Center for Bio/Molecular Science and Engineering , Naval Research Laboratory , Code 6900, 4555 Overlook Avenue SW , Washington , D.C. 20375 , United States.
Optical Sciences Division , Naval Research Laboratory , Code 5600, 4555 Overlook Avenue SW , Washington , D.C. 20375 , United States.
Bioconjug Chem. 2018 Aug 15;29(8):2701-2714. doi: 10.1021/acs.bioconjchem.8b00374. Epub 2018 Jul 24.
Current challenges in photodynamic therapy (PDT) include both the targeted delivery of the photosensitizer (PS) to the desired cellular location and the maintenance of PS efficacy. Zinc phthalocyanine (ZnPc), a macrocyclic porphyrin and a potent PS for PDT, undergoes photoexcitation to generate reactive singlet oxygen that kills cells efficiently, particularly when delivered to the plasma membrane. Like other commonly employed PS, ZnPc is highly hydrophobic and prone to self-aggregation in aqueous biological media. Further, it lacks innate subcellular targeting specificity. Cumulatively, these attributes pose significant challenges for delivery via traditional systemic drug delivery modalities. Here, we report the development and characterization of a liquid crystal nanoparticle (LCNP)-based formulation for the encapsulation and targeted tethering of ZnPc to the plasma membrane bilayer. ZnPc was coloaded with the organic fluorophore, perylene (PY), in the hydrophobic polymeric matrix of the LCNP core. PY facilitated the fluorescence-based tracking of the LCNP carrier while also serving as a Förster resonance energy transfer (FRET) donor to the ZnPc acceptor. This configuration availed efficient singlet oxygen generation via enhanced excitation of ZnPc from multiple surrounding PY energy donors. When excited in a FRET configuration, cuvette-based assays revealed that singlet oxygen generation from the ZnPc was ∼1.8-fold greater and kinetically 12 times faster compared to when the ZnPc was excited directly. The specific tethering of the LCNPs to the plasma membrane of HEK 293 T/17 and HeLa cells was achieved by surface functionalization of the NPs with PEGylated cholesterol. In HeLa cells, LCNPs coloaded with PY and ZnPc, when photoexcited in a FRET configuration, mediated 70% greater cell killing compared to LCNPs containing ZnPc alone (direct excitation of ZnPc). This was attributed to a significant increase of the oxidative stress in the cells during the PDT. Overall, this work details the ability of the LCNP platform to facilitate (1) the specific tethering of the PY-ZnPc FRET pair to the plasma membrane and (2) the FRET-mediated, augmented singlet oxygen generation for enhanced PDT relative to the direct excitation of ZnPc alone.
当前光动力疗法（PDT）面临的挑战包括光敏剂（PS）靶向递送至所需细胞位置和维持 PS 疗效。锌酞菁（ZnPc）是一种大环卟啉，也是 PDT 的一种有效 PS，它经历光激发产生有效的活性单线态氧，有效地杀死细胞，特别是当递送至质膜时。像其他常用的 PS 一样，ZnPc 具有很强的疏水性，并且容易在水性生物介质中自聚集。此外，它缺乏先天的亚细胞靶向特异性。累积起来，这些特性给通过传统的全身药物输送方式输送带来了重大挑战。在这里，我们报告了一种基于液晶纳米颗粒（LCNP）的制剂的开发和表征，用于将 ZnPc 封装并靶向固定到质膜双层。ZnPc 与有机荧光团苝（PY）共载入 LCNP 核心的疏水性聚合物基质中。PY 促进了 LCNP 载体的荧光跟踪，同时也作为 ZnPc 受体的Förster 共振能量转移（FRET）供体。这种配置通过来自多个周围 PY 能量供体的 ZnPc 的增强激发，提供了有效的单线态氧生成。在 FRET 配置下激发时，比色皿测定法显示，来自 ZnPc 的单线态氧生成增加了约 1.8 倍，动力学上快了 12 倍，与直接激发 ZnPc 相比。通过用 PEG 化胆固醇对 NPs 进行表面功能化，实现了 LCNP 对 HEK 293 T/17 和 HeLa 细胞质膜的特异性固定。在 HeLa 细胞中，当以 FRET 构型光激发时，共载有 PY 和 ZnPc 的 LCNP 介导的细胞杀伤比单独含有 ZnPc 的 LCNP 增加了 70%（直接激发 ZnPc）。这归因于 PDT 期间细胞内氧化应激的显著增加。总体而言，这项工作详细说明了 LCNP 平台的能力，即（1）促进 PY-ZnPc FRET 对质膜的特异性固定，以及（2）通过 FRET 介导的，增强的单线态氧生成，从而增强 PDT 相对于单独直接激发 ZnPc。
