Multiphoton-Absorption-Induced-Luminescence (MAIL) Imaging of

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Multiphoton-Absorption-Induced-Luminescence (MAIL) Imaging of Tumor-Targeted Gold Nanoparticles Matthew B. Dowling,† Linjie Li,‡ Juhee Park,‡ George Kumi,‡ Anjan Nan,§ Hamid Ghandehari,| John T. Fourkas,‡,⊥ and Philip DeShong*,†,‡ Department of Bioengineering, Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742-2111, United States, School of Pharmacy, University of Maryland, Baltimore, Maryland 21201, United States, and Department of Bioengineering, University of Utah, Salt Lake City, Utah 84101, United States. Received March 3, 2010; Revised Manuscript Received September 16, 2010

We demonstrate that multiphoton-absorption-induced luminescence (MAIL) is an effective means of monitoring the uptake of targeted nanoparticles into cells. Gold nanoparticles (AuNPs) with diameters of 4.5 and 16 nm were surface-functionalized with monocyclic RGDfK, an RGD peptide analogue that specifically targets the Rvβ3 integrin, a membrane protein that is highly overexpressed in activated endothelial cells during tumor angiogenesis. To determine whether cyclic RGD can enhance the uptake of the functionalized AuNPs into activated endothelium, human umbilical vein endothelial cells (HUVECs) were used as a model system. MAIL imaging of HUVECs incubated with AuNPs demonstrates differential uptake of AuNPs functionalized with RGD analogues: RGDfKmodified nanoparticles are taken up by the HUVECs preferentially compared to AuNPs modified with linear RGD (GRGDSP) conjugates or with no surface conjugates. The luminescence counts observed for the AuNPRGDfK conjugates are an order of magnitude greater than for AuNP-GRGDSP conjugates. Transmission electron microscopy shows that, once internalized, the AuNP-RGDfK conjugates remain primarily within endosomal and lysosomal vesicles in the cytoplasm of the cells. Significant aggregation of these particles was observed within the cells. MAIL imaging studies in the presence of specific uptake inhibitors indicate that AuNP-RGDfK conjugate uptake involves a specific binding event, with Rvβ3 integrin-mediated endocytosis being an important uptake mechanism.

INTRODUCTION The development of techniques for targeting of multifunctional nanostructures to specific locations in vivo is a major goal in nanomedicine (1-4). Once these nanostructures reach their destination, they should be able to perform additional tasks, such as releasing a payload or being taken up into specific cells via endocytosis. The design of effective nanovectors for targeting applications (diagnostics, imaging, or drug delivery) would benefit greatly from methods that allow the direct probing of these processes. Within the field of single molecule/particle tracking, there are two classes of probes, (1) organic fluorophores and (2) quantum dots, which have generated the majority of the interest. However, despite the clear advances in their associated imaging techniques, both classes present major disadvantages that limit their practical applications as trackable probes within biological systems. Organic fluorophores are subject to photobleaching within time scales that are much shorter than many important biological processes (5, 6). Quantum dots experience blinking upon sustained excitation, and they are synthesized from cytotoxic materials (7, 8). * Corresponding author. Philip DeShong, 2101 Building 091, College of Chemical and Life Sciences, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742. Phone: (301) 405-1892, Fax: (301) 314-9121. E-mail: [email protected]. † Department of Bioengineering, University of Maryland. ‡ Department of Chemistry and Biochemistry, University of Maryland. § School of Pharmacy, University of Maryland. | University of Utah. ⊥ Prof. John T. Fourkas is also affiliated with (1) Institute of Physical Science and Technology, (2) Center for Nanophysics and Advanced Materials, (3) Maryland NanoCenter, University of Maryland, College Park, MD 20742.

Additionally, the chemistries involved in attaching targeting agents and/or therapeutic molecules to quantum dots are generally complex and not well-understood (9, 10). Noble metal nanostructures such as gold and silver represent a third major class of nanovectors for direct probing. Unlike organic fluorophores or quantum dots, these structures do not experience photobleaching or blinking upon sustained excitation (5). Furthermore, the chemistries of noble metals are wellunderstood, and they present minimal toxicity and/or inflammatory concerns. Hence, photoinduced luminescence from noble metals has attracted attention for use in biomedical imaging, particularly as this method has become increasingly efficient over the past few decades. In 1986, Boyd et al. discovered that gold surfaces exhibit a 106-fold enhancement in luminescence intensity when subjected to two-photon excitation as opposed to single-photon excitation (11). More recently, it has been demonstrated that noble metal nanoparticles also exhibit efficient luminescence upon multiphoton excitation. Farrer et al. demonstrated that multiphoton-absorption-induced luminescence (MAIL1 ) from AuNPs is generated efficiently with 800 nm laser intensities that are lower than those typically used for twophoton imaging of living cells (5). For AuNPs, this luminescence spans the visible spectrum and was shown to arise from threephoton absorption when excited with 800 nm light (5). Similar results have been found for silver nanoparticles (12). We have recently shown, by correlating MAIL intensity with SEM 1

Abbreviations: MAIL (multiphoton-absorption-induced-luminescence), HUVECs (human umbilical vein endothelial cells), RGD (arginine-glycine-aspartic acid peptide), RGDfk (arginine-glycineaspartic acid-phenylalanine-lysine monocylic peptide), AuNPs (gold nanoparticles).

10.1021/bc100115m  2010 American Chemical Society Published on Web 10/21/2010

MAIL Imaging of Tumor-Targeted Au NPs

images, that aggregates of AuNPs show even stronger MAIL signals than individual particles (13). MAIL from gold nanostructures shows considerable promise for biomedical applications. Durr et al. have shown that luminescence from gold nanorods can be orders of magnitude brighter than autofluorescence from cancer cells (14). Wang et al. were able to image gold nanorods within cancer cells in vitro and flowing in a mouse ear blood vessel in vivo using MAIL (15). Park et al. used MAIL to perform three-dimensional imaging of gold nanoshells within mouse tumor tissue (16). Qu et al. have used MAIL to image AuNPs targeted to lymphoma cells (17). In addition, Nagesha et al. have used MAIL to visualize AuNPs in Dictyostelium discoideum cells and murine embryonic stem cells (6). Here, we also use MAIL, but instead to monitor the targeting and endocytosis of AuNPs of different sizes to human umbilical vein endothelial cells (HUVECs), which serve as an in vitro model for the activated endothelium of tumor angiogenesis. The targeting moiety of interest in this study is RGDfK, a cyclic RGD analogue. This peptide has a large binding affinity for the Rvβ3 integrin, a membrane protein that is overexpressed in the activated endothelium and thus represents a potential target for chemotherapy (18-23). Fani et al. showed that free RGDfK peptide displays approximately 4-fold preferential uptake into tumor tissue as compared to a linear RGD control peptide (24). Additionally, we have previously shown that water-soluble polymers with side chains terminated in RGDfK localize efficiently within solid tumors in vivo (19-23). A number of other groups have demonstrated that the RGD peptide is generally an effective targeting moiety for therapeutic molecules and nanoparticles to the activated endothelium (25-28). AuNPs provide a platform for targeted drug delivery and tumor ablation. However, the fate of AuNP-RGDfK systems in endothelial cells is unknown (18, 23). In this study, we conjugate AuNPs with the targeting peptide RGDfK or, as a control, with a linear RGD peptide. AuNPs are attractive for targeting applications because they are highly stable and biocompatible, have well-established surface chemistry, and can exhibit strong MAIL signals. We demonstrate that MAIL imaging methodology can monitor the uptake of AuNPs functionalized with RGD peptide analogues. The collected images indicate that the AuNPs undergo differential uptake into HUVECs based upon both particle size and surface conjugate, and that the uptake is largely a receptor-mediated process. In addition, we benchmark the MAIL imaging with transmission electron microscopy, and demonstrate that the two techniques give complementary results. This suggests that MAIL may be used for rapid, routine screening of the efficacy of a given targeting moiety attached to AuNPs. It is also worth noting that the MAIL imaging reported in this manuscript uses low power and thus is capable of real-time, 3D imaging of living cells.

EXPERIMENTAL PROCEDURES Chemicals. Linear GRGDSP was obtained from Calbiochem, Inc. (La Jolla, CA). RGDfK (Mw 604.5) was obtained from Anaspec, Inc. (San Jose, CA). Fibrinogen, sodium azide, bovine serum albumin, paraformaldehyde, MTT, and doxorubicin were each purchased from Sigma. Hydrogen tetrachloroaurate (III) was obtained from Alfa Aesar. All chemicals for the preparation of AuNPs and surface functionalization with peptide conjugates were purchased from commercial suppliers and used without further purification unless noted otherwise. Milli-Q water (Millipore, 18 MΩ) was used throughout. Preparation of Citrate-Stabilized AuNPs. Water-soluble AuNPs were prepared according to the method reported by Natan (29) for 4.5 nm size and a modification of the method reported by Frens (30) for 16 nm size.

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4.5-nm-Diameter Gold Nanoparticles. A solution of HAuCl4 (1.00 mL, 29.4 mM in H2O, Alfa Aesar) was added into 90 mL of H2O at room temperature. After the solution had been stirred for 1 min, a solution of sodium citrate (2.00 mL, 38.8 mM in H2O) was added, followed by a fresh solution of NaBH4 (1.00 mL, 0.075% in 38.8 mM of sodium citrate). The red colloidal suspension was stirred for 10 min and stored at 4 °C. 16-nm-Diameter Gold Nanoparticles. To a refluxing solution of HAuCl4 (100 mL, 1.47 mM in H2O) was added a solution of sodium citrate (2.00 mL, 0.340 M in H2O). The resulting red colloidal suspension was stirred at reflux for 20 min, cooled to room temperature, and stored at 4 °C. General Procedure for the Synthesis of RGD Peptide Conjugates. Peptide conjugates were prepared using a modification of the reported conjugation procedure developed in the DeShong lab (31). A solution of RGD peptide (1.0 equiv) in HEPES buffer (0.1 M, pH 7.4) was mixed with a solution of the N-hydroxy succinimidyl thioctic ester (1.3 equiv) in acetone. The resulting mixture was stirred at room temperature for 24 h, and the acetone was removed in vacuo. The aqueous layer was acidified with aq HCl (0.2 M) to pH 3, washed with organic solvent (EtOAc or ether), and neutralized with aq NaOH (0.2 M). The remaining organic solvent was removed in vacuo, and the final solution was filtered through a 200 nm membrane filter. This RGD peptide conjugate solution in HEPES buffer was used for the surface functionalization of AuNPs without further purification. Linear RGD Peptide Conjugate. Linear RGD peptide (5.0 mg, GRGDSP, Calbiochem) in 4.0 mL of buffer was treated with N-hydroxy succinimidyl thioctic ester (3.4 mg) in 2.0 mL of acetone. After purification as described above in the General Procedure, the final solution was filtered with a total volume of 5.0 mL. Cyclic RGD Peptide Conjugate. Cyclic RGD peptide (2.4 mg, RGDfK, Anaspec) in 2.0 mL of buffer was treated with N-hydroxy succinimidyl thioctic ester (1.6 mg) in 2.0 mL of acetone. After purification as described above in the General Procedure, the final solution was filtered as described above with a total volume of 7.0 mL. General Procedure for the Surface Functionalization of AuNPs with RGD Peptide Conjugates. A solution of citratestabilized AuNPs was mixed with a solution of RGD peptide conjugate. Self-assembly of peptide conjugate onto the surface of AuNPs was accomplished by leaving the solution at room temperature for 24 h. The functionalized particles were then purified by centrifugal filtering (Centriplus YM-30, Amicon). The process was repeated two times with H2O. The residue in the centriplus filter was additionally purified by gel filtration (prepacked column, PD-10, GE Healthcare). The final volume of particle solution was adjusted with H2O and stored at 4 °C. 4.5-nm-Diameter AuNPs Functionalized with Linear RGD Peptide. To a solution of citrate-stabilized AuNPs (5.0 mL) was added a solution of linear RGD peptide conjugate in HEPES buffer (0.25 mL). After purification as described above in the General Procedure, the final volume of purified functionalized nanoparticle solution is 5.0 mL in H2O. 4.5-nm-Diameter AuNPs Functionalized with Cyclic RGD Peptide. To a solution of citrate-stabilized AuNPs (5.0 mL) was added a solution of cyclic RGD peptide conjugate in HEPES buffer (0.50 mL). After purification as described above in the General Procedure, the final volume of purified functionalized nanoparticle solution is 5.0 mL in H2O. 16-nm-Diameter AuNPs Functionalized with Linear RGD Peptide. A solution of linear RGD peptide conjugate in HEPES buffer (0.5 mL) was diluted with HEPES buffer (7.5 mL, 20 mM, pH 7.4), and the resulting solution was then mixed with a solution of citrate-stabilized AuNPs (4.0 mL). After purification

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as described above in the General Procedure, the final volume of purified functionalized nanoparticle solution is 16 mL in H2O. 16-nm-Diameter AuNPs Functionalized with Cyclic RGD Peptide. A solution of cyclic RGD peptide conjugate in HEPES buffer (0.5 mL) was diluted with HEPES buffer (3.5 mL, 20 mM, pH 7.4), and the resulting solution was then mixed with a solution of citrate-stabilized AuNPs (2.0 mL). After purification as described above in the General Procedure, the final volume of purified functionalized nanoparticle solution is 8.0 mL in H2O. Characterization of AuNPs. UV-visible absorption spectra were recorded using an Ocean Optics USB 2000 spectrometer. Transmission electron microscopy (TEM) measurements were performed using a ZEISS EM10 CA. Samples were prepared by drop casting 5 µL of the sample onto a Formvar coated copper grid. The distribution of particle sizes was determined using ImageJ. Cell Lines. HUVECs (human umbilical vein endothelial cells, [Lonza] Cambrex Biosciences, Walkersville, MD) were cultured in endothelial growth media at 37 °C in a humidified atmosphere of 5% CO2 (v/v). The growth media consisted of 500 mL of endothelial cell basal media (EBM) supplemented with 10 ng/ mL recombinant epidermal growth factor (hEGF), 1 µg/mL hydrocortisone, 12 µg/mL bovine brain extract, 25 U/mL heparin, 50 µg/mL amphotericin B, and 2% fetal bovine serum. The cells were detached and harvested using 0.05% trypsin/ 0.02% EDTA in PBS. Cell Viability. To determine cell cytotoxicitiy/viability, the cells were plated at a density of 1 × 104 cells/well in a 96-well plate at 37 °C in 5% CO2 atmosphere. After 24 h of culture, the medium in the wells was replaced with fresh medium containing nanoparticles in varying concentrations. After 24 h, 100 µL of MTT solution (5 mg/mL in phosphate buffer pH 7.4, MTT Sigma-Aldrich) was added to each well. After 3 h incubation at 37 °C and 5% CO2 for exponentially growing cells and 15 min for steady-state confluent cells, the medium was removed and formazan crystals were solubilized with 100 µL of DMSO, and the solution was vigorously mixed to dissolve the reacted dye. The absorbance of each well was read on a microplate reader at 560 nm. The spectrophotometer was calibrated to zero absorbance, using culture medium without cells. The relative cell viability (%) related to control wells containing cell culture medium without nanoparticles was calculated by [A]test/[A]control × 100. As a positive control, 100 µM doxorubicin (DOX) was added to an extra set of plated culture wells in conjunction with each test sample. Transmission Electron Microscopy (TEM) of Cells. The medium containing the gold nanoparticles not taken up by the cells was discarded, and the cells were thoroughly washed with PBS buffer. Cells were scraped from the culture dish and centrifuged at 5000 g for 5 min, and the supernatant was removed. The cell pellets were fixed in a 0.1 M PBS solution containing 2.5% glutaraldehyde and 4% paraformaldehyde for 1 h. The pellets were then rinsed with 0.1 M PBS, embedded in 2% agarose gel, postfixed in 4% osmium tetroxide solution for 1 h, rinsed with distilled water, stained with 0.5% uranyl acetate for 1 h, dehydrated in a graded series of ethanol (30%, 60%, 70%, 90%, and 100%), and embedded in epoxy resin. The resin was polymerized at 60 °C for 48 h. Ultrathin sections (50-70 nm) obtained with a LKB ultramictrotome were stained with 5% uranyl acetate and 2% aqueous lead citrate and imaged via transmission electron microscopy. One representative image out of 30 captured images at the same magnification (61600×) is shown in the results and discussion sections for each experimental condition studied. Amino Acid Analysis. Amino acid content analysis of the AuNP-peptide conjugates was performed by Commonwealth Biotechnologies, Inc. (Richmond, VA), by chromatographic

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measurement of derivatized peaks after hydrolysis of the sample in 6 N HCl at 110 °C for 20 h. MAIL Imaging of Cells. Cells were plated at a density of 5 × 104 cells/mL in four-chamber tissue culture slides (BD Falcon); each chamber had been treated with 1 mL of fibrinogen (200 µg/mL) for 24 h at 4 °C prior to seeding the cells. After 24 h of culture, the medium in the chambers was replaced with fresh medium containing nanoparticles of three varieties, separately: (1) bare Au citrate NPs, (2) linear RGD conjugated AuNPs, and (3) monocyclic RGD conjugated AuNPs. One chamber per slide was refilled with fresh media containing no nanoparticles to serve as a control. After incubation for a particular time interval (e.g., 2 h, 6 h, 24 h), the media were removed from the chambers and cells were fixed by treatment with paraformaldehyde (4% v/v in PBS) for 10 min. The cells were then washed twice with 25 mM glycine in PBS to remove unreacted paraformaldehyde. Again, one representative image out of a set of 30 captured images at the same magnification (40×) is displayed in the results and discussion section for each experimental condition studied. Apparatus. The multiphoton microscope has been described in detail previously (32), and so we present only brief details here. The excitation source was a Coherent Mira 900-F Ti:sapphire laser that produced pulses with a duration of approximately 150 fs at a center wavelength of 800 nm. The laser was focused into the sample through a 40× microscope objective with a numerical aperture of 0.75 (Zeiss Plan Neofluar). An excitation power of 1.5 mW was used. MAIL emission was collected by the same objective and was detected by single-photon-counting avalanche photodiodes. Light with wavelengths of 750 nm and longer was filtered out before detection. Images were collected by scanning the laser beam across the sample using a pair of galvanometric mirrors. A program written in LabView was used to control the scanning and to monitor the luminescence counts as a function of position on the sample. In the resulting MAIL images, a color is assigned to each pixel based upon the luminescence counts collected at that pixel position. Hence, the images give an immediate indication of the location and intensity of AuNP structures present within the captured frame. Integration of MAIL Image Intensities. In order to generate semiquantitative data from each MAIL image, pixel values were integrated in a program written in LabView. The program discarded values lower than 15, which were considered to be random noise present even on sample chamber slides to which no gold had been added. All remaining pixel values were summed and assigned to the corresponding image. Blocking of AuNP Uptake into HUVECs. Cells were cultured in fibrinogen-treated four-chamber slides for 24 h. After that period, three wells were treated with different blocking agents and one was left as an untreated control. The blocking agents used were sodium azide (0.1%), bovine serum albumin (3%), and free cyclic RGDfK (1 µM). Cells were treated with blocking agent for 10 min and then washed twice with PBS. AuNPs with surface-conjugated RGDfK were added to all wells in 1 mL of growth media. After an additional 24 h, cells were fixed with paraformaldehyde and subsequently imaged by MAIL.

RESULTS AND DISCUSSION We studied AuNPs of two different diameters, 4.5 and 16 nm, to determine whether the size of the particle, and correspondingly the number of peptide conjugates attached, would have an effect on the uptake results. Figure 1 shows TEM images of these particles ex vivo and corresponding UV absorption spectra. Figures 1b and 1e show the cyclic RGD conjugates for 4.5 and 16 nm particles, respectively, demonstrat-



Figure 1. TEM images and UV-vis absorption spectra of AuNPs: (a) citrate-stabilized AuNPs (average diameter, 4.5 ( 1.4 nm); (b) cyclic RGD peptide-functionalized AuNPs; (c) 4.5 nm citrate-stabilized AuNPs (in black, λmax ) 520 nm), linear RGD peptide-functionalized AuNPs (in blue, λmax ) 523 nm), and cyclic RGD peptide-functionalized AuNPs (in red, λmax ) 523 nm); (d) citrate-stabilized AuNPs (average diameter, 15.8 ( 1.6 nm); (e) cyclic RGD peptide-functionalized AuNPs; (f) 15.8 nm citrate-stabilized AuNPs (in black, λmax ) 520 nm), linear RGD peptidefunctionalized AuNPs (in blue, λmax ) 526 nm), and cyclic RGD peptide-functionalized AuNPs (in red, λmax ) 526 nm).

ing a significant amount of aggregation relative to their bare citrate counterparts in Figures 1a and 1d. The increased aggregation of the peptide-functionalized AuNPs can be attributed to hydrogen bonding occurring between surface conjugates on adjacent particles, as water was removed from the system by vacuum prior to TEM analysis. Aggregation of functionalized AuNPs on TEM grids has been observed for many derivatives. Analysis of the solutions prior to deposition by dynamic light scattering indicated that aggregation in solution is minimal. The wavelength of maximum absorption for both particle sizes remains constant at 525 nm for bare citrate AuNPs, linear RGD conjugated AuNPs, and cyclic RGD conjugated AuNPs, although the absorbance values increase slightly over the measured wavelength range for both the linear and cyclic RGDconjugated AuNPs. Absorption peaks for aggregated samples are broadened and shifted significantly to longer wavelengths. As our observed UV absorbance peaks are sharp and are consistent with values reported previously for stable AuNP suspensions, aggregation in solution is likely to be insignificant. Furthermore, it has been previously demonstrated that AuNPs do not aggregrate in serum-completed cell growth media (33). Figure 2 summarizes the results of the MTT cytotoxicity assay for 4.5 nm AuNPs incubated with HUVECs. Even at a concentration of 39 µg/mL of AuNPs, which is significantly higher than what would be possible in a biologically relevant setting, the AuNPs were nontoxic to the HUVECs. However, DOX mixed with any AuNP sample diminishes cell survival to below 2%. There are two key points to take from the results of this experiment. First, 4.5 nm AuNPs, which are comparable in size to many cellular proteins, do not harm the target cells. Second, the addition of targeting peptides to the surfaces of the AuNPs does not lead to measurable toxicity of the nanovector under the conditions used in this study. This preliminary toxicological screening result is promising for the potential use of surface-functionalized particles as in vivo targeting agents. We also note that similar results were found for the 16 nm

Figure 2. MTT assay of peptide-targeted AuNPs incubated with HUVECs.4.5 nm particles of three different types, (1) AuNP-RGDfK conjugates (maroon bars), (2) AuNP-GRGDSP conjugates (orange bars), and (3) bare citrate-stabilized AuNPs (gold bars), were each incubated with HUVECs for a 72 h MTT assay. Doxorubicin (DOX; 100 µM) was incubated with the HUVECs as a positive control (green bars).

AuNPs, both bare and with the targeting peptides conjugated to the particle surfaces (data not shown for brevity). Table 1 summarizes the results of amino acid analysis on the AuNP-peptide conjugates for the 4.5 nm AuNPs. The grafting density of linear RGD peptides on the AuNPs was approximately 5 times as large as that of cyclic RGD peptides. This finding is reasonable, since the cyclic peptide conjugate takes up a significantly larger effective area than the linear peptide counterparts. Since the peptide conjugates were reacted with the AuNPs in large excess, it is assumed that saturation of the particle surfaces was achieved, and so the greater surface


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Table 1. Amino Acid Analysis of Peptide-Conjugated AuNPs (4.5 nm)a conjugate type

(avg no. of conjugated peptides)/particle

(avg no. of conjugated peptides)/nm2

cyclic RGD peptide linear RGD peptide

124.6 ( 29.3 610.7 ( 92.9

2.0 ( 0.5 9.6 ( 1.5

a Particle/peptide conjugates were treated in highly acidic conditions to hydrolyze the bound amino acids, whose mass was subsequently measured. It is shown that the average number of peptide conjugates per particle is nearly five times as high for the linear RGDs as compared to the cyclic RGDfKs (indicated error is s.d.).

density of linear peptides resulted. The average number of conjugated peptides per unit area for 4.5 nm AuNPs is shown

in Table 1, and the surface densities for 16 nm AuNPs are expected to be similar, but were not determined in this study. MAIL Detection of Particle Uptake into Cells. MAIL imaging was performed on HUVECs incubated for 6 and 24 h with 4.5 nm AuNPs and with 16 nm AuNPs. Note that all MAIL images in this study show one representative cell from a single image out of the correspondingly treated cell population, although many images were collected. Shown in Figure 3 are MAIL images and corresponding transmission optical images at 6 h incubation with 4.5 nm AuNPs. Figure 4 shows HUVECs incubated with the same AuNPs for 24 h. The cells incubated with the AuNPs with cyclic RGD conjugates (Figures 3a and 4a) exhibit a greater luminescence signal than those cells

Figure 3. MAIL/transmission optical (40×) images for 6 h uptake of targeted 4.5 nm AuNPs into HUVECs. (a) MAIL image of 4.5 nm AuNPs with cyclic RGD conjugates; (b) MAIL image of 4.5 nm AuNPs with linear RGD conjugates; (c) MAIL image of bare citrate 4.5 nm AuNPs; (b) MAIL image of controls, i.e., no AuNPs incubated with HUVECs; (e) transmission image of 4.5 nm AuNPs with cyclic RGD conjugates; (f) transmission image of 4.5 nm AuNPs with linear RGD conjugates; (g) transmission image of bare citrate 4.5 nm AuNPs; (h) transmission image of control sample. The scale bar is 10 µm.

Figure 4. MAIL/transmission optical (40×) images for 24 h uptake of targeted 4.5 nm AuNPs into HUVECs. (a) MAIL image of 4.5 nm AuNPs with cyclic RGD conjugates; (b) MAIL image of 4.5 nm AuNPs with linear RGD conjugates; (c) MAIL image of bare citrate 4.5 nm AuNPs; (b) MAIL image of controls, i.e. no AuNPs incubated with HUVECs; (e) transmission image of 4.5 nm AuNPs with cyclic RGD conjugates; (f) transmission image of 4.5 nm AuNPs with linear RGD conjugates; (g) transmission image of bare citrate 4.5 nm AuNPs; (h) transmission image of control sample. The scale bar is 10 µm.



Figure 5. MAIL/transmission optical (40×) images for 6 h uptake of targeted 16 nm AuNPs into HUVECs. (a) MAIL image of 16 nm AuNPs with cyclic RGD conjugates; (b) MAIL image of 16 nm AuNPs with linear RGD conjugates; (c) MAIL image of bare citrate 16 nm AuNPs; (b) MAIL image of controls, i.e., no AuNPs incubated with HUVECs; (e) transmission image of 16 nm AuNPs with cyclic RGD conjugates; (f) transmission image of 16 nm AuNPs with linear RGD conjugates; (g) transmission image of bare citrate 16 nm AuNPs; (h) transmission image of control sample. The scale bar is 10 µm.

Figure 6. MAIL/transmission optical (40×) images for 24 h uptake of targeted 16 nm AuNPs into HUVECs. (a) MAIL image of 16 nm AuNPs with cyclic RGD conjugates; (b) MAIL image of 16 nm AuNPs with linear RGD conjugates; (c) MAIL image of bare citrate 16 nm AuNPs; (b) MAIL image of controls, i.e., no AuNPs incubated with HUVECs; (e) transmission image of 16 nm AuNPs with cyclic RGD conjugates; (f) transmission image of 16 nm AuNPs with linear RGD conjugates; (g) transmission image of bare citrate 16 nm AuNPs; (h) transmission image of control sample. The scale bar is 10 µm.

incubated with either AuNPs functionalized with linear RGD conjugates (Figures 3b and 4b) or bare citrate AuNPs (Figures 3c and 4c). Figures 5 and 6 show images of HUVECs incubated with 16 nm AuNPs after 6 h and 24 h of incubation, respectively. Again, a greater luminescence signal is observed in the samples containing the AuNPs with cyclic RGD conjugates (Figures 5a and 6a), and uptake increases significantly over the 24 h period. The MAIL signals for 16 nm AuNPs are stronger than those for 4.5 nm AuNPs. This effect could be intrinsic to the AuNPs or could be due to a more effective exocytotic mechanism for smaller particles by the HUVECs. Note that the concentration of particles incubated with the HUVECs was 39 µg/mL, which was the upper limit value for the 72 h MTT assay. Thus, a large

number of the targeted AuNPs are getting into the cells, but they do not appear to harm the cells in any significant way. Figure 7 displays a histogram of the integrated MAIL counts from treated cell populations corresponding to representative images displayed in Figures 3-6. While the intensity values in this figure do not give an absolute quantitative measure of the number AuNPs within the cell, the data clearly indicate that differential uptake is observed among the experimental conditions. The 16 nm AuNP-RGDfK conjugates at 24 h displayed the highest intensity value (1.31 × 106) among the tested samples. The data also indicate that large increase in intensity was observed over an 18 h period (between the 6 and 24 h test points) for the 4.5 nm AuNP-RGDfK conjugates and the 16 nm AuNP-RGDfK conjugates at 12.36-fold and 13.17-fold,


Figure 7. Integrated counts from MAIL images of targeted AuNPs (Corresponding representative images shown in Figures 3-6).

respectively. Again, as anticipated from the images at 24 h, the AuNP-RGDfK conjugate samples had significantly higher average luminescence counts (1.31 × 106 [16 nm] and 6.75 × 105 [4.5 nm]) than the AuNP-GRGDSP (4.18 × 105 [16 nm] and 1.08 × 105 [4.5 nm]) or citrate-stabilized AuNP (1.01 × 105 [16 nm] and 5.23 × 104 [4.5 nm]) controls of the same particle size. For the 6 h time interval, preferential uptake of the 4.5 nm citrate-stabilized AuNPs compared to the 4.5-nm AuNP-GRGDSP conjugate was unexpected. There are three possible explanations for this observation. First there should be a large difference in zeta potential (surface charge) between the functionalized and unfunctionalized gold nanoparticles that may influence uptake. Alternatively, preferential uptake of the citrate nanoparticles may arise from the saturation of the linear RGD peptide on surface of the gold nanoparticles, which would interfere with single peptide-receptor binding events via steric hindrance. Finally, the 4.5 nm particles may be more susceptible to exocytosis mechanisms during the initial uptake period (33). At this point, it is not possible to differentiate among these three

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explanations without significant experimentation. These additional studies are underway and will be reported in due course. Determination of the Cellular Uptake Mechanism. Figures 8 and 9 show HUVEC cells incubated with AuNP-RGDfK conjugates for 24 h in the presence of different uptake-blocking agents for 4.5 and 16 nm AuNPs, respectively. The leftmost images are controls in which the HUVECs have not been treated with a blocking agent. We observe significantly greater luminescence in these images (Figures 8a and 9a), indicating that the blocking agents alter the uptake of the AuNP-RGDfK conjugates. In Figures 8b and 9b, the cells have been treated with 0.1 wt % sodium azide, an endocytosis inhibitor, for 10 min prior to addition of AuNP-RGDfKs. The luminescence signal is diminished significantly for both 4.5 and 16 nm AuNPs, although some small patches of signal are still observed for these samples. The cells in Figures 8c and 9c have been treated for 10 min with 3% BSA, which blocks nonspecific binding to the cells. For both samples, a smaller luminescence signal was observed, although this signal was more comparable to that of the untreated controls. This inhibition study indicates that a large fraction of the functionalized AuNPs is taken up into the cells via specific binding to a cell-surface receptor followed by receptor-mediated endocytosis. Figures 8d and 9d demonstrate that treatment of the cells with 1 µM RGDfK to block Rvβ3 integrins significantly decreases the intensity of the luminescent signals relative to the untreated controls. This result implies that the majority of uptake must involve endocytosis via the Rvβ3 integrin. Figure 10 displays a histogram of the integrated MAIL counts from the treated cell populations corresponding to the representative images shown in Figures 8 and 9. As expected, the untreated HUVEC samples had the highest integrated counts at 5.28 × 105 and 1.09 × 106 for 4.5 and 16 nm AuNPs, respectively. AuNP-RGDfK conjugates taken up into HUVECs pretreated with 3% BSA displayed the next highest intensity values, with a 64% drop-off in intensity for 4.5 nm AuNPRGDfKs (1.91 × 105 counts) and a 68% drop-off in intensity for 16 nm AuNP-RGDfKs (3.54 × 105 counts) relative to the untreated HUVEC controls. For both 4.5 nm and 16 nm

Figure 8. MAIL/transmission optical (40×) images for 24 h uptake of targeted 4.5 nm AuNPs into HUVECs. (a) MAIL image 4.5 nm AuNPs with cyclic RGD conjugates (no treatment); (b) MAIL image of 4.5 nm AuNPs with cyclic RGD conjugates, HUVECs treated with sodium azide; (c) MAIL image of 4.5 nm AuNPs with cyclic RGD conjugates, HUVECs blocked with BSA; (d) MAIL image of 4.5 nm AuNPs with cyclic RGD conjugates, HUVECs treated with free RGDfK; (e) transmission image of 4.5 nm AuNPs with cyclic RGD conjugates (no treatment); (f) transmission image of 4.5 nm AuNPs with cyclic RGD conjugates, HUVECs treated with sodium azide; (g) transmission image of 4.5 nm AuNPs with cyclic RGD conjugates, HUVECs blocked with BSA; (h) transmission image of 4.5 nm AuNPs with cyclic RGD conjugates, HUVECs treated with free RGDfK. The scale bar is 10 µm.



Figure 9. MAIL/transmission optical (40×) images for 24 h uptake of targeted 16 nm AuNPs into HUVECs. (a) MAIL image 16 nm AuNPs with cyclic RGD conjugates (no treatment); (b) MAIL image of 16 nm AuNPs with cyclic RGD conjugates, HUVECs treated with sodium azide; (c) MAIL image of 16 nm AuNPs with cyclic RGD conjugates, HUVECs blocked with BSA; (d) MAIL image of 16 nm AuNPs with cyclic RGD conjugates, HUVECs treated with free RGDfK; (e) transmission image of 16 nm AuNPs with cyclic RGD conjugates (no treatment); (f) transmission image of 16 nm AuNPs with cyclic RGD conjugates, HUVECs treated with sodium azide; (g) transmission image of 16 nm AuNPs with cyclic RGD conjugates, HUVECs blocked with BSA; (h) transmission image of 16 nm AuNPs with cyclic RGD conjugates, HUVECs treated with free RGDfK. The scale bar is 10 µm.

Figure 10. Integrated counts from MAIL images of AuNP-RGDfK conjugates pretreated with blocking agents (corresponding representative images in Figures 8-9, a-d).

AuNP-RGDfKs taken up in HUVECs pretreated with BSA, intensity values 2.5 to 4 times higher were measured relative to the sodium azide and free RGDfK pretreated HUVECs incubated with the same AuNPs. The individual values suggest that this preferential uptake is due to a specific binding event between the AuNP-RGDfKs and cell surface receptors, while smaller amounts of signal may be attributed to nonspecific uptake (free RGDfK blocking) and nonspecific binding to the cell surface (free RGDfK blocking and sodium azide treatment). Detection of Particle Uptake into Cells via TEM imaging. Figure 11 shows TEM images of HUVECs incubated with 4.5 and 16 nm AuNPs with surface conjugated RGDfK for a period of 72 h. These images demonstrate that the nanoparticles are taken primarily into endosomal and lysosomal vesicles. Aggregation of the AuNPs is observed within the vesicle. This intracellular destabilization may arise from ligand exchange reactions within the cell, as suggested by Rotello et al., (35) or may be the result of digestion of the ligand shell by proteases within the endosomes and lysosomes, a mechanism proposed by Nativo et al. in an AuNP uptake study on human fibroblasts

Figure 11. TEM images of AuNPs taken up by HUVECs. (a) 16 nm AuNPs with surface conjugated cyclic RGD peptide incubated with HUVECs for 72 h; AuNPs are seen to be uptaken into an endosomal compartments. (b) 4.5 nm AuNPs with surface conjugated cyclic RGD peptide incubated with HUVECs for 72 h; AuNPs are also observed clustered within endosomal compartments.


(36). These TEM studies serve as a benchmark to demonstrate that MAIL imaging can provide similar information to electron microscopy, but with the potential of performing imaging in real time and on live cells.

CONCLUSIONS We have shown via MAIL imaging that AuNPs of two different diameters, 4.5 and 16 nm, can be targeted specifically to activated endothelial cells by covalent conjugation of RGD peptides to the surface of the nanoparticle. Detected signal from these particles within the cells depended on two factors: (1) the size of the particle, 4.5 nm vs 16 nm, and (2) the structure of the RGD analog. AuNP-RGDfK conjugates exhibit highly differential nanoparticle uptake when compared to AuNPs conjugated with linear RGD or bare AuNPs. The integrated counts for MAIL images of the targeted HUVECs were an order of magnitude higher for the AuNP-RGDfK conjugates than for the AuNP-GRGDSP controls. Also, cells incubated with 16 nm AuNPs exhibit significantly higher MAIL signals than those incubated with the smaller 4.5 nm AuNPs. Additional experiments in which uptake into HUVECs was blocked with BSA, sodium azide, or free RGDfK, indicated that AuNPRGDfK conjugates are internalized following a specific binding event and that Rvβ3 integrin-mediated endocytosis is an important uptake mechanism. Results from MAIL studies were verified by comparison to TEM images, which revealed that the endocytosed AuNPs remain primarily within endosomal and lysosomal vesicles. The studies reported here demonstrate that MAIL is a powerful tool for monitoring targeting and determining uptake mechanisms of surface-functionalized nanostructures. Given the capability of MAIL for rapid, three-dimensional imaging with high resolution, this technique shows great promise for applications in nanomedicine.

ACKNOWLEDGMENT P.D. and H.G. thank the NSF-NIRT (CHE 0511219478) for generous financial support. In addition, M.B.D. would like to thank the Fischell Fellowship in Biomedical Engineering for graduate support. The authors would also like to acknowledge Dr. Ru-Ching Hsia at the University of Maryland Baltimore School of Dentistry EM facility for assistance in TEM sample preparation and imaging.

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