Recombinant phosphatidylserine-binding nanobodies for targeting of extracellular vesicles to tumor cells: a plug-and-play approach.
Recombinant phosphatidylserine-binding nanobodies for targeting of extracellular vesicles to tumor cells: a plug-and-play approach.
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DOI：
10.1039/c7nr06966a
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发表时间：
2018-02-01
期刊：
影响因子：
6.7
通讯作者：
Vader P
中科院分区：
文献类型：
--
作者：
Kooijmans SAA ;Gitz-Francois JJJM ;Schiffelers RM ;Vader P
用重组磷脂酰丝氨酸结合纳米抗体修饰分离的细胞外囊泡增加了肿瘤细胞对它们的摄取。细胞外囊泡（EV）由于其在细胞之间功能性地转移生物货物的能力而越来越被认为是候选药物递送系统。然而，通过生产细胞的工程化来操纵EV的靶向性质可能是具有挑战性的并且是耗时的。作为一种赋予分离的EV肿瘤靶向特性的新方法，我们产生了针对表皮生长因子受体（EGFR）的纳米抗体与乳凝集素（C1 C2）的磷脂酰丝氨酸（PS）结合结构域融合的重组融合蛋白。C1 C2-纳米抗体融合蛋白在HEK 293细胞中表达，并从培养基中分离，如通过SDS-PAGE确定的具有接近完全的纯度。融合蛋白特异性结合PS和其他常见的EV膜脂质没有表现出亲和力。此外，与融合至非靶向对照纳米抗体的C1 C2（R2-C1 C2）相反，融合至抗EGFR纳米抗体的C1 C2（EGa 1-C1 C2）以高亲和力结合EGFR并与其天然配体EGF的结合竞争。这两种蛋白质都容易自缔合到来自红细胞和Neuro 2A细胞的EV的膜上，而不影响EV的大小和完整性。EV结合的R2-C1 C2不影响EV-细胞相互作用，而EV结合的EGa 1-C1 C2剂量依赖性地增强EGFR过表达肿瘤细胞对EV的特异性结合和摄取。总之，我们开发了一种新的策略，在分离后有效和普遍地赋予PS暴露EV肿瘤靶向特性，而不影响EV特征，避免了修饰EV分泌细胞的需要。这种策略也可以用于用其他部分（包括成像探针或治疗性蛋白质）装饰EV。
Decoration of isolated extracellular vesicles with recombinant phosphatidylserine-binding nanobodies increases their uptake by tumor cells. Extracellular vesicles (EVs) are increasingly being recognized as candidate drug delivery systems due to their ability to functionally transfer biological cargo between cells. However, manipulation of targeting properties of EVs through engineering of the producer cells can be challenging and time-consuming. As a novel approach to confer tumor targeting properties to isolated EVs, we generated recombinant fusion proteins of nanobodies against the epidermal growth factor receptor (EGFR) fused to phosphatidylserine (PS)-binding domains of lactadherin (C1C2). C1C2-nanobody fusion proteins were expressed in HEK293 cells and isolated from culture medium with near-complete purity as determined by SDS-PAGE. Fusion proteins specifically bound PS and showed no affinity for other common EV membrane lipids. Furthermore, C1C2 fused to anti-EGFR nanobodies (EGa1-C1C2) bound EGFR with high affinity and competed with binding of its natural ligand EGF, as opposed to C1C2 fused to non-targeting control nanobodies (R2-C1C2). Both proteins readily self-associated onto membranes of EVs derived from erythrocytes and Neuro2A cells without affecting EV size and integrity. EV-bound R2-C1C2 did not influence EV–cell interactions, whereas EV-bound EGa1-C1C2 dose-dependently enhanced specific binding and uptake of EVs by EGFR-overexpressing tumor cells. In conclusion, we developed a novel strategy to efficiently and universally confer tumor targeting properties to PS-exposing EVs after their isolation, without affecting EV characteristics, circumventing the need to modify EV-secreting cells. This strategy may also be employed to decorate EVs with other moieties, including imaging probes or therapeutic proteins.
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影响因子：
46.9
作者：
Alvarez-Erviti, Lydia;Seow, Yiqi;Wood, Matthew J. A.
通讯作者：
Wood, Matthew J. A.
影响因子：
12.4
作者：
Bellavia D;Raimondo S;Calabrese G;Forte S;Cristaldi M;Patinella A;Memeo L;Manno M;Raccosta S;Diana P;Cirrincione G;Giavaresi G;Monteleone F;Fontana S;De Leo G;Alessandro R
通讯作者：
Alessandro R
影响因子：
10.8
作者：
Kooijmans, S. A. A.;Fliervoet, L. A. L.;Schiffelers, R. M.
通讯作者：
Schiffelers, R. M.
DOI：
10.1111/jth.12767
发表时间：
2015-02
期刊：
Journal of thrombosis and haemostasis : JTH
影响因子：
--
作者：
Arraud N;Gounou C;Linares R;Brisson AR
通讯作者：
Brisson AR
影响因子：
21.3
作者：
Costa-Silva B;Aiello NM;Ocean AJ;Singh S;Zhang H;Thakur BK;Becker A;Hoshino A;Mark MT;Molina H;Xiang J;Zhang T;Theilen TM;García-Santos G;Williams C;Ararso Y;Huang Y;Rodrigues G;Shen TL;Labori KJ;Lothe IM;Kure EH;Hernandez J;Doussot A;Ebbesen SH;Grandgenett PM;Hollingsworth MA;Jain M;Mallya K;Batra SK;Jarnagin WR;Schwartz RE;Matei I;Peinado H;Stanger BZ;Bromberg J;Lyden D
通讯作者：
Lyden D