Cellular uptake and transport of gold nanoparticles

23 downloads 0 Views 952KB Size Report
conventional delivery methods, with the nanoparticles residing in lysosomes within 40 minutes of ... uptake of Au NPs for applications in radiation therapy.
Available online at www.sciencedirect.com

Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 161 – 169 www.nanomedjournal.com

Original Article

Cellular uptake and transport of gold nanoparticles incorporated in a liposomal carrier, Devika B. Chithrani, PhDa,⁎, Michael Dunne, BAScb , James Stewart, MASca , Christine Allen, PhDb , David A. Jaffray, PhDa,c a

Department of Radiation Physics, Princess Margaret Hospital, University Health Network, Toronto, Ontario, Canada b Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada c Departments of Radiation Oncology and Medical Biophysics, University of Toronto, Toronto, Ontario, Canada Received 6 February 2009; accepted 29 April 2009

Abstract Recent interest in using gold nanoparticles (Au NPs) for therapy in radiation medicine has motivated development of a liposome-based system to enhance their delivery to cells. In this study, liposomes were demonstrated to perform like a “Trojan Horse” to deliver small (1.4 nm) Au NPs into tumor cells by overcoming the energetically unfavorable endocytosis process for small NPs. The results reveal that the liposomal approach provides a thousand-fold enhancement in the cellular uptake of the small Au NPs. Real-time intracellular tracking of the Au NP–liposomes revealed an average speed of 12.48 ± 3.12 μm/hr for their intracellular transport. Analysis of the time-dependent intracellular spatial distribution of the Au NP–liposomes demonstrated that they reside in lysosomes (final degrading organelles) within 40 minutes of incubation. Knowledge gained in these studies opens the door to pursuing liposomes as a viable strategy for delivery of Au NPs in radiation therapy applications. From the Clinical Editor: Gold nanoparticles (Au NPs) as part of an optimized liposome-based delivery system have been proposed for therapy in radiation medicine. The approach resulted in a thousand-fold enhancement in the cellular uptake of Au NPs compared to conventional delivery methods, with the nanoparticles residing in lysosomes within 40 minutes of incubation. © 2010 Elsevier Inc. All rights reserved. Key words: Cell uptake; Gold nanoparticles; Intracellular transport; Liposomes

There is tremendous interest in the design of multifunctional nanotechnology for applications in medicine. Recent advances in engineering and technology have led to the development of many new nanoscale platforms, including quantum dots, nanoshells, gold nanoparticles (Au NPs), paramagnetic NPs, carbon nanotubes, and improvements in traditional, lipid-based platforms.1-9 There has been great interest in using Au NPs in the field of radiation medicine, and these NPs have been shown to provide therapeutic enhancement in radiation therapy.10-13 The efficacy of Au NPs as an enhancer for radiation therapy relies on the successful delivery of the particles to the tumor site

No conflict of interest was reported by the authors of this article. This work was supported by the Orey and Mary Fidani Family Chair in Radiation Physics/Princess Margaret Hospital Foundation and by the Ontario Institute for Cancer Research (OICR), Canada. ⁎Corresponding author: University Health Network, STTARR Program, 7th Floor, 101, College Street, Toronto, ON M5G 1L7, Canada. E-mail address: [email protected] (D.B. Chithrani).

and internalization into tumor cells. There are significant challenges with regard to the delivery of Au NPs at the whole body and cellular levels. For example, in vivo evaluation of Au NPs in preclinical animal models has revealed short circulation lifetimes and only limited accumulation of the particles at the tumor site.11,14 One of the approaches for improving the in vivo stability, circulation lifetime, and cellular uptake of small Au NPs has been to incorporate the particles into or on the surface of liposomes.11,15 Liposomes are the most established of the advanced delivery technologies and consist of a lipid bilayer that envelops an internal aqueous compartment. Three general types of Au-liposome complexes have been reported.16-20 Those complexes were prepared by reducing Au ions in liposomes,16 by mixing lipid and Au NPs possessing hydrophobic surfaces,17-19 and via physical absorption of Au NPs onto the surface of liposomes.20 In the current study, phospholipids conjugated with small Au NPs (diameter = 1.4 nm) were exchanged into preformed liposomes to produce Au NP–incorporated liposomes with a well-defined size and narrow

1549-9634/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2009.04.009 Please cite this article as: D.B. Chithrani, M. Dunne, J. Stewart, C. Allen, D.A. Jaffray, Cellular uptake and transport of gold nanoparticles incorporated in a liposomal carrier. Nanomedicine: NBM 2010;6:161-169, doi:10.1016/j.nano.2009.04.009

162

D.B. Chithrani et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 161–169

size distribution. We have selected the combination of small Au NPs and liposomes for the following reasons: (a) Addition of small NPs to the surface of liposomes, in comparison with addition of larger-sized Au NPs, results in only minor changes to liposome surface properties and stability. (b) Small NPs can be targeted to the nucleus after the cellular internalization process. The radiobiological effects of Au NPs will likely be dependent on their intracellular distribution and benefit from perinuclear/ nuclear localization. (c) The core of the liposome can be used as a carrier for conventional imaging contrast agents such as iodine and gadolinium or therapeutic agents creating multifunctional systems. (d) The liposomes are a platform that has already been optimized to result in extended circulation lifetime in vivo, and circulation lifetime of smaller NPs can be increased by placing them on a liposome. In addition, each liposome can carry hundreds of smaller NPs, whereas if larger Au NPs were used, it would be necessary to modify the surface of each particle to increase the circulation lifetime. In addition, once NPs are used for a particular application, excretion of smaller Au NPs (b5 to 6 nm) is easier than excretion of larger Au NPs.21 In this article, we report the physicochemical characterization including the zeta potential, morphology, and energy-dispersive X-ray spectroscopy of Au NP–liposomes. The cellular internalization of liposomes, with varying amounts of surface conjugated Au NP, was also evaluated in HeLa cells. Most importantly, we have tracked the intracellular transport of Au NP–incorporated liposomes in vitro for determination of their intracellular speed and diffusion coefficient. Findings from this research demonstrate the potential of using liposomes to improve the intracellular uptake of Au NPs for applications in radiation therapy. Methods Preparation of liposomes 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol (CH) (Northern Lipids, Vancouver, Canada) in a mole ratio of 55:40 were dissolved in ethanol at 70°C and hydrated in 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) buffered saline (HBS) such that once the ethanol was evaporated, the final lipid concentration was 100 mM. Liposomes were formed by five repetitions of extrusion through two stacked membranes with 200-nm pores using a Lipex Extruder (Northern Lipids Inc.) followed by a further five cycles through two 80-nm pore membranes. Incorporation of Au NPs onto preformed liposomes Au NPs with a diameter of 1.4 nm and conjugated to DPPE lipid (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-NNanogold; DPPE-Nanogold; Nanoprobes, Yaphank, New York) were first dissolved in ethanol. The DPPE-Nanogold

was mixed with preformed liposomes of size ∼90 nm at specific ratios (ie, DPPE-Nanogold:liposome = 2000:1, 1000:1, 500:1). The mixture was heated for 8 hours at 40°C to allow for transfer of the DPPE-Nanogold into the liposome bilayer. The resulting Au NP–incorporated liposomes were then incubated with 1,2distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (PEG2000-DSPE) comprising 5 mol % of the total amount of lipid. The liposomes were dialyzed overnight against a 250-fold volume excess of HBS using 100,000 molecular weight cutoff membranes to remove any free Au NPs or DPPE-Nanogold. Characterization of Au NP–liposomes Dynamic light scattering Liposome samples were diluted to 0.25 mM lipid in phosphate-buffered saline (PBS) solution. Size and size distribution were then measured using a particle size analyzer (90Plus; Brookhaven, Holtsville, New York) at 25°C. Reported results are an average of five distinct 3-minute measurements taken at a 90-degree detection angle. This was repeated for three different samples (n = 3), and the difference between mean values was not statistically significant [P N .10, using analysis of variance (ANOVA)]. Zeta potential A zeta potential analyzer (ZetaPALS; Brookhaven) was used to determine the electrokinetic surface potential of liposomes both with and without Au NPs. Liposomes were diluted to 2.5 mM lipid in water, and the zeta potential was analyzed at 25°C. In each case the average of five separate measurements is reported. This was repeated for three different samples, and the difference between mean values was not statistically significant (P N .10, using analysis of variance ANOVA). Transmission electron microscopy The size and morphology of the liposomes were studied using a Hitachi 7000 microscope (Hitachi, Tokyo, Japan) operating at an accelerating voltage of 80 kV. The liposome samples were first diluted (1:10) in distilled water, and a 20 μL aliquot was applied onto a transmission electron microscopy (TEM) grid. The solution was then left for 1 minute, and the excess was removed from the grid using filter paper. For negative staining of the sample, 10 μL 2% phosphotungstic acid (PTA) was applied immediately (before the sample was dried) and left for 30 seconds prior to removal of excess PTA. The grids were placed in the grid box for several hours or overnight for drying before imaging. To analyze the size distribution, the TEM micrographs were segmented in MATLAB. Segmentation was accomplished with marker controlled watershed transform. Once the contours were delineated, it was possible to measure the individual diameters of the particles. Three batches of 50 Au NP–liposomes were selected for analysis of their size. Particle size was reported as the mean diameter. Scanning transmission electron microscopy To visualize small Au NPs (size 1.4 nm) on the surface of the liposome, a high-resolution microscope (STEM HD2000; Hitachi, Tokyo, Japan) with 1.8 Å resolution was used. The samples were prepared as described above. The same instrument

D.B. Chithrani et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 161–169

was used in the energy dispersive X-ray spectrometers mode for elemental analysis of Au. Quantification of number of Au NPs per liposome The concentration of liposomes was estimated from the known concentration of phospholipid, 15 mM, and the assumption that each liposome was composed of 73,000 phospholipids. The number of phospholipids per liposome was calculated from the diameter of the vesicles (ie, 90 nm) and the known headgroup surface area for phospholipids in fluid bilayers (ie, due to cholesterol content).22 The Au-conjugated phospholipids were exchanged into the preformed liposomes to produce Au NP–liposomes. The liposomes were dialyzed to remove any free Au-conjugated phospholipid. After preparation, a known concentration of Au NP–liposomes was processed at 120°C in nitric acid for 2 hours. The concentration of Au NPs in the sample was measured using ICP-AES (inductively coupled plasma atomic emission spectroscopy) technique, which will be discussed in detail later. If the number of Au NPs present is N for a sample with M liposomes, the number of NPs per liposome is given by N/M. Five samples were analyzed for each formulation, and the mean value is given with the standard deviation. This was repeated for three separate experiments, and the difference in values was not found to be statistically significant (P N .10, using ANOVA). Preparation of fluorescent-labeled Au NP–liposomes for four-dimensional confocal microscopy For optical microscopy, liposomes were tagged with a fluorescent lipid [1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine-rhodamine-B-sulfonyl)] with excitation and emission at 550/590 nm. In this case, preformed liposomes were mixed with both DPPE-Nanogold and fluorescent lipid at 0.15 and 0.1 mol%, respectively. Measurement of cellular uptake of Au NP–liposomes HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% FBS (fetal bovine serum). Cells were incubated at 37°C in a water-saturated incubator with 5% CO2. For cellular uptake studies, cells were grown in Petri dishes (Fisherbrand; Somerville, New Jersey, 60 × 15 mm) until they reached 80% confluency and were incubated with fresh media for 3 hours before introducing Au NP– liposomes. Cells were incubated with Au NP–liposomes (1 × 109 liposomes/mL) for 8 hours, and at the end of the incubation period, cells were washed with PBS three times and trypsinized to remove them from the bottom for quantification purposes. Cells were counted before processing with HNO3 at 200°C in an oil bath for ICP-AES analysis to quantify the number of Au NPs internalized per cell. Four-dimensional analysis of cellular transport of Au NP–liposomes For four-dimensional analysis of the cellular uptake of Au NP–liposomes, HeLa cells were grown in glass-bottom microwell dishes (MatTek Corporation, Ashland, Massachusetts). Cells were incubated with Au NP–liposomes labeled with a

163

fluorescent lipid with excitation/emission at 550/590 nm (lissamine rhodamine) for 30 minutes for their initial internalization. To clearly distinguish the individual vesicles containing Au NP–liposomes, the concentration of liposomes in the media was reduced by a factor of 100, from the method described in the previous section, to 1 × 107 liposomes/mL. The dish was placed on the stand of the microscope (Zeiss Two-Photon Confocal Microscope, LSM 510 Meta, Göttingen, Germany) in such a way that the coverslip area was in contact with the × 63 waterimmersed microscope objective. Sequences of Z-stacks were taken for a group of selected cells in 5-minute time intervals for real-time analysis of transport within the cell cytoplasm. Cell permeable Lyso Tracker (Lyso Tracker Green DND-26; Molecular Probes-L7526, Eugene, Oregon) was employed for live cell staining of lysosomes. To study the distribution of the Au NP–liposomes in organelles, the cells were incubated with 60 μL Lyso Tracker (50 nM) (excitation/emission at 504/511 nm) along with the liposomes labeled with rhodamine fluorescent dye (excitation/emission at 550/590 nm) for 30 minutes. Quantification of cellular uptake of Au NPs using ICP-AES technique Cells were washed with PBS twice, detached from the Petri dish using trypsin (1X) (Sigma, St. Louis, Missouri), counted, and processed at 120°C in nitric acid for 2 hours. The concentration of Au was measured using ICP-AES. The following equations were then used to convert the number of Au atoms, as determined by ICP-AES, to the number of Au NPs. For a sphere of diameter D, the number of atoms (U) in each volume of Au NPs was determined. In the calculation, a refers to the length of the one side of the unit cell, which has a value of 4.076 Å, and there are four Au atoms per unit cell due to the facecentered cubic structure [U = (2/3)π(D/a)3].23,24 If M is the measured number of Au atoms from ICP-AES and N is the number of NPs for the analyzed sample, the number of NPs per cell can be determined by dividing N by the total number of cells for that sample. This calculation assumes a homogeneous intracellular distribution of NPs in the cell population. Three dishes were used for each experimental data point, and the mean value was given with the standard deviation. The experiment was repeated three times, and the difference in mean values was not statistically significant (P N .1, ANOVA). Determination of position of vesicles containing Au NP–liposomes using centroid calculation For quantitative determination of the intracellular motion of the vesicles, three-dimensional image stacks were acquired at 5minute intervals. Each stack consists of eight slices with a slice thickness of 0.8 μm. To correct for any macroscopic translation errors between acquisition times, each image stack was rigidly registered to the first acquired stack using a three-dimensional phase correlation technique.25 After registration, connected three-dimensional fluorescent regions from the first stack were segmented using a flood-filling algorithm. By appropriate choices of the threshold arguments, effects caused by fluorescence background noise can be removed.26 Details of this technique have been provided elsewhere.27

164

D.B. Chithrani et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 161–169

Figure 1. Schematic depicting preparation of Au NP–liposomes. (A) Liposomes 90 nm in diameter are preformed. (B) Au NP–phospholipids are then exchanged into the lipid bilayer of the preformed liposomes.

Figure 2. Characterization of Au NP–liposomes. (A) A bright-field TEM image of preformed liposomes. (B) A bright-field TEM image of Au NP–liposomes; the darker color of the liposomes is due to the presence of Au NPs on the surface. (C) Data obtained from DLS (dynamic light scattering) and zeta potential analysis of liposomes with/without Au NPs. (D) A high-resolution dark-field TEM image of an Au NP–liposome ∼90 nm in diameter with Au NPs of 1.4 nm diameter, seen as small white, bright dots, localized on the surface of the liposome.

Results The Au NP–liposomes, illustrated in Figure 1, were prepared by exchanging the Au NP conjugated phospholipid (ie, DPPENanogold) into liposomes having a diameter of 90 nm and a uniform size distribution. Figure 2, A is a bright-field TEM image of liposomes prior to insertion of the Au NP– phospholipid. Figure 2, B is a bright-field TEM image of the

liposomes after insertion of the Au NP–lipid, with the darker color of the liposomes being due to the presence of Au NPs on their surface. Figure 2, C summarizes the data obtained for analysis of the size, polydispersity, and zeta potential of the liposomes before and after insertion of the Au NP–phospholipid. Furthermore, as shown in Figure 2, D, high-resolution scanning transmission electron microscopy, with a resolution of ∼1 nm, enabled visualization of the small Au NPs on the surfaces of the

D.B. Chithrani et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 161–169

Figure 3. Energy-dispersive X-ray spectroscopy measurements across a liposome to further verify the presence of Au NPs. (A) Bright-field TEM image of the liposome selected for analysis. The data was collected along the line marked across the liposome. (B) Data obtained from elemental analysis showing the increase in signal for Au across the liposome (line WZ) compared with that for two other positions away from the liposome surface (WX, YZ).

liposomes. The individual Au NPs of size 1.4 nm located on the surfaces of the liposomes are clearly seen as small bright dots. The presence of the Au NPs on the liposomes was further verified by performing elemental analysis using energy-dispersive X-ray spectroscopy across the liposome (along the line marked in red as shown in Figure 3, A). Elemental analysis shows the presence of Au on the liposome (line XY) in comparison with that of two other positions away from the liposome surface (WX, YZ; Figure 3, B). To evaluate the cellular internalization of the Au NP– incorporated liposomes, three distinct formulations were prepared that varied in terms of the initial mixing ratio of Au NP– phospholipid to liposome (ie, 2000:1, 1000:1, 500:1 Au NP: liposome). The dark-field TEM images show a decrease in the number of Au NPs per liposome with a decrease in the initial mixing ratio (Supplementary Material, Figure S1). As determined by ICP-AES, the number of Au NPs per liposome were 550, 400, and 200 for initial Au NP-lipid:liposome mixing ratios of 2000:1, 1000:1, and 500:1, respectively. Incorporation of the Au NP–lipid into the liposome did not significantly affect the size, size distribution, or zeta potential of the liposomes (Supplementary Material, Figure S2).

165

Figure 4, A summarizes the results for evaluation of cellular uptake of the three Au NP–liposome formulations in HeLa cells. According to the figure, the cellular uptake of the Au NP– liposomes is independent of the number of Au NPs present per liposome. The number of Au NPs internalized per cell was also calculated and is shown in Figure 4, B. The intracellular motion of the Au NP–incorporated liposomes was captured by acquiring three-dimensional image stacks at 5-minute intervals on a two-photon confocal microscope. Previous studies have shown that liposomes are mostly internalized by endocytosis and transported via the endolysosomal pathway.16,28-30 Similarly, in this study cellular uptake of the Au NP–liposomes was found to be temperature dependent with cellular uptake being decreased by 80% as the incubation temperature was decreased from 37° to 4°C. In this study, the transport of 80 different endocytic vesicles was examined in detail across 10 populations of cells. Figure 5 shows the details of vesicle motion for 11 vesicles containing the Au NP–liposomes from one of the groups of cells. Figure 5, A highlights the chosen 11 vesicles selected for the study, and it was necessary to use five image planes to index those vesicles as they were localized in different Z-planes relative to the reference frame chosen. The trajectories of the 11 vesicles for a period of 30 minutes are illustrated in Figure 5, B. The real-time motion of the 11 vesicles is shown in Figure 5, C, where the green dot represents the starting point and the red dot indicates the finish. Transport of vesicles in the cytoplasm was heterogeneous. Most of the vesicles showed upward motion toward the upper cell membrane. The average vesicle speed and diffusion coefficient for each of the vesicles are shown in Figure 6, A and Figure 6, B, respectively. The average vesicle speed and diffusion coefficient for the 11 vesicles shown in Figure 5, A were 12.5 ± 3.1 μm/hr and 5.1 ± 2.1 μm2/hr, respectively. Real-time motion of a single vesicle (vesicle no. 11) is demonstrated in a schematic in Figure 6, C and in several image stacks acquired during a period of 30 minutes shown in Figure 6, D. Finally, the intracellular pathway or fate of the Au NP– liposomes was evaluated at the cellular level. Figure 7 shows confocal images of a group of cells after nuclear and lysosomal staining. Importantly, the composite image shows that most of the Au NP–liposomes have accumulated in lysosomes after only 45 minutes of incubation. The overlap of the red and green regions represents liposomes in lysosomes. It is also evident that the Au NP–liposomes were not localized in the nucleus even though most of them were localized close to the nuclear membrane.

Discussion There has recently been widespread interest in the use of Au NPs for a range of medical applications including drug delivery and radiation therapy.10,11,31 In most of these studies, Au NPs with diameters of 2 to 30 nm have been used. However, recent theoretical and experimental data have shown that there is an ideal size for endocytotic uptake of NPs into cells.32-35 According to these studies, NPs with diameters of 50 nm have the highest degree of cellular uptake. The optimal particle size is a result of the competition between the thermodynamic driving

166

D.B. Chithrani et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 161–169

Figure 4. Cellular uptake of Au NP–liposomes. (A) Cellular uptake of liposomes with different numbers of Au NPs on their surfaces. The number of Au NPs per liposome was 550, 390, and 200 for initial mixing ratios of 1:2000, 1:1000, and 1:500, respectively. (B) The number of Au NPs internalized per cell after incubation with the three Au NP–liposome formulations. Inset: Comparison between cellular uptake of small Au NPs alone and Au NPs conjugated to liposome surface.

Figure 5. Details of intracellular transport properties of vesicles containing Au NP–liposome nanostructures. (A) Eleven vesicles were selected for analysis, and five fields of view in the panel were used to show their locations within the cell. The vesicles (labeled in yellow) were localized in different focal planes across the cell. (B) Model diagram showing a single cell placed inside a three-dimensional box with positive X, Y, and Z directions labeled with reference to the image frame. Path of vesicles was tracked by imaging the same field of view (XY plane) across several Z-planes along the body of the cell as a function of time. (C) Trajectories of 11 vesicles within a period of 30 minutes. The starting position is marked with a green dot, and final position is marked with a red dot.

force that is necessary for membrane wrapping of the NP and receptor diffusion kinetics.17 When the particle size is smaller than the optimal value, the membrane wrapping causes an unfavorable increase in free energy decreasing the degree of cell uptake. Hence in this study, liposomes were used as a “Trojan Horse” to overcome the energetically unfavorable endocytotic uptake of small Au NPs and to deliver large numbers of the Au NPs into cells. The Au-conjugated phospholipids were exchanged into preformed liposomes to produce Au NP–liposomes 90 nm in diameter with a uniform size distribution. Evaluation of cellular internalization in HeLa cells revealed that the uptake was independent of the number of Au NPs present on the liposome surface. Therefore, it is likely that it is the size and surface properties of the liposomes that played the most significant role

in influencing the cellular uptake of the Au NP–liposomes. Importantly, the use of liposomes facilitated delivery of significant quantities of Au NPs into cells. Specifically the liposomes provided a means to deliver up to 850,000 Au NPs per cell. As mentioned previously, the delivery of small NPs into cells is energetically unfavorable. For example, as shown in Appendix C, in a study that examined the effect of NP size on cell uptake, 50 nm was found to be the optimum size. An increase (ie, N50 nm) or decrease (ie, b50 nm) in NP size was found to result in a significant decrease in cell uptake. Indeed, a decrease in the diameter of the NPs from 50 nm to 6 nm resulted in an 18-fold decrease in cell uptake.32,33 Theoretically, cell uptake for NPs with a diameter of 1.4 nm, which is equivalent to the diameter of Au NPs employed in this study, is predicted to be effectively zero (Supplementary Material, Figure S3). The

D.B. Chithrani et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 161–169

167

Figure 6. The transport characteristics of vesicles identified in Figure 5. (A, B) Average speed and diffusion coefficients of the 11 selected vesicles. The ensemble average speed and diffusion coefficient of the 11 vesicles were 12.5 ± 3.1 μm/hr and 5.1 ± 2.1 μm2/hr, respectively. (C) Schematic illustrating the intracellular motion of vesicle no. 11 as a function of time. (D) The motion of vesicle no. 11 is displayed using a series of confocal images. Panels Z1, Z2, Z3, Z4, and Z5 display five planes across the cell (from top to bottom of the cell). Each panel consists of images of the same field of view as a function of time. Vesicle no. 11 showed motion downwards followed by upwards motion toward the cell periphery as illustrated by a yellow dotted line. When the vesicle is localized in between two planes (vesicle is identified with a yellow-colored loop around it), its actual position is estimated using centroid calculation (see Supplement S1). Initially, vesicle no. 11 is localized in between slices Z3 and Z4 but localized more toward the Z4 slice.

Figure 7. Distribution of liposomes in endosomes and lysosomes after internalization into HeLa cells. The panel consists of five sections from the same field of view. From left: phase-contrast image; nucleus stained with DAPI, 4′,6-diamidino-2-phenylindole, (marked with blue); rhodamine-labeled liposomes (marked in red); lysosomes stained with Lyso Tracker Green (marked in green); and a composite image.

average size of the liposomes used in this study was 90 nm, and an average of 1520 liposomes were internalized per cell. Based on the experimental data for smaller NPs, a 1000-fold increase in the uptake of smaller Au NPs can be achieved by incorporation onto the surface of liposomes (Figure 4, B, inset). Therefore, we believe that this liposome-based platform can be used for effective intracellular delivery of small NPs by overcoming the challenges faced during their endocytosis process. Many of the applications require transport of small NPs to specific cell and tissue compartments in vivo. This

liposome-based delivery technology also has the capability to circulate in vivo for prolonged periods and accumulate at tumor sites via enhanced permeation and retention effects.36 The intracellular transport and subcellular distribution of the Au NP–liposomes were also monitored by four-dimensional confocal microscopy. The ensemble average speed and diffusion coefficients of vesicles containing the Au NP– liposomes were 12.5 ± 3.1 μm/hr and 5.1 ± 2.1 μm2/hr, respectively. In addition, the detailed confocal studies on intracellular organelle distribution of the Au NP–liposomes

168

D.B. Chithrani et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 161–169

showed that they become trapped in endosomes prior to being fused with lysosomes for processing. Analysis of the timedependent distribution of the Au NP–liposomes demonstrated that the fraction of liposomes in lysosomes increased with time. Furthermore, no Au NP–liposomes were localized within the nucleus. The slow transport of these nanostructures is in accordance with previous reports that have indicated slower transport of cellular organelles such as secretory granules. The diffusion coefficient for secretory granules has been reported to be 6.84 μm2/hr, which is closer to the value obtained for the endolyso vesicles in the current study.37 A recent study by Huff et al also suggested that transport of Au nanorods was vesicular, and the diffusion coefficient for the Au NPs was found to be 1.5 μm2/ hr.38 Al-Jamal et al have used four-dimensional confocal microscopy to estimate the diffusion coefficient of dendrimer nanostructures (size 2.5 to 10 nm), and the value obtained for the diffusion coefficient was 22.7 μm2/hr. The value obtained for the diffusion coefficient for Au NP–liposomes (size 90 nm) in this study was 5.1 μm2/hr, and the differences in values may suggest that the transport kinetics of nanostructures could also be dependent on the temperature, size, and morphology of the NPs, as well as cell type. There were no significant differences in the transport properties of liposomes with no Au NPs and Au NP– liposomes (Supplementary Material, Figure S4). Liposomes have been used as an effective carrier for smallmolecule drugs and other therapeutic agents. Details on the intracellular speed and diffusion coefficient of liposomes as well as their intracellular fate can be used to further exploit this technology for delivery of therapeutic agents. In addition, the current four-dimensional microscopy technique may be pursued for real-time monitoring of liposomal delivery of drugs at the cellular level. These findings demonstrate that liposomes provide an efficient method of intracellular delivery of small Au NPs. The cellular uptake of smaller NPs was enhanced 1000-fold using liposomes as a carrier. According to the real-time intracellular tracking studies, the Au NP–liposomes were transported with an average speed of 12.48 ± 3.12 μm/hr. In addition, the Au NP– liposomes were shown to reside in lysosomes within 40 minutes of incubation. Future studies will evaluate the pharmacokinetics and biodistribution of the Au NP–liposomes in vivo as well as their efficacy to enhance radiation therapy. Considering the wellcharacterized biodistribution of liposomes in vivo, the use of this technology for delivery of Au NPs is likely to result in improved biodistribution properties for the NPs in vivo, such as increased tumor accumulation. In the future, other contrast agents for imaging applications or anticancer drugs for therapy may be incorporated into the aqueous interior of the Au NP–liposomes to create truly multifunctional systems. Furthermore, targeting ligands can be easily conjugated to the surfaces of the Au NP– liposomes to pursue active targeting to specific cell populations.

Acknowledgments The authors would like to thank Dr. Geof Aers, Mr. Nicolas Gonzalez, Mr. Batista Calvieri, and Mr. Dan Mathers for their technical assistance in sample preparation and analysis.

Appendix A. Supplementary material Supplementary material associated with this article can be found in the online version, at doi:10.1016/j.nano.2009.04.009.

References 1. Alivisatos P. The use of nanocrystals in biological detection. Nature Biotechnol 2003;22:47-51. 2. Colvin VL, Schlamp MC, Alivisatos P. Light-emitting diodes made from cadmium selenide nanocrystals and a semiconducting polymer. Nature 1994;370:354-7. 3. Souza GR, Christianson DR, Staquicini FI, Ozawa MG, Snyder EY, Sidman RL, et al. Networks of gold nanoparticles and bacteriophage as biological sensors and cell-targeting agents. Proc Natl Acad Sci U S A 2006;103:1215-20. 4. Jin H, Heller DA, Strano MS. Single-particle tracking of endocytosis and exocytosis of single-walled carbon nanotubes in NIH-3T3 cells. Nano Lett 2008;8:1577-85. 5. Ruenraroengsak P, Al-Jamal KT, Hartell N, Braeckmans K, Smedt SCD, Florence AT. Cell uptake, cytoplasmic diffusion and nuclear access of a 6.5nm diameter dendrimer. Int J Pharm 2007;331:215-9. 6. Kneipp JK, Kneipp H, McLaughlin M, Brown D, Kneipp K. In vivo molecular probing of cellular compartments with gold nanoparticles and nanoaggregates. Nano Lett 2006;6:2225-31. 7. Sokolov K, Follen M, Aaron J, Pavlova I, Malpica A, Lotan R, et al. Real-time vital optical imaging of precancer using anti-epidermal growth factor receptor antibodies conjugated to gold nanoparticles. Cancer Res 2003;63:1999-2004. 8. Mulde WJM, Strijkers GJ, Van Tilborg GAF, Griffioen AW, Nicolay K. Lipid-based nanoparticles for contrast-enhanced MRI and molecular imaging. NMR Biomed 2006;19:142-64. 9. Nativo P, Prior IA, Brust M. Uptake and intracellular fate of surfacemodified gold nanoparticles. ACS Nano 2008;2:1639-44. 10. Zheng Y, Hunting DJ, Ayotee P, Sanche L. Radiosensitization of DNA by gold nanoparticles irradiated with high-energy electrons. Radiat Res 2008;169:19-27. 11. Hainfeld JF, Slatkin DN, Smilowitz HM. The use of gold nanoparticles to enhance radiotherapy in mice. Phys Med Biol 2004; 49:N309-15. 12. Herold DM, Das IJ, Stobbe CC, Iyer RV, Chapman JD. Gold microspheres: a selective technique for producing biologically effective dose enhancement. Int J Radiat Biol 2000;76:1357-64. 13. Chen W, Zhang J. Using nanoparticles to enable simultaneous radiation and photodynamic therapies for cancer treatment. J Nanosci Nanotechnol 2006;6:1159-66. 14. Kim D, Park S, Lee JH, Jeong YY, Jon S. Antibiofouling polymercoated gold nanoparticles as a contrast agent for in vivo X-ray computed tomography imaging. J Am Chem Soc 2008;129:7661-5. 15. Zheng J, Perkins G, Kirilova A, Allen CJ, Jaffray DA. Multimodal contrast agent for combined CT and MR imaging applications. Invest Radiol 2006;41:339-48. 16. Hong K, Friend DS, Glabe CG, Papahadjopoulos D. Liposome containing colloidal gold are a useful probe of liposome-cell interations. Biochim Biophys Acta 1983;732:320-3. 17. Paasonen L, Laaksonen T, Johans C, Yliperttula M, Kontturi K, Urtti A. Gold nanoparticles enable selective light-induced contents release from liposomes. J Controlled Release 2007;122:86-93. 18. Park SH, Oh SG, Mun JY, Han SS. Loading of gold nanoparticles inside the DPPC bilayers of liposome and their effects on membrane fluidities. Colloids Surf B 2006;48:112-8. 19. Li X, Li Y, Yang C, Li Y. Liposome induced self-assembly of gold nanoparticles into hollow spheres. Langmuir 2004;20:3734-9. 20. Kojima C, Hirano Y, Yuba E, Harada A, Kono K. Preparation and characterization of complexes of liposomes with gold nanoparticles. Colloids Surf B 2008;66:246-52.

D.B. Chithrani et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 161–169 21. Choi HS, Liu W, Misra P, Tanaka E, Zimmer JP, Ipe BI, et al. Renal clearance of quantum dots. Nature Biotechnol 2007;25:1165-70. 22. Kumar S, Harrison N, Richards-Kortum R, Sokolov K. Plasmonic nanosensors for imaging intracellular biomarkers in live cells. Nano Lett 2007;7:1338-43. 23. Thomas JM. Colloidal metals: past, present and future. Pure Appl Chem 1988;60:1517-28. 24. Davey WP. Precision measurements of the lattice constants of twelve common metals. Phys Rev 1925;25:753-61. 25. Hoge WS, Westin CF. Identification of translational displacements between N-dimensional data sets using the high-order SVD and phase correlation. IEEE Trans Image Process 2005;14:884-9. 26. Patterson GH, Piston DW. Photobleaching in two-photon excitation microscopy. Biophys J 2000;78:2159-62. 27. Chithrani BD, Stewart J, Christine CJ, Jaffray DA. Intracellular uptake, 490 transport, and processing of nanostructures in cancer cells. Nanomedicine: Nanotechnology, Biology, and Medicine 2009;5: 118-27. 28. Lee KD, Nir S, Papahadjopoulos D. Quantitative analysis of liposomecell interations in vitro. Biochemistry 1993;32:889-99. 29. Straubinger RM, Hong K, Friend DS, Papahadjopoulos D. Endocytosis of liposomes and intracellular fate of encapsulated molecules: encounter with a low pH compartment after internalization in coated vesicles. Cell 1983;32:1069-79.

169

30. Matthay KK, Abai AM, Cobb S, Hong K, Papahadjopoulos D, Straubinger RM. Role of ligand in antibody-directed endocytosis of liposomes by human T-leukemia cell. Proc Natl Acad Sci U S A 1989; 49:4879-86. 31. Ghosh P, Han G, De M, Kim CK, Rotello VM. Gold nanoparticles in delivery applications. Adv Drug Deliv Rev 2008;60:1307-15. 32. Zhang S, Li J, Lykotrafitis G, Bao G, Suresh S. Size-dependent endocytosis of nanoparticles. Adv Mater 2008;21:419-24. 33. Gao H, Shi W, Freund LB. Mechanics of receptor-mediated endocytosis. Proc Natl Acad Sci U S A 2005;102:9469-74. 34. Chithrani BD, Ghazani AA, Chan WCW. Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells. Nano Lett 2006;6:662-8. 35. Chithrani BD, Chan WCW. Elucidating the mechanism of cellular uptake and removal of protein-coated gold nanoparticles of different sizes and shapes. Nano Lett 2007;7:1542-50. 36. Xiong XB, Huang Y, Lu WL, Zhang X, Zhang H, Nagai T, et al. Intracellular delivery of doxorubicin with Rgd-modified sterically stabilized liposomes for an improved antitumor efficacy: in vitro and in vivo. J Pharm Sci 2005;94:1782-93. 37. Kaether C, Gerdes HH. Monitoring of protein secretion with green fluorecent protein. Methods Enzymol 1999;302:11-9. 38. Huff TB, Hansen MN, Zhao Y, Chen JX, Wei A. Controlling the cellular uptake of gold nanorods. Langmuir 2007;23:1596-9.