这项工作是由德意志研究联合会授予 HI-698 / 10-1和RU-1652 / 1-1的支持。我们感谢琳五月优秀的技术援助和公司DYOMICS有限公司，耶拿的盛情支持。

注：所有的程序都是由区域动物委员会，并按照有关伦理使用动物的国际准则认可。 1.准备材料和仪器<ol><li>自发形成囊泡分散液的制备（SFV） <ol><li>溶解和制备原液以下磷脂：214毫克/毫升的蛋磷脂酰胆碱（EPC）134毫克/毫升胆固醇，122毫克/毫升1,2- distearoyl- SN -glycero -3- phosphoethanolamine- -N - [甲氧基（聚乙二醇）-2000]（铵盐）（MPEG 2000 -DSPE）和2毫克/毫升1,2-dioleoyl- SN -glycero -3- phosphoethanolamine- -N - （7-硝基2-1,3苯并恶-4-基）（铵盐）的氯仿，并存储在玻璃小瓶（NBD-DOP​​E）。 </li><li>提供大约3毫升氯仿在一个圆底烧瓶中并磷脂原液适当量转移到圆底烧瓶中，制备脂质体的EP组成C：澈：的mPEG 2000 -DSPE以6.5的摩尔比：3：0.5。脂质体双荧光标记添加0.3％（摩尔）NBD掺杂到脂质的解决方案。 </li><li>蒸发从减压（300毫巴）在55℃下使用旋转蒸发器将有机磷脂溶液中的氯仿。 </li><li>后，形成均匀的磷脂膜，降低压力至10毫巴1-2小时，以除去残留的氯仿存款。 </li><li>而氯仿蒸发，溶解DY-676-COOH（6.181微米）在10毫米的Tris缓冲液pH 7.4，并填写与液氮杜瓦瓶。接通超声波浴设定在50℃。 </li><li>传送DY-676-COOH（6.181微米）溶液到圆底烧瓶水合干燥的磷脂膜和涡大力直到自发形成囊泡（SFV）分散形式的适当体积（0.5-1毫升）中。确保所有的磷脂的分散，以避免脂质损失。 </li><li>仔细TRAnsfer圆底烧瓶含有SFV分散到液氮中并冷冻的分散3-5分​​钟。放置在圆底烧瓶中进在50℃的超声波浴中解冻该分散体然后旋涡振荡分散大力为1-2分钟。重复此步骤，六次，总共七个冻融循环。 </li></ol></li><li> SFV挤压，形成均匀的脂质体囊<ol><li>转移SFV分散到1ml注射器（注射器-a）和使用LiposoFast-基本挤出成注射器-b至100纳米的聚碳酸酯膜挤出的分散体。 </li><li>挤出从注射器-b中的分散性，回注射器一个，然后重复该循环10次。由于挤压，在从一个模糊的外观，以清晰的色散随时间的变化注射器的解决方案。经过十次（20单挤压步骤）从设备中取出注射器b和挤出分散从注射器直接上一次进入无菌1.5毫升反应管中。 </li></ol></li><li>脂质体的净化封装DY-676-COOH免费染料<ol><li>准备使用浸泡在10mM Tris缓冲液pH 7.4（柱长28厘米，直径0.8厘米）G25珠的柱色谱纯化。 </li><li>传送0.5毫升挤出囊泡分散体到凝胶床并且让样品漏极到凝胶基质中。 </li><li>洗脱用10mM Tris缓冲液pH为7.4（ 图1A）的脂质体和完全洗柱直至游离染料下水道出列。如果需要的话，收集并根据制造商的说明通过脱盐和脱水循环的游离染料。 </li><li>通过超速离心（200000×g离心2小时，在8℃）浓缩该洗脱的脂质体，然后它们分散在无菌的10mM Tris缓冲液的pH值，7.4足够体积。 </li></ol></li><li>封装DY-676-COOH浓度的定量<ol><li>通过溶解DY-676-COOH（0，82，124准备校准曲线，247，494，988纳米）在10mM Tris缓冲液，pH 7.4中含有0.1％的Triton X100。 </li><li>溶解的脂质体的2微升（的50毫摩尔/升的库存100纳摩尔最终脂质）中5分钟，在室温下，在含有1％Triton X100的破坏囊泡和释放包封的染料加入100μl的Tris缓冲液中。然后稀释用10mM Tris缓冲，pH为7.4至0.1％的最终的Triton-X100浓度样品（V / V）制备1毫升的总体积。准备好所有的样品一式两份。 </li><li>测量所有样品的吸收和发射（免费DY-676-COOH和Triton-X100处理的脂质体）在激发λ= 645 nm和发射λ= 700纳米。建立和使用游离染料的校准曲线来确定包封染料的浓度。 </li></ol></li><li>脂质体的表征<ol><li>确定脂质体通过动态光散射的大小和ζ电位。稀释的脂质体的样品与过滤灭菌（0.2微米）的10mM Tris缓冲液pH 7.4到交流oncentration 100-300微米（脂）。传送稀释的样品置于低体积一次性杯，并根据制造商的说明测量样品。 </li><li>通过电子显微镜表征脂质体根据标准方法来证明脂质体囊泡的大小，完整性和均匀性。 </li></ol></li></ol> 2.验证荧光淬灭，并准备脂质体的激活<ol><li>荧光猝灭和激活的理化分析<ol><li>准备两个1.5毫升管的唇-Q和2管免费DY-676-COOH。传输100纳摩尔总脂质（2.38微升的42毫摩尔/ L的唇-Q原液，含有包封的DY-676-COOH的138微克/毫升）成相应的管中。自由转会DY-676-COOH相当于唇-Q的染料含量（0.38微克导致例如，从138微克/ 1000微升2.38 X微升唇-Q使用）。孵育1桶每个探针在4℃下的e和冻结第二管在-80℃下过夜（16小时）。 </li><li>升温的加热块至30℃。填用碎冰冷却箱和平衡的10毫摩尔Tris缓冲液pH 7.4的等分试样至室温。 </li><li>从4℃除去探针和在室温下平衡（用铝箔包裹以避光），并迅速解冻探针-80℃，在30℃下进行5分钟。将它们转移至室温（也包在铝箔避光保护）之前寒意在冰上解冻的探针1分钟。 </li><li>添加的10mM Tris缓冲液（pH7.4）中，以各探针的100微升最终体积并平衡所有的探针在RT 10分钟。 </li><li>吸管80微升每种探针的进入低容积的玻璃比色杯中，并从上一个分光计400-900毫微米测量每个探测器的吸收。将探头返回到其相应的管中。 </li><li>转移80微升每个探针到合适的玻璃比色杯并测量通过激励探针上的荧光分光光度计的荧光发射在674 nm和从694-800纳米测量荧光。 </li></ol></li><li>细胞吸收和荧光激活<ol><li>根据标准条件（37℃，5％CO 2和95％潮湿气氛）得到并培养在其相应的培养基以下细胞系。这里，使用鼠巨噬细胞系J774A.1（Dulbecco改良的Eagle培养基补充有10％（V / V）胎牛血清），人成胶质细胞瘤细胞系，U-118MG（含有必需的维生素和10％MEM（体积/ ⅴ）胎牛血清）和人纤​​维肉瘤细胞系HT-1080（RPMI，用5％FCS）中。 </li><li>外套8孔腔室载玻片用聚L-赖氨酸（添加100微升0.001％聚-L-赖氨酸，以每孔，并孵育在37℃下进行10分钟。吸溶液并让室载玻片干涸至少4小时，在RT在无菌工作台，冲洗玻片3用200μlHank氏缓冲盐溶液次直到需要再密封它们与石蜡和铝箔，并储存在4℃。 </li><li>而该腔室载玻片枯竭，直到需要过滤消毒脂质体和染料溶液，并储存于4℃。 </li><li>探头摄取分析全身体内 NIR荧光成像系统，准备5个小培养瓶每个3测试细胞系（15瓶计）。种子2×10 6 J774A.1 U-118MG和每个培养瓶中的HT-1080细胞用5ml各自的培养基中（在五元组），并生长16-24小时。平行于培养瓶中分别接种30,000个细胞每个细胞系（J774A.1和HT-1080）或20,000个细胞（U-118MG）至2孔腔室载玻片中生长和在500μl培养基中16-24小时。 </li><li>第二天，加入唇-Q 100纳摩尔（脂质终量），以每细胞系，并在腔室载玻片每个细胞系的一个孔2烧瓶中。立即传输每个细胞系1烧瓶至4℃，并在第二个烧瓶回培养箱。 注：该探针的量添加到该烧瓶中，并在腔室玻片是相同的，使得上腔室玻片的浓度高10倍（5毫升是500微升培养基）。这是必要的，因为微观检测比在来自瓶中的细胞沉淀被成像的近红外荧光成像系统较不敏感。 </li><li>添加游离DY-676-COOH在相当于唇-Q的染料含量的细胞中每个细胞系，并在腔室载玻片每个细胞系的一个孔2烧瓶的浓度，然后立即传送每个细胞系1烧瓶至4℃（能量消耗），并一起另一烧瓶与腔滑动回到培养箱中。孵育所有细胞24小时，在相应的条件。将细胞在烧瓶中不含探针用作未处理的对照。 </li></ol></li><li> NIRF成像和半定量分析<ol><li>经过24小时培养的持续时间，通过用Hanks洗涤细胞2次收获细胞在烧瓶缓冲在500μl管盐溶液（HBSS），然后通过离心（5分钟，在200×g离心）刮去细胞在500μl的HBSS和沉淀。 </li><li>放置管子与细胞小球（和HBSS）在一个NIRF成像和图像使用滤波器对激励（615-665纳米）和发射（切&gt; 700纳米）。 </li><li>扣除自体荧光，并根据制造商的说明书评估靶的强度与自体荧光。这会给荧光强度作为平均信号（定标计数/秒），这代表缩放对于曝光时间，相机增益，分级和比特深度后计数的水平，从而使该测量是在彼此之间可比较的半定量水平。 </li></ol></li><li>共聚焦显微镜分析<ol><li>经过24小时培养后，洗涤2次，用500μL的HBSS收获上腔室载玻片中的细胞。 </li><li>修复了CELLS用含有3.7％（V / V）的甲醛进行30分钟，在室温加入200μlHBSS中。 </li><li>而固定是怎么回事，淡化色斑DNA，赫斯特-33258 1:50安装解决方案。 </li><li>固定后，洗涤细胞2次用HBSS，然后从玻璃载片分开的腔室。添加包含在对应于所述腔室玻片的孔中的每个点的DNA的染色50μl的安装方案。覆盖细胞用玻璃盖玻片，密封透明指甲油和风干10分钟边在室温（暗）。 </li><li>在合适的荧光显微镜或共焦显微镜图像的细胞。用于相应部件的可视化下面的激发和发射设置：原子核（赫斯特-33258：激发405纳米，发射420-480纳米）。 NBD-DOP​​E（脂质体脂质：激发488纳米，发射530纳米）。 DY-676-COOH（NIR荧光染料：激发波长633-645，650-700的发射波长）。 注：确保荧光显微镜vailable配备合适的过滤器，允许激发波长和发射超过630纳米高。 </li></ol></li></ol> 3.脂质体为基础的炎症活体荧光成像<ol><li>动物和材料的制备<ol><li>房子8-12周龄雄性NMRI小鼠体重与食物和水随意在标准条件下约36克</li><li>实验开始七天前，让所有小鼠的低脱镁叶绿饮食，以减少组织自发荧光。 </li><li>每个实验开始二十四小时之前，剃小鼠在期望区域（ 例如，整个如果后腿水肿成像所需的背部区域）。 </li><li>称重动物并计算探针的量将每只小鼠（LIP-Q和唇dQ的以10微摩尔（脂质浓度喷射）每公斤体重和自由DY-676-COOH（相当于染料的唇-Q使用的内容） 。 </li><li>图像中的动物全身NIR荧光成像器与用于细胞沉淀相同的设置。这种测量提供了动物的自发荧光。 </li><li>溶解10毫克酵母聚糖-A在1ml等渗盐水溶液和过夜储存于4℃。 </li></ol></li><li>诱导炎症和体内 NIRF成像<ol><li>准备每只小鼠3注射器包含以下解决方案。填充1注射器用50μl酵母聚糖A溶液（10毫克/毫升）和第二注射器用50μl等渗盐水溶液。与探针，即唇-Q和唇-DQ（10微摩尔/ kg体重（脂肪））被指定为试验动物和免费DY-676-COOH（集中在唇-Q）控制动物填补第三注射器。确保探针用无菌HBSS稀释至150微升最终体积。 </li><li>适用于动物的眼睛眼霜以避免干燥，麻醉动物用2％异氟醚，直到他们都深深睡着了，在爪子触摸时没有反应（这大约需要2分钟）。 </li><li>放置在鼠标上的暖垫（仍在麻醉）和注入的右后腿的酵母聚糖A溶液皮下和在左后腿的盐溶液。立即静脉注射探头和图像的动物之后再注入/测量记录时间（T = 0小时）。保存生成的图像作为图像数据集，并重复上述步骤，对所有其他动物和相应的探针。 </li><li>图像中的动物，每2小时，10小时后喷射，然后在24小时后喷射，确保在测量室的阶段是温暖（例如，通过将暖垫下方），为了避免低温。每次测量后，将动物的食物和水随意一个笼子里，放在笼中温带动物室。安乐死的动物通过首先麻醉用2％异氟烷，直到动物不再反应以触摸，然后用二氧化碳安乐死5-10分钟，使得确保动物停止呼吸完全尸僵发生。 </li><li>根据该可在线评估器官的标准协议（http://www.freebookez.com/mouse-dissection-lab-report/）和图像解剖小鼠。 </li><li>根据制造商的说明，通过首先扣除动物（去混合），然后分配的自体荧光的兴趣（左后腿与盐水）和发炎区域的目标荧光（右后腿与酵母聚糖-A）中的区域的整体荧光评估测量结果。 </li></ol></li></ol>

The authors have nothing to disclose.

因为脂质体还可以作为递送系统用于荧光染料，它们使目标疾病的成像。高浓度的荧光染料如NIRF染料，这里使用DY676-COOH的封装导致截留染料的荧光淬灭的高水平。荧光猝灭，这种现象以高浓度看到的许多荧光团可被利用在几种体内成像应用，其中，具有高灵敏度的目标区域的可靠的检测被征求。使用脂质体还提供了保护，这是不可缺少的用于体内应用的染料。 ...

脂质体已被广泛研究，并作为最生物相容性生物医学的药物递送系统的临床应用1,2之一。它们主要由磷脂和胆固醇，这两者都是生物相容的化合物模仿天然细胞膜份。而亲水性物质可在含水内部被截留，亲脂性试剂可以脂质体磷脂双层3内被引入。脂质体的含水内部中的封装的物质给予保护，防止降解在体内 ，也防止了主机系统从用于疾病的治疗的细胞毒性药物，例如化学治疗旨在破坏肿瘤细胞的毒性作用。脂质体表面与像聚乙二醇聚合物的修饰（PEG化）进一步延伸体内的脂质体的血液循环时间，由于空间位稳定4。 Moreov呃脂质体可隔离的高浓度的几种物质，如蛋白质5,6，亲水性物质7,8和酶9。因此，它们作为可靠的临床治疗和诊断工具，值得他们的批准用于递送细胞毒性药物，如阿霉素治疗癌症4。由于其灵活性，脂质体还可以装载有荧光染料用于诊断和图像引导手术目的。 荧光成像提供了一个高性价比的和非侵入体内诊断工具，但需要一些基本要求。它可以证明，这适合最适合于体内成像的荧光染料具有特征吸收和发射最大值在光分散和散射，以及组织自发荧光从水始发和血红蛋白是低的范围内。因此，这样的探针具有其650和900纳米之间10 ABS /青霉最大值。<html>除此之外，无论是在体外和体内的荧光染料的稳定性是至关重要的，如调理作用和快速清除可大大限制了它们用于体内成像应用11。其他效果，例如差的稳定性和低灵敏度或观察与吲哚花青绿（ICG）12-16对靶器官的细胞毒作用，是不希望的，并用于体内成像设计探针时，必须考虑到。这些观察导致了若干临床前的近红外荧光染料，纳米颗粒以及用于体内成像的炎性过程，癌症和用于图像引导手术17-20新技术的积极发展。尽管大多数的临床前NIRF（近红外荧光）的稳定性的染料在体外 ，其通过肝脏和肾脏快速灌注和清除阻碍它们在疾病和炎症过程的体内光学成像的使用。 ntent“&gt;因此，我们提出了一个协议对荧光染料的封装，如良好表征的近红外荧光染料DY-676-COOH，在脂质体以其倾向自淬火，在相对 ​​高浓度的21，在高浓度的H-二聚体形成和/或PI-层叠荧光团分子的增加之间位于彼此的福斯特半径结果在荧光分子之间荧光共振能量转移（FRET）内的荧光团的分子之间的相互作用，在低浓度的空间中，从而防止PI-堆积作用和H-二聚体形成并导致高的荧光发射。高，低浓度和伴随荧光猝灭和激活之间的开关是一种很有前途的策略，可被利用为光学成像22上 。在这方面，封装的高浓度的NIRF染料的DY-676-COOH在脂质体的含水内部更发vorable用于体内成像比游离染料。该方法的挑战在于在正确的封装首先和其次，在从包封高浓度的染料产生的利益的验证。与游离染料与非淬火脂质体制剂与低浓度的染料的比较猝灭脂质体的成像特性，也是必不可少的。我们显示通过简单的，但高度有效膜水化和挤压协议结合交替冻结和解冻循环是DY-676-COOH的淬火浓度的脂质体中的封装是可行的。用于制备脂质体的其它方法，如逆相蒸发法23以及乙醇注入法24使脂质体制剂具有高包封率的许多亲水性的物质。然而，要被封装的物质的性质可以影响所述包封效率。实际上，这里介绍的薄膜水化和挤压协议揭示了DY-676-COOH的封装效率最高。为了说明DY-676-COOH，一个酵母多糖诱发的水肿模型，它允许在几个小时内的炎症过程的研究的脂质体包封的好处，被使用。这里，它被证明脂质体与高浓度的包封的DY-676-COOH是在炎症过程中比游离染料或未淬火脂质体制剂具有低染料浓度的体内光学成像更适合于全身。因而底层协议提供了一种简单而快速的方法，以产生荧光猝灭脂质体和在体外和体内的活化和成像潜在的有效性。

<table border="0" cellpadding="0" cellspacing="0" width="872">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Name of Material/ Equipment</td>
			<td style="width:179px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:267px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:309px;">Materials and equipments for preparation of liposomes</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">egg phospahtidylcholine</td>
			<td>Avanti Polar Lipids</td>
			<td>840051P</td>
			<td>Dissolve in Chloroform and store in glass vials (214 mg/ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cholesterol</td>
			<td>Sigma</td>
			<td>C8667</td>
			<td>Dissolve in Chloroform and store in glass vials (134 mg/ml)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt)</td>
			<td>Avanti Polar Lipids</td>
			<td>880120P</td>
			<td>Dissolve in Chloroform and store in glass vials (122 mg/ml)</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:309px;">1,2-dioleoyl-snglycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt)</td>
			<td>Avanti Polar Lipids</td>
			<td>810145P</td>
			<td>Dissolve in Chloroform and store in glass vials (2mg/ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Sartorius MC1 (d = 0.01 mg)</td>
			<td>Sartorius AG</td>
			<td>Research RC 210 P</td>
			<td>used for weighing the phospholipids</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Rotavapor</td>
			<td>B&#252;chi Labortechnik AG</td>
			<td>R-114</td>
			<td>used for hydration of phospholipid film</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Waterbath</td>
			<td>B&#252;chi Labortechnik AG</td>
			<td>R-481</td>
			<td>used for hydration of phospholipid film</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Vacuum Controller</td>
			<td>B&#252;chi Labortechnik AG</td>
			<td>B-720</td>
			<td>used for hydration of phospholipid film</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Vacobox</td>
			<td>B&#252;chi Labortechnik AG</td>
			<td>B-177</td>
			<td>used for hydration of phospholipid film</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Circulation Chiller</td>
			<td style="width:179px;">LAUDA DR. R. WOBSER 
			GMBH &#38; CO. KG</td>
			<td>WKL 230</td>
			<td>used for hydration of phospholipid film</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DY-676-COOH</td>
			<td>Dyomics GmbH</td>
			<td>676-00</td>
			<td>Dissolve in 10 mM Tris and store stock at -20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-(Hydroxymethyl)-aminomethan</td>
			<td>Applichem</td>
			<td>A1086</td>
			<td>buffer 10 mM, pH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trichlormethan</td>
			<td>Carl Roth GmbH + Co. KG</td>
			<td>Y015.2</td>
			<td>used for liposome preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sonicator</td>
			<td>Merck Eurolab GmbH</td>
			<td>USR 170 H</td>
			<td>used for liposome preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex Genie 2 (Pop-off Cup, No. 146-3011-00)</td>
			<td>Scientific Industries Inc.</td>
			<td>SI-0256</td>
			<td>used for liposome preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sephadex G25 medium&#160;</td>
			<td>GE Healthcare Europe GmbH</td>
			<td>17-0033-01</td>
			<td>used for liposome purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X100</td>
			<td>Ferak Berlin GmbH</td>
			<td>505002</td>
			<td>used to destruct liposomes&#160; for dye quantification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LiposoFast-Basic</td>
			<td>Avestin Inc.</td>
			<td style="width:119px;"></td>
			<td>used for the extrusion of liposomes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polycarbonate filter membrane, 100 nm (Whatman Nucleopore Trans Etch Membrane, NUCLEPR PC 19 MM, 0.1 U)</td>
			<td>VWR</td>
			<td style="width:119px;"></td>
			<td>used for the extrusion of liposomes via LiposoFast-Basic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluostar Optima</td>
			<td>BMG Labtech</td>
			<td style="width:119px;"></td>
			<td>used for dye quantification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zetasizer Nano ZS</td>
			<td>Malvern</td>
			<td style="width:119px;"></td>
			<td>used for the determination of liposome size and zetapotential</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Ultracentrifuge&#160;</td>
			<td style="width:179px;">Beckmann Coulter GmbH</td>
			<td style="width:119px;">XL 80</td>
			<td>used for concentration of the samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Rotor</td>
			<td style="width:179px;">Beckmann Coulter GmbH</td>
			<td style="width:119px;">SW 55 TI</td>
			<td>used for concentration of the samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;"></td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:309px;">Materials and equipments for the evaluation of liposome and optical imaging&#160;</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;"></td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Zymosan-A from Saccharomyces cereviciae</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:119px;">Z4250-250MG</td>
			<td>used for induction of inflammation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Isotonic Saline (0.9)</td>
			<td style="width:179px;">Fresenius GmbH</td>
			<td style="width:119px;">PZN-2159621</td>
			<td>used for the dilution of Zymosan-A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Isoflurane vaporizer</td>
			<td>Ohmeda Isotec 4</td>
			<td style="width:119px;"></td>
			<td>used for anesthesizing animals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Isoflurane</td>
			<td>Actavis GmbH&#160;</td>
			<td style="width:119px;">PZN-7253744</td>
			<td>anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermo Mat Pro 20 W</td>
			<td style="width:179px;">Lucky Reptile</td>
			<td style="width:119px;">61202-HTP-20</td>
			<td>used to keep animals warm during anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Omnican-F (1 ml injection)&#160;</td>
			<td style="width:179px;">Braun</td>
			<td style="width:119px;">PZN-3115465</td>
			<td>used for subcutaneous and intravenous application of probes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Panthenol eye cream</td>
			<td style="width:179px;">Jenapharm</td>
			<td style="width:119px;">PZN-3524531</td>
			<td>used to prevent dryness of the eyes of animals during anesthesia</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:309px;">Hanks buffered saline solution</td>
			<td style="width:179px;">PAA Laboratories /Biochrom AG</td>
			<td style="width:119px;">L2045</td>
			<td>w/o Mg2+, Ca2+ and phenol red. For dilution of probes and for washing of cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">8-Well chamber slides</td>
			<td style="width:179px;">BD Biosciences</td>
			<td style="width:119px;">354108</td>
			<td>used for cell culture followed by microscopy&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Cell culture flasks</td>
			<td style="width:179px;">Greiner BioOne</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture media</td>
			<td colspan="2">Gibco (life technologies GmbH)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal calf serum&#160;</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-Lysine solution (0,01% - 50&#160;ml)</td>
			<td>Sigma</td>
			<td style="width:119px;">P4832</td>
			<td>used to coat cell culture chamber slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mountant Permafluor</td>
			<td>ThermoScientific&#160;</td>
			<td>S21022-3</td>
			<td>Mounting solution for microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Hoechst-33258</td>
			<td style="width:179px;">AppliChem</td>
			<td style="width:119px;"></td>
			<td>DNA stain for microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;"></td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Hera-Safe</td>
			<td>Heraeus Instruments</td>
			<td style="width:119px;"></td>
			<td>sterile work bench used for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HERA cell</td>
			<td>Heraeus Instruments</td>
			<td style="width:119px;"></td>
			<td>Incubator used for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">LSM510-Meta</td>
			<td style="width:179px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td>used for confocal microscopy</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:309px;">Maestro-TM in vivo fluorescence imaging system</td>
			<td style="width:179px;">CRi, Woburn</td>
			<td style="width:119px;"></td>
			<td>used for whole body fluorescence imaging of small animals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Spectrophotometer (Ultrospec 4300 pro UV)</td>
			<td style="width:179px;">GE Healthcare</td>
			<td style="width:119px;"></td>
			<td>used for measurement of absorption</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Spectrofluorometer (Jasco FP-6200)</td>
			<td style="width:179px;">Jasco</td>
			<td style="width:119px;"></td>
			<td>used for measurement of fluorescence emission</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;"></td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Animals</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NMRI mice (8-12 weeks old, male)</td>
			<td style="width:179px;">Elevage Janvier, France</td>
			<td style="width:119px;"></td>
			<td>used for inflammation trials</td>
		</tr>
	</tbody>
</table>

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.

高浓度的荧光染料，如这里所使用的脂质体的含水内部的NIRF染料DY676-COOH的封装导致荧光猝灭的一个高的水平。荧光猝灭，这种现象可见有许多荧光团以高浓度，可以利用几种体内成像应用，其中一个高灵敏度的目标区域的可靠的检测要求较高。使用脂质体还提供了保护，这是不可缺少的用于体内应用的染料。脂质体的彻底的表征是必要的，包括几个因素，如染料包封，稳定性和脂?...

Watch this Scientific Journal Video about Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging at JoVE.com

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 ...

fluorescence-quenching-liposomal-encapsulated-near-infrared

Research

JoVE Journal

Bioengineering

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

16.1K 次观看.  Jena University Hospital. 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. 

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

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

三维光学分辨率的光声显微镜

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

科研

生物工程

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

阿的Cre - LOX P为检测细胞融合重组方法在体内</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

病毒粒子为<em在体内</em&gt;肿瘤显像

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

成像变性的胶原股<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;通过光引发杂交笼胶原蛋白仿生肽

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

从混合三维培养使用近红外Photoimmunotherapy技术选择性细胞消除

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

RGD功能化水凝胶作为治疗应用的工具合成

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

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

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

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. ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

DNA 纳米管作为研究半聚合物的万能工具

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.