Mechanistic studies of Gemcitabine-loaded nanoplatforms in resistant pancreatic cancer cells
© Papa et al.; licensee BioMed Central Ltd. 2012
Received: 19 June 2012
Accepted: 20 September 2012
Published: 22 September 2012
Pancreatic cancer remains the deadliest of all cancers, with a mortality rate of 91%. Gemcitabine is considered the gold chemotherapeutic standard, but only marginally improves life-span due to its chemical instability and low cell penetrance. A new paradigm to improve Gemcitabine’s therapeutic index is to administer it in nanoparticles, which favour its delivery to cells when under 500 nm in diameter. Although promising, this approach still suffers from major limitations, as the choice of nanovector used as well as its effects on Gemcitabine intracellular trafficking inside pancreatic cancer cells remain unknown. A proper elucidation of these mechanisms would allow for the elaboration of better strategies to engineer more potent Gemcitabine nanotherapeutics against pancreatic cancer.
Gemcitabine was encapsulated in two types of commonly used nanovectors, namely poly(lactic-co-glycolic acid) (PLGA) and cholesterol-based liposomes, and their physico-chemical parameters assessed in vitro. Their mechanisms of action in human pancreatic cells were compared with those of the free drug, and with each others, using cytotoxity, apoptosis and ultrastructural analyses.
Physico-chemical analyses of both drugs showed high loading efficiencies and sizes of less than 200 nm, as assessed by dynamic light scattering (DLS) and transmission electron microscopy (TEM), with a drug release profile of at least one week. These profiles translated to significant cytotoxicity and apoptosis, as well as distinct intracellular trafficking mechanisms, which were most pronounced in the case of PLGem showing significant mitochondrial, cytosolic and endoplasmic reticulum stresses.
Our study demonstrates how the choice of nanovector affects the mechanisms of drug action and is a crucial determinant of Gemcitabine intracellular trafficking and potency in pancreatic cancer settings.
KeywordsGemcitabine Pancreatic cancer Liposome Poly(lactic-co-glycolic acid) Transmission electron microscopy
Pancreatic ductal adenocarcinoma (PDA) kills 37,680 Americans per year, with a mortality rate of 91%, making it the deadliest of all cancers [1–4]. Since 1998, the nucleoside analogue Gemcitabine has been the drug of choice for treating PDA, often in conjunction with radiotherapy and/or a cocktail of other chemotherapeutics [2, 5–7]. However, Gemcitabine only marginally improves lifespan, mainly due to its chemical instability and poor cellular uptake, resulting in an extremely short half-life and bioavailability [8–12]. This translates into frequent administrations of Gemcitabine at high doses, culminating in significant systemic toxicity and associated resistance, thus overshadowing the drug’s promising pharmacological effects. Recent attempts to remedy this problem by trying new drug combinations have not made it past Phase II clinical trials and are very poorly tolerated by the patient, as the lacunae due to the drug’s low half-life, low cellular uptake and high systemic toxicity remain [5, 10, 13, 14]. These conundrums explain why treatment options have remained stagnant for over the past five decades and create an unparalleled need to find a different modality to deliver Gemcitabine to eradicate pancreatic cancer.
Nanotechnology has made exceptional headway in this regard during the past decade, emerging as a revolutionary platform to treat a wide variety of tumors, mainly due to prolonged drug release, as well as increased cell internalization when under 500 nm in diameter [15–21]. As such, we and others have shown that using biodegradable poly(lactic-co-glycolic acid) (PLGA) or liposomal nanovectors to deliver chemotherapeutics results in significant improvement of tumor burden in a wide variety of cancers, including those of the breast and the skin [17, 22–24]. Recently, Gemcitabine liposomal nanoformulations have shown promising results in the Laboratory, including prolonged drug release and attenuation of tumor burden [25–27]. However, the rational for choosing the liposomal nanovector for Gemcitabine, as opposed to other nanoformulations, remains unclear. Furthermore, although it has been clearly documented that nanoplatforms increase endocytosis of Gemcitabine, their underlying mechanisms of action once the drug has entered the cell remains uncharacterized [26, 28]. Also unknown is whether the type of nanovector used affects this intracellular trafficking of Gemcitabine. Most nanoparticle studies thus far have only alluded to the intracellular delivery of their payload by indirect means, such as by cytotoxicity and apoptotic studies. A more robust analysis can be performed by combining these studies with ultrastructural characterization by transmission electron microscopy . Elucidation of these factors would allow for more robust nanoparticle engineering.
The aims of this study were two-fold. Firstly, to compare whether nanovector type affects the physico-chemical and biological effects of Gemcitabine and secondly, to directly investigate, at the ultrastuctural level, the underlying mechanisms of action imparted by these nanovectors on Gemcitabine trafficking in pancreatic cancer cells, as opposed to free drug. Towards these aims, Gemcitabine was encapsulated in PLGA or liposomal nanovectors, and the resulting nanoplatforms termed PLGem and GemPo, respectively. Both nanoparticles were around 150 nm in diameter and provided sustained Gemcitabine release for at least a week. Using a human resistant pancreatic cell line , we demonstrated that PLGem promoted more cytotoxicity and apoptosis than both GemPo and free Gemcitabine, which translated into strikingly different mechanisms of action at the ultrastructural level.
All the solvents were purchased from Sigma-Aldrich (St-Louis, MO) and Fisher Scientific (Pittsburgh, PA), unless otherwise noted, and used without further purification. L-α-Phosphatidylcholine (PC), cholesterol (chol) and polyvinyl alcohol (PVA) were obtained from Sigma-Aldrich, whereas 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG-2000) was purchased from Avanti Polar Lipids (Alabaster, AL). The poly(lactic-co-glycolic acid) (M. W. ≈ 4.2 kDa) having a lactic/glycolic molar ratio of 50:50 was purchased from Lakeshore Biomaterials (Birmingham, AL). Gemcitabine hydrochloride was purchased from Tocris (Ellisville, MO).
Synthesis of Gemcitabine-encapsulated liposomes (GemPo)
Synthesis of Gemcitabine-encapsulated PLGA nanoparticles (PLGem)
Transmission electron microscopy (TEM) of the nanoparticles
Samples were deposited onto a carbon membrane supported by a copper grid and left until complete drying was achieved. Next, a drop of 2% uranyl acetate solution was applied to improve the contrast of the sample. TEM observations were performed on a JEOL JEM 200 CX microscope for PLGem, achieving a lattice and a point-to-point resolution of 1.4 Å and 3.5 Å. The acceleration voltage was set to 120 kV. GemPo was imaged with a JEOL 1200-EX operating at 80 kV.
In vitro drug release profiles
PLGem or GemPo were suspended in 500 μL of PBS or PANC1 cell lysates, and sealed in a dialysis bag (MWCO ≈ 1,000 Da). The dialysis bag was incubated in 1 mL of PBS buffer at room temperature with gentle shaking, in a humidified chamber to prevent evaporation, and the dialysis was kept for up to a month. 10 μL of aliquots were extracted from the incubation medium at predetermined time intervals, dissolved in 90 μL DMSO and the released Gemcitabine was quantified by UV-visible spectroscopy at the characteristic wavelength of λ = 268 nm. After withdrawing each aliquot, the incubation medium was replenished with 10 μL of fresh PBS.
The human pancreatic carcinoma cell line, PANC1, was obtained from American Type Tissue Culture Collection (Rockville, MD) and was maintained in DMEM supplemented with 10% FBS and antibiotic/antimycotic (all from Invitrogen, Carlsbad, CA). PANC1 cells were grown on 100 mm dishes, subcultured using trypsin (0.25%) and EDTA (0.01%) treatment and replated at 2,500 cells.cm-2. Cells were incubated with serum-deprived medium prior to drug addition, which itself was in complete medium. For all experiments, cells were treated with PLGem, GemPo or free Gemcitabine, with solvent- or empty nanovector-treated cells serving as internal controls.
MTS cytotoxicity assay
PANC1 cells were seeded at a density of 10,000 cells per well in 96-well plates overnight. Cells were incubated with drugs for 3 days. Solvent-treated cells served as internal controls. The percentages of viable cells were then quantified with 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium (MTS) from the CellTiter 96 AQueous One Solution kit (Promega Corporation, Madison, WI). MTS is reduced by mitochondrial dehydrogenases of live cells, yielding a colored adduct that can be read spectrophotometrically. Briefly, the cells were washed with PBS, incubated with 0.3 mg.mL-1 of MTS, in basal medium without phenol red, for 2 h at 37°C and absorbance was then measured at 490 nm in a microplate spectrophotometer (Epoch, Biotek Instruments, Winooski, VT). Final absorbance, corresponding to cell proliferation, was plotted after removing background values from each data point and divided by the mean of solvent-treated cells.
Apoptosis study by AnnexinV-FITC and propidium iodide staining
Cells grown in 6-well plates were treated with 1 μM drugs for 2 days, and incubated with 5 μL of AnnexinV-Alexa Fluor 488 in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) for 15 min in the dark, according to the manufacturer’s protocol. Cells were then washed with binding buffer, counterstained with propidium iodide and immediately processed for FITC and propidium iodide detection using an Accuri C6 flow cytometer (ex/em 488/499 and 535/617 nm, respectively). AnnexinV-Alexa Fluor 488, propidium iodide, or both, were omitted for the negative controls.
Ultracharacterization studies of pancreatic cells by TEM
PANC1 cells treated with 1 μM of drugs for 1 day were fixed in 2.5% gluteraldehyde, 1.25% paraformaldehyde and 0.03% picric acid in 0.1 M sodium cacodylate buffer (pH = 7.4). The cells were then postfixed for 30 min in 1% Osmium tetroxide (OsO4)/1.5% Potassium ferrocyanide (K4Fe(CN)6), washed in water 3 times and incubated in 1% aqueous uranyl acetate for 30 min, followed by 2 washes in water and subsequent dehydration in grades of alcohol (5 min each: 50%, 70%, 95%, 2x 100%). Cells were removed from the dish in propyleneoxide, pelleted at 3,000 rpm for 3 min and infiltrated for 2 h in a 1:1 mixture of propyleneoxide and TAAB Epon (Marivac Canada Inc. St. Laurent, Canada). The samples were subsequently embedded in TAAB Epon and polymerized at 60°C for 2 days. Ultrathin sections (about 60 nm) were cut on a Reichert Ultracut-S microtome, picked up on to copper grids stained with lead citrate and examined in a Tecnai G2 Spirit BioTWIN microscope operating at 80 kV and images were recorded with an AMT 2 k CCD camera. Solvent- and empty nanovector-treated cells served as internal controls.
All results were expressed as mean ± SEM of at least quadruplate samples. Statistical comparisons were obtained using one-way ANOVA, followed by the Newman-Keuls test. Probability (p) values less than 0.05 were considered significant.
Physico-chemical parameters of PLGem and GemPo
Synthesis of Gemcitabine-loaded nanoplatforms
Nanoparticles morphologies, sizes and encapsulation efficiencies
Gemcitabine release profiles are altered by nanovector type
Gemcitabine-mediated cytotoxicity is improved in the PLGA nanovector
Gemcitabine-mediated apoptosis is improved in the PLGA nanovector
Ultracharacterization studies show differing internalization mechanisms between free Gemcitabine, PLGem and GemPo
There were significant differences between free Gemcitabine and both nanoplatforms with respect to off-target effects. Only PLGem- and GemPo-treated cells showed significant dilatation/swelling of the endoplasmic reticulum (ER) (Figures 6C5 and D4), which correlated with an overproduction of glycogen, localizing to the cytoplasm for PLGem (Figure 6D2) and to the cytoplasm, ribosomes and vesicles for GemPo (Figures 6C1 and C6). In addition, significant loss of electron density in the cytoplasm was observed for both nanoplatforms (Figures 6C2, D6). Lastly, only GemPo (Figures 6C7, C8) and PLGem (results not shown) treatments significantly enhanced mitochondrial pseudo-inclusions into the nucleus.
Differences between both nanoplatforms were also observed, as PLGem caused additional perturbations. As such, PLGem treatment led to an intracellular trafficking pathway not observed with GemPo, namely the presence of larger transport vesicles (mean size around 2μm, figure 6D7) adjacent to the membranal invaginations and leading up to the nucleus (Figure 6D8). PLGem also provoked a strong morphological perturbation of mitochondrial cristae into a concentric configuration (Figure 6D6), as well as ER hypertrophy (Figure 6D5).
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.
In conclusion, our study uncovers novel mechanisms of action employed by Gemcitabine-loaded nanoplatforms, as opposed to free drug, and confirms that the choice of nanovector is a crucial parameter that should be taken into consideration for delivering Gemcitabine to resistant pancreatic cancer cells. Although these results need to be validated in vivo, they represent the first study of its kinds in pancreatic cancer research and could serve as an intermediate step before passing to mice studies. Interestingly, the ultrastructural analyses reported herein uncovered potential new targets which could be combined with Gemcitabine for pancreatic cancer treatment, namely the ER and mitochondria.
Dynamic Light Scattering
Fluorescence Activated Cell Sorting
Pancreatic Ductal Adenocarcinoma
Transmission Electron Microscopy.
This work was supported by a CIHR Fellowship to Rania Harfouche, as well as a Department of Defense BCRP Era of Hope Scholar Award and a Mary Kay Ash Charitable Foundation Grant to Shiladitya Sengupta. Anne-Laure Papa and Sudipta Basu are grateful for the support of the Carnot Foundation and the Charles A. King Trust Postdoctoral Fellowships, respectively. We acknowledge Louise Trakimas, Maria Ericsson and Elizabeth Benecchi from the Department of Cell Biology at Harvard Medical School for their advice and technical support during TEM imaging of cells. Dr Aaron Goldman is acknowledged for his assistance with Figure 1. The authors wish to thank Dr Zoltan Szabo for his editorial guidance.
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