M Garcia Angélica, de Alwis Weerasekera Hasitha, Pitre Spencer P, McNeill Brian, Lissi Eduardo, Edwards Ana M, Alarcon Emilio I
Facultad de Ciencias Básicas, Escuela de Ciencias Químicas, Universidad Pedagógica y Tecnológica de Colombia-UPTC, Colombia.
Department of Chemistry and Centre for Catalysis Research and Innovation, University of Ottawa, 10 Marie Curie, Ottawa, K1N 6N5, Canada.
J Photochem Photobiol B. 2016 Oct;163:385-90. doi: 10.1016/j.jphotobiol.2016.09.002. Epub 2016 Sep 4.
Comparable intracellular concentrations (≈30pmol/10(6) cells) of bovine serum albumin-ZnPc (BSA) adduct outperformed dipalmitoyl-phosphatidyl-choline (DPPC) liposomes containing ZnPc at photodynamic-killing of human cervical cancer cells (HeLa) after only 15min of irradiation using red light (λ>620nm, 30W/cm(2)). This result could not be simply explained in terms of dye aggregation within the carrier, since in the liposomes the dye was considerably less aggregated than in bovine serum albumin, formulation that was capable to induce cell apoptosis upon red light exposure. Thus, using specific organelle staining, our cumulative data points towards intrinsic differences in intra-cellular localization depending on the cargo vehicle used, being ZnPc:BSA preferentially located in the near vicinity of the nucleus and in the Golgi structures, while the liposomal formulation ZnPc:DPPC was preferentially located in cellular membrane and cytoplasm. In addition to those differences, using real-time advanced fluorescence lifetime imaging of HeLa cells loaded with the photosensitizer contained in the different vehicles, we have found that only for the ZnPc:BSA formulation, there was no significant changes in the fluorescence lifetime of the photosensitizer inside the cells. This contrasts with the in situ≈two-fold reduction of the fluorescence lifetime measured for the liposomal ZnPc formulation. Those observations point towards the superiority of the protein to preserve dye aggregation, and its photochemical activity, post-cell uptake, demonstrating the pivotal role of the delivery vehicle at determining the ultimate fate of a photosensitizer.
在使用红光（λ>620nm，30W/cm²）照射仅15分钟后，牛血清白蛋白 - 锌酞菁（BSA）加合物的细胞内浓度相当（≈30pmol/10⁶个细胞），在对人宫颈癌细胞（HeLa）的光动力杀伤方面优于含有锌酞菁的二棕榈酰磷脂酰胆碱（DPPC）脂质体。这个结果不能简单地用载体中染料的聚集来解释，因为在脂质体中染料的聚集程度比在牛血清白蛋白中低得多，而牛血清白蛋白制剂在红光照射下能够诱导细胞凋亡。因此，通过特定细胞器染色，我们的累积数据表明，根据所使用的载药载体不同，细胞内定位存在内在差异，即ZnPc:BSA优先位于细胞核附近和高尔基体结构中，而脂质体制剂ZnPc:DPPC优先位于细胞膜和细胞质中。除了这些差异，通过对负载不同载体中所含光敏剂的HeLa细胞进行实时先进荧光寿命成像，我们发现只有对于ZnPc:BSA制剂，细胞内光敏剂的荧光寿命没有显著变化。这与脂质体ZnPc制剂原位测量的荧光寿命约降低两倍形成对比。这些观察结果表明蛋白质在保持染料聚集及其细胞摄取后的光化学活性方面具有优越性，证明了载药载体在决定光敏剂最终命运方面的关键作用。
