Name: evaluation of antimicrobial activities of nanoparticles and nanostructured surfaces in vitro
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作者感谢美国国家科学基金会（NSF CPET奖1512764和NSF PIRE 1545852），美国国立卫生研究院（NIH NIDCR 1R03DE028631），加州大学（UC）摄政教师发展奖学金，研究种子资助委员会（Huinan Liu）和加州大学河滨分校研究生研究指导计划授予Patricia Holt-Torres的资助。作者感谢加州大学河滨分校高级显微镜和微量分析中央设施（CFAMM）为使用SEM / EDS和Perry Cheung博士使用XRD提供的帮助。作者还要感谢Morgan Elizabeth Nator和Samhitha Tumkur在实验和数据分析方面的帮助。本文中表达的任何意见、发现、结论或建议均为作者的观点，并不一定反映美国国家科学基金会或美国国立卫生研究院的观点。

为了介绍直接共培养和直接暴露方法，我们使用氧化镁纳米颗粒（nMgO）作为模型材料来证明细菌相互作用。为了介绍直接培养和聚焦接触暴露方法，我们以具有纳米结构表面的镁合金为例。
1. 纳米材料的灭菌
注意：在微生物培养之前，所有纳米材料必须经过灭菌或消毒。可以使用的方法包括热、压力、辐射和消毒剂，但必须在 体外 实...

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我们提出了四种体外方法（A-D）来表征纳米颗粒和纳米结构表面的抗菌活性。虽然这些方法中的每一种都量化了细菌随时间推移对纳米材料的生长和活力，但用于测量初始细菌接种密度、生长和活力随时间变化的方法存在一些差异。其中三种方法，直接共培养方法（A）17，直接培养方法（C）14和聚焦接触暴露方法（D）16，通过计算每单位体积<sup class="xref...

仅在美国，每年就有 170 万人发生医院获得性感染 （HAI），其中每 17 例感染中就有 1 例导致死亡1。此外，据估计，HAI的治疗费用每年从280亿美元到450亿美元不等1，2。这些 HAI 主要由耐甲氧西林金黄色葡萄球菌 （MRSA）3，4 和铜绿假单胞菌 4 为主，它们通常从慢性伤口感染中分离出来，通常需要广泛的治疗和时间才能产生良好的患者预后。
在过去的几十年中，已经开发了多种抗生素类别来治疗与这些和其他致病菌相关的感染。例如，利福霉素类似物已被用于治疗MRSA，其他革兰氏阳性和革兰氏阴性菌感染以及分枝杆菌属感染5。在1990年代，为了有效治疗越来越多的结核分枝杆菌感染，额外的药物与利福霉素类似物联合使用以提高其有效性。然而，大约 5% 的结核分枝杆菌病例仍然对利福平5，6 耐药，并且人们越来越关注多重耐药细菌7。 目前，单独使用抗生素可能不足以治疗HAI，这引发了对替代抗菌疗法的持续寻找1。
重金属，如银（Ag）8，9，10和金（Au）11，以及陶瓷，如二氧化钛（TiO 2）12和氧化锌（ZnO）13，以纳米颗粒（NP）形式（分别为AgNP，AuNP，TiO2 NP和ZnONP）已被检查其抗菌活性，并已被确定为潜在的抗生素替代品。此外，生物可吸收材料，如镁合金（Mg合金）14，15，16，氧化镁纳米颗粒17，18，19，20，21和氢氧化镁纳米颗粒[分别为nMgO和nMg（OH）2]22，23，24，也进行了检查。然而，以前对纳米颗粒的抗菌研究使用了不一致的材料和研究方法，导致数据难以或不可能比较，有时本质上是矛盾的18，19。例如，银纳米颗粒的最低抑菌浓度（MIC）和最小杀菌浓度（MBC）在不同的研究中差异很大。Ipe等人25评估了平均粒径为~26nm的AgNPs的抗菌活性，以确定MIC对革兰氏阳性和革兰氏阴性细菌的抵抗力。鉴定出的铜绿假单胞菌、大肠杆菌、金黄色葡萄球菌和MRSA的MIC分别为2 μg/mL、5 μg/mL、10 μg/mL和10 μg/mL。相比之下，Parvekar等人26评估了平均粒径为5nm的AgNPs。在这种情况下，发现AgNP MIC和0.625 mg / mL的MBC对金黄色葡萄球菌有效。此外，Loo等人27评估了尺寸为4.06nm的AgNP。当大肠杆菌暴露于这些纳米颗粒时，报告的MIC和MBC为7.8μg/ mL。最后，Ali等人28研究了平均尺寸为18 nm的球形AgNPs的抗菌性能。当铜绿假单胞菌、大肠杆菌和MRSA暴露于这些纳米颗粒时，MIC的鉴定分别为27 μg/mL、36 μg/mL、27 μg/mL和36 μg/mL，MBC分别为36 μg/mL、42 μg/mL和30 μg/mL。
尽管近几十年来纳米颗粒的抗菌活性已被广泛研究和报道，但用于直接比较研究的材料和研究方法没有标准。出于这个原因，我们提出了两种方法，直接共培养方法（方法A）和直接暴露方法（方法B），以表征和比较纳米颗粒的抗菌活性，同时保持材料和方法的一致性。
除纳米颗粒外，还研究了纳米结构表面的抗菌活性。这些包括碳基材料，如石墨烯纳米片、碳纳米管和石墨29，以及纯镁和镁合金。这些材料中的每一种都表现出至少一种抗菌机制，包括碳基材料对细胞膜的物理损伤，以及当镁降解时通过释放活性氧（ROS）对代谢过程或DNA的损害。此外，当锌（Zn）和钙（Ca）结合形成镁合金时，镁基体晶粒尺寸的细化增强，与仅镁样品相比，这导致细菌对基材表面的粘附减少14。为了证明抗菌活性，我们提出了直接培养方法（方法C），该方法通过定量具有直接和间接表面接触的细菌菌落形成单位（CFU）来确定细菌随时间推移对纳米结构材料及其周围的粘附。
表面纳米结构的几何形状，包括尺寸、形状和方向，可能会影响材料的杀菌活性。例如，Lin等人16 通过阳极氧化和电泳沉积（EPD）在镁衬底表面制备了不同的纳米结构MgO层。在 体外暴露于纳米结构表面一段时间后，与未处理的Mg相比， 金黄色葡萄球菌 的生长显着减少。这表明纳米结构表面对细菌粘附的效力高于未处理的金属镁表面。为了揭示各种纳米结构表面抗菌性能的不同机制，本文讨论了一种确定感兴趣区域内细胞-表面相互作用的聚焦接触暴露方法（方法D）。
本文的目的是介绍四种适用于不同纳米颗粒、纳米结构表面和微生物物种的体外方法。我们讨论了每种方法的关键考虑因素，以产生一致、可重复的数据以提高可比性。具体地，直接共培养方法17和直接暴露方法用于检查纳米颗粒的抗菌特性。通过直接共培养法，可以确定单个物种的最小抑菌浓度和最小杀菌浓度（MIC和MBC90-99.99），并且可以确定多个物种的最有效浓度（MPC）。通过直接暴露方法，纳米颗粒在最小抑制浓度下的抑菌或杀菌作用可以通过随时间推移的实时光密度读数来表征。直接培养方法14适用于直接和间接检查与纳米结构表面接触的细菌。最后，提出聚焦接触曝光16方法，通过直接应用细菌和表征细胞-纳米结构界面上的细菌生长来检查纳米结构表面上特定区域的抗菌活性。该方法从日本工业标准JIS Z 2801：200016修改而来，旨在关注微生物-表面相互作用，并排除微生物培养中大宗样品降解对抗菌活性的影响。

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		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>Milipore Sigma</td>
			<td>Z336777</td>
			<td></td>
		</tr>
		<tr>
			<td>80 L NTRL Certified Convection Drying Oven&#160;</td>
			<td>MTI Corporation</td>
			<td>BPG-7082</td>
			<td>https://www.mtixtl.com/BPG-7082.aspx</td>
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		<tr>
			<td>(hydroxymethyl) aminomethane buffer pH 8.5; Tris buffer&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>42457</td>
			<td></td>
		</tr>
		<tr>
			<td>AnaSpec&#160;THIOFLAVIN T ULTRAPURE GRADE</td>
			<td>Fisher Scientific</td>
			<td>50-850-291</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron-multiplying charge-coupled device digital camera&#160;</td>
			<td>Hamamatsu</td>
			<td>C9100-13</td>
			<td></td>
		</tr>
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			<td>Falcon 15 mL conical tubes</td>
			<td>Fisher Scientific</td>
			<td>14-959-49B</td>
			<td></td>
		</tr>
		<tr>
			<td>Gluteraldehyde</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>G5882</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Brightline, Hausser Scientific</td>
			<td>1492</td>
			<td></td>
		</tr>
		<tr>
			<td>Inductively coupled plasma - optical emission spectrometry (ICP-OES)</td>
			<td>PerkinElmer</td>
			<td>8000</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverse microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria Bertani Broth</td>
			<td>Sigma Life Science&#160;</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria Bertani Broth + agar</td>
			<td>Sigma Life Science&#160;</td>
			<td>L2897</td>
			<td></td>
		</tr>
		<tr>
			<td>MacroTube 5.0&#160;&#160;</td>
			<td>Benchmark Scientific</td>
			<td>C1005-T5-ST</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium oxide nanoparticles</td>
			<td>US Research Nanomaterials, Inc</td>
			<td>Stock #:&#160;US3310&#160;&#160;&#160;M</td>
			<td>MgO, 99+%, 20 nm</td>
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		<tr>
			<td>MS Semi-Micro Balance</td>
			<td>Mettler Toledo</td>
			<td>MS105D</td>
			<td></td>
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		<tr>
			<td>Nitrocellulose paper</td>
			<td>Fisherbrand</td>
			<td>09-801A</td>
			<td></td>
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			<td>Non-tissue treated 12-well polystyrene plate</td>
			<td>Falcon Corning Brand&#160;</td>
			<td>351143</td>
			<td></td>
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			<td>Non-tissue treated 48-well polystyrene plate</td>
			<td>Falcon Corning Brand&#160;</td>
			<td>351178</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-tissue treated 96-well polystyrene plate</td>
			<td>Falcon Corning Brand&#160;</td>
			<td>351172</td>
			<td></td>
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		<tr>
			<td>Petri dish 100 mm</td>
			<td>VWR</td>
			<td>470210-568</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish, 15 mm</td>
			<td>Fisherbrand</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>VWR</td>
			<td>SP70P</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning electron microscopy (SEM)</td>
			<td>TESCAN&#160;</td>
			<td>Vega3 SBH</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>VWR</td>
			<td>97043-936</td>
			<td></td>
		</tr>
		<tr>
			<td>Table top centrifuge</td>
			<td>Fisher Scientific</td>
			<td>accuSpin Micro 17</td>
			<td></td>
		</tr>
		<tr>
			<td>Table top centrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>Centrifuge 5430</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic Soy Agar</td>
			<td>MP</td>
			<td>1010617</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic Soy Broth</td>
			<td>Sigma-Aldrich</td>
			<td>22092-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Vis spectrophotometer&#160;</td>
			<td>Tecan</td>
			<td>Infinite 200 PRO</td>
			<td>https://lifesciences.tecan.com/plate_readers/infinite_200_pro</td>
		</tr>
		<tr>
			<td>VWR Benchmark Incu-shaker 10L</td>
			<td>VWR</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>X-ray power defraction</td>
			<td>&#160;Panalytical</td>
			<td>N/A</td>
			<td>PANalytical Empyrean Series 2</td>
		</tr>
	</tbody>
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evaluation of antimicrobial activities of nanoparticles and nanostructured surfaces in vitro

纳米颗粒和纳米结构表面（如银、氧化锌、二氧化钛和氧化镁）的抗菌活性以前已在临床和环境环境以及消费品中探索过。然而，所使用的实验方法和材料缺乏一致性，最终导致了相互矛盾的结果，即使在相同纳米结构类型和细菌种类的研究中也是如此。对于希望在产品设计中使用纳米结构作为添加剂或涂层的研究人员来说，这些相互矛盾的数据限制了它们在临床环境中的使用。 
为了应对这一困境，在本文中，我们提出了四种不同的方法来确定纳米颗粒和纳米结构表面的抗菌活性，并讨论了它们在不同场景中的适用性。调整一致的方法有望产生可重复的数据，这些数据可以在研究中进行比较，并针对不同的纳米结构类型和微生物物种实施。我们介绍了两种确定纳米颗粒抗菌活性的方法和两种纳米结构表面抗菌活性的方法。 
对于纳米颗粒，直接共培养方法可用于确定纳米颗粒的最小抑制和最小杀菌浓度，直接暴露培养方法可用于评估纳米颗粒暴露引起的实时抑菌与杀菌活性。对于纳米结构表面，直接培养方法用于确定与纳米结构表面间接和直接接触的细菌的活力，并且聚焦接触暴露方法用于检查纳米结构表面特定区域的抗菌活性。我们讨论了在确定纳米颗粒和纳米结构表面的抗菌特性时要考虑的 体外 研究设计的关键实验变量。所有这些方法的成本都相对较低，采用相对容易掌握和可重复的技术以实现一致性，并且适用于广泛的纳米结构类型和微生物物种。

氧化镁纳米颗粒和纳米结构表面的抗菌活性鉴定已使用适用于不同材料类型和微生物物种的四种 体外 方法提出。
方法A和方法B在滞后期（方法A）和对数期（方法B）暴露于纳米颗粒时检查细菌活性24小时或更长时间。方法A提供了有关MIC和MBC的结果，而方法B确定了纳米颗粒的抑制作用与杀菌效果。方法C检查与纳米结构表面直接和间接接触的细菌活性，方法D检查细胞 - ?...

Watch this Scientific Journal Video about Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro at JoVE.com

Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro

我们介绍了四种使用 体外 技术评估纳米颗粒和纳米结构表面的抗菌活性的方法。这些方法可以适用于研究不同纳米颗粒和纳米结构表面与各种微生物物种的相互作用。

体外评估纳米颗粒和纳米结构表面的抗菌活性

The antimicrobial activities of nanoparticles and nanostructured surfaces, such as silver, zinc oxide, titanium dioxide, and magnesium oxide, have been ...

该协议提供了一种从初步阶段研究到更复杂的表面检查的纳米颗粒和纳米结构表面的抗菌活性的方法。与先前描述的方法不同，这些方法导致跨研究的数据不可比，这些方法在不同的纳米颗粒材料，纳米结构材料和微生物物种中产生一致和可比较的结果。在方法A和B中保持纳米颗粒的均匀分布可能很困难。彻底涡旋样品将确保纳米颗粒悬浮液，等分试样之间移液三到四次将保持悬浮液通过将一毫升过夜生长的细菌培养物等分到 1.5 毫升微量离心管中开始收集培养的细菌。在产生足够数量的等分试样后，将它们离心以达到所需的座位密度。将上清液收集到收集容器中。并将细胞沉淀重新悬浮在0.5毫升新鲜生长培养基中。合并来自两个管的悬浮液，并重复离心。离心后，用新鲜生长培养基进行第二次洗涤，并在离心管之前重复将两个管中的内容物合并为一个。用0.33毫升三缓冲液或羟乙基氨基甲烷缓冲液进行第三次洗涤。将三种悬浮液（每种体积为0.33毫升）合并到一台1.5毫升微量离心机中。离心后，从沉淀的细胞中除去上清液，并将细胞沉淀重新悬浮在一毫升新鲜的tris缓冲液中，该缓冲液将称为细胞悬液或滞后期细菌接种培养物。为了形成细菌和纳米颗粒培养物，将两毫升细菌接种培养物等分到12孔非组织处理的聚苯乙烯板的每个孔中。对于每五毫升含有预先称重的氧化镁颗粒的微量离心管，加入三毫升细菌接种培养物。并短暂涡旋管以将氧化镁纳米颗粒与细菌混合。混合后，将一毫升氧化镁纳米颗粒悬浮液等分到三个单独的孔中，以创建每个预先测量重量的氧化镁纳米颗粒的三份样品。将样品在 37 摄氏度和 120 rpm 下孵育过夜。并使用移液器将三毫升样品转移到单独的 15 毫升锥形管中。然后在96孔板中进行1至10次连续稀释，方法是向B行至G行的每个孔中加入180微升tris缓冲液以获得适当数量的列。短暂涡旋含有细菌样品的15毫升锥形管，并将50微升细菌样品加入A行的单个孔中.接下来，从A行将20微升细菌样品转移到B行的相应孔中，短暂混合。使用无菌移液器吸头，将20微升从B行的孔转移到C行中的相应孔.继续直到G行以完成从10到B行中的负到10到G行中的负6的系列稀释.完成后，将每个孔的100微升移液到适当的生长琼脂平板中， 并铺展在平板上以分散细胞培养物。将孔板放入37摄氏度的培养箱振荡器中过夜，并将琼脂平板在37摄氏度下孵育24小时。检查板，并计数具有大约25至300个菌落的平板。如果可能，选择具有相同稀释值的板。对于要测试的每种材料，通过将三个 200 微升等分试样添加到生长培养基中到三个孔中，将三个 200 微升等分试样的细菌培养物添加到其他孔中来稀释过夜细菌样品。将96孔板放入读板器并扫描。如有必要，继续加入肉汤或过夜细菌培养物，直到达到600纳米或OD 600处的光密度约为0.01。为了制备细菌纳米颗粒悬浮液和无菌培养基纳米颗粒悬浮液，从一式三份的15毫升锥形管中取出一毫升细菌培养物或培养基。并将其加入含有预先测量的纳米颗粒的五毫升离心管中。涡旋试管以混合溶液。通过将一毫升等分试样从五毫升离心管转移到 15 毫升锥形管中，均匀分布纳米颗粒。完成后，将每个样品的200微升等分到96孔板的各个孔中。在确定前面描述的座密度后，将样品和对照添加到48孔非组织处理聚苯乙烯板的各个孔中。接下来，将0.75毫升细胞悬液等分到包含样品和对照的每个孔中。将48孔板放入培养箱振荡器中，在37摄氏度下振荡24小时，以120RPM振荡。孵育后，从每组中收集两个样品，并将它们单独转移到标记的五毫升微量离心管中。向每个试管中加入两毫升修订的模拟体液或RSBF。通过将灭菌的亚硝基纤维素纸修剪成直径一厘米来制备它们。将切好的硝酸纤维素纸放在含有适当培养基的琼脂平板上。接下来，将50微升稀释的细菌培养物添加到滤纸上，然后将50微升适当的培养基添加到每个样品表面的中心。使用无菌镊子，从螺旋钻表面拿起硝酸纤维素纸。小心地翻转硝酸纤维素纸并将其放在样品表面上，使细菌与50微升培养基和感兴趣的纳米结构表面接触。如果样品在培养物中可降解，则在其下方放置一个支架以提起它，以避免与tris缓冲液接触。通过在含有良好样品的样品中加入一毫升tris缓冲液来保持湿度。孵育过夜后，从每个样品表面收集硝酸纤维素纸，并将它们放入五毫升tris缓冲液中。涡旋收集的滤纸和纳米表面材料样品五秒钟。从样品中收集tris缓冲液悬浮液，并将它们放入单独的新鲜收集管中。使用方法A的抗菌作用确定了每毫升氧化镁纳米颗粒1毫克的最小和抑制浓度或MIC，以及革兰氏阴性大肠杆菌和铜绿假单胞菌的最低杀细菌浓度或MBC 99.9分别为每毫升氧化镁纳米颗粒1.0和1.6毫克。革兰氏阳性表皮葡萄球菌、金黄色葡萄球菌和耐甲氧西林金黄色葡萄球菌（MRSA）分别证明了MIC的值为每毫升氧化镁纳米颗粒0.5、0.7和1毫克。表皮链球菌和金黄色葡萄球菌的NBC 99.99值分别为每毫升1.6和1.2毫克。虽然MRSA没有降低到NBC 90以上。在药物敏感和耐药念珠菌属、白色念珠菌和白色念珠菌氟康唑耐药或FR中分别检测到1.2和1毫克/毫升的MIC。相比之下，氧化镁纳米颗粒表现出每毫升1和0.7毫克的MIC值，或分别抗光滑梭菌和光滑棘白菌素，或ER。每个候选物种的NBC 90达到每毫升0.7至1.2毫克，但只有C.glabrata ER在每毫升1.2毫克时降低到NBC 99.9。在方法B中，暴露于纳米颗粒的MRSA呈指数增长，OD为0.85。暴露于每毫升1.2毫克的氧化镁纳米颗粒和每毫升6.25毫克的甲氧苄啶分别导致80.2%和81.6%的细菌生长减少。同样，每毫升2.9毫克氢氧化镁纳米颗粒将细菌生长降低到70.3%暴露于每毫升万古霉素一微升导致MRSA生长减少99.99%，表明氧化镁和氢氧化镁纳米颗粒的抑菌活性。方法C显示间接接触对细菌生长没有抑制作用。结果表明，在所有样品中，ZC21对MRSA的粘附和生长具有最强的抗菌活性。在方法D中，在1.9A、1.9 AA和EPD样品或其成对的滤纸中未发现活的金黄色葡萄球菌。然而，跪下后暴露于EPD样品可减少纳米结构表面和削皮滤纸上少数细胞的细菌生长。鉴定纳米颗粒和纳米结构表面抗菌活性导致了生物工程应用中的其他研究，包括与慢性伤口和植入物相关感染相关的研究。

evaluation-antimicrobial-activities-nanoparticles-nanostructured

Research

JoVE Journal

Bioengineering

Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Micropatterned表面透明质酸与肿瘤细胞相互作用的研究

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

科研

生物工程

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 ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

高分子基因传递纳米粒子的纳米粒子跟踪分析和高通量流式细胞仪评价

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

一机能磁性纳米纤维素的细菌与氧化铁纳米粒子的制备

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

眼刺激试验（EIT）使用重构型人角膜上皮样（RHCE）组织模型眼刺激性化学品危险性鉴定

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

温敏纳米结构表面的组织工程准备

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

液体中脉冲激光烧蚀制备金属纳米粒子：研究纳米粒子抗菌性能的工具

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

一个<em&gt;体外</em鼠类的椎间盘&gt;器官培养模型

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver MWCNTs were prepared by the reductive deposition of silver from an aqueous solution of AgNO3 on the oxidized MWCNTs. Given the unique color of the CNTs, it was not possible to apply them to the minimum inhibitory concentration or mitochondrial toxicity assays to evaluate the toxicity and antibacterial properties, since they would interfere with the assays. The inhibition zone and minimum bactericidal concentration for the Ag-MWCNTs were measured and Live/Dead and Trypan Blue assays were used to measure the toxicity and antibacterial properties without interfering with the color of the CNTs.

In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

银纳米粒子改性多壁碳纳米管的合成及其抗菌活性和细胞毒性的评价

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...

Antimicrobial Characterization of Advanced Materials for Bioengineering Applications

The development of new advanced materials with enhanced properties is becoming more and more important in a wide range of bioengineering applications. Thus, many novel biomaterials are being designed to mimic specific environments required for biomedical applications such as tissue engineering and controlled drug delivery. The development of materials with improved properties for the immobilization of cells or enzymes is also a current research topic in bioprocess engineering. However, one of the most desirable properties of a material in these applications is the antimicrobial capacity to avoid any undesirable infections. For this, we present easy-to-follow protocols for the antimicrobial characterization of materials based on (i) the agar disk diffusion test (diffusion method) and (ii) the ISO 22196:2007 norm to measure the antimicrobial activity on material surfaces (contact method). This protocol must be performed using Gram-positive and Gram-negative bacteria and yeast to cover a broad range of microorganisms. As an example, 4 materials with different chemical natures are tested following this protocol against Staphylococcus aureus, Escherichia coli, and Candida albicans.The results of these tests exhibit non-antimicrobial activity for the first material and increasing antibacterial activity against Gram-positive and Gram-negative bacteria for the other 3 materials. However, none of the 4 materials are able to inhibit the growth of Candida albicans.

We present a protocol for the antimicrobial characterization of advanced materials. Here, the antimicrobial activity on material surfaces is measured by two methods that complement each other: one is based on the agar disk diffusion test, and the other is a standard procedure based on the ISO 22196:2007 norm.

生物工程应用先进材料的抗菌性能研究

The development of new advanced materials with enhanced properties is becoming more and more important in a wide range of bioengineering applications. ...