Nanotechnology has made immense progress in the last decade, providing novel treatments as a last resort for hard-to-treat cancers, and more recently, in the pancreatic cancer field . An advantage of nanotechnology lies in the ability to engineer tailor-made formulations to meet ones need. However, the underlying mechanisms explaining the beneficial effect of nanoplatforms versus those of free drugs on drug trafficking remain unclear, as are the effects of the nanovectors themselves. Elucidating these mechanisms could provide crucial insights into engineering novel cancer therapeutics yielding more potent and selective nanoformulations. In the current study, we investigated the mechanisms of free Gemcitabine and two Gemcitabine nanoplatforms, namely PLGem and GemPo, in a resistant human pancreatic cancer cell line, PANC1, which most closely mimics the PDA phenotype and hence, is ideally suited to investigate the role of nanovectors in circumventing drug resistance . In non-resistant pancreatic cells, our preliminary data shows that Gemcitabine and the nanoparticles elicited almost complete cell death, hence making the comparison between free and encapsulated Gemcitabine difficult. Our data also indicates that this inherent resistance pertains to the presence of membrane efflux pumps selectively in the PANC1 cell line. The exact mechanisms by which nanoparticles are able to evade this resistance, however, remains to be investigated.
In order for nanoparticles to deliver proper payload and thus achieve pharmacological effects, drug release and subsequent sustained degradation of each nanoplatform are critical steps . The rates of degradation mainly depend on diffusion of drugs through the nanovectors, as well as erosion of these nanoparticles . Based on the Gemcitabine release profiles studies herein, although both nanoplatforms exhibited sustained drug release, PLGem delivered a higher payload than GemPo in PANC1 cells, which correlated with the increased cytotoxicity and apoptosis observed with PLGem. Higher hydrolytic degradation rates reported for PLGA backbones as opposed to lipids have previously been reported [35, 36]. These results indicate that PLGA is a more-suited nanovector for administering Gemcitabine to resistant pancreatic cancer cells as it preferentially prolongs Gemcitabine’s release.
Thus far, studies which investigated the mechanisms of nanoparticle internalization in cancer cells mainly focused on endocytotic mechanisms including clathrin-mediated endocytosis, which is supported in this study , and phagocytosis, [19, 38, 39]. However, subsequent intracellular trafficking studies were only based on inferences from biological assays (e.g. MTS, apoptosis), or labeling of the nanovector with a fluorochrome. Although the later approach successfully allows monitoring of nanovector internalization in real-time, it does not take into account that the drug has already been release from the nanovector, nor does it allow for precise investigation of the drug’s effect on organelles. In this study, we have overcome these limitations by performing ultrastructural analysis in order to track Gemcitabine (free or encapsulated) internalization inside PANC1 cells.
Ultrastructural analysis provides a straightforward mean of investigating whole-cell effects of Gemcitabine at high resolution. Effectively, both on-target (eg. nuclear) and off-target (eg. ER, cytoplasmic and mitochondrial) differences in intracellular trafficking between treatment groups were readily observed. As such, TEM investigation shows that free drug was internalized by a 5-fold smaller membranal invaginations than both nanoplatforms, indicating that an additional internalization pathway occurs in the presence of the nanovectors, which is supported by several studies [38, 40]. Furthermore, the degree of nuclear fenestrations implies that PLGem and GemPo reach their nuclear target more robustly than Gemcitabine, which might explains why GemPo treatment yielded slightly more apoptosis.
Mechanisms proper to the nanoplatforms include significant mitochondrial pseudo-inclusions into the nucleus, dilatation/swelling of the ER and loss of electron density in the cytoplasm. These effects have been reported in instances of severe apoptosis and necrosis [41–44]. Interestingly, there were major differences between both nanoplatforms, as PLGem elicited more off-target effects than GemPo. PLGem administration resulted in the appearance of large vacuoles adjacent to the membranal invaginations and leading up to the nucleus, implying a more robust pathway by which PLGem reaches its genomic target as opposed to GemPo. PLGem treatment also led to significant ER hypertrophy and rearrangement of mitochondrial cristae into concentric rings. These cristae rearrangements are thought to be a defense mechanism during instances of cellular deterioration, and have been reported in a case of hypertrophic cardiomyopathy [45, 46]. Furthermore, the appearance of ER hypertrophy reflects a more advanced state of ER stress and apoptosis as opposed to GemPo treatment , which is corroborated by both MTS and FACS data. These results constitute, to the best of our knowledge, novel findings, and might represent a strategy deployed by PLGem to overcome drug resistance in resistant pancreatic cells, although this remains to be investigated.
Taken together, ultrastructural mechanistic studies in the PANC1 cell line confirm the preferential biological effects observed with PLGem versus GemPo and free Gemcitabine and further indicate that, when Gemcitabine is delivered in PLGA, it can more potently target several organelles asides from the nucleus, including the ER and mitochondria.