巨噬細胞(Macrophages)是一種位於組織內的白血球，屬於吞噬型細胞，主要能對病原體或外來物進行巨噬作用(Phagocytosis)，將其消化，同時能引發其他免疫細胞，對病原體做出更強烈的免疫反應，所以在細胞治療相關的研究中，常以巨噬細胞為主要研究之標的，透過生物工法的改造，增加巨噬細胞對特定疾病/病原的專一性，以達到治療疾病的目的。然而，以藥物(蛋白質或核酸分子)輸送至巨噬細胞內來達到改造細胞目的的方式，常常會因為巨噬作用強大的分子吸收與代謝的能力，而使得被吸收的藥物分子在細胞體內輸送途中就被降解而無法完成細胞轉染(Transfection)。為了克服這一難題，我們利用超聲波駐波場搭配聚乳酸-聚甘醇酸(Poly(D,L-lactide-co-glycolide); PLGA)藥物微球載體探索一種新式的藥物傳遞至巨噬細胞之方法。本研究中，以Calcein-AM螢光化合物作為模擬的藥物，並以有機溶劑揮發法製備出包覆Calcein-AM的PLGA微球載體。我們以靜態光散射儀分析出微球成品平均粒徑為2.23m並且以動態光散射儀量測出微粒平均電位為-91.82.82mV。再者，我們比較有無PLGA包覆的Calcein-AM兩者於細胞體內降解的情形，結果顯示，攝取微球載體的DH82犬單核巨噬細胞可連續發出螢光長達72小時，而Calcein-AM化合物僅能給予細胞48小時螢光逐漸衰退，證明PLGA微球載體能保護被包覆的物質於細胞體內中並可將其緩慢釋放至少72小時。此外，為了瞭解微球載體與巨噬細胞在超聲波駐波場下之影響，我們以高解析度CCD連續觀察20分鐘，訂定出1 MHz、10 W之超聲波輸出能量、作用時間10分鐘為超聲波駐波場系統設定的最佳參數。最後，為了要驗證超聲波駐波場能否提升微球載體輸送至巨噬細胞體內的效率並且探討兩者間的比例關係，我們設計不同細胞微球數目比例(細胞：微球= 2:1、1:1、1:2)，在經/未經超聲波駐波場處理後，以顯微鏡觀察並搭配流式細胞儀測量各組螢光數量和螢光強度以量化分析其藥物輸送之效率。實驗結果顯示以細胞與微球比2：1可獲得最高的藥物輸送效率，其系統在超聲波駐波場處理過後24小時螢光數量和螢光強度分別明顯提升了2(P< 0.05)和8倍(P< 0.05)。綜合整體數據分析結果，本研究成果證明了利用超聲波駐波場和PLGA微球載體所建構出的藥物輸送系統，可提高藥物分子傳遞至巨噬細胞內的效率。;Inherent resistance of professional phagocytes for drug delivery has long been considered as one of the major barriers for development of immunocellular therapy. To overcome this challenge, a novel in vitro molecular delivery approach to macrophages using ultrasound standing wave field (USWF) associated with microspheres made by poly(lactic-co-glycolic acid) (PLGA) as drug carrier was explored.In this study, fluorescent compound of Calcein-AM was employed as the model drug and the Calcein-AM-loaded PLGA microspheres (CAPMs) were fabricated by organic solvent evaporation method. The mean size and surface charge of the CAPMs was 2.23 m and -91.8 2.82 mV detected using static and dynamic light scattering techniques, respectively. The CAPMs-internalized cells (canine macrophage DH82 cells) may continuously exhibit fluorescent expression for 72 h while most of cells treated with free Calcein-AM molecules lose fluorescence after 48 h, indicating that the encapsulated substance (e.g., Calcein-AM) can be protected from degradation by PLGA microcarriers and constantly released into cytoplasm for at least 72 h. After determining the optimal USWF parameters of 1-MHz frequency, 10-W acoustic output energy and 10-min exposure time, the efficiency of CAPMs internalization by DH82 cells (Cells # : CAPMs # = 2:1, 1:1, 1:2) with and/or without USWF treatment were examined. In this study, both number and fluorescence intensity of cells with and without USWF treatment were quantitatively analyzed by using flow cytometry. Our results showed that the optimal ratio of cell to CAPM was 2:1 in which the drug delivery efficiency of USWF-treated groupsignificantly enhanced about 2- (P< 0.05) and 8-fold (P< 0.05) in terms of fluorescence-expressed cell number and fluorescence intensity emitted, respectively as compared to the setting without USWF treatment. Overall the synthetic system of USWF in association with PLGA drug microcarriers developed in this study provided a feasible means for enhancement of molecular transfer efficiency of macrophages in vitro.
