这项工作是由夺标基地的经费计划（工作单位MA041-06-41-4943）的支持。 ON由国家研究理事会博士后研究会士的支持。

1. DIO-LCNP和DIO-LCNP-PEG-澈的制备<ol><li>溶解液晶二丙烯酸酯交联剂（DACTP11，45毫克），3,3&#39;- dioctadecyloxacarbocyanine高氯酸（DIO，2毫克），和自由基引发剂（偶氮二异丁腈，1毫克）聚合于2ml氯仿中。此添加到丙烯酸酯官能化的表面活性剂（AC10COONa，13毫克7毫升）的水溶液。 </li><li>搅拌1小时，并在80％振幅的混合物超声处理5分钟以产生由通过聚合表面活性剂在水包围的有机材料的小滴的细乳液。 </li><li>加热该混合物至64℃的油浴中 以引发两者交联剂和表面活性剂的聚合作为氯仿慢慢蒸发，留下一个由表面活性剂稳定的含DIO-NP悬浮液。 </li><li>通过0.2μm注射过滤器过滤的NP悬浮液（3次），以去除任何聚集。申通再在4℃下将过滤的NP溶液直至进一步使用。 </li><li> PEG-澈的偶联通过EDC耦合DIO-LCNP。 <ol><li>溶解澈-PEG-NH 2·HCl的（PEG-澈，0.9毫摩尔）在25mM HEPES缓冲液（pH7.0）中。 </li><li>制备含有N-羟基磺基琥珀酰亚胺钠盐（NHSS，40毫摩尔），并在从浓缩储备溶液HEPES缓冲液1-乙基-3-（3-（二甲基氨基） -丙基）碳化二亚胺盐酸盐（EDC，400毫摩尔）的工作溶液。 </li><li>立即加入20微升NHSS / EDC的新鲜制备的工作溶液到1.0 DIO-LCNP毫升在HEPES缓冲液中，并搅拌5分钟。 </li><li>加入20μl的澈-PEG-NH 2·HCl的原液至该混合物并搅拌2小时。 </li><li>用台式小型离心机，并通过与平衡的PD-10尺寸排阻层析柱13传递上清液短暂离心以最大速度（〜2000 xg离心）将反应混合物进行30秒Dulbecco氏磷酸盐缓冲盐水（DPBS，0.1倍）。 </li></ol></li></ol> 2. DIO-LCNP和DIO-LCNP-PEG-澈的表征<ol><li> 确认通过凝胶电泳PEG-澈成功的共价结合到DIO-LCNP表面。 <ol><li>溶解0.5克琼脂糖在三 - 硼酸盐电泳50毫升（1×）（TBE; 89毫摩尔Tris（pH值7.6），89毫硼酸，和2mM EDTA）缓冲液中。加热微波炉中的溶液以溶解琼脂糖。让溶液稍微冷却并倒入内容到在电泳箱的凝胶板。将凝胶梳子创建样品孔。 </li><li>一旦凝胶固化，添加所需的量（足以淹没在腔室中的凝胶）电泳缓冲液的室TBE的。 </li><li>添加35微升DIO-LCNP样品（修订与甘油，5％体积/体积）至凝胶的孔中，并在110伏的电压20分钟运行</li><li>图片使用凝胶成像系统无线凝胶个激发和发射滤光片分别在488纳米和500-550纳米。 </li></ol></li><li>通过稀释在PBS中DIO-LCNP溶液（〜200倍稀释）（pH约8，0.1×）和一DLS仪器14测量评估通过动态光散射（DLS）的颗粒尺寸和分布。测量使用合适的zeta电位的测量仪器的DIO-LCNPs的ζ电位。 </li></ol> 3.细胞培养皿中准备交货的实验和成像注:DIO-LCNP标签上的HEK 293T / 17的人胚胎肾细胞（通道5和15之间），为先前4描述的是培养的进行。执行输送实验和如下所述的随后的细胞成像。 <ol><li>通过用牛纤连蛋白（涂覆它们在20微克的浓度制备直径35 mm组织培养皿（装有14性mm 1号盖玻片插入）〜100微升/ ml）的DPBS为在37℃下2小时。 </li><li>除去从培养皿纤连蛋白涂布溶液。收获HEK 293的T1 / 7细胞从T-25烧瓶中由第一洗涤用3ml的DPBS中，然后通过加入2ml胰蛋白酶-EDTA（0.5％胰蛋白酶0.25％的EDTA）的细胞单层。 </li><li>在37℃下〜3分钟的烧瓶中。除去胰蛋白酶-EDTA将烧瓶返回到培养箱中。一旦细胞从烧瓶中脱离，通过加入4 ml完全培养基中和胰蛋白酶（; DMEM; Dulbecco改良的Eagle培养基修正，以含有10％胎牛血清，5％丙酮酸钠和5％抗生素/抗真菌剂）向烧瓶中。通过在细胞计数器计数他们确定在悬浮液中的细胞浓度。 </li><li>调节细胞浓度至〜8×10 4个细胞/用生长培养基毫升。加入3毫升细胞悬浮液，以在孵化过夜盘和文化;第二天，细胞应在约70％汇合，并应准备使用。充足细胞密度为细胞的高百分比的健壮和有效的标记是至关重要的。 </li></ol> 4. DIO和DIO-LCNPs的细胞交付和固定细胞成像<ol><li>在送介质准备DIO，DIO-LCNP和DIO-LCNP-PEG-澈1毫升的解决方案（HEPES-DMEM，含25毫米的HEPES DMEM）稀释DIO 自由和DIO-LCNPs股票方案;适当的DIO浓度对培养细胞会〜1-10微米（表示为在DIO DIO-LCNP的形式免费或DIO任浓度）。 </li><li>使用血清吸管除去从培养皿生长培养基洗细胞单层（参见步骤3）的HEPES的DMEM（每次2毫升清洗）两次。轻轻添加和使用移液管向/从在餐具的边缘除去的HEPES的DMEM进行洗涤。 </li><li>增加±毫升准备好的DIO 免费或DIO-LCNP交付解决方案的培养皿中心和菜回到我ncubator为一段适当的时间（通常为15或30分钟，这取决于实验的需要）。潜伏期较长时间增加的非膜领域更非特异性细胞标记（ 如细胞质）。 </li><li>潜伏期后，取出使用血清吸管的交付解决方案。用DPBS（每次2毫升洗涤）洗涤细胞单层两次。轻轻从盘的边缘添加和删除DPBS到/执行洗涤。 </li><li>固定用4％多聚甲醛（在DPBS中制备）在室温下15分钟的细胞单层。 注意:多聚甲醛是易燃品，对呼吸道有刺激性，并且可能致癌。 </li><li>除去用吸管的多聚甲醛溶液，并通过加入并使用移液管向/从在餐具的边缘除去DPBS轻轻洗涤细胞1次用DPBS（2毫升）中。 </li><li>固定细胞准备使用共聚焦激光扫描被成像为膜状荧光信号的存在显微镜（CLSM）。立即执行的摄像，或者，替换用含有0.05％NaN 3的DPBS介质和菜肴存储在4℃。 注意:为NaN 3是有毒的;使用NaN的3时谨慎操作。 注:以这种方式存储固定样本应48小时为最佳结果中进行成像。 </li></ol>活细胞DIO和DIO-LCNPs和FRET成像5.细胞交付注:在该方法中，将细胞colabeled 6μM每个DIO-LCNP-PEG-澈（其中DIO是FRET供体）和1,1&#39;-双十八烷基-3,3,3&#39;，3&#39;- tetramethylindocarbocyanine高氯酸（DII 免费 ，其中dii是FRET受体）。 DIO从DIO-LCNP-PEG-澈和其掺入释放到质膜是通过在从DIO施主的能量转移到所述的DiI受体的观察增加确认。 <ol><li>与DIO-LCNP-PEG-澈和FRE的DiI顺序标签细胞e。使用在步骤4中描述的方法。 </li><li>染色后，清洗细胞用血清吸管1次用DPBS（2ml）中，并用2毫升活细胞成像溶液（LCIS）更换洗涤缓冲液。 </li><li>在装有加热的孵育室，图像通过在488纳米的4小时的时间内通过激励DIO施主使用共聚焦显微镜（60X物镜）具有FRET成像配置以30分钟的间隔，并收集全发射的活细胞样本的显微镜阶段无论是DIO捐助者和从490-700纳米的DiI受体配备了32通道光谱探测器的光谱。 </li><li>注:有关FRET成像的更多信息，请参考15。 </li><li>确定两个DIO给体和从与DIO-LCNP-PEG-澈和的DiI染色的细胞的DiI受体的时间分辨发射强度。计算时间分辨受主/施主FRET比率（DII EMI / DIO EMI）的图像，这将稳步增加，并最终板盟一旦DIO给体划分成的膜的数量已经达到最大16。 </li></ol> 6. DIO的细胞毒性检测和DIO-LCNPs到HEK 293T / 17细胞注:DIO-LCNP材料的细胞毒性使用基于染料四唑鎓增殖测定17评估。细胞，在不同浓度的物质的存在下模拟交付/标记条件下多孔板中培养。然后将细胞72小时培养以允许增殖。的染料（MTS，（3-（4,5-二甲基-2-基）-5-（3-羧基甲氧基）-2-（4-磺苯基）-2H-四唑）然后被加入到孔中，以及代谢活性细胞将染料成蓝色甲产物。颜色形成的量成正比的活细胞的数量。 <ol><li>收获HEK 293的T1 / 7细胞从T-25烧瓶中通过以下步骤3.2.Seed中描述的方法HEK 293T / 17细胞（5,000个细胞/100μl/孔），以24小时96孔组织培养处理板与文化的井。 </li><li>完全从使用微量孔除去培养基和添加的HEPES的DMEM中加入50μl含有游离 DIO，DIO-LCNPs，或在递增浓度复制井DIO-LCNP-PEG-澈。孵育的细胞在37℃下进行30分钟。 注意:通常情况下，以一式三份或一式四份重复做是足以产生统计学上可靠的数据。 <ol><li>培养后，除去含有使用微量的物料的输送介质并用100微升生长培养基的更换。培养细胞72小时。 </li></ol></li><li>加入20μl四唑基片的至每个孔，将板返回孵化器，并允许颜色形成，以在37℃下继续进行4小时。读出的甲臜产物的吸光度（ABS）在570nm（吸收极小对该STU使用的DIO-LCNPs使用微量滴定板读数器DY）和650纳米（对于非特异性背景吸收的减法）。 </li><li>积微分吸光度值（绝对570 -绝对650）相对于材料浓度，其结果作为控制细胞增殖（在仅细胞培养基的细胞增殖的程度）百分之报告。 </li></ol> 7.数据分析<ol><li>统计学分析数据以方差的单变量分析（ANOVA）。多重比较，应用邦费罗尼的事后检验。提供所有的平均值作为除另有说明外（SEM）的平均值±标准误差。 注:意义可接受的概率P &lt;0.05。 </li></ol>

<ol>
	<li>Nag, O. K., Field, L. D., Chen, Y., Sangtani, A., Breger, J. C., Delehanty, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+actuation+of+therapeutic+nanoparticles:+an+update+on+recent+progress.">Controlled actuation of therapeutic nanoparticles: an update on recent progress.</a> Ther. Deliv. 7 (5), 335-352 (2016).</li><li>Galvao, J., Davis, B., Tilley, M., Normando, E., Duchen, M. R., Cordeiro, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unexpected+low-dose+toxicity+of+the+universal+solvent+DMSO.">Unexpected low-dose toxicity of the universal solvent DMSO.</a> FASEB J. 28 (3), 1317-1330 (2014).</li><li>Spillmann, C. M., Naciri, J., Anderson, G. P., Chen, M. S., Ratna, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+tuning+of+organic+nanocolloids+by+controlled+molecular+interactions.">Spectral tuning of organic nanocolloids by controlled molecular interactions.</a> ACS Nano. 3 (10), 3214-3220 (2009).</li><li>Spillmann, C. M., Naciri, J., Algar, W. R., Medintz, I. L., Delehanty, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifunctional+Liquid+Crystal+Nanoparticles+for+Intracellular+Fluorescent+Imaging+and+Drug+Delivery.">Multifunctional Liquid Crystal Nanoparticles for Intracellular Fluorescent Imaging and Drug Delivery.</a> ACS Nano. 8 (7), 6986-6997 (2014).</li><li>Timmers, M., Vermijlen, D., Vekemans, K., De Zanger, R., Wisse, E., Braet, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+DiO-labelled+tumour+cells+in+liver+sections+by+confocal+laser+scanning+microscopy.">Tracing DiO-labelled tumour cells in liver sections by confocal laser scanning microscopy.</a> J. Microsc. 208 (Pt 1), 65-74 (2002).</li><li>Mufson, E. J., Brady, D. R., Kordower, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+neuronal+connections+in+postmortem+human+hippocampal+complex+with+the+carbocyanine+dye+DiI.">Tracing neuronal connections in postmortem human hippocampal complex with the carbocyanine dye DiI.</a> Neurobiol Aging. 11 (6), 649-653 (1990).</li><li>K&#246;bbert, C., Apps, R., Bechmann, I., Lanciego, J. L., Mey, J., Thanos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+concepts+in+neuroanatomical+tracing.">Current concepts in neuroanatomical tracing.</a> Prog. Neurobiol. 62 (4), 327-351 (2000).</li><li>Honig, M. G., Hume, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dil+and+DiO:+versatile+fluorescent+dyes+for+neuronal+labelling+and+pathway+tracing.">Dil and DiO: versatile fluorescent dyes for neuronal labelling and pathway tracing.</a> Trends Neurosci. 12 (9), 333-341 (1989).</li><li>Gan, W. B., Bishop, D. L., Turney, S. G., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vital+imaging+and+ultrastructural+analysis+of+individual+axon+terminals+labeled+by+iontophoretic+application+of+lipophilic+dye.">Vital imaging and ultrastructural analysis of individual axon terminals labeled by iontophoretic application of lipophilic dye.</a> J. Neurosci. Methods. 93 (1), 13-20 (1999).</li><li>Godement, P., Vanselow, J., Thanos, S., Bonhoeffer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+in+developing+visual+systems+with+a+new+method+of+staining+neurones+and+their+processes+in+fixed+tissue.">A study in developing visual systems with a new method of staining neurones and their processes in fixed tissue.</a> Development. 101 (4), 697-713 (1987).</li><li>Ragnarson, B., Bengtsson, L., Haegerstrand, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+with+fluorescent+carbocyanine+dyes+of+cultured+endothelial+and+smooth+muscle+cells+by+growth+in+dye-containing+medium.">Labeling with fluorescent carbocyanine dyes of cultured endothelial and smooth muscle cells by growth in dye-containing medium.</a> Histochemistry. 97 (4), 329-333 (1992).</li><li>Korkotian, E., Schwarz, A., Pelled, D., Schwarzmann, G., Segal, M., Futerman, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevation+of+intracellular+glucosylceramide+levels+results+in+an+increase+in+endoplasmic+reticulum+density+and+in+functional+calcium+stores+in+cultured+neurons.">Elevation of intracellular glucosylceramide levels results in an increase in endoplasmic reticulum density and in functional calcium stores in cultured neurons.</a> J. Biol. Chem. 274 (31), 21673-21678 (1999).</li><li>Garrett, R. H., Grisham, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biochemistry. , (2013).</li><li>Berne, B. J., Pecora, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dynamic Light Scattering. , 41155-41159 (2000).</li><li>Kremers, G. J., Piston, D. W., Davidson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Basics of FRET Microscopy. , (2016).</li><li>Chen, H., Kim, S., Li, L., Wang, S., Park, K., Cheng, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Release+of+hydrophobic+molecules+from+polymer+micelles+into+cell+membranes+revealed+by+Fo&#776;rster+resonance+energy+transfer+imaging.">Release of hydrophobic molecules from polymer micelles into cell membranes revealed by Fo&#776;rster resonance energy transfer imaging.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 6596-6601 (2008).</li><li>Campling, B. G., Pym, J., Galbraith, P. R., Cole, S. P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+MTT+assay+for+rapid+determination+of+chemosensitivity+of+human+leukemic+blast+cells.">Use of the MTT assay for rapid determination of chemosensitivity of human leukemic blast cells.</a> Leukemia Res. 12, 823-831 (1988).</li><li>Nag, O. K., Naciri, J., Oh, E., Spillmann, C. M., Delehanty, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+raft-mediated+membrane+tethering+and+delivery+of+hydrophobic+cargos+from+liquid+crystal-based+nanocarriers.">Lipid raft-mediated membrane tethering and delivery of hydrophobic cargos from liquid crystal-based nanocarriers.</a> Bioconjug. Chem. 27 (4), 982-993 (2016).</li><li>Karve, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revival+of+the+abandoned+therapeutic+wortmannin+by+nanoparticle+drug+delivery.">Revival of the abandoned therapeutic wortmannin by nanoparticle drug delivery.</a> Proc. Natl. Acad. Sci. U. S. A. 109 (21), 8230-8235 (2012).</li></ol>

作者宣称，他们没有竞争的经济利益。

NMDD的一个持续目标是控制目标和药物制剂的细胞和组织的递送，并同时提高药物疗效相结合。对于此已经构成了显著挑战的一种特定类的药物分子是有节制地在水介质中不溶于疏水性药物/显像剂。这个问题一直困扰的强效药物的转变，从体外细胞培养系统，临床上并导致了一些有前途的药物分子被"搁置"，或遗弃，而不是在临床上进一步推行。渥曼青霉素（Wtmn），例如是磷脂酰肌醇3&#39;?...

由于与活细胞的接口的纳米材料（材料≤100纳米的至少一个维度）的出现，持续目标是采取纳米颗粒（纳米颗粒），用于各种应用的独特大小依赖特性的优势。这些应用包括细胞和组织的标记/成像（ 在体外和在体内 ），实时检测和的药物和其它货物1的受控递送。这种相关的NP性质的实例包括半导体纳米晶体的尺寸依赖性发射（量子点，量子点）;金纳米颗粒的光热特性;脂质体的水性核心的大负荷能力;和碳同素异形体，例如单壁碳纳米管和石墨烯的弹道导电性。 最近，显著的兴趣已经出现在药品和其他货物，如对比度/成像的控制调制使用核动力源男厕所。在这里，基本原理是显著提高/通过提供它作为一个NP制剂优化整体溶解度，递送剂量，循环时间，并最终清除药物的货物。这已经到了被称为NP介导的药物递送（NMDD），并有目前有7种FDA批准的药物NP制剂在临床上用于治疗各种癌症和数百个临床试验的各个阶段。在本质上，目标是"实现事半功倍;"也就是说，要使用NP作为支架通过取较大的表面积的优点用较少的给药施用以提供更多的药物:体积（ 如 ，硬颗粒，如量子点和金属氧化物）纳米粒子的或它们的大的内部容积为装载大货的有效载荷（ 例如 ，脂质体或胶束）。这里的目的是为了减少多全身递送给药方案的必要性，而在同一时间促进水溶液稳定性和增强的循环，特别是用于有挑战性疏水药物货物，虽然高效，是在水性介质中微溶。 因此，本文所描述的工作的目的是确定使用一种新的NP支架，用于向亲脂质膜双层的具体和受控递送疏水货物的生存能力。对工作的动机是固有的有限的溶解度和难于从含水介质递送疏水性分子的细胞。典型地，这样的疏水性分子的递送需要使用的有机溶剂（ 例如 ，DMSO）或两亲性表面活性剂（ 例如 ，泊洛沙姆），这可能是有毒的和妥协的细胞和组织的生存力2，或胶束载体，其可以具有有限的内部负载能力。这里所选择的NP载体是一种新型液晶的NP（LCNP）先前开发3制剂和先前已示出，实现了〜40倍<html>改进，在抗癌药物阿霉素的培养细胞4的功效。 在本文所述的工作中，所选择的代表性的货物是电位膜染料，3,3&#39;- dioctadecyloxacarbocyanine高氯酸（DIO）。 DIO是已经用于生活和固定神经细胞，膜电位测量顺行和逆行追踪不溶于水的染料，和一般的膜标记5，6，7，8，9。由于其疏水特性，DIO通常直接加入到细胞单层或组织中的结晶形式10，或它在非常高的浓度下培养 （〜1-20μM）从浓度原液11,12稀释后。 内容"&gt;这里，方法是利用以LCNP平台，一个多功能的NP，其内芯是完全疏水性的，其表面是同时的亲水性和适合于生物耦合，作为递送载体用于DIO。DIO掺入合成期间LCNP芯和对NP表面，然后用聚乙二醇化的胆固醇部分官能化，以促进膜的DIO-LCNP合奏的质膜的结合。这种方法产生了一个输送系统，该系统划分的DIO到质膜以更大的保真度和膜滞留时间比DIO的游离形式从本体溶液递送（DIO 免费 ）。另外，该方法表明，DIO的LCNP介导的递送基本调制并驱动染料的特定分区的速率进入亲脂质膜双层。这是实现同时伴随减少游离药物的细胞毒作用〜40％，通过提供它作为一个LCNP配方。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-ethyl-3-(3-(dimethylamino)-propyl)carbodiimide hydrochloride (EDCA)</td>
			<td>ThermoFisher</td>
			<td>E2247</td>
			<td></td>
		</tr>
		<tr>
			<td>3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO)</td>
			<td>Sigma Aldrich</td>
			<td>D4292-20MG</td>
			<td>Hazardous/ make stock solution in DMSO</td>
		</tr>
		<tr>
			<td>Cholesterol poly(ethylene glycol) amine hydrochloride</td>
			<td>Nanocs, Inc.</td>
			<td>PG2-AMCS-2k</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess automated cell counter</td>
			<td>ThermoFisher</td>
			<td>C10227</td>
			<td></td>
		</tr>
		<tr>
			<td>Dioctadecyl-3,3,3&#8242;,3&#8242;-tetramethylindocarbocyanine perchlorate (DiI)</td>
			<td>Sigma Aldrich</td>
			<td>468495-100MG</td>
			<td>Hazardous/ make stock solution in DMSO</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>ThermoFisher</td>
			<td>21063045</td>
			<td>Warm in 37&#176;C before use</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (DPBS)</td>
			<td>ThermoFisher</td>
			<td>14040182</td>
			<td>Warm in 37&#176;C before use</td>
		</tr>
		<tr>
			<td>Dynamic light scattering instrument</td>
			<td>ZetaSizer NanoSeries (Malvern Instruments Ltd., Worcestershire, UK)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin Bovine Protein, Plasma</td>
			<td>ThermoFisher</td>
			<td>33010018</td>
			<td>Make stock solution 1mg/mL using DPBS. Use 20-30 &#181;g/mL for coating MetTek dish, 2 h@ 37&#176;C</td>
		</tr>
		<tr>
			<td>Formaldehyde (16%, W/V)</td>
			<td>ThermoFisher</td>
			<td>28906</td>
			<td>Hazardous, dilute to 4% using DPBS</td>
		</tr>
		<tr>
			<td>Human embryonic kidney cells (HEK 293T/17)</td>
			<td>American Type Culture Collection</td>
			<td>ATCC&#174; CRL-11268&#8482;</td>
			<td></td>
		</tr>
		<tr>
			<td>Live cell imaging solution (LCIS)</td>
			<td>ThermoFisher</td>
			<td>A14291DJ</td>
			<td>Warm in 37&#176;C before use</td>
		</tr>
		<tr>
			<td>MatTek 14 mm # 1.0 coverglass insert cell culture dish</td>
			<td>MatTek corporation</td>
			<td>P35G-1.0-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Modified Eagle Medium (DMEM) containing 25 mM HEPES</td>
			<td>ThermoFisher</td>
			<td>21063045</td>
			<td>Warm in 37&#176;C before use</td>
		</tr>
		<tr>
			<td>N-hydroxysulfosuccinimide sodium salt (NHSS)</td>
			<td>ThermoFisher</td>
			<td>24510</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1si spectral confocal microscope</td>
			<td>Nikon Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain (0.4%)&#160;</td>
			<td>ThermoFisher</td>
			<td>T10282</td>
			<td>mix as a 50% to the cell suspension before counting the cells</td>
		</tr>
		<tr>
			<td>Zeta potential instrument</td>
			<td>ZetaSizer NanoSeries (Malvern Instruments Ltd., Worcestershire, UK)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic Processor</td>
			<td>Sonics and Materials Inc</td>
			<td>GEX 600-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Cetntrifuge</td>
			<td>Benchmark</td>
			<td>Mini-fuge-04477</td>
			<td></td>
		</tr>
		<tr>
			<td>PD-10 Sephadex&#8482; G-25 Medium</td>
			<td>GE Healthcare</td>
			<td>17-0851-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad ChemiDoc XRS Imaging System</td>
			<td>Bio-RAD</td>
			<td>76S/07434</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA(0.25%), phenol red</td>
			<td>ThermoFisher</td>
			<td>25200056</td>
			<td></td>
		</tr>
	</tbody>
</table>

targeted plasma membrane delivery of a hydrophobic cargo encapsulated in a liquid crystal nanoparticle carrier

药物/成像剂对细胞的受控递送是治疗学的发展和对细胞信号过程的研究是至关重要的。最近，纳米粒子（NPS）都表现出这样的运载系统的发展显著的承诺。这里，液晶的NP（LCNP）系递送系统已被用于不溶于水的染料，3,3&#39;- dioctadecyloxacarbocyanine高氯酸（DIO）的受控递送，从NP芯内的等离子体的疏水区双层膜。在纳米颗粒的合成中，将染料有效地掺入疏水LCNP芯，如确认由多个光谱分析。 PEG化的胆固醇衍生物的NP表面（DIO-LCNP-PEG-澈）的缀合使染料加载纳米粒的结合在HEK 293T / 17细胞质膜。时间分辨激光扫描共聚焦显微镜和荧光共振能量转移（FRET）成像证实通从LCNP芯和其插入质膜双层香港专业教育学院DIO的流出。最后，迪欧作为LCNP-PEG-澈交付衰减DIO的细胞毒作用;相比DIO 无批量交付解决方案DIO的NP形式展出少〜30％-40％的毒性。这种做法表明了LCNP平台的效用的有效模式疏水分子货物的具体膜交付和调制。

制备LCNPs其中对NP的疏水核心中装入一个代表膜标记探针来演示LCNP的效用作为有效递送载体为疏水货物。为此目的，选择的货物是高度不溶于水的电位膜标记染料，DIO。 DIO-装载LCNPs（DIO-LCNPs）用与化学成分DACTP11，AC10COONa，和DIO两相微乳液技术合成， 如图 1 18。在这个NP系统中，结晶交联剂DACTP11的共价连接的聚合物网络提供其中DIO驻留的交联网络中的间?...

Watch this Scientific Journal Video about Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier at JoVE.com

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

一种液晶纳米颗粒（LCNP）纳米载体被利用作为用于受控递送疏水货物的活细胞的质膜的车辆。

疏水货物封装有针对性的质膜交付在液晶纳米颗粒载体

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

targeted-plasma-membrane-delivery-hydrophobic-cargo-encapsulated

Research

JoVE Journal

Bioengineering

7.5K 次观看.  Naval Research Laboratory. 一种液晶纳米颗粒（LCNP）纳米载体被利用作为用于受控递送疏水货物的活细胞的质膜的车辆。 

视频: Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the microvasculature are controlled by varying ultrasound pulsing parameters.  Specifically, we are testing whether such approaches may be used to treat malignant brain tumors through drug delivery and microvascular ablation.  Preliminary studies have been performed to determine whether targeted drug-bearing nanoparticle delivery can be facilitated by the ultrasound mediated destruction of "composite" delivery agents comprised of 100nm poly(lactide-co-glycolide) (PLAGA) nanoparticles that are adhered to albumin shelled microbubbles. We denote these agents as microbubble-nanoparticle composite agents (MNCAs). When targeted to subcutaneous C6 gliomas with ultrasound, we observed an immediate 4.6-fold increase in nanoparticle delivery in MNCA treated tumors over tumors treated with microbubbles co-administered with nanoparticles and a 8.5 fold increase over non-treated tumors. Furthermore, in many cancer applications, we believe it may be desirable to perform targeted drug delivery in conjunction with ablation of the tumor microcirculation, which will lead to tumor hypoxia and apoptosis. To this end, we have tested the efficacy of non-theramal cavitation-induced microvascular ablation, showing that this approach elicits tumor perfusion reduction, apoptosis, significant growth inhibition, and necrosis. Taken together, these results indicate that our ultrasound-targeted approach has the potential to increase therapeutic efficiency by creating tumor necrosis through microvascular ablation and/or simultaneously enhancing the drug payload in gliomas.

Insonation of microbubbles is a promising strategy for tumor ablation at reduced time-averaged acoustic powers, as well as for the targeted delivery of therapeutics. The purpose of the present study is to develop low duty cycle ultrasound pulsing strategies and nanocarriers to maximize non-thermal microvascular ablation and payload delivery to subcutaneous C6 gliomas.

超声造影有针对性的治疗胶质瘤的小鼠通过药物轴承纳米粒子交付和微血管消融

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the ...

科研

生物工程

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological research. Perhaps the most widely used physical force to achieve noninvasive manipulation of small particles has been dielectrophoresis(DEP).1 However, DEP on its own lacks the versatility and precision that are desired when manipulating cells since it is traditionally done with stationary electrodes. Optical tweezers, which utilize a three dimensional electromagnetic field gradient to exert forces on small particles, achieve this desired versatility and precision.2 However, a major drawback of this approach is the high radiation intensity required to achieve the necessary force to trap a particle which can damage biological samples.3 A solution that allows trapping and sorting with lower optical intensities are optoelectronic tweezers (OET) but OET's have limitations with fine manipulation of small particles; being DEP-based technology also puts constraint on the property of the solution.4,5
This video article will describe two methods that decrease the intensity of the radiation needed for optical manipulation of living cells and also describe a method for orientation control. The first method is plasmonic tweezers which use a random gold nanoparticle (AuNP) array as a substrate for the sample as shown in Figure 1. The AuNP array converts the incident photons into localized surface plasmons (LSP) which consist of resonant dipole moments that radiate and generate a patterned radiation field with a large gradient in the cell solution. Initial work on surface plasmon enhanced trapping by Righini et al and our own modeling have shown the fields generated by the plasmonic substrate reduce the initial intensity required by enhancing the gradient field that traps the particle.6,7,8 The plasmonic approach allows for fine orientation control of ellipsoidal particles and cells with low optical intensities because of more efficient optical energy conversion into mechanical energy and a dipole-dependent radiation field. These fields are shown in figure 2 and the low trapping intensities are detailed in figures 4 and 5. The main problems with plasmonic tweezers are that the LSP's generate a considerable amount of heat and the trapping is only two dimensional. This heat generates convective flows and thermophoresis which can be powerful enough to expel submicron particles from the trap.9,10 The second approach that we will describe is utilizing periodic dielectric nanostructures to scatter incident light very efficiently into diffraction modes, as shown in figure 6.11 Ideally, one would make this structure out of a dielectric material to avoid the same heating problems experienced with the plasmonic tweezers but in our approach an aluminum-coated diffraction grating is used as a one-dimensional periodic dielectric nanostructure. Although it is not a semiconductor, it did not experience significant heating and effectively trapped small particles with low trapping intensities, as shown in figure 7. Alignment of particles with the grating substrate conceptually validates the proposition that a 2-D photonic crystal could allow precise rotation of non-spherical micron sized particles.10 The efficiencies of these optical traps are increased due to the enhanced fields produced by the nanostructures described in this paper.

Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles.

为增强微型和纳米操纵利用电浆和光子晶体纳米结构

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

有针对性的稳定同位素稀释液相色谱 - 质谱法分析细胞脂质提取

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

微型涡辅助电穿孔的顺序传递分子

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

血浆或尿液的水凝胶纳米粒子收获检测低丰度蛋白

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

荧光淬一个脂质体包封的近红外荧光团的作为一个工具<em&gt;在体内</em&gt;光学成像

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

交变磁场敏感的混合明胶微凝胶药物控释

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and as soft robots. Here, we demonstrate the preparation of shape-responsive liquid crystal elastomers (LCEs) and LCE nanocomposites along with characterization of their shape-responsiveness, mechanical properties, and microstructure. Two types of LCEs &#8212; polysiloxane-based and epoxy-based &#8212; are synthesized, aligned, and characterized. Polysiloxane-based LCEs are prepared through two crosslinking steps, the second under an applied load, resulting in monodomain LCEs. Polysiloxane LCE nanocomposites are prepared through the addition of conductive carbon black nanoparticles, both throughout the bulk of the LCE and to the LCE surface. Epoxy-based LCEs are prepared through a reversible esterification reaction. Epoxy-based LCEs are aligned through the application of a uniaxial load at elevated (160 &#176;C) temperatures. Aligned LCEs and LCE nanocomposites are characterized with respect to reversible strain, mechanical stiffness, and liquid crystal ordering using a combination of imaging, two-dimensional X-ray diffraction measurements, differential scanning calorimetry, and dynamic mechanical analysis. LCEs and LCE nanocomposites can be stimulated with heat and/or electrical potential to controllably generate strains in cell culture media, and we demonstrate the application of LCEs as shape-responsive substrates for cell culture using a custom-made apparatus.

We demonstrate the preparation of siloxane-based and epoxy-based liquid crystal elastomers (LCEs) and LCE nanocomposites. The LCEs are characterized with respect to reversible strain, liquid crystal ordering, and stiffness. As a potential application, we demonstrate their use as shape-responsive substrates in a custom device for active cell culture.

单域液晶的弹性体和液晶橡胶纳米复合材料的制备

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

生物相容的液晶弹性体泡沫的合成中用作细胞支架的三维空间细胞培养

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread chondrocyte death, potentially leading to irreversible breakdown of the joint and the onset of osteoarthritis. Additionally, maintenance of chondrocyte viability is important in osteochondral graft procedures for optimal surgical outcomes. We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine synovial joints after application of controlled mechanical loads or impacts. This method can be used in comparative studies to investigate the effects of different mechanical loading regimens, different environmental conditions or genetic manipulations, as well as different stages of cartilage degeneration on short- and/or long-term vulnerability of in situ articular chondrocytes. The goal of the protocol introduced in the manuscript is to assess the spatial extent of cell injury/death on the articular surface of murine synovial joints. Importantly, this method enables testing on fully intact cartilage without compromising native boundary conditions. Moreover, it allows for real-time visualization of vitally stained articular chondrocytes and single image-based analysis of cell injury induced by application of controlled static and impact loading regimens. Our representative results demonstrate that in healthy cartilage explants, the spatial extent of cell injury depends sensitively on load magnitude and impact intensity. Our method can be easily adapted to investigate the effects of different mechanical loading regimens, different environmental conditions or different genetic manipulations on the mechanical vulnerability of in situ articular chondrocytes.

We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine joints after application of controlled mechanical loads or impacts. This method can be used to investigate how osteoarthritis, genetic factors and/or different loading regimens affect the vulnerability of in situ chondrocytes.

全完整型小鼠软骨外种机械负荷对软骨细胞损伤的实时可视化与分析

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread ...