Nontransformed and Cancer Cells Can Utilize Different Endocytic

Nontransformed and Cancer Cells Can Utilize Different Endocytic Pathways To Internalize Dendritic Nanoparticle Variants: Implications on Nanocarrier D...
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Nontransformed and Cancer Cells Can Utilize Different Endocytic Pathways To Internalize Dendritic Nanoparticle Variants: Implications on Nanocarrier Design Nelson K. Y. Wong,† Rajesh A. Shenoi,‡,§ Srinivas Abbina,‡ Irina Chafeeva,‡ Jayachandran N. Kizhakkedathu,‡ and Mohamed K. Khan*,†,⊥,∥ †

Department of Experimental Therapeutics, British Columbia Cancer Research Centre; Radiation Oncology, British Columbia Cancer Agency − Vancouver Centre, 675 West 10th Avenue, Vancouver, BC, Canada V5Z 1L3 ‡ Centre for Blood Research, Department of Pathology and Laboratory Medicine, Department of Chemistry, University of British Columbia, Vancouver, Canada V6T 2B5 ⊥ Radiation Oncology, Banner MD Anderson Cancer Center, Gilbert, AZ 85234, USA S Supporting Information *

ABSTRACT: Three hyperbranched polyglycerol nanoparticle (HPG NP) variants were synthesized and fluorescently labeled for the study of their cellular interactions. The polymeric nanoparticle that contains a hydrophobic core and a hydrophilic HPG shell, HPG-C10-HPG, is taken up faster by HT-29 cancer cells than nontransformed cells, while similar uptake rates are observed with both cell types for the nanoparticle HPG-C10PEG that contains a hydrophobic core and a polyethylene glycol shell. The nanoparticle HPG-104, containing neither the hydrophobic core nor the polyethylene glycol shell, is taken up faster by nontransformed cells than HT-29 cells. Importantly, cancer and normal cells can utilize different endocytic mechanisms for the internalization of these HPG NPs. Both HPG-C10-HPG and HPG-C10-PEG are taken up by HT-29 cells through clathrin-mediated endocytosis and macropinocytosis. Nontransformed cells, however, take up HPG-C10-HPG and HPG-104 through macropinocytosis, while these cells utilize both clathrin-mediated endocytosis and macropinocytosis to internalize HPG-C10-PEG.

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INTRODUCTION

Polymeric nanoparticles based on hyperbranched polyglycerol are highly versatile and biocompatible; they have potential for a variety of biomedical applications,6−11 including drug delivery.12−16 Although the cellular uptake of unmodified (nondrug-binding) hyperbranched polyglycerol nanoparticles (HPG NPs) of different sizes and molecular weights has been reported,17 there is no literature on how the interaction and uptake differ between normal and cancer cells and how the chemical composition of the NPs influence the uptake. Moreover, we have recently reported the delivery of docetaxel using two of these HPG NPs and found that these formulations widen the therapeutic index of the drug at the cellular level, and we hypothesized that the HPG NP:drug complex interacts with cancer and normal cells differently.14,16 To investigate how the chemical composition of HPG NPs affects cellular interaction, we labeled three HPG NP variants with Alexa 488 and followed their interaction with cancer and normal cells. In this study, we found that cancer and normal cells utilize different endocytic

The clinical utility of many anticancer drugs has been hampered by their narrow therapeutic index, which is due to their inability to impart selective toxicity to cancer cells while sparing the normal healthy cells. Research efforts into nanotechnology have been invested with the goal of achieving specific drug delivery with anatomical precision to enhance efficacy and reduce toxicity. The reduction of cardiotoxicity of the liposomal formulation of doxorubicin testifies the ability in managing drug toxicity, using nanotechnology, through diverting drug accumulation at the organ level.1 To further enhance the specificity of payload delivery, be it small-molecule inhibitors or siRNAs, the nanoparticle carriers should be designed to enhance the interaction of the payload with its appropriate cellular target.2,3 To this end, the influence of physicochemical properties of nanoparticles such as size, shape, charge, and surface hydrophobicity on cellular uptake has been studied.4,5 However, there are limited reports on how polymeric nanoparticles differ in their interaction with normal and cancer cells,4,5 the insight of which will facilitate the optimal design of polymeric NP carriers. © 2017 American Chemical Society

Received: April 25, 2017 Revised: June 23, 2017 Published: June 27, 2017 2427

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water for 2 days with periodic changes in water in order to remove any unbound Alexa 488. Alexa labeling of HPG-C10-HPG and HPG-104 was performed using the above-described procedure. Tagging of the fluorophore to the HPG NPs was confirmed by measuring the fluorescence intensity using a Cary fluorescence spectrometer (Agilent Technologies, Santa Clara, CA, USA). Measuring the Hydrophobicity of the Alexa 488-conjugated Polymers by Octanol/Water Partition. The hydrophobicity of the HPG NPs was determined quantitatively using octanol/water partition coefficients (Kow).18 Briefly, a small amount of each Alexa 488-labeled HPG NP (0.3−0.5 mg) was dissolved in a mixture (V/V-1:1) of HPLC grade octanol (1.5 mL) and water. The solution was stirred for 2 h at 25 °C. The resulting solution was centrifuged at 13,000 rpm at 4 °C for 30 min to separate the organic and aqueous layers. The concentration of the nanoparticles in each isolated layer was determined by UV spectroscopy. The absorbance at 495 nm, the characteristic absorbance peak of Alexa 488, was assessed. The molar absorptivity (ε) of the fluorophore tagged HPG NP in water was determined as 74,122 L mol−1 cm−1. The octanol/water partition coefficient (Kow) was determined using the following formula:

mechanisms to internalize these nanoparticles. In addition, we found that the hydrophobicity of the HPG NPs was one of the parameters that influences their cellular uptake: cancer cells preferentially take up the most hydrophobic HPG NP, while the normal cells preferentially take up the least hydrophobic HPG NP. Therefore, the results of this work have design implications in the development of HPG NPs for targeting cancer cells.

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EXPERIMENTAL SECTION

Cell Culture. Human dermal fibroblasts, neonatal (HDFn), were purchased from Invitrogen (Carlsbad, CA, USA) and were cultured in Medium 106 with low serum supplement (Invitrogen). HT-29, MDAMB-231, and BxPC-3 cells were purchased from ATCC and were kept in McCoy’s 5A, Dulbecco’s Modified Eagles Medium, or RPMI 1640 medium (Hyclone, San Angelo, TX, USA), respectively, with 10% heat-inactivated fetal bovine serum (Invitrogen). Synthesis of Hyperbranched Polyglycerol Nanoparticles (HPG NPs). The syntheses and physicochemical characterization of these HPG NPs were reported previously.16 HPG-C10-HPG is a nanoparticle that contains a hydrophobic core of C10 alkyl chains with a hydrophilic HPG shell. HPG-C10-PEG is similar to HPG-C10-HPG, except that the polyethylene glycol (PEG) shell was grafted onto the surface of the nanoparticle. HPG-104 does not contain any C10 or PEG chain. The molecular weights of the NPs were determined using gel permeation chromatography coupled with a multiangle laser light scattering detector (GPC-MALLS) in 0.1 M NaNO3. The hydrodynamic radii of the NPs were measured using quasi-elastic light scattering (QELS) in 0.1 M NaNO3. These three HPG NPs have similar hydrodynamic radii and molecular weights (Table 1).

Kow =

Measuring the Zeta Potential of the Polymers. The zeta potential of the polymers was measured using a Zetasizer Nano ZS (Malvern Instruments Ltd., UK). The Nano ZS has a 4 mW helium− neon laser operating at a wavelength of 633 nm. The polymers were dissolved in Milli-Q water, and measurements were taken at a scattering angle of 173° at 25 °C. Cellular Uptake Experiments. Cells were seeded in 12-well plates (BD Falcon) at 150,000 cells/well 1 day before the experiments were carried out. On the experimental day, the media were removed and Alexa 488-labeled HPG NPs suspended in growth media (200 μg/ mL, unless otherwise stated) were applied to the cells. After the incubation for different durations (1, 2, and 4 h) at 37 °C, the cells were washed twice with PBS before trypsinization. The cells were centrifuged at 200g at 4 °C for 5 min before they were resuspended in ice-cold 2% fetal bovine serum/PBS containing propidium iodide (Sigma-Aldrich, St. Louis, MO, USA). The centrifugation and resuspension of the cells were repeated once again before the uptake of HPG NPs was assessed with flow cytometry. For experiments with the endocytic inhibitors, the toxicities of the inhibitors were first determined with titrations. The selected concentrations of the inhibitors were cytotoxic to HPGC10-PEG > HPG-104 (Table 1), indicating that the presence of 2430

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Figure 3. (A) Flow cytometry histograms of HDFn and HT-29 cells incubated with HPG NPs for 4 h at 4 or 37 °C. (B) Normalized levels of uptake of HPG NPs by HDFn and HT-29 cells from three independent experiments. Error bars are standard errors of the mean.

Uptake of HPG NPs by Nontransformed and Cancer Cells. A direct comparison of the extent of cellular uptake by the three HPG NPs by flow cytometry was enabled due to their similar fluorescence properties. In order to examine the interaction of these nanoparticles with normal (nontransformed cells) and cancer cells, human neonatal dermal fibroblasts cells (HDFn) and colorectal cancer cells (HT-29) respectively were incubated with each of these fluorophore-labeled nanoparticles for 1, 2, or 4 h at 37 °C and analyzed. The cellular uptake of the nanoparticles increased in the first 4 h of incubation for all three nanoparticles, as evidenced by the shift in the fluorescence intensities to the right over time (Figure 2A) for

both the nontransformed and the cancer cells. The uptake rates of the HPG NPs between 1 and 4 h followed a linear relationship in all cases with R2 > 0.99 (Figure 2B), and the uptake rates are indicated by the slopes of the linear regression. The cellular uptake rate was found to depend on the nature of the HPG NP as well as on the cell type used. Thus, while the uptake rate for HPG-C10-HPG, the most hydrophobic HPG NP, was about 3 times faster for the HT-29 cells than the HDFn cells (slope: 34.2 ± 2.3 vs 11.2 ± 3.3, Figure 2B), the PEGylated nanoparticle with intermediate hydrophobicity, HPG-C10-PEG, was taken up by both cell types at similar rates (12.1 ± 1.2 for HDFn vs 14.2 ± 1.4 for HT-29, Figure 2431

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results not only reveal the energy dependence of cellular uptake but also confirm that the HPG NPs were not simply attached to the cell surface. For if the HPG NPs were attached only on the cell surface, the same mean fluorescence intensities would have been observed with the incubations at both temperatures. Cellular internalization of the HPG NPs was further confirmed using confocal microscopy. Alexa 488-labeled HPG-C10-HPG and HPG-C10-PEG were readily detectable in the cytoplasm of both HDFn and HT-29 cells after 4 h of incubation (Figure 4). However, HPG-104 within the cells

2B). Intriguingly, the nontransformed cells took up HPG-104, the least hydrophobic NP among the three, about 3 times faster than the cancer cells (3.2 ± 0.9 vs 1.1 ± 0.1, Figure 2B), albeit the uptake rates were much slower compared to the other two HPG NP variants. The uptake rates of HPG-C10-HPG and HPG-C10-PEG by HDFn cells were the same, while HPG-104 was taken up at a much slower rate (Figure 2C). For the HT-29 cells, the uptake rate followed the order HPG-C10-HPG > HPG-C10-PEG ≫ HPG-104 (Figure 2C), which reflects the order of the hydrophobicity of these NPs. Reichert and co-workers have previously reported that cellular uptake of unmodified HPG NPs by lung cancer cells A549 was size-dependent, with uptake rates increasing with polymer molecular weights.17 The HPG NPs in the present study share very similar hydrodynamic sizes and molecular weights,16 and therefore, it is possible that the interaction of these NPs with normal and cancer cells is dictated primarily by the differences in their hydrophobic character. The most hydrophobic among the three, HPG-C10-HPG, was taken up preferentially by the cancer cells. The engraftment of the PEG shell in HPG-C10-PEG, instead of the HPG shell, lowers the overall hydrophobicity of the NP (Table 1), and this renders similar uptake rates of this NP by the fibroblasts and the cancer cells. The least hydrophobic HPG-104 was taken up more effectively by the normal cells. These data suggest that the cellular uptake of HPG NPs by cancer cells is enhanced with increasing hydrophobic character of the NPs. Most of the studies to date have focused on how physicochemical characteristics of NPs affect the uptake by cancer cells;5 however, there are very few studies that address the difference in NP uptake between cancer and normal cells.4 Knowledge of the differential uptake of NPs by normal and cancer cells, however, is critical for designing drug nanocarriers that selectively target cancer cells. Perevedentseva and coworkers have reported that 100 nm nanodiamond was taken up more effectively by a lung cancer cell line, when compared to noncancerous lung cells.19 In addition, polymeric micelles with cross-linked ionic cores were shown to be taken up more efficiently by cancer cells of epithelial origin when compared to nontransformed lung epithelial cells.20 The authors reported that the uptake difference observed with cancerous epithelial cells was due to the lack of tight junctions,20 which were present in normal epithelial cells. Since the dermal fibroblasts (HDFn) used in the present study do not have tight junctions but have adheren junctions,21 we hypothesized that the difference in the uptake rates of the HPG NPs was mainly due to different uptake pathways that were utilized by normal and cancer cells. Uptake of HPG NPs Is Energy-Dependent. It is wellknown that endocytosis is an energy-dependent process and is inhibited at lower temperatures.22 To determine if the uptake of the HPG NPs requires energy expenditure, which implies the utilization of endocytic pathway(s), uptake of Alexa 488-labeled NPs incubated with HDFn and HT-29 cells for 4 h at 37 and 4 °C was compared using flow cytometry. Cellular uptake of the NPs was significantly lower at 4 °C when compared to that at 37 °C, as indicated by the significant reduction in the mean fluorescence intensities (Figure 3A). The energy dependence was observed with the uptake of all three HPG NPs and with both the normal and cancer cell types. Quantification of uptake levels from independent experiments showed that the uptake levels of HPG NPs incubated with cells at 4 °C was an order of magnitude less than those incubated at 37 °C (Figure 3B). The

Figure 4. Confocal microscopy images of HDFn and HT-29 cells after incubation with Alexa488-labeled HPG NPs. Signals from Alexa 488 were depicted in green. Cell nuclei were labeled with DAPI and depicted as blue. Negative controls (cells not incubated with HPG NPs) were included to ascertain that cellular autofluorescence was not interpreted as a positive signal. Scale bars depict a length of 10 μm.

could not be visualized in both cell types due to the very low uptake level of this NP (Figure 2A and C). We concluded that the sensitivity of confocal microscopy was not high enough to detect such low uptake levels, while the energy dependence of this NP uptake was clearly demonstrated by flow cytometry (Figures 2 and 3). These results show that all HPG NPs are internalized actively by the cells. This has also been demonstrated with various NP platforms, including lipid-based nanoparticles,23,24 polymeric nanoparticles,25−27 and inorganic nanoparticles.28,29 However, it is known that some NPs can enter cells through a passive process, which does not require energy expenditure.30,31 From a drug delivery perspective, we believe that it is unfavorable for NP drug carriers to penetrate into cells through a passive process, for this may confer a nonspecific payload delivery. On the contrary, it is the difference in the active uptake of the NP carriers by normal and cancer cells that presents a unique property that can be exploited for cancer cell-specific payload delivery, as demonstrated here with HPG NPs. Uptake of HPG NPs by Different Endocytosis Pathways. NPs are known to be internalized by cells through employing various endocytosis pathways such as macropinocytosis, phagocytosis, and receptor-mediated endocytosis.5 In order to decipher the endocytic pathways operating in the uptake of different HPG NPs and their relative contributions toward the cellular internalization process, uptake studies were performed with HDFn and HT-29 cells in the presence of various specific endocytic inhibitors. To assess the contribution of macropinocytosis in the internalization of the different HPG NPs, HDFn and HT-29 cells were pretreated with 5-(N-ethyl-N-isopropyl) amiloride (EIPA), a specific inhibitor of this endocytotic pathway,32 and incubated with the Alexa 488-labeled HPG NPs in the presence of the inhibitor for 4 h (Figure 5A). Experiments with vehicle 2432

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Figure 5. (A) Histograms from flow cytometry analyses of cells incubated with HPG NPs in the presence of DMSO (vector control) or macropinocytosis inhibitor EIPA. (B) Fluorescence intensities of Alexa 488-labeled HPG NPs were quantified and normalized from three independent experiments. Error bars are standard errors of the mean.

this mechanism, while HT-29 did not utilize this endocytic pathway to internalize this NP. The colorectal cancer cell line HT-29 has been reported to carry wild-type K-ras genes;35 however, this cell line contains BRAF V600E mutation, which is a constitutively active downstream effector of Ras.36 Therefore, it is uncertain if HT-29 cells exhibit a higher degree of dependence on using this endocytic pathway, as in Rastransformed cells.34 HT-29 has also been reported to take up other polymeric NPs through macropinocytosis;37 however, the authors did not examine if a difference exists between the utilization of this pathway between the cancer cell line and nontransformed cells. It has also been reported that lipid NPs can be internalized by cancer cells through macropinocytosis.23,24 To determine if clathrin-mediated endocytosis plays a role in the uptake of the different HPG NPs, the cells were pretreated with 25 μM chlorpromazine (CPZ) for 30 min before the incubation of Alexa 488-labeled HPG NPs with the cells in the presence of the inhibitor. Intriguingly, the uptake of HPG-C10HPG by cancerous HT-29 cells was significantly hampered with CPZ treatment, while no inhibition was observed with the

control, DMSO, were performed at the same time for comparison. For HPG-C10-HPG and HPG-C10-PEG, the presence of EIPA significantly reduced their uptake by HDFn and HT-29 cells (Figure 5B). The inhibitory effect was more prominent with HT-29 cells for these two NPs (extent of inhibition was statistically significant, p < 0.05), suggesting that these cells rely more on utilizing macropinocytosis to take up these NPs. For the unmodified HPG-104, a reduction of ∼30% in uptake was observed with the inhibition of macropinocytosis by HDFn cells, while no significant reduction in the uptake was observed with the HT-29 cells (Figure 5A and B). Macropinocytosis is an actin-dependent fluid uptake pathway utilized by amoebae, as well as mammalian cells, for feeding purposes.33 This mechanism is particularly active in Rasmutated cancer cells,33 and Ras-transformed cells utilize this mechanism for the internalization of extracellular proteins.34 Our results reveal that both nontransformed fibroblasts and the colorectal cancer cells internalize HPG-C10-HPG and HPGC10-PEG through macropinocytosis, perhaps as part of the cellular feeding process. On the other hand, HPG-104 was internalized by nontransformed fibroblasts partially through 2433

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Figure 6. (A) Flow cytometry analyses of cells incubated with HPG NPs in the presence of DMSO (vector control) or clathrin-mediated endocytosis inhibitor CPZ. (B) Fluorescence intensities of Alexa 488-labeled HPG NPs quantified and normalized from three independent experiments. Error bars are standard errors of the mean.

29 through this mechanism. More studies are required to reveal the reasons for such a difference. Nonetheless, this observation suggests that HPG NPs can be designed to trigger clathrinmediated endocytosis by cancer cells with improved specificity for payload delivery. Filipin is a sterol-binding agent that binds to cholesterol, and it inhibits caveolin-mediated endocytosis.40 Recently, filipin has also been shown to inhibit endocytosis through the clathrin independent carrier/GPI-anchored protein enriched early endosomal compartment (CLIC/GEEC) pathway.41 Since the inhibitory action of filipin is due to its cholesterol-binding property, these internalization mechanisms are collectively referred to as cholesterol-sensitive endocytosis here. When the fibroblasts and the HT-29 cells were treated with filipin, no inhibition of uptake of any of the HPG NPs was observed by both cell types (Figure 7). Interestingly, however, the uptake of HPG-C10-HPG was increased by more than 2.5-fold (Figure 7) in the nontransformed cells but not in the cancer cells. Perhaps the inhibition of caveolin-mediated endocytosis and/or the CLIC/GEEC pathway in the fibroblasts activates some other endocytic pathways due to potential cross-talks,42 thereby

nontransformed HDFn (Figure 6). On the other hand, the uptake of HPG-C10-PEG was inhibited to comparable levels in both HDFn and HT-29 while the uptake of HPG-104 was not hindered at all in the presence of CPZ (Figure 6). Clathrin-mediated endocytosis is a cellular process that is essential for intercellular signaling, nutrient uptake, and membrane recycling.38 As the name suggests, this form of endocytic pathway depends on clathrin, which forms a coated pit that is eventually pinched off from the surface for the internalization of extracellular cargo. This form of cellular uptake mechanism has been studied for more than 40 years;39 however, the initiation event of this cellular uptake mechanism is still elusive.38 The current literature suggests that clathrinmediated endocytosis does not require ligand−receptor interaction at the cell membrane, and it has been proposed that the nucleation of sites for clathrin-mediated endocytosis can be a stochastic event.38 If that is the case, HPG NPs can randomly engage any preformed nucleation site and subsequently be internalized. However, it is intriguing that HPGC10-HPG is not internalized by nontransformed fibroblasts through this endocytic pathway, while it is internalized by HT2434

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Figure 7. (A) Flow cytometry analyses of cells incubated with HPG NPs in the presence of DMSO (vector control) or Filipin, an inhibitor of cholesterol-sensitive endocytosis, including caveolin-mediated endocytosis and clathrin independent carrier/GPI-anchored protein enriched early endosomal compartment (CLIC/GEEC) pathway. (B) Fluorescence intensities of Alexa 488-labeled HPG NPs were quantified and normalized from three independent experiments. Error bars are standard errors of the mean.

causing the increased internalization of HPG-C10-HPG. Dombu et al. have also observed that treatment of epithelial cells with filipin increased the internalization of polysaccharide cationic NPs.43 Further experimentation revealed that the filipin treatment inhibited exocytosis of these NPs.43 Therefore, another explanation for the increased uptake of HPG-C10-HPG is the inhibition of exocytosis of the NP. This, in turn, suggests that the accumulation of HPG-C10-HPG observed is a net effect of the endocytosis and exocytosis of this NP. The relative contributions of the examined endocytic pathways toward the uptake of HPG NPs are summarized in Table 2. The relative contribution of each endocytic pathway was estimated by the extent of the inhibition observed with the specific inhibitors; i.e., the greater extent of dependence on a given endocytic mechanism is suggested by the greater extent of inhibition by the corresponding inhibitor. For HPG-C10HPG, the cancer cells HT-29 utilize both macropinocytosis and clathrin-mediated endocytosis to internalize the NP, while the primary fibroblasts HDFn utilize macropinocytosis as a major uptake mechanim without utilizing clathrin-mediated endocy-

Table 2. Relative Contributions of Different Endocytic Mechanisms Utilized by the Nontransformed Fibroblasts HDFn and HT-29 Cancer Cells with Different HPG NPsa macropinocytosis HT-29 HDFn

++++ ++

HT-29 HDFn

++++ +++

HT-29 HDFn

++

clathrin-mediated endocytosis HPG-C10-HPG +++ HPG-C10-PEG +++ ++ HPG-104 -

caveolin-mediated endocytosis -

a

Dependence on endocytic mechanism: ++++, >60% uptake decrease by inhibitor; +++, 40−60% uptake decrease by inhibitor; ++, 20−40% uptake decrease by inhibitor; -, no dependence.

tosis. This may explain the different uptake rates that were observed with this NP between the two cell lines (Figure 2B) 2435

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Figure 8. Uptake of HPG NPs by MDA-MB-231 (breast cancer) and BxPC-3 (pancreatic cancer) cells. Mean fluorescence intensities of the internalized HPG NPs by the cancer cells over 1−4 h are shown (A). The levels of uptake of HPG-C10-HPG in the absence (DMSO control) or presence of macropinocytosis inhibitor EIPA (B) or clathrin-mediated endocytosis inhibitor CPZ (C) were compared. Data presented are from at least three independent experiments. Error bars are standard errors of the mean.

mean fluorescence intensities followed a linear pattern in the first 4 h (Figure 8A) with R2 > 0.99. Moreover, both cell lines took up the HPG NPs in the same order as observed with HT29, although the rates of uptake of the HPG NPs are different (Table 3). Both cancer cell lines preferentially took up the most

and the differential killing effect of the docetaxel/HPG-C10HPG formulation that we reported before.16 On the other hand, when the HPG NP was PEGylated (HPG-C10-PEG), both cell lines utilized macropinocytosis and clathrin-mediated endocytosis to internalize this NP, and the uptake rates of this NP by these cell lines are comparable (Figure 2B). Interestingly, when docetaxel was loaded into HPG-C10-PEG, this formulation also preferentially killed cancer cells, when compared to the cytotoxic effect on primary cells.14 Perhaps the relative contributions of macropinocytosis and clathrinmediated endocytosis are different in the normal cells and cancer cells, thereby leading to different processing of the drug formulation and the availability of the drug to the cellular target. More experiments will be required, however, to test this hypothesis. Although HPG-104 was taken up very slowly by both cell types, the internalization of this NP was energydependent (Figure 3). None of the examined endocytic pathways was utilized by HT-29 to take up this NP, but the fibroblasts utilize macropinocytosis, in part, to take up HPG104. Although none of the HPG NPs was taken up through caveolin-mediated endocytosis or through the CLIC/GEEC pathway in this study, there are reports that polymeric NP or monosaccharide functionalized NPs can be taken up through caveolin-mediated endocytosis.27,44 To determine if the HPG NP uptake pattern observed with HT-29 cells is reproduced in other cancer cell lines, we repeated the experiment with two unrelated cancer cell lines: MDA-MB-231, a breast cancer cell line, and BxPC-3, a pancreatic cancer cell line. Similar to the cellular uptake of HPG NPs observed before, the increase of the normalized

Table 3. Uptake Rates of HPG NPs by MDA-MB-231 and BxPC-3 Cells MDA-MB-231 BxPC-3

HPG-C10-HPG

HPG-C10-PEG

HPG-104

22.1 ± 7.2 7.5 ± 2.2

9.6 ± 1.4 3.0 ± 0.7

2.3 ± 0.7 0.9 ± 0.3

hydrophobic HPG NP, i.e., HPG-C10-HPG, and these cancer cell lines took up HPG-104, the least hydrophobic HPG NP, with the lowest rates. Another difference of the HPG NP uptake between the nontransformed HDFn cells and the HT-29 cancer cells was that HPG-C10-HPG was taken up by HT-29 through macropinocytosis and clathrin-mediated endocytosis, while HDFn does not utilize clathrin-mediated endocytosis. To determine if MDA-MB-231 and BxPC-3 internalize HPG-C10HPG using the same mechanisms as HT-29, uptake studies were conducted with the absence or presence of EIPA or CPZ. The cells were incubated with 100 μg/mL of HPG-C10-HPG for 4 h with DMSO or the inhibitors after a 30 min pretreatment with the inhibitors. Similar to HT-29, the uptake of HPG-C10-HPG by MDA-MB-231 cells was inhibited by both EIPA (Figure 8B) and CPZ (Figure 8C), indicative of the utilization of micropinocytosis and clathrin-mediated endocy2436

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Campus, Thalappady, Rubber Board P O, Kottayam-686 009, Kerala, India. ∥ M.K.K.: Banner MD Anderson Cancer Center, Radiation Oncology, 2946 E. Banner Gateway Drive, Gilbert, AZ 85234, USA.

tosis for the uptake of this HPG NP. Interestingly, BxPC-3, which exhibited a much lower uptake rate of HPG-C10-HPG, was only inhibited by EIPA (Figure 8B) but not by CPZ (Figure 8C). Together, these observations reveal that all cancer cell lines examined preferentially internalize the most hydrophobic HPG NP. Moreover, the cancer cells can utilize different endocytic pathways to preferentially take up an NP, as demonstrated by the uptake of HPG-C10-HPG by HT-29 and MDA-MB-231, when compared to the nontransformed HDFn. At the moment, we do not know the exact mechanism(s) that dictate(s) the uptake difference. One possibility is that the difference may lie in the protein corona formed around the NPs when they encounter serum proteins,45 leading to different interactions with the cancer and normal cells. Another possible contributing factor is the intrinsic preferential usage of a specific endocytic mechanism by certain cancer cells. An example is the preferential usage of macropinocytosis by Ras-mutated cancer cells, as discussed above.32,34 We have previously demonstrated that the presence of a PEG shell enhances the biodistribution profile of HPG NPs.16 However, the presence of a PEG shell, on the other hand, lowers the cellular uptake rate by cancer cells, as shown here. These observations, together, suggest that a PEG shell of HPG NP that can be shed in the tumor microenvironment may be a favorable design, as some of the researchers have already begun to explore.46

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by funding from University of British Columbia, British Columbia Cancer Agency, Michael Smith Foundation for Health Research, Canadian Institute of Health Research, Natural Sciences and Engineering Research Council of Cancer, and Pancreatic Cancer Canada. The authors would like to acknowledge the start-up funds from University of British Columbia and BC Cancer Agency for M.K.K. J.N.K. is a recipient of the Michael Smith Foundation for Health Research Scholar award. J.N.K. also acknowledges the funding from Canadian Institutes of Health Research and Natural Sciences and Engineering Research Council of Canada. This work was also partially funded by Pancreatic Cancer Canada. The authors would also like to thank Dr. Jingsong Wang of the Imaging Facility at Child & Family Research Institute for the excellent technical help.

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CONCLUSIONS Although many studies have been invested into understanding how the physicochemical properties of NPs affect their internalization by cancer cells, there is limited information available on the differences of NP internalization between cancer and nontransformed cells. In our opinion, this is an inadequately explored territory, and a better understanding of this phenomenon will help design a NP carrier to exploit this observation for better payload specificity, for instance, specific delivery to cancer cells. Here we report that structural variation of HPG NPs leads to different interactions with normal and cancer cells. The HPG NPs are actively taken up by cells through energy-dependent processes, and the different internalization rates are associated with their hydrophobic character. We trust that the observations made in this study will help design HPG NPs for cancer-targeted payload delivery.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.7b00590. Figures showing 1H NMR analysis and gel permeation chromatography spectra (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Mohamed K. Khan: 0000-0003-2789-7623 Present Addresses §

R.A.S.: Inter University Centre for Biomedical Research & Super Speciality Hospital, Mahatma Gandhi University 2437

DOI: 10.1021/acs.biomac.7b00590 Biomacromolecules 2017, 18, 2427−2438

Article

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DOI: 10.1021/acs.biomac.7b00590 Biomacromolecules 2017, 18, 2427−2438