，用于定制组织培养小室

Replicating tissue barriers&#160;is critical for generating relevant in vitro models for evaluating novel therapeutics. Today, this is commonly done using ...

纳米丝绸制造和药物输送应用

摘要

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使用微流体装置生产siRNA负载的脂质纳米颗粒

在 vivosilk/aav 薄膜在光基因表达中的靶向表达

纳米原纤维基底膜模拟物，由重组功能化蜘蛛丝制成，用于定制组织培养小室

纳米原纤维基底膜模拟物，由重组功能化蜘蛛丝制成，用于定制组织培养小室

纳米原纤维基底膜模拟物，由重组功能化蜘蛛丝制成，用于定制组织培养小室