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Fluorescence lifetime microscopy unveils the supramolecular organization of liposomal Doxorubicin
Nanoscale ( IF 5.8 ) Pub Date : 2022-06-17 , DOI: 10.1039/d2nr00311b Paolo Tentori 1, 2 , Giovanni Signore 3 , Andrea Camposeo 4 , Annalisa Carretta 1 , Gianmarco Ferri 1 , Pasqualantonio Pingue 1, 4 , Stefano Luin 1 , Daniela Pozzi 5 , Enrico Gratton 6 , Fabio Beltram 1, 2 , Giulio Caracciolo 5 , Francesco Cardarelli 1, 4
Nanoscale ( IF 5.8 ) Pub Date : 2022-06-17 , DOI: 10.1039/d2nr00311b Paolo Tentori 1, 2 , Giovanni Signore 3 , Andrea Camposeo 4 , Annalisa Carretta 1 , Gianmarco Ferri 1 , Pasqualantonio Pingue 1, 4 , Stefano Luin 1 , Daniela Pozzi 5 , Enrico Gratton 6 , Fabio Beltram 1, 2 , Giulio Caracciolo 5 , Francesco Cardarelli 1, 4
Affiliation
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The supramolecular organization of Doxorubicin (DOX) within the standard Doxoves® liposomal formulation (DOX®) is investigated using visible light and phasor approach to fluorescence lifetime imaging (phasor-FLIM). First, the phasor-FLIM signature of DOX® is resolved into the contribution of three co-existing fluorescent species, each with its characteristic mono-exponential lifetime, namely: crystallized DOX (DOXc, 0.2 ns), free DOX (DOXf, 1.0 ns), and DOX bound to the liposomal membrane (DOXb, 4.5 ns). Then, the exact molar fractions of the three species are determined by combining phasor-FLIM with quantitative absorption/fluorescence spectroscopy on DOXc, DOXf, and DOXb pure standards. The final picture on DOX® comprises most of the drug in the crystallized form (∼98%), with the remaining fractions divided between free (∼1.4%) and membrane-bound drug (∼0.7%). Finally, phasor-FLIM in the presence of a DOX dynamic quencher allows us to suggest that DOXf is both encapsulated and non-encapsulated, and that DOXb is present on both liposome-membrane leaflets. We argue that the present experimental protocol can be applied to the investigation of the supramolecular organization of encapsulated luminescent drugs/molecules all the way from the production phase to their state within living matter.
中文翻译：
荧光寿命显微镜揭示脂质体多柔比星的超分子结构
使用可见光和荧光寿命成像相量法 (phasor-FLIM) 研究标准 Doxoves® 脂质体制剂 (DOX®) 中多柔比星 (DOX) 的超分子结构。首先，DOX® 的 phasor-FLIM 特征被分解为三种共存荧光物质的贡献，每种荧光物质都有其特有的单指数寿命，即：结晶 DOX (DOX c , 0.2 ns)、游离 DOX (DOX f , 1.0 ns), DOX 与脂质体膜结合 (DOX b , 4.5 ns)。然后，通过将 phasor-FLIM 与 DOX c、 DOX f和 DOX b的定量吸收/荧光光谱相结合，确定三种物质的精确摩尔分数纯标准。DOX® 的最终图片包含大部分结晶形式的药物（~98%），其余部分分为游离（~1.4%）和膜结合药物（~0.7%）。最后，在 DOX 动态猝灭剂存在下的 phasor-FLIM 允许我们提出 DOX f既是封装的又是非封装的，并且 DOX b存在于两个脂质体膜小叶上。我们认为，本实验方案可应用于研究封装发光药物/分子的超分子组织，从生产阶段一直到其在生命物质中的状态。
更新日期：2022-06-17
中文翻译：
荧光寿命显微镜揭示脂质体多柔比星的超分子结构
使用可见光和荧光寿命成像相量法 (phasor-FLIM) 研究标准 Doxoves® 脂质体制剂 (DOX®) 中多柔比星 (DOX) 的超分子结构。首先，DOX® 的 phasor-FLIM 特征被分解为三种共存荧光物质的贡献，每种荧光物质都有其特有的单指数寿命，即：结晶 DOX (DOX c , 0.2 ns)、游离 DOX (DOX f , 1.0 ns), DOX 与脂质体膜结合 (DOX b , 4.5 ns)。然后，通过将 phasor-FLIM 与 DOX c、 DOX f和 DOX b的定量吸收/荧光光谱相结合，确定三种物质的精确摩尔分数纯标准。DOX® 的最终图片包含大部分结晶形式的药物（~98%），其余部分分为游离（~1.4%）和膜结合药物（~0.7%）。最后，在 DOX 动态猝灭剂存在下的 phasor-FLIM 允许我们提出 DOX f既是封装的又是非封装的，并且 DOX b存在于两个脂质体膜小叶上。我们认为，本实验方案可应用于研究封装发光药物/分子的超分子组织，从生产阶段一直到其在生命物质中的状态。
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