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Bioengineering

Griffithsin 修饰纤维支架预防性传播感染的制备与表征

Published: October 31, 2017
doi:
10.3791/56492
, ,
1Department of Chemistry,University of Louisville, 2Department of Pharmacology and Toxicology,University of Louisville, 3Center for Predictive Medicine,University of Louisville, 4Department of Microbiology and Immunology,University of Louisville, 5Department of Bioengineering,University of Louisville

Summary

这份手稿描述了制造和表征 Griffithsin 修饰聚 (乳酸-羟基酸) 纺纤维的程序, 证明有效的粘合剂和抗病毒活性对抗人体免疫缺陷病毒1型感染体外。本文介绍了用于合成、表面修饰和表征表面改性纤维 Griffithsin 的形态、共轭和解吸的方法。

Abstract

纺纤维 (EFs) 已广泛应用于各种治疗应用中;然而, 它们只是最近才被用作预防和治疗性传播感染 (sti) 的技术。此外, 许多 EF 技术的重点是封装活性剂, 相对于利用表面传授 biofunctionality。在这里, 我们描述了一种方法制造和表面修饰聚 (乳酸-羟基) 酸 (PLGA) 纺纤维, 具有强大的抗病毒凝集素 Griffithsin (GRFT)。PLGA 是一种经 FDA 批准的聚合物, 由于其优异的化学和生物相容性, 已被广泛应用于药物的传递。GRFT 是一种天然的、有效的、安全的凝集素, 对包括人体免疫缺陷病毒1型 (HIV-1) 在内的多种病毒具有广泛的活性。当结合, GRFT 改性纤维已经证明了强力灭活的 HIV-1在体外。这份手稿描述的方法来制造和表征 GRFT 修改的 EFs。首先, PLGA 是纺制造纤维支架。纤维随后表面修饰与 GRFT 使用 1-乙基 3-(3-dimethylaminopropyl) 二 (和 n-羟基琥珀酰亚胺 (NHS) 化学。扫描电子显微镜 (SEM) 用于评估表面修饰的配方的大小和形貌。此外, 基于 gp120 或血 (HA) 的 ELISA 可以用来量化 GRFT 共轭的数量, 以及 GRFT 解吸从纤维表面。该协议可以更广泛地应用于制造表面修饰的各种不同蛋白质的纤维。

Introduction

使用 EFs 作为一个专题传送平台有可能大大减少性传播感染。目前, 有超过3600万人感染艾滋病毒, 仅在2015年就有超过200万新病例报告为1,2。此外, 单纯疱疹病毒2型 (HSV-2) 感染影响到全世界数亿人, 并已证明, 以提高艾滋病毒的获得 2-5 倍3。由于 HSV-2 感染和艾滋病毒的获取之间的这种关系, 有很大的兴趣开发新的活性药物, 同时提供预防多种性传播疾病的保护。此外, 开发新的车辆, 以改善这些抗病毒药物的运送提供了进一步加强保护和治疗功效的潜力。为了达到这个目标, EFs 被作为一个新的交付平台来进行研究, 以减少 HIV-1 和 HSV-2 感染的流行。

在过去的二十年中, EFs 在药物传递和组织工程领域中得到了广泛的应用4。通常, 生物相容性聚合物被选择容易转化为治疗性应用。为了制造聚合物 EFs, 所选的聚合物在有机溶剂或水溶液中溶解, 这取决于聚合物疏水性的程度5。在静电纺丝过程之前, 有兴趣的活性剂被添加到溶剂或水溶液中。然后, 聚合物溶液被吸入到注射器中, 并在受到电流的同时慢慢喷射出来。这个过程通常会导致聚合物纤维与板料或圆柱形宏观 (图 1) 和光纤直径不等, 从微米到纳米级的6。对于大多数的治疗性应用, 活性剂在纤维纺丝过程中被整合在一起, 通过扩散和随后的纤维降解从纤维中释放出来。通过使用不同类型的聚合物或聚合物共混物来改变降解或释放的速率, 以建立一个理想的释放剖面, 传授独特的化学和物理性质7, 并促进几乎所有的封装复合.因此, EFs 已证明有利于小分子药物和生物制剂的交付, 包括蛋白质、肽、寡核苷酸和生长因子6,8,9。

在 STI 预防领域, 最近使用 EFs 来合并和提供抗病毒药物的持续或诱导释放10,11,12,13,14 ,15,16,17,18,19。在最早的一项研究中, pH 反应纤维的开发, 以释放活性剂, 以响应环境变化的女性生殖道 (首次登记税), 作为一种按需保护的方法, 反对 HIV-111。因为, 其他研究已经调查了由聚乙烯氧化物和聚 l-乳酸 (PLLA) 组成的聚合物共混物, 以评估抗病毒药物和避孕剂在 HIV-1 预防和避孕方面的可调谐释放,体外12. 其他研究显示了 EFs 提供以下内容的可行性: 小分子抗病毒药物的长时间释放14, 强大和灵活的机械性能20, 3 维交付体系结构21, 抑制精子穿透性12, 以及与其他交付技术合并的能力13。最后, 以前的工作已经评估了聚合物纤维的持续交付抗病毒剂抗常见 co-infective 病毒, HSV-2 和 HIV-114。在这项研究中, 高分子纤维通过保持其结构长达1月, 并为病毒进入提供了物理屏障, 从而为抗病毒药物的传递提供了补充活性。从这些结果中可以看出, EFs 可能被用于物理和化学上阻碍病毒感染。

虽然可调谐释放特性使聚合 efs 成为一个诱人的杀菌剂交付平台, 在其他应用中开发了 efs 作为表面修饰的支架7。EFs 已被用来模仿细胞外基质 (ECM) 的形态学, 通常充当支架以改善细胞再生22, 并增强其在组织工程中的效用23,24。由聚ε-内 (PCL) 和 PLLA 等聚合物组成的纤维经过表面修饰后, 在静电纺丝作用下, 通过生长因子和蛋白质来传授类似于 ECM 的特性, 包括增加细胞黏附和增殖25,26. 此外, 还对抗菌表面改性的 EFs 进行了评估, 以防止特定致病细菌的生长,27,28。由于这种多功能性和诱导生物效应的能力, EF 技术继续在各种领域扩展, 以提供 multi-mechanistic 的功能。然而, 尽管它们在多种应用中都有效用, 但表面改性纤维最近才在杀菌剂领域29中进行了探索。

在开发新的预防和治疗性传播疾病的新技术的同时, 还开发了新型的生物疗法。最有前途的杀菌剂候选者之一是粘合剂抗病毒凝集素, GRFT30。最初来源于一种红藻, GRFT 已证明活动作为一个强有力的抑制剂的艾滋病毒, HSV-2, SARS, 以及丙肝病毒31,32,33,34,35,36. 事实上, 在 biologically-based 抑制剂中, GRFT 具有最有力的抗 HIV 活性, 几乎在接触30时就立即灭活 HIV-1, 同时在培养基中维持稳定和活动, 从阴道微生物最多10天37。最近, 0.1% GRFT 凝胶被证明保护小鼠免受阴道 HSV-2 的挑战, 使它成为对 HSV-2 和 HIV-1 的第一行保护的有希望的候选者32,38. 对于 HIV 病毒, GRFT 抑制感染的物理结合 gp120 或终端甘露糖 n-链接的糖残留病毒的信封表面, 以防止进入38,39,40,41 ,42。这种抑制是非常强大的, 与 IC50的接近 3 ng/mL43。除了抑制艾滋病毒感染, 研究还表明, GRFT 通过抑制病毒的细胞传播来防止 HSV-2 感染 (32。在所有情况下, GRFT 已经证明是对病毒颗粒的粘合剂, 同时显示出高抗变性。最后, GRFT 已经证明了协同活动与诺福韦 (TFV) 和其他抗病毒药物的组合44, 使它可行, 并可能有利于 co-administer 与 EFs。GRFT 的强大特性使它成为一种极好的 biologically-based 抗病毒候选物, 通过 EF 技术可以提高分娩的效率。

利用这一知识的粘合剂和固有的抗病毒性能的 GRFT, 一个高分子纤维脚手架设计, 整合这些属性, 提供第一层病毒进入抑制29。找到灵感的方式, 宫颈粘液阻碍病毒传输主要通过粘粘蛋白相互作用, 我们假设, 通过使用 EFs 作为脚手架和共价修改表面与 GRFT, 高密度表面共轭 GRFT 会在其入口45,4647中削弱和禁用病毒。在这里, EFs 被开发为一个固定的脚手架, 提供一个蛋白质, 病毒性的粘合剂钝化屏障平台。我们试图结合的强大的抗病毒性能的 GRFT 与一个生物相容, 可修改, 耐用的高分子平台, 创造一个新的病毒 “陷阱”。

为了实现这些目标, 由 PLGA 组成的纤维是纺, 并使用琥珀酰亚胺化学, 以随后修改 EF 表面与 GRFT。PLGA 作为一个模型聚合物, 由于其广泛使用的静电纺丝48, 结合其生物相容性和 cost-effectiveness。此外, 表面修改利用了 EFs 的大面积, 并提供了一个有用的替代方案, 可与封装结合, 以最大程度地提高光纤实用程序49。与传统的封装方法不同的是, 只有部分 GRFT 是可用的 (而且只有在首次登记时才会出现), 表面修饰可以使 GRFT 在整个治疗期间保持最大的生物活性。此外, 采用传统的静电纺丝方法, 如蛋白质等亲水性化合物的加入, 可能会导致较低的封装效率和蛋白质活性的丧失50。因此, GRFT 表面改性纤维可以提供一种有前途的替代交付方法, 可以单独使用或与静电纺丝结合, 以加强预防性病感染。

Protocol

1. 纺纤维脚手架的制备和制造 警告: 所有与溶剂或聚合物溶液的工作应在化学油烟机上执行 。在开始协议之前, 请参阅每个试剂的材料安全数据表. electrospin 3 毫升 15% w/w PLGA 聚合物溶液, 重720毫克50:50 聚 (乳酸-羟基酸) (plga; 0.55 到 0.75 dL/克, 31-57 kDa) 成10毫升闪烁瓶。解决方案的体积是基于当前研究中使用的典型批次大小. 注: 要添加到给定体积的溶剂中的聚?…

Representative Results

纤维形态对表面改性 EFs 的抗病毒能力有显著影响。虽然静电纺丝是一种方便而直接的方法, 但优化聚合物的配方可能会导致不规则的纤维形态 (图 5B-C)。静电纺丝条件的改变, 形成串珠状或无定形的垫样形态, 通常是由溶剂-聚合物不相容, 低聚合物粘度, 流速, 或其他静电纺丝条件造成的。由此产生的纤维结构变化可能导致不同的药物合并…

Discussion

由于其多孔结构和大面积, EFs 已发现在医疗保健的各种应用, 其中包括服务作为治疗性分娩的车辆。在 EFs 中可以将药物和其他活性剂纳入可调谐的分娩, 而生物制剂和化学配体可以被共轭到纤维表面, 用于特定于单元的目标52或传感53。这里描述了 GRFT 表面修饰的 PLGA EFs 的制作, 作为一个预防 HIV 感染的递送支架。采用静电纺丝法合成了 GRFT-EFs, 相对于其他纤维生…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢犹太遗产基金为这项研究提供资金。我们感谢 Dr. 斯图尔特 II 慷慨地提供使用静电纺丝系统。我们还感谢 Dr. 为我们提供 Griffithsin。此外, 我们还感谢 Dr. 之场和他的实验室在 GRFT ELISA 工作中训练我们。

Materials

Poly(Lactide-co-Glycolide) (PLGA) 50:50 Lactel B6013-2P
1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) Thermo Scientific  147541000
Blunt Dispensing Needle 18g X 1/2 Brico Medical Supplies BN1815
BD 3mL Syringe Luer-lok tip VWR 309657
Parafilm (plastic film) Sigma Aldrich P7793
2-(N-Morpholino)ethanesulfonic acid (MES Buffer) Sigma Aldrich M3671 
Sodium Chloride Sigma Aldrich S7653
Potassium Chloride Sigma Aldrich P9333
Sodium phosphate dibasic Sigma Aldrich S7907
Potassium phosphate monobasic Sigma Aldrich P0662
Hydroxysuccinimide (NHS) Thermo Scientific  24500
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) Thermo Scientific  22980
2-Mercaptoethanol Fisher BP176
Griffithsin (GRFT) Kentucky Bioprocessing NA custom made, no product number
Dimethyl Sulfoxide Milipore 317275
Polyethylene glycol sorbitan monolaurate (Polysorbate, Tween 20) Sigma Aldrich P9416 
Tris EDTA Buffer Sigma Aldrich 93283
Flat-Bottom Immuno Nonsterile 96-Well Plates Thermo Scientific  3355
Influenza Hemagglutinin (HA) Kentucky Bioprocessing NA custom made, no product number
Goat Anti-GRFT (Primary Antibody) Kentucky Bioprocessing NA custom made, no product number
 Donkey anti-goat IgG-HRP (Secondary Antibody) Santa Cruz 2056
Sure Blue TMB Microwell Peroxidase Substrate KPL 52-00-00
DOWNLOAD MATERIALS LIST

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FabricationCharacterizationGriffithsinFiber ScaffoldsSexually Transmitted InfectionsPolylactic AcidCo-glycolic AcidElectrospun FibersAntiviral ProteinDrug DeliveryHIV-1ProphylacticTherapeuticPLGAHFIPElectrospinningSyringe PumpPBSMESEDCNHS

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Vuong, H. R., Tyo, K. M., Steinbach-Rankins, J. M. Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections. J. Vis. Exp. (128), e56492, doi:10.3791/56492 (2017).
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