以外覆紅血球膜之磁性奈米顆粒標的與分離流行性感冒病毒

Abstract

與目標細胞之表面受體結合是病毒感染的首要過程，在這個過程中，病毒會藉由其表面蛋白特異性地鍵結細胞受體，進而入侵細胞。根據病毒與宿主細胞之間有選擇性的結合力，因此可以透過將細胞膜連同表面受體一同包覆在奈米顆粒表面來標的病毒。本研究選用流行性感冒病毒株 A/Puerto Rico/8/34 (H1N1) 作為模式病毒，將紅血球的細胞膜黏附在具有磁性的奈米顆粒表面 (簡稱RBC-mNP)，進行流行性感冒病毒的標的與分離。首先，藉由奈米顆粒追蹤分析，得知作為核心的奈米顆粒與紅血球膜囊泡之間最佳的比例關係，再以蔗糖密度梯度離心方式將外覆紅血球膜之奈米顆粒純化，以提高病毒標的與分離的效率。以穿透式電子顯微鏡觀察，當奈米顆粒外覆具有高密度唾液酸分布的紅血球細胞膜，與流行性感冒病毒混合後會形成穩定結合狀態。接著將超順磁氧化鐵裝載於已建立好的紅血球膜奈米顆粒之中，以此具有磁力感受性之RBC-mNP用來處理含有流感病毒的尿囊液，發現RBC-mNP能有效標的病毒，經過磁力吸引聚集，可達到分離且濃縮流感病毒的效果；RBC-mNP濃縮分離過的尿囊液，不僅在qRT-PCR分析中有顯著較高的病毒基因量，在細胞培養中，流感病毒的感染力價亦有顯著提升，經過RBC-mNP的前處理並可增強病毒樣本在流感快篩試片上的陽性訊號，有助於病毒診斷的敏感度。本研究利用流感病毒與紅血球的血球凝集特性，製備外覆紅血球膜之磁性奈米顆粒，成功應用於流感病毒的標的與分離，提高偵測效率。未來此平台將可繼續應用於遞送治療流感病毒的藥物，或用於標的其他重要的病原，有助於傳染病的診斷與防治。

Attachment to cellular surfaces is a major attribute among infectious pathogens for initiating disease pathogenesis. In viral infections, viruses exploit receptor-ligand interactions to latch onto cellular exterior prior to subsequent entry and invasion. In light of the selective binding affinity between viral pathogens and cells, nanoparticles cloaked in cellular membranes are herein employed for virus targeting. Using influenza virus A/Puerto Rico/8/34 (H1N1) as a model, erythrocyte membrane-cloaked nanoparticles are prepared and modified with magnetic functionalities (RBC-mNP) for virus targeting and isolation. To maximize targeting and isolation efficiency, density gradient centrifugation and nanoparticle tracking analysis were applied to minimize presence of uncoated particles and membrane vesicles. The resulting nanoparticles possess a distinctive membrane corona, a sialylated surface, and form colloidally stable clusters with influenza viruses. Magnetic functionality is bestowed to the nanoparticles through encapsulation of superparamagnetic iron-oxide particles, which enables influenza virus enrichment via magnetic extraction. Viral samples enriched by the RBC-mNPs are compatible with downstream analysis by qRT-PCR, immunochromatographic strip test, and cell-based titering assays. The demonstration of pathogen targeting and isolation by RBC-mNPs highlights a biologically inspired approach towards improved treatment and diagnosis against infectious disease threats. The work also sheds light on the efficient membrane cloaking mechanism that bestows nanoparticles with cell mimicking functionalities.