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. Author manuscript; available in PMC: 2016 Jan 3.
Published in final edited form as: Small. 2009 Mar;5(3):370–376. doi: 10.1002/smll.200800972

Cancer Cell Phenotype Dependent Differential Intracellular Trafficking of Unconjugated Quantum Dots

Sutapa Barua 1, Kaushal Rege 1,(*)
PMCID: PMC4698342  NIHMSID: NIHMS100173  PMID: 19089841

Abstract

A diverse array of nanoparticles, including quantum dots, metals, polymers, liposomes, and dendrimers, is being investigated as therapeutics and imaging agents in cancer disease. However, the role of the cancer cell phenotype on the uptake and intracellular fate of nanoparticles in cancer cells remains poorly understood. Here we report that differences in cancer cell phenotype can lead to significant differences in intracellular sorting, trafficking and localization of nanoparticles. Unconjugated anionic quantum dots demonstrated dramatically different intracellular profiles in three closely related human prostate cancer cells used in the current investigation: PC3, PC3-flu, and PC3-PSMA. Quantum dots demonstrated punctuated intracellular localization throughout the cytoplasm in PC3 cells. In contrast, the nanoparticles localized mainly at a single juxtanuclear location (‘dot-of-dots’) inside the perinuclear recycling compartment (PNRC) in PC3-PSMA cells, where they colocalized with transferrin and the Prostate-Specific Membrane Antigen. Our results indicate that nanoparticle sorting and transport is influenced by changes in cancer cell phenotype, and can have significant implications in the design and engineering of nanoscale drug delivery and imaging systems for advanced tumors.

Keywords: Quantum Dots, Nanoparticle Trafficking, Intracellular Transport, Microtubules, Perinuclear Recycling Compartment

1. INTRODUCTION

Cellular uptake of exogenous material relies on a number of different internalization mechanisms including, phagocytosis, macropinocytosis, receptor-mediated endocytosis, clathrin-mediated, endocytosis, caveolin-mediated endocytosis, and clathrin-and caveolin-independent endocytosis.[1-3] Internalized material is then sorted, and trafficked to different locations inside cells depending on cellular polarity, expression of sorting and motor proteins, protein-protein interactions, and cytoskeletal organization. Genetic and phenotypic alterations can lead to pronounced differences in transport of cargo inside cells, and can be both, a cause and result of a number of disease states[4-7], including cancer.[8]

Cancer disease progression to the aggressive metastatic state is a result of the accumulation of various genetic changes. Malignant cells undergo significant differences in their cellular phenotype as a consequence of these genetic changes; characteristic of these include, alterations in intra- and extra-cellular protein expression, cell polarity, and cell survival. While the polarized phenotype of non-malignant epithelial cells results in different trafficking mechanisms at their apical and basolateral regions,[9] malignant cells are typically characterized by loss in polarity which influences intracellular sorting and trafficking of cargo in these cells.[10] In addition, due to the heterogeneous nature of epithelial tumors, phenotypic differences in cancer cells play a significant role in the uptake, intracellular sorting, trafficking, and localization of internalized cargo.[11]

Nanoscale therapeutics, diagnostics, and imaging agents hold great promise in the detection and treatment of advanced cancer disease.[12-14] An understanding of the processing and fate of targeted and untargeted nanoparticles in cancer cells can facilitate the design and engineering of novel nanoscale agents that possess higher efficacies and selectivities for targeting specific intracellular locations. Receptor expression profiles on cancer cells influence the intracellular trafficking of targeted nanoparticles.[15-17] While some reports describe the uptake of unconjugated nanoparticles,[18] most reports describe the role of conjugated molecules including polymers[19], cell penetrating peptides,[20-22] and / or serum proteins[23] in receptor-independent (untargeted) uptake and trafficking of nanoparticles in cancer cells. Recent reports have described the role of nanoparticle size and surface chemistry on their uptake and intracellular fate in cancer cells. For example, 25-50 nm Herceptin-conjugated gold nanoparticles demonstrated the highest uptake in SK-BR-3 breast cancer cells among nanoparticles ranging from 2-100 nm in size.[24] Similarly, 50 nm particles demonstrated greatest uptake in HeLa cells among unconjugated gold particles ranging from 14-100 nm in diameter; adsorption of serum-containing proteins on the surface of the anionic nanoparticles was thought to promote the non-specific uptake of the nanoparticles by cancer cells.[23] Polymeric particles less than 25 nm in diameter were reported to be taken up by a non-degradative, cholesterol independent, and non-clathrin and non-caveolae dependent endocytosis leading to their transport as punctate structures in the perinuclear region of HeLa cells; larger sized (40 nm) nanoparticles did not demonstrate this behavior. [25]

Prostate cancer (PCa) is the most frequently diagnosed malignancy in men in the United States with over 210,000 cases diagnosed in 2007; approximately 30,000 men die every year due to the disease.[26-28] Lowering androgen levels results in tumor shrinkage or decelerated tumor growth in approximately 90% of treated cases.[29] Unfortunately, these results are usually transient and a large number of patients subsequently undergo disease progression to aggressive, androgen-independent, and chemo and radiation therapy resistant prostate cancer disease. Consequently, in addition to the discovery of novel molecular therapeutics, nanoparticle-mediated ablation of prostate cancer cells is also being currently investigated. [30-32]

In this report, we investigate the role of cancer cell phenotype on the uptake and intracellular routing of unconjugated anionic nanoparticles in bone metastasis-derived PC3, PC3-flu,[33] and PC3-PSMA[33] human prostate cancer cells. Differences in these closely related cell lines can be indicative of phenotypic differences that occur during disease progression and different cancer cell populations existing in tumors. These cells were employed to investigate the role of cellular heterogeneity on nanoparticle fate in cancer cells. Quantum dots (QDs) are of interest in biomedical imaging applications due to their greater photostability, broader excitation and narrower, symmetric emission wavelengths, compared to traditional organic dyes; [34, 35] we chose quantum dots as model nanoparticles for our current investigation. We demonstrate that unconjugated anionic quantum dots are taken up spontaneously by prostate cancer cells, and that their intracellular fate is dramatically influenced by the cancer cell phenotype.

2. RESULTS AND DISCUSSION

Various novel therapeutic interventions, including those based on nanoparticles, are being pursued with an eye towards increasing survival in cases of aggressive, drug-resistant, metastatic, and androgen-independent prostate cancer. We hypothesized that an investigation into the role of the advanced cancer cell phenotype on intracellular trafficking and localization of nanoparticles can eventually aid the design of efficacious nanoscale therapeutics. As a result, we investigated the uptake, sorting, trafficking and localization of unconjugated quantum dots in advanced prostate cancer cells. While a number of cell lines, including LNCaP, C4-2, and DU-145, have been employed for the in vitro evaluation of nanoscale therapeutics for prostate cancer,[36-38] we chose bone-metastasis derived PC-3 cells for the current investigation. PC-3 cells are androgen independent and therefore, represent the advanced form of prostate cancer disease. This is in contrast to LNCaP cells, which are androgen responsive and represent a more ‘manageable’ form of the disease. In addition, the availability of sub-clones of PC3 cells (PC3-flu and PC3-PSMA) cells makes it convenient for investigating nanoparticle trafficking in closely related advanced prostate cancer cells.

Quantum dots (0.2 nM) demonstrated punctated intracellular localization throughout the cytoplasm in PC3 cells (Figure 1a) characteristic of lysozomal localization.[39] In contrast, QDs localized mainly at a single juxtanuclear location (‘dot-of-dots’) inside PC3-PSMA cells (Figures 1c and 1d). Kinetic experiments indicated that the dot-of-dots formation was complete in 5h in a concentration-dependent fashion in PC3-PSMA cells (Supporting Figures S1 and S2) and the structure remained intact for at least 72h (not shown). Higher concentrations of the quantum dots (1 nM) were required for the formation of the ‘dot-of-dots’ structure in the presence of serum under similar conditions (Supporting Figure S3) indicating that the presence of serum proteins inhibited nanoparticle uptake at lower concentrations. PC3-flu cells demonstrated trafficking profiles similar to both, PC3 and PC3-PSMA cells (Figure 1b); while QDs formed the ‘dot-of-dots’ structure as seen in PC3-PSMA cells, they also localized throughout the cytoplasm similar to PC3 cells, and along the cellular periphery.

Figure 1.

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Differential intracellular localization of QDs in human prostate cancer cells. (a) PC3, (b) PC3-flu, (c) PC3-PSMA, (d) overlay of phase contrast and fluorescence microscopy image of ‘dot-of-dots’ formation in PC3-PSMA cells.

Following the above observations, we investigated the factors that influence the uptake of nanoparticles leading to the formation of the dot-of-dots structure in PC3-PSMA cells. Different mechanisms including, lipid raft-mediated, clathrin-mediated, and adsorptive endocytosis, play a role in the cellular entry of exogenous material. Lipid rafts are cholesterol-rich membrane platforms that have been implicated in the entry of viruses in mammalian cells. We found that extraction of cholesterol using methyl-β-cyclodextrin from the surface of PC3-PSMA cells resulted in no change in the uptake and trafficking of quantum dots (Figures 2a and 2b) which indicated that disruption of lipid rafts did not inhibit the endocytosis of quantum dots. Clathrin-mediated endocytosis constitutes an important mechanism in the uptake of exogeneous material, including nanoparticles, in both, polarized[40] and nonpolarized[17, 23] epithelial cells. Treatment with the clathrin inhibitor chlorpromazine resulted in complete inhibition of nanoparticle uptake (Figures 2c and 2d) indicating that clathrin-mediated endocytosis was responsible for the entry of QDs in these cells.

Figure 2.

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Role of lipid rafts and clathrin on quantum dot internalization and ‘dot-of-dots’ formation in PC3-PSMA cells. PC3-PSMA cells were treated (a) with or (b) without the cholesterol extracting agent, methyl-β-cyclodextrin for evaluating the role of lipid rafts and (c) with or (d) without the clathrin inhibiting agent, chlorpromazine for evaluating the role of clathrin on the uptake of quantum dots.

Motor proteins kinesins and dyenins transport cargo-containing vesicles to the plus (cell periphery) and minus (microtubule organizing center) ends of microtubules, respectively. In order to investigate the role of microtubules on the formation of dot-of-dots structure following clathrin-mediated endocytosis, we disrupted microtubule transport by treating cells with the microtubule depolymerizing agent, nocodazole. Microtubule disruption in PC3 cells (figure 3a) resulted in reduced intracellular uptake and trafficking of quantum dots in these cells; however, the nanoparticles still localized as punctated dots throughout the cytoplasm. Nocodazole treatment resulted in complete disruption of the dot-of-dots formation in both, PC3-flu (Figure 3b) and PC3-PSMA (Figure 3c) cells, indicating that a functional microtubule network is necessary for the intracellular trafficking of nanoparticles in these cells. Interestingly, quantum dots were transported closer to the cell periphery and away from the nucleus in PC3-PSMA cells indicating that microtubule disruption results in mis-sorting and altered trafficking, a phenomenon previously observed in both, malignant and untransformed primary cells.[41, 42] The punctated nanoparticle distribution in the cytoplasm of PC3-PSMA cells (Figure 3c) after nocodazole treatment was qualitatively similar to the nanoparticle distribution observed in PC3 cells without microtubule disruption.

Figure 3.

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Effect of microtubule disruption on quantum dot trafficking in (a) PC3, (b) PC3-flu, and PC3-PSMA cells. Cells were treated with the microtubule polymerizing agent, nocodazole, for 1h prior to treatment with quantum dots for 5h. In the figure, scale bar = 20 μm.

Following uptake by clathrin-mediated endocytosis, molecular and / or nanoscale cargo are sorted in sorting endosomal complexes and are trafficked on microtubules along one of three different pathways: degradative lysozomal pathway, retrograde transport, or to the perniuclear recycling compartment (PNRC).[3] We investigated the intracellular fate of quantum dots in all three prostate cancer cell lines using confocal fluorescence microscopy. Figure 4 shows intracellular colocalization of quantum dots with DAPI, FITC-transferrin, Lysotracker® Green DND-26, and FITC-labeled antibody against the Prostate-Specific Membrane Antigen (PSMA) which are markers for cell nuclei, recycling endosomes, acidic compartments (late endosomes / lysozomes), and PSMA, respectively. In the case of PC3 (Figure 4a. i) and PC3-flu (Figure 4a.ii) cells, punctated dots were observed throughout the cytoplasmic space around the nucleus (shown in blue). Figure 4a. iii, however, shows that the dot-of-dots formation (red) was found at a juxtanuclear location inside PC3-PSMA cells (nucleus shown in blue) which indicates that following uptake from the entire cell surface, QDs were trafficked along microtubules to a centralized juxtanuclear location, the microtubule organizing center (MTOC), in these cells. This behavior was also observed in some PC3-flu cells (Figure 4a ii).

Figure 4.

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Intracellular localization of QDs in (i) PC3, (ii) PC3-flu, and (iii) PC3-PSMA cells determined by colocalization for organelle / vesicle specific markers and quantum dots using confocal fluorescence microscopy. Confocal microscopy images show colocalization of QDs with:
  1. Cell nuclei using DAPI (blue stain)
  2. Recycling endosomes using FITC-transferrin
  3. Acidic vesicles (late endosomes & lysosomes) using LysoTracker Green DND-26
  4. PSMA using FITC labeled anti-PSMA antibody
Colocalization of green-fluorescent markers (dyes) and red-fluorescent QDs is shown in yellow color in the images. Scale bars = 20 μm

The dot-of-dots structure colocalized with FITC-transferrin in PC3-PSMA (Figure 4b. iii) and PC3-flu (Figure 4b. ii) cells as seen from the yellow color from an overlay of the red fluorescent quantum dots and green-fluorescent FITC-transferrin. Transferrin is a known marker for the perinuclear recycling endosomal compartment (PNRC) which, in nonpolarized cells, is a long-lived structure found close to the nucleus and near the MTOC.[3, 41, 42] Near-complete colocalization of transferrin and quantum dots indicated that the nanoparticles localized almost exclusively as ‘dot-of dots’ in the PNRC in PC3-PSMA cells. As in the case of QDs, microtubule disruption in PC3-PSMA cells led to disruption of the PNRC and mis-sorting of transferrin. Following microtubule disruption, transferrin was trafficked to cytoplasmic compartments where only partial colocalization of the protein with QDs was seen (Supporting Figure S4). These results are in agreement with literature reports[41, 42] and were qualitatively similar to those observed with PC3 cells that had not been treated with nocodazole leading to the observation that microtubule disruption ‘reverted’ trafficking in PC3-PSMA back to that observed in the parental PC3 cells. While some QDs colocalized with transferrin in cytoplasmic recycling endosomes in PC3 cells (Figure 4b. i), a significant number of QDs did not colocalize with transferrin indicating their localization in early / sorting endosomes in addition to cytoplasmic recycling endosomes[41]. In the case of PC3-flu cells, vesicles containing both transferrin and QDs were seen in the cytoplasm in PC3-flu cells, in addition to the PNRC.

Colocalization analyses were also carried out with LysoTracker® Green DND-26 and all three prostate cancer cell lines; LysoTracker® stains acidic vesicles (e.g. late endosomes and lysozomes) inside cells. As with transferrin, a portion of intracellular QDs colocalized with acidic vesicles in PC3 cells (Figure 4c. i) further indicating the presence of these nanoparticles in different cytoplasmic compartments. Colocalization experiments also revealed the acidic nature of the QD-containing PNRC in both, PC3-PSMA (Figure 4c. ii) and PC3-flu cells (Figure 4c. iii). While some reports indicate that the PNRC is only mildly acidic with a pH range of 6.0- 6.5,[3] other reports indicate that the compartment has a pH value of 5.6.[41] The latter is in agreement with LysoTracker® staining of the compartment; however, we reason that the acidic nature of the cargo present in these vesicles, i.e. carboxylated quantum dots, also contributes to the acidification of these vesicles which in turn, results in strong staining with the reagent. Interestingly, a significant fraction of quantum dots was also observed in non-acidic vesicles in PC3-flu cells (Figure 4c. ii); the nature of these compartments is not known at this point. In contrast, while quantum dots were observed in the PNRC in PC3-PSMA cells, acidic compartments without quantum dots were observed all along the periphery of these cells.

The Prostate-Specific Membrane Antigen (PSMA) is a 750-amino acid type II membrane glycoprotein which is abundantly expressed in all stages of prostate cancer disease; the expression of the protein increases in cases of hormone-refractory and metastatic disease. The receptor is over-expressed in approximately 70% of cases with aggressive metastatic disease and is a reliable marker of disease progression. PSMA overexpression correlates with poor prognosis[43, 44] and has been employed for the targeted ablation of prostate cancer cells.[37, 45-48] The extracellular region of the PSMA receptor possesses 26% and 28% homology with transferrin receptors (TfR), TfR1 and TfR2, respectively.[49-51] Following both constitutive and antibody binding-induced internalization from clathrin-coated pits, PSMA is known to co-localize with transferrin in the PNRC mediated by a cytoplasmic internalization sequence and filamin.[52] Given that QDs colocalized with transferrin in PC3-PSMA cells, we asked if the nanoparticles also colocalized with PSMA in these cells. Colocalization of QDs with the FITC-labeled PSMA antibody was indeed seen in PC3-PSMA cells (Figure 4d. iii) further indicating that the nanoparticles reside in the PNRC in PC3-PSMA cells. It is unclear at this point if PSMA has any role to play in the uptake and subsequently, trafficking of QDs. It is more likely that PSMA undergoes the same fate of clathrin-mediated uptake, microtubule-mediated transport, and localization in the perinuclear recycling compartment[53] independent of the nanoparticles. In addition, the partial localization of QDs in the PNRC of PSMA non-expressing PC3-flu cells further indicates that QD trafficking to the PNRC may occur independently of the PSMA. PC3 (Figure 4d. i) and PC3-flu (Figure 4d. ii) cells did not show staining for the anti-PSMA antibody which is consistent with the lack of receptor expression on these cells.

3. CONCLUSIONS

In summary, our results demonstrate that unconjugated anionic quantum dots are spontaneously taken up by closely related cancer cells which then sort and traffic these to dramatically different fates (Figure 5). Serum proteins and conjugated molecules such as targeting antibodies or cell penetrating peptides are not necessary for the uptake and trafficking of nanoparticles in these cells. Following internalization from clathrin-coated pits, nanoparticles are trafficked in vesicles along microtubules to the sorting endosomal complex in these non-polarized cells. At this stage, nanoparticles are destined for different fates depending on the cell phenotype. Nanoparticles can be either trafficked in vesicles along the ‘default’ lysozomal degradation pathway as in PC3 cells or they can be sorted and transported along microtubules to the perinuclear recycling compartment (PNRC) as observed in PC3-PSMA cells. These results underscore the importance of investigating intracellular mechanisms for delivered nanoparticles, both as therapeutics and diagnostics. Future work will elucidate molecular mechanisms underlying the decisions that lead to differential sorting of nanoparticles in different cancer cells which, in turn, will lead to information that can be exploited to manipulate the delivery of nansocale cargo to predetermined locations inside cells.

Figure 5.

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Schematic of uptake, sorting, trafficking, and intracellular localization of quantum dots in cancer cells. Following clathrin-mediated uptake into early endosomes (EE), quantum dots are trafficked to the sorting endosomal complex (SEC). At this stage, vesicles are either sent back to the cell surface via recycling endosomes (RE), trafficked towards degradative late endosomes (LE) and lysozomes, or are trafficked to the perinuclear recycling compartment (PNRC) near the microtubule organizing center (MTOC).

4. METHODS

4.1 Cell culture

The PC3 human prostate cancer cell line was obtained from the American Type Culture Collection (ATCC, VA). PC3-PSMA and PC3-flu cells, derived from PC3 cells, were generous gifts from Dr. Michael Sadelain, Memorial Sloan Kettering Cancer Center, New York, NY. The PC3-PSMA cell line is a sub clone of PC3 cells retrovirally transduced to stably express the PSMA receptor[33]; PC-3 flu cells are obtained by retrovirally transducing PC3 cells with the flu peptide[33]. All cells were grown in RPMI-1640 (HyClone®, UT) containing 10% fetal bovine serum (HyClone®, UT) and 1% penicillin / streptomycin (HyClone®, UT) in 5% CO2 at 37 °C in an incubator.

4.2 Quantum Dot Treatment

Qdot® 655 nanocrystals (ZnS-capped CdSe quantum dots; 8.2 μM) were purchased from Invitrogen, CA. The particle size of quantum dots in PBS was determined as 27 nm using Dynamic Light Scattering; data not shown) PC3, PC3-PSMA, and PC3-flu human prostate cancer cells were plated in 24-well cell culture plates (Corning Inc., NY) with or without glass coverslips (12 mm circle diameter; Fisher) at a density of 50,000 cells per well and allowed to attach overnight. For uptake experiments, cells were treated with QDs (0.2 nM) in serum-free media for 5h, fixed with 4% p-formaldehyde, and imaged using fluorescence microscopy (AxioObserver D1, Carl Zeiss MicroImaging Inc., Germany). The fluorescence excitation of the Zeiss inverted microscope was equipped with an objective of 40X / 0.6 numerical aperture (NA) (without immersion) incorporating a cover glass correction ring with it. Images were captured using filters consisting of 550/670 nm excitation/ emission for quantum dots, using a spot CCD camera. For kinetics experiments, cells were treated with QDs for different times, fixed, and imaged using fluorescence microscopy. Fluorescence microscopy was carried out with cells in 24-well plates using a Zeiss AxioObserver D1 (Carl Zeiss MicroImaging, Inc.) for all other experiments.

4.3 Uptake, Trafficking and Intracellular Localization of Quantum Dots

Cells (50,000/well) were treated with the lipid raft extracting agent methyl-β-cyclodextrin (8 mM for 30 min) or clathrin disrupting agent chlorpromazine (10 mg/ml for 1h), following which, cells were washed with PBS and treated with quantum dots (0.2 nM) for 5h. The microtubule depolymerizing agent, nocodazole (Sigma-Aldrich) was employed in order to investigate the role of microtubule-mediated transport of QDs in cells. Cells grown on glass coverslips placed in 24-well plates were treated with 40 μM nocodazole for 1 h at 37 °C. Nocodazole-containing medium was then removed and cells were washed with PBS, and incubated with QDs for 5 h. Cells were treated with 1 μl FITC-transferrin (stock concentration 5 mg/mL; Invitrogen, CA) for the last one hour of the incubation in order to investigate the role of microtubule disruption on localization in recycling endosomes.

In order to visualize localization of QDs with respect to the nucleus, cells were treated with QDs for 5 h, following which, cells were fixed with 4% paraformaldehyde, and stained with the nucleic acid stain, 4’,6-diamidino-2-phenylindole (DAPI; 1 mg/ml stock concentration; 1.5 μl; Invitrogen, CA), in PBS (500 μL) for 15 min at room temperature (22°C). The cells were then washed three times with PBS and visualized using fluorescence microscopy (Zeiss AxioObserver D1 inverted microscope).

For co-localization experiments, cells were settled on coverslips placed in 24-well plates for 24 h following which QDs (0.2 final concentration in 500 μL) were added. After 4 h, the cells were incubated with FITC-labeled transferrin (1 μl of 5 mg/mL stock ; Invitrogen, CA), LysoTracker® Green DND-26 (2 μl of 1 mM stock; Invitrogen, CA), or FITC labeled anti- PSMA antibody (Ab) (15 μl of 50 μg/ml stock; Marine Biological Laboratory, MA) for 1 h in order to visualize recycling endosomes / perinuclear recycling compartment, lysosomes, and the internalized Prostate-Specific Membrane Antigen (PSMA), respectively. After 5 h total treatment with QDs, cells were washed twice with phosphate-buffered saline (PBS; 500 μl/ well) and fixed with 4% paraformaldehyde for 10 min at room temperature (22°C). Cells were then gently washed with PBS three times, the coverslips were removed from the 24-well plates and mounted in permount mounting media (Fisher, NJ) on glass slides for confocal fluorescence microscopy.

Laser scanning confocal microscopy was carried out with a Leica SP2 microscope (Leica Microsystems, Heidelberg, Germany) in the W. M. Keck Bioimaging Laboratory, Arizona State University, AZ in order to determine the intracellular localization of QDs. Confocal images were obtained in a z-series using an upright microscope equipped with 40X / 1.25 NA oil immersion objective lens, Ar laser (488 nm) and a transmitted light detector photomultiplier (PMT). Light emitted at 525 nm and 650 nm was recorded for the green channel (FITC and LysoTracker) and red channel (QDs), respectively. Images were acquired with dual-channel scanning at 512×512 pixels using PMTs along with image acquisition and analysis software (Leica confocal software, version 2.61, Leica Microsystems, Mannheim, Germany). Images were then stacked in RGB color using Image Processing and Analysis in Java (ImageJ) 1.38X software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/, 1997-2005); the average intensity was used to compare different images.

Supplementary Material

Supporting Information

Acknowledgments

The authors thank Gertrude Gunset and Dr. Michel Sadelain of the Memorial Sloan-Kettering Cancer Center for PC3-flu and PC3-PSMA cells. We thank Bret Judson of the W. M. Keck Bioimaging Laboratory at Arizona State University for his invaluable assistance with confocal microscopy. This work was supported by National Institutes of Health Grant Number 1R21CA133618-01 and start-up funds from the state of Arizona to KR.

Footnotes

Author Contributions: KR planned and designed experiments, SB and KR carried out the experimental work, analyzed results, and wrote the manuscript.

Competing Financial Interests: None

Supporting Information: Additional figures describing QD dose -dependent uptake, internalization kinetics, and the effect of microtubule disruption on transferrin-QD colocalization are included in the Supporting Information section.

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Supporting Information
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