Label-Free Liposome & ExosomeHyperspectral Microscopy

納米尺度囊泡(如脂質體和外泌體)作為藥物傳遞載體的研究取得了顯著進展，使其在FDA的臨床試驗數量不斷增加。大多數早期臨床試驗工作都集中在攜帶藥物或基因治療負載的工程脂質體上。然而，天然外泌體載體正在迅速取得進展。

Research of nano-scale vesicles such as liposomes and exosomes as drug delivery vectors has progressed significantly leading to increasing numbers of FDA clinical trials. Most of these early clinical trial efforts have focused on engineered liposomes carrying a drug or gene therapy load. However, rapid progress is now being made with natural exosomal vectors.

關鍵挑戰仍然是了解這些納米級載體靶向腫瘤細胞的功效，這通常通過內吞事件或直接細胞膜融合發生，最終將藥物釋放到細胞中。由于囊泡的熒光標記會干擾其任務，因此需要一種無標記的體外成像方法來確認各種囊泡-藥物負載組合的這種功效。成像方法還應有助于確定囊泡樣品中的適當藥物負荷，并確認其與活細胞的相互作用和攝取。

Key challenges remain in understanding the efficacy of tumor cell targeting with these nanoscale carriers, which most often happens through endocytic events or direct cell membrane fusion for eventual release of the drugs to the cell. Since fluorescent labeling of the vesicle can interfere with its task, a label-free, in-vitro imaging method is required to confirm this efficacy with a variety of vesicle-drug load combinations. The imaging method should also help

determine proper drug load across the vesicle sample and confirm its interaction and uptake with live cells.

CytoViva 的增強型暗場高光譜顯微鏡在幫助研究人員了解囊泡載體靶向細胞以及裝載藥物的定時釋放方面非常有效。使用這種無標簽成像技術，可以：

CytoViva's Enhanced Darkfield Hyperspectral Microscope is proving to be highly effective in helping researchers understand both the targeting of vesicle carriers to cells as well as the timed release of their drug cargo. With this label free imaging technique it is possible to:

• 在體外和溶液中成像<100 nm 的無標記外泌體或脂質體

• 區分具有不同有效載荷的囊泡和空囊泡

• 使用光譜圖對囊泡藥物構建體進行無標記細胞運輸，以識別與活細胞或固定細胞的相互作用

• Image label-free exosomes and liposomes < 100 nm in-vitro and in solution• Distinguish vesicles with differing payloads as well as empty vesicles• Conduct label-free cell trafficking of the vesicle-drug construct with spectral mapping to identify interaction with live or fixed cells

上面顯示了用于此應用的 CytoViva 系統的具體示例。在此示例中，將前列腺癌特異性肽添加到負載多柔比星的脂質體中。借助 CytoViva 增強型暗場高光譜顯微鏡系統，研究人員能夠創建負載阿霉素的脂質體（圖 2）獨有的參考光譜庫（圖 1）。請注意，圖 1 中所示的參考光譜在 575nm 處有一個非常明顯的峰，這與阿霉素的光譜特性一致。圖 3 顯示了已與脂質體構建體一起孵育的無標記腫瘤細胞。圖 4 中的紅色像素展示了參考光譜的光譜映射，確認了脂質體構建體在腫瘤細胞內的存在和位置。

A specific example of the CytoViva system being used for this application is shown above. In this example, a prostate cancer-specific peptide is added to a doxorubicin-loaded liposome. With the CytoViva Enhanced Darkfield Hyperspectral Microscope system, the researcher is able to create a reference spectral library (figure 1) unique to the doxorubicin-loaded liposome (figure 2). Note that the reference spectra illustrated in figure 1 has a very distinct peak at ~575nm that is consistent with the spectral properties of doxorubicin. Figure 3 shows a label-free tumor cell that has been incubated with the liposome construct. The red pixels in figure 4 demonstrate the spectral mapping of the reference spectrum confirming the presence and location of the liposomal construct within the tumor cell.

CytoViva 系統的一個主要優點是它不需要熒光標記或改變細胞及脂質體的結構即可有效的成像和分析。此外，這些樣本可以使用 SynVivo for CytoViva 活細胞微流控系統進行活細胞成像。使用該系統，納米藥物遞送結構可以添加到活細胞培養物中，并且可以隨著時間的推移捕獲圖像，記錄動態相互作用。該系統還可用作“模擬體內”系統，用于觀察生物環境中的納米粒子命運。

A key benefit of the CytoViva system is that it does not require fluorescent labeling or other alteration of the cell structure or the liposomes for effective imaging and analysis. Additionally, these samples can be imaged as live cells using the SynVivo for CytoViva live-cell microfluidics chamber. With this system, the nano-drug delivery construct can be added to the live cell culture and images can be captured over time, recording the dynamic interaction. This system can also be used as a "simulated in-vivo" system for observing nanoparticle fate in the biological environment.
 

CytoViva是由美國Auburn大學與Aetos技術有限公司合作成立，具有高校和軍事公司背景，CytoViva納米高光譜成像技術最初是由美國國防部和美國宇航局空間衛星航空成像開發的技術發展而來，該公司創造性的將該技術與增強型暗場技術結合并應用于微觀層面，使其成為一個專有、集成的系統，能夠在納米尺度上對材料、藥物、生命單元、活性大分子、環境污染物等進行高光譜成像及定性定量分析。

CytoViva納米高光譜成像技術2005年一經面市，就在2006年和2007年連續兩屆獲得著名的R&D100大獎，07年同年獲得Nano50TM獎，在09年獲得了兩項美國專利，專利號7542203和7564623，并迅速得到全球各個國家重點實驗室、科研機構及大型制藥企業的認可，包括FDA， NASA， NIST， NIH， EPA， USDA， NIOSH， Lawrence Berkeley Labs， Dow Chemical，Merck， Johnson& Johnson， Stanford， Duke， Harvard等等。