Name: characterization of intra-cartilage transport properties of cationic peptide carriers
Uploaded: 2020-08-10T13:00:00
Description: Watch this Scientific Journal Video about Characterization of Intra-Cartilage Transport Properties of Cationic Peptide Carriers at JoVE.com

这项工作由美国国防部通过国会指导的医学研究计划（CDMRP）根据W81XWH-17-1-0085合同和国家卫生研究院R03 EB025903-1提供资金。AV由东北大学工程学院院长奖学金资助。

大学批准进行死组织实验。牛关节是从屠宰场商业获得的。
1. 软骨外植提取
<ol><li>使用手术刀（#10刀片），切除脂肪，肌肉，韧带，肌腱和所有其他结缔组织，以暴露软骨从牛膝盖关节的毛骨。</li>
	<li>使用 3 mm 和 6 mm 皮肤冲孔，将垂直冲孔放入软骨以提取圆柱形塞。立即将插头插入包含 500 μL 1x 磷酸盐缓冲盐水 （PBS） 的 48 孔板的单个孔中，并辅以 1% v/v 抗生素抗霉菌。</...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritis+-+An+untreatable+disease.">Osteoarthritis - An untreatable disease.</a> Nature Reviews Drug Discovery. 4 (4), 331-344 (2005).</li><li>Martel-Pelletier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+osteoarthritis.">Pathophysiology of osteoarthritis.</a> Osteoarthritis and Cartilage. 7 (4), 371-373 (1999).</li><li>Sophia Fox, A. J., Bedi, A., Rodeo, S. 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B., 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=A+randomized,+double-blind+study+of+AMG+108+(a+fully+human+monoclonal+antibody+to+IL-1R1)+in+patients+with+osteoarthritis+of+the+knee.">A randomized, double-blind study of AMG 108 (a fully human monoclonal antibody to IL-1R1) in patients with osteoarthritis of the knee.</a> Arthritis Research and Therapy. 13 (4), 125 (2011).</li><li>Evans, C. H., Kraus, V. B., Setton, L. 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K., 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=Avidin+grafted+dextran+nanostructure+enables+a+month-long+intra-discal+retention.">Avidin grafted dextran nanostructure enables a month-long intra-discal retention.</a> Scientific Reports. 10.1, 1-14 (2020).</li><li>Troeberg, L., Nagase, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteases+involved+in+cartilage+matrix+degradation+in+osteoarthritis.">Proteases involved in cartilage matrix degradation in osteoarthritis.</a> Biochimica et Biophysica Acta - Proteins and Proteomics. 1824 (1), 133-145 (2012).</li><li>Kirk, T. B., Wilson, A. S., Stachowiak, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+dehydration+on+the+surface+morphology+of+articular+cartilage.">The effects of dehydration on the surface morphology of articular cartilage.</a> Journal of Orthopaedic Rheumatology. 6 (2-3), 75-80 (1993).</li><li>Ateshian, G. A., Maas, S., Weiss, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solute+transport+across+a+contact+interface+in+deformable+porous+media.">Solute transport across a contact interface in deformable porous media.</a> Journal of Biomechanics. 45 (6), 1023-1027 (2012).</li><li>Arbabi, V., Pouran, B., Weinans, H., Zadpoor, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphasic+modeling+of+charged+solute+transport+across+articular+cartilage:+Application+of+multi-zone+finite-bath+model.">Multiphasic modeling of charged solute transport across articular cartilage: Application of multi-zone finite-bath model.</a> Journal of Biomechanics. 49 (9), 1510-1517 (2016).</li><li>Arbabi, V., Pouran, B., Zadpoor, A. A., Weinans, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+and+finite+element+protocol+to+investigate+the+transport+of+neutral+and+charged+solutes+across+articular+cartilage.">An experimental and finite element protocol to investigate the transport of neutral and charged solutes across articular cartilage.</a> Journal of Visualized Experiments. 2017 (122), (2017).</li><li>Sampson, S. L., Sylvia, M., Fields, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+dynamic+loading+on+solute+transport+through+the+human+cartilage+endplate.">Effects of dynamic loading on solute transport through the human cartilage endplate.</a> Journal of Biomechanics. 83, 273-279 (2019).</li><li>Bajpayee, A. G., Scheu, M., Grodzinsky, A. J., Porter, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrostatic+interactions+enable+rapid+penetration,+enhanced+uptake+and+retention+of+intra-articular+injected+avidin+in+rat+knee+joints.">Electrostatic interactions enable rapid penetration, enhanced uptake and retention of intra-articular injected avidin in rat knee joints.</a> Journal of Orthopaedic Research : Official Publication of the Orthopaedic Research Society. 32 (8), 1044-1051 (2014).</li><li>Bajpayee, A. G., 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=Sustained+intra-cartilage+delivery+of+low+dose+dexamethasone+using+a+cationic+carrier+for+treatment+of+post+traumatic+osteoarthritis.">Sustained intra-cartilage delivery of low dose dexamethasone using a cationic carrier for treatment of post traumatic osteoarthritis.</a> European Cells &amp; Materials. 34, 341-364 (2017).</li><li>Malda, J., 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=Of+Mice,+Men+and+Elephants:+The+Relation+between+Articular+Cartilage+Thickness+and+Body+Mass.">Of Mice, Men and Elephants: The Relation between Articular Cartilage Thickness and Body Mass.</a> PLoS One. 8 (2), 57683 (2013).</li><li>Frisbie, D. D., Cross, M. W., McIlwraith, C. 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此处描述的方法和协议对靶向带负电组织靶向药物输送领域非常重要。由于这些组织中存在带负电的聚集体高密度，因此会形成屏障，从而阻止药物到达其位于基质深处的细胞靶点。为面对这个突出的挑战，药物可进行改性，以纳入带正药的药载体,，可提高,,,药物在组织1、3、4、14、15、16、17、18、19,3,<sup class="...

药物输送到体内带负电的组织仍然是一个挑战，因为药物无法深入组织以达到细胞和基质靶点1。其中几个组织由密集包装的负电荷聚集组成，在组织内产生高负固定电荷密度（FCD）2，并充当大多数大分子3，4,的传递屏障。然而，在带正电荷的药物携带者的协助下，这种带负电荷的组织屏障实际上可以通过静电电荷相互作用转化为药物库，以持续输送药物1、5、6、7（,5,6,7图1）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61340/61340fig01.jpg"> 图1：基于电荷的软骨内软骨交付CPC。将CPC在关节空间的关节内注射。带正电荷的CPC和带负电荷的聚集组之间的静电相互作用，通过软骨实现快速和全深度渗透。这个数字已经修改了从维达达瓦米等人4。<a href="https://www.jove.com/files/ftp_upload/61340/61340fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
最近，短长度的阳离子肽载体（CPC）被设计为创建小型阳离子域，能够携带更大的治疗，以交付给负充电软骨4。为了有效地将药物输送到软骨，用于治疗流行的,8、9和退行性疾病，如骨关节炎（OA）10，药物的治疗浓度必须渗透到组织深处，其中大多数软骨细胞（胆囊细胞）位于组织深处。虽然有几个潜在的疾病修改药物可用，没有一个已获得FDA批准，因为这些不能有效地瞄准软骨12，13。12,因此，评估药物载体的运输特性是预测药物在诱导治疗反应方面的有效性所必需的。在这里，我们设计了三个独立的实验，可用于评估CPC4的均衡吸收率、渗透深度和非均衡扩散率。
为了确保软骨内有足够的药物浓度，可以提供最佳的治疗反应，吸收实验被设计成量化软骨4中的平衡CPC浓度。在此设计中，在软骨与其周围浴池之间的平衡之后，可以使用吸收比确定软骨内（与矩阵绑定或自由）的溶质总量。此比率的计算方法是将软骨内的溶解物浓度与平衡浴的溶解度标准化。原则上，中性溶解体，其通过软骨的扩散不由电荷相互作用辅助，其吸收比将小于1。相反，通过静电相互作用增强的阳离子溶解剂的吸收比大于1。但是，如 CPC 所示，使用最佳正电荷会导致更高的吸收比率（大于 300）4。
虽然软骨内的药物浓度高对于获得治疗效果很重要，但药物在软骨全部厚度中扩散也至关重要。因此，需要进行显示渗透深度的研究，以确保药物到达软骨深处，以便达到基质和细胞靶点，从而提供更有效的治疗。该实验旨在评估溶解液通过软骨的双向扩散，模拟药物在体内关节注射后扩散到软骨。使用共和显微镜进行荧光成像，可以评估渗透到软骨的深度。净粒子电荷在调节药物在基质中扩散的深度方面起着关键作用。需要基于组织 FCD 的最佳净电荷，以便进行阳离子颗粒和阳离子组织基质之间的弱可逆结合相互作用。这意味着任何相互作用都足够弱，使粒子可以脱离矩阵，但可逆的性质，以便它可以绑定到另一个矩阵结合位点在组织4的更深。相反，粒子的正净电荷过多会损害扩散，因为过强的基质结合会防止粒子从软骨表面区域的初始结合位点分离。这将导致生物反应不足，因为大多数目标位点位于组织11的深处。
为了进一步量化结合相互作用的强度，通过软骨分析药物扩散率是有利的。非平衡扩散研究允许比较不同溶解体之间的实时扩散速率。当药物扩散到软骨的浅层、中间和深层区域时，结合相互作用的存在可以极大地改变扩散率。当药物和软骨基质之间存在结合相互作用时，它被定义为有效的扩散性（DEFF）。在这种情况下，一旦所有结合位点被占用，药物的扩散速率由稳态扩散 （DSS ） 控制。不同溶质的DEFF 的比较决定了溶质与基质的相对结合强度。对于给定的溶质，如果 DEFF 和 DSS 在同一数量级内，则意味着在扩散过程中药物和基质之间存在最小的结合。但是，如果DEFF 大于DSS，则存在大量粒子与矩阵结合。
设计的实验单独允许通过软骨对溶质运输进行表征，但是，设计一个带最佳电检的药物载体需要包括所有结果的整体分析。电荷相互作用的弱和可逆性质控制粒子扩散率，并允许高平衡吸收和快速全深度穿透软骨。通过均衡吸收实验，应寻找通过非均衡扩散率研究可以验证的电荷相互作用能表现出高吸收的载体。然而，这些结合相互作用应该是弱和可逆的性质，以允许通过软骨的溶质全厚渗透。理想的药物载体将拥有最佳电荷，使强的结合吸收和高软骨内药物浓度，但不是太强，以阻碍全厚度扩散4。提出的实验将有助于针对药物携带者的电荷组织的设计特征。这些协议用于描述通过软骨4的C CPC运输，但是，这些协议也可以应用于各种药物和药物载体，通过软骨和其他带负电的组织。

<table>
	<tbody>
		<tr>
			<td>316 Stainless Steel SAE Washer</td>
			<td>McMaster-Carr</td>
			<td>91950A044</td>
			<td>For number 5 screw size, 0.14&#34; ID, 0.312&#34; OD</td>
		</tr>
		<tr>
			<td>96-Well Polystyrene Plate</td>
			<td>Fisherbrand</td>
			<td>12566620</td>
			<td>Black</td>
		</tr>
		<tr>
			<td>Acrylic Thick Gauge Sheet</td>
			<td>Reynolds Polymer</td>
			<td>N/A</td>
			<td>For non-equilibrium diffusion and 1-D diffusion transport chamber</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td>100x</td>
		</tr>
		<tr>
			<td>Bovine Cartilage</td>
			<td>Research 87</td>
			<td>N/A</td>
			<td>2-3 weeks old, femoropatellar groove</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher BioReagents</td>
			<td>BP671-1</td>
			<td></td>
		</tr>
		<tr>
			<td>CPC+14</td>
			<td>LifeTein</td>
			<td>LT1524</td>
			<td>Custom designed peptide</td>
		</tr>
		<tr>
			<td>CPC+20</td>
			<td>LifeTein</td>
			<td>LT1525</td>
			<td>Custom designed peptide</td>
		</tr>
		<tr>
			<td>CPC+8</td>
			<td>LifeTein</td>
			<td>LT1523</td>
			<td>Custom designed peptide</td>
		</tr>
		<tr>
			<td>Delicate Task Wipers</td>
			<td>Kimberly-Clark Professional</td>
			<td>34155</td>
			<td></td>
		</tr>
		<tr>
			<td>Dermal Punch</td>
			<td>MedBlades</td>
			<td>MB5-1</td>
			<td>3, 4 and 6 mm</td>
		</tr>
		<tr>
			<td>Economy Plain Glass Microscope Slides</td>
			<td>Fisherbrand</td>
			<td>12550A3</td>
			<td></td>
		</tr>
		<tr>
			<td>Flat Bottom Cell Culture Plates</td>
			<td>Corning Costar</td>
			<td>3595</td>
			<td>Clear, 96 well</td>
		</tr>
		<tr>
			<td>Flexible Wrapping Film</td>
			<td>Bemis Parafilm M Laboratory</td>
			<td>1337412</td>
			<td></td>
		</tr>
		<tr>
			<td>Gold Seal Cover Glass</td>
			<td>Electron Microscopy Sciences</td>
			<td>6378701</td>
			<td># 1.5, 18x18 mm</td>
		</tr>
		<tr>
			<td>Hammer-Driven Hole Punch</td>
			<td>McMaster-Carr</td>
			<td>3427A15</td>
			<td>1/2&#34; Diameter</td>
		</tr>
		<tr>
			<td>Hammer-Driven Hole Punch</td>
			<td>McMaster-Carr</td>
			<td>3427A19</td>
			<td>3/4&#34; Diameter</td>
		</tr>
		<tr>
			<td>Laser</td>
			<td>Chroma Technology</td>
			<td>AT480/30m</td>
			<td>Spectrophotometer Laser Light</td>
		</tr>
		<tr>
			<td>Low-Strength Steel Hex Nut</td>
			<td>McMaster-Carr</td>
			<td>90480A007</td>
			<td>6-32 Thread size</td>
		</tr>
		<tr>
			<td>LSM 700 Confocal Microscope</td>
			<td>Zeiss</td>
			<td>LSM 700</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Magnetic Stirring Bars</td>
			<td>Bel-Art Spinbar</td>
			<td>F37119-0007</td>
			<td>7x2 mm</td>
		</tr>
		<tr>
			<td>Multipurpose Neoprene Rubber Sheet</td>
			<td>McMaster-Carr</td>
			<td>1370N12</td>
			<td>1/32&#34; Thickness</td>
		</tr>
		<tr>
			<td>Non-Fat Dried Bovine Milk</td>
			<td>Sigma Aldrich</td>
			<td>M7409</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish</td>
			<td>Chemglass Life Sciences</td>
			<td>CGN1802145</td>
			<td>150 mm diameter</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline</td>
			<td>Corning</td>
			<td>21-040-CMR</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Plate Shaker</td>
			<td>VWR</td>
			<td>89032-088</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitors</td>
			<td>Thermo Scientific</td>
			<td>A32953</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor Blades</td>
			<td>Fisherbrand</td>
			<td>12640</td>
			<td></td>
		</tr>
		<tr>
			<td>R-Cast Acrylic Thin Gauge Sheet</td>
			<td>Reynolds Polymer</td>
			<td>N/A</td>
			<td>Black transport chamber inserts</td>
		</tr>
		<tr>
			<td>RTV Silicone</td>
			<td>Loctite</td>
			<td>234323</td>
			<td>Epoxy, Non-corrosive, clear</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>TedPella</td>
			<td>549-3</td>
			<td>#10, #11 blades</td>
		</tr>
		<tr>
			<td>Signal Receiver</td>
			<td>Chroma Technology</td>
			<td>ET515lp</td>
			<td>Spectrophotometer Laser Signal Receiver</td>
		</tr>
		<tr>
			<td>Snap-Cap Microcentrifuge Tubes</td>
			<td>Eppendorf</td>
			<td>22363204</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Spatula</td>
			<td>TedPella</td>
			<td>13508</td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy H1 Microplate Reader</td>
			<td>Biotek</td>
			<td>H1M</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc-Plated Alloy Steel Socket Head Screw</td>
			<td>McMaster-Carr</td>
			<td>90128A153</td>
			<td>6-32 Thread size, 1&#34; Long</td>
		</tr>
	</tbody>
</table>

characterization of intra-cartilage transport properties of cationic peptide carriers

体内一些带负电的组织，如软骨，由于其负充电的聚类药物密度高，对靶向药物的输送提出了障碍，因此，需要改进靶向方法，以增加其治疗反应。由于软骨具有高负固定电荷密度，因此药物可以用正电荷药物载体进行改性，以利用静电相互作用，从而增强软骨内药物的运输能力。因此，研究药物携带者的运输对于预测药物在诱导生物反应方面的有效性至关重要。进行了三个实验的设计，可以量化软骨外植中阳离子肽载体的均衡吸收、渗透深度和非均衡扩散率。均衡吸收实验提供了软骨内溶质浓度与周围浴池相比的测量，这对于预测药物载体在提高软骨药物的治疗浓度方面的潜力非常有用。使用共物显微镜进行渗透深度研究，允许从软骨表面扩散到深区对1D溶质扩散的视觉表示，这对于评估溶质是否到达其基质和细胞靶点非常重要。使用定制设计的运输室进行非均衡扩散率研究，通过描述在组织内荧光标记的溶解剂的扩散速率，可以测量与组织基质结合相互作用的强度;这有利于设计软骨的最佳结合强度载体。三种运输实验的结果共同为设计最佳电荷药物载体提供了指导，这些药物载体利用了药物输送应用中的弱和可逆电荷相互作用。这些实验方法也可以用于评价药物和药物载体结合的运输。此外，这些方法可以适用于瞄准其他带负功能的组织，如半月板，角膜和玻璃幽默。

在软骨对CPC进行平衡吸收后，当溶质被组织吸收时，浴荧光会减少。但是，如果最终浴池的荧光值仍与初始值相似，则表示没有/最小溶质吸收。溶质吸收的另一个确认是，如果组织已经明显改变颜色的荧光染料的颜色。在荧光值转换为浓度后，使用荧光值使用吸收比（RU）确定软骨中溶解的定量吸收率。使用初始浴浓度 （C浴，i）和平衡浴浓度 （C浴）， 软骨内的溶质浓?...

Watch this Scientific Journal Video about Characterization of Intra-Cartilage Transport Properties of Cationic Peptide Carriers at JoVE.com

Characterization of Intra-Cartilage Transport Properties of Cationic Peptide Carriers

该协议确定软骨中阳离子肽载体的均衡吸收、渗透深度和非平衡扩散率。运输特性的特性对于确保有效的生物反应至关重要。这些方法可用于设计最佳充电药物载体，以定位带负充电的组织。

阳性肽载体软骨内运输特性的特征

Several negatively charged tissues in the body, like cartilage, present a barrier to the targeted drug delivery due to their high density of negatively ...

描述治疗药物及其载体的运输特性对于确保有效的生物反应至关重要。这些方法有助于设计具有最佳充电功能的药物载体，以定位带负充电的组织。这些技术的主要优点是，它们能够通过一系列体外实验更好地预测体内的疗效，这些实验通过组织来描述溶质运输。软骨疾病，如骨关节炎，仍然未经治疗，因为药物无法穿透密集的血管软骨基质，需要药物载体来序曲疗效。首先使用精细的任务擦拭，轻轻地去除表面的三毫米直径，一毫米厚软骨外植多余的PBS。使用平衡快速记录每个外植的湿重，并立即将外植放入 PBS 浴池以防止脱水。接下来，在每口孔的96孔板内井中加入300微升新鲜准备的30微摩尔荧光标记阳离子肽载体溶液，并使用铲子在每口溶液中添加一个外植剂。用 300 微升 PBS 填充每个周围油井，并盖上板盖。用柔性薄膜密封板的边缘，以尽量减少蒸发，并将板在37摄氏度的培养箱中，以每分钟50转的速度将板放在摇床上24小时，轨道为15毫米。在孵育结束时，将平衡浴液从每一井转移到单独的聚丙烯管中，然后从库存30微摩尔阳离子肽载体溶液中进行连续稀释，以产生标准曲线。然后，将每种溶液和标准200微升转移到黑色96孔板的单个孔中，并基于荧光标签的激发和发射波长获得每个样品和标准的荧光读数。要确定软骨外植中阳离子肽载体渗透的深度，请使用手术刀将直径六毫米、一毫米厚的软骨外植切成两半，并使用蛋白酶抑制剂补充 PBS 来滋润由此产生的半圆盘片。将环氧树脂涂抹在定制设计的一维运输室的井中心，并在井内固定一个半盘外植，外植的表层朝向井的上游。从井中去除多余的胶水，防止与软骨扩散表面积接触，并在外植两侧加入80微升蛋白酶抑制剂补充PBS。将液体向上和向下移液，检查液体泄漏到另一侧。如果没有泄漏，请用标有阳离子肽载体溶液的80微升30微摩尔荧光溶液取代上游端补充的蛋白酶抑制剂PBS，并小心地将运输室放入细胞培养皿中。用PBS覆盖菜底，避免阳离子肽载体溶液蒸发。注意上游和下游腔室的解决方案之间没有直接接触。将覆盖的盘放在摇床上，在室温下限制颗粒沉积4或24小时，每分钟50转，轨道为15毫米。在孵育结束时，从腔室中去除外植，从每个外植中心切出约 100 微米厚的切片。将每片外植物放在玻璃滑梯和盖滑之间，用新鲜的蛋白酶抑制剂补充PBS来滋润切片。将幻灯片固定到共合显微镜的舞台上，以 10 倍放大倍率通过切片的全厚获得一叠 Z-stack 荧光图像。打开图像文件和图像 J，单击"图像"，然后从下拉菜单中选择"堆栈"和"Z 项目"。然后，将切片编号从 1 输入到最终切片和"投影类型"下，选择平均强度并单击"确定"。为了评估非平衡阳离子肽载体软骨扩散率，组装运输室的每一半，包括一个大型橡胶垫片、一个聚甲基丙烯酸酯刀片和一个小型橡胶垫片。测量软骨外植的厚度，然后将外植物放在塑料刀片的孔中，表面表面朝向上游腔室，将两半夹在一起完成组装。使用扳手将两半拧紧拧在一起，然后用两毫升蛋白酶抑制剂补充PBS填充上游腔室。检查下游腔室有无来自上游腔室的泄漏。如果未检测到泄漏，则用两毫升蛋白酶抑制剂补充PBS填充下游腔室。在上流和腔室中添加一个迷你搅拌棒，并将腔室放在搅拌板上，使分光光度计的激光聚焦到下游腔室的中心。在下游腔室后面分光光度计的信号接收器部分，收集稳定、实时的下游荧光发射读数至少五分钟。获得稳定读数后，在上游腔室中加入荧光标记阳离子肽载体库存溶液的预计算体积，最终浴液浓度为三微摩尔，并监测下游荧光信号，同时使溶质运输达到坡度的稳步增加。达到稳定状态后，将 20 微升溶液从上游腔室转移到下游腔室进行尖峰测试，并收集实时下游荧光读数。过高的正电荷将限制溶质穿透到表面区域，因为载体与软骨基质的负电荷聚集群结合太强。相反，能够利用弱和可逆电荷相互作用的载体渗透到组织的深层区域。然而，具有最佳充电功能的药物载体不仅会穿透组织深处，而且会表现出高组织内吸收率，如所示，可以通过平衡浴和组织湿重的荧光测量确定。非平衡扩散传输实验产生具有逐渐增加斜率的数据生成曲线。曲线的初始部分表示溶质基质结合相互作用时通过软骨的溶质扩散。一旦溶解物到达下游腔室，曲线的斜率会随着荧光读数随时间的增加而增加。然后，曲线的第二部分达到一个稳定的斜率，表示稳定状态扩散。在曲线稳定状态部分绘制的切线 X 截距表示达到稳定状态扩散或 tau 滞后所用的时间。溶液从上游转移到下游腔室后，观察到荧光的峰值，此时可以使用稳定的荧光强度将荧光与浓度关联。然后可以计算具有代表性的有效扩散率和稳定状态扩散值，如所示。请注意，由于所有基于电荷的绑定站点被占用，稳定状态扩散率比有效扩散率高两个数量级。因此，此时，扩散基于大小而不是电荷，导致相同大小的 CPC 之间的稳定状态扩散值相似。在整个实验中保持外植水分并尽量减少溶液蒸发，以防止软骨形态和溶液浓度的变化，并确保准确和可重复的数据采集。在设计出具有最佳充电的药载体后，可以利用各种结合技术来改变药物，增强组织靶向，并促进评估其生物功效。

Research

JoVE Journal

Bioengineering

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

量化的内皮细胞糖复合物的力学性能，用原子力显微镜

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

科研

生物工程

Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

新型原子力显微镜基于生物淘选的形态特殊试剂对TDP-43变型肌萎缩侧索硬化症隔离

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

在生物膜的力学性能原位制图粒子追踪Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Transport Properties of Ibuprofen Encapsulated in Cyclodextrin Nanosponge Hydrogels: A Proton HR-MAS NMR Spectroscopy Study

布洛芬封装的输运性质环糊精Nanosponge凝胶：质子HR-MAS NMR光谱研究

The chemical cross-linking of &#946;-cyclodextrin (&#946;-CD) with ethylenediaminetetraacetic dianhydride (EDTA) led to branched polymers referred to as ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

治疗的siRNA递送至CNS使用阳离子和阴离子脂质体

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Synthesis of Monocyte-targeting Peptide Amphiphile Micelles for Imaging of Atherosclerosis

单核靶向肽 Amphiphile 胶束合成动脉粥样硬化的研究

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

三维肽支架中哺乳动物细胞的培养

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

阳离子纳米脂质体的生成, 用于高效输送体外转录信使 rna

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

多孔硅薄膜作为神经生长因子载体的设计

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...