NEST Laboratory, Scuola Normale Superiore, Piazza San Silvestro 12, 56127 Pisa, Italy.
NEST Laboratory, Istituto Nanoscienze-CNR, Piazza San Silvestro 12, 56127 Pisa, Italy.
Molecules. 2024 May 4;29(9):2137. doi: 10.3390/molecules29092137.
Fluorescence lifetime imaging microscopy (FLIM) has proven to be a useful method for analyzing various aspects of material science and biology, like the supramolecular organization of (slightly) fluorescent compounds or the metabolic activity in non-labeled cells; in particular, FLIM phasor analysis (phasor-FLIM) has the potential for an intuitive representation of complex fluorescence decays and therefore of the analyzed properties. Here we present and make available tools to fully exploit this potential, in particular by coding via hue, saturation, and intensity the phasor positions and their weights both in the phasor plot and in the microscope image. We apply these tools to analyze FLIM data acquired via two-photon microscopy to visualize: (i) different phases of the drug pioglitazone (PGZ) in solutions and/or crystals, (ii) the position in the phasor plot of non-labelled poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs), and (iii) the effect of PGZ or PGZ-containing NPs on the metabolism of insulinoma (INS-1 E) model cells. PGZ is recognized for its efficacy in addressing insulin resistance and hyperglycemia in type 2 diabetes mellitus, and polymeric nanoparticles offer versatile platforms for drug delivery due to their biocompatibility and controlled release kinetics. This study lays the foundation for a better understanding via phasor-FLIM of the organization and effects of drugs, in particular, PGZ, within NPs, aiming at better control of encapsulation and pharmacokinetics, and potentially at novel anti-diabetics theragnostic nanotools.
荧光寿命成像显微镜(FLIM)已被证明是一种用于分析材料科学和生物学各个方面的有用方法,例如(略)荧光化合物的超分子组织或未标记细胞中的代谢活性;特别是,FLIM 相分析(phasor-FLIM)有可能直观地表示复杂的荧光衰减,从而表示分析的性质。在这里,我们介绍并提供了充分利用这一潜力的工具,特别是通过色调、饱和度和强度对相图和显微镜图像中的相点位置及其权重进行编码。我们将这些工具应用于通过双光子显微镜获取的 FLIM 数据分析,以可视化:(i)溶液和/或晶体中药物吡格列酮(PGZ)的不同相,(ii)未标记的聚(乳酸-共-乙醇酸)(PLGA)纳米粒子(NPs)在相图中的位置,以及(iii)PGZ 或含有 PGZ 的 NPs 对胰岛素瘤(INS-1 E)模型细胞代谢的影响。PGZ 因其在治疗 2 型糖尿病中的胰岛素抵抗和高血糖方面的疗效而备受认可,而聚合物纳米粒子由于其生物相容性和控制释放动力学,为药物输送提供了多功能平台。这项研究为通过 phasor-FLIM 更好地理解药物,特别是 PGZ,在 NPs 中的组织和作用奠定了基础,旨在更好地控制封装和药代动力学,并有可能开发出新型的抗糖尿病治疗诊断纳米工具。
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