Uptake and Metabolism of a Dual Fluorochrome Tat-nanoparticle in

Bioconjugate Chem. , 2003, 14 (6), pp 1115–1121 ..... Feasibility of Multimodal Detection Using 3T MRI, Small Animal PET, and Fluorescence Imaging...
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Bioconjugate Chem. 2003, 14, 1115−1121

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Uptake and Metabolism of a Dual Fluorochrome Tat-nanoparticle in HeLa Cells A. M. Koch,† F. Reynolds,‡ M. F. Kircher,‡ H. P. Merkle,† R. Weissleder,‡ and L. Josephson*,‡ Department of Chemistry and Applied BioSciences, Drug Formulation & Delivery, Swiss Federal Institute of Technology Zurich (ETHZ), Winterthurerstrasse 190 8057 Zurich, Switzerland, and Center for Molecular Imaging Research, Massachusetts General Hospital/Harvard Medical School, Building 149, 13th Street, Charlestown, Massachusetts 02129. Received July 16, 2003

The ability to use magnetic nanoparticles for cell tracking, or for the delivery of nanoparticle-based therapeutic agents, requires a detailed understanding of probe metabolism and transport. Here we report on the development and metabolism of a dual fluorochrome version of our tat-CLIO nanoparticle termed Tat(FITC)-Cy3.5-CLIO. The nanoparticle features an FITC label on the tat peptide and a Cy3.5 dye directly attached to the cross-linked coating of dextran. This nanoparticle was rapidly internalized by HeLa cells, labeling 100% of cells in 45 min, with the amount of label per cell increasing linearly with time up to 3 h. Cells loaded with nanoparticles for 1 h retained 40-60% of their FITC and Cy3.5 labels over a period of 72 h in label-free media. Over a period of 144 h, or approximately 3.5 cell divisions, the T2 spin-spin relaxation time of cells was not significantly changed, indicating retention of the iron oxide among the dividing cell population. Using confocal microscopy and unfixed cells, both dyes were nuclear and perinuclear (broadly cytoplasmic) after Tat(FITC)-Cy3.5-CLIO labeling. Implications of the rapid labeling and slow excretion of the Tat(FITC)-Cy3.5-CLIO nanoparticle are discussed for cell tracking and drug delivery applications.

INTRODUCTION

The ability of membrane-translocating peptides to move materials through the plasma membrane or even cell layers and tissues has the potential to lead to the development of new types of pharmaceuticals, which can overcome cellular barriers for the delivery of current compounds. The membrane-translocating sequence of the tat protein of HIV (tat-peptide) has been used to ferry diagnostic and therapeutic agents across membranes and has led to new forms of cyclosporine (1), improved radiotherapies (2), and new methods for intracellular delivery of DNA and enzymes (3-5). However, the development of tat peptide-based pharmaceuticals would be furthered, not merely by studies on the efficacy of such compounds, but by a thorough understanding of the transport and degradation of tat-like probes. The relationship between probe utility on one hand and probe metabolism and transport on the other can be illustrated with tat-CLIO (tat-cross-linked iron oxide), a magnetic nanoparticle to which tat peptides have been attached (6, 7). Tat-CLIO-loaded cells have been used to study cell movement in vivo in a variety of medically important applications including stem cell engraftment (7) and T cell infiltration in adoptive immunotherapy (8). In magnetic resonance (MR) cell tracking applications, excretion of the nanoparticle after internalization, if it occurs, limits the observation time of labeled cells. On the other hand, for the tat-mediated delivery of therapeutic agents, particularly for delivery of agents across epithelial or endothelial barriers, internalization without externalization would not permit tat-drug conjugates to * Corresponding author. Phone: 617-726-6478, Fax: 617-7265788, e-mail: [email protected]. † Swiss Federal Institute of Technology Zurich. ‡ Massachusetts General Hospital/Harvard Medical School.

pass through cell layers and achieve tissue penetration. In the liver tat-CLIO crosses endothelial barriers in the vasculature and is found in the parenchyma (9). The tatmediated delivery of a diverse type of molecules including fluorochromes, peptides, and enzymes through tissues in vivo has been reported (10, 11). With the interrelated, long-term objectives of understanding the fate of Tat-CLIO-loaded cells followed by magnetic resonance (MR) tracking in vivo, and determining the ability of Tat-CLIO to undergo transepithelial or transendothelial transport, we examined the uptake and excretion of dual fluorochrome, multimodal (fluorescent and magnetic) tat-nanoparticles. Here we describe a Tat-CLIO that is detectable by its effects on MR with one fluorochrome, fluorescein isothiocyanate (FITC), attached to each tat peptide, Tat(FITC), and a second fluorochrome, Cy3.5, is attached to the coating of nanoparticle (cross-linked iron oxide, CLIO). We term this form of tat-CLIO Tat(FITC)-Cy3.5-CLIO, to denote its dual fluorochrome labels. On the basis of fluorescent activated cell sorter (FACS) analysis, Tat(FITC)-Cy3.5CLIO was rapidly internalized and slowly excreted by HeLa cells. On the basis of effects on the cellular T2 (spin-spin relaxation time), the superparamagnetic iron oxide nanoparticle cores were not eliminated during the 7-day observation period. Implications of this pharmacokinetic pattern are described for cell tracking and pharmaceutical targeting applications. MATERIALS AND METHODS

Nanoparticle Synthesis. To synthesize dual fluorochrome-labeled magnetic nanoparticles, the dye Cy3.5 was first attached to the amines of a cross-linked dextrancoated nanoparticle termed amino-CLIO, to yield Cy3.5CLIO. The tat peptide, denoted Tat(FITC), was attached to the nanoparticle using the bifunctional reagent, suc-

10.1021/bc034123v CCC: $25.00 © 2003 American Chemical Society Published on Web 10/28/2003

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Figure 1. Synthesis and features of Tat(FITC)-3.5-CLIO. (A) Synthetic scheme. (B) Schematic diagram showing features of Tat(FITC)-3.5-CLIO. Tat(FITC) is the peptide GRKKRRQRRRGYK(FITC)C-NH2. Succinimidyl iodoacetic acid (SIA) gives a thioether (S-C) linkage between the peptide and the nanoparticle. Table 1. Physical Properties of Tat(FITC)-Cy3.5-CLIO nanoparticle

Cy3.5/nanoparticle

Tat(FITC)/nanoparticle

size (nm)

Tat(FITC)-Cy3.5-CLIO

1.4

24

49.1

cinimidyl iodoacetate (SIA), as shown in Figure 1A. A schematic representation of the structure of Tat(FITC)Cy3.5-CLIO is shown in Figure 1B. A tat peptide, termed Tat(FITC), with the sequence GRKKRRQRRRGYK(FITC)C-NH2 was synthesized and purified as described (9). The amino-CLIO nanoparticle was prepared as described (6, 12). Cy 3.5-CLIO was prepared by adding 175 µL of aminoCLIO (2 mg Fe) to one tube of monofunctional Cy3.5 dye as supplied by the manufacturer (Amersham Biosciences, Little Chalfont, UK). The mixture was vortexed and allowed to sit for a minimum of 2 h. Reaction with Cy3.5 consumes 1.4 amino groups per nanoparticle, out of a total of 40-60 amino groups per nanoparticle. Cy 3.5CLIO was added to N-succinimidyl iodoacetate (SIA, 4 mg, 14 µmol, Molecular Bioscience, Boulder, CO) in 100 µL of DMSO. The mixture was vortexed, allowed to sit for 1 h, and purified with a PD-10 desalting column (Sigma) in 0.02 M citrate, 0.15 M NaCl, pH 8. A single separation step was used to remove unreacted Cy3.5 and SIA. Tat(FITC)-Cy3.5-CLIO was prepared by adding Tat(FITC) (60 µL, 5 mM) in 0.1% v/v trifluroacetic acid to SIA activated Cy3.5-CLIO (2 mg Fe). The mixture was vortexed and allowed to react for 1-2 h, and unreacted

peptide was removed by ultrafiltration (Minicon YM-30, Millipore, Bedford, MA). The number of attached Cy3.5 and FITC dyes was determined spectrophotometrically, for fluorescein (494 ) 73000) and for Cy3.5 (581 ) 150000). Iron was determined spectrophotometrically(6), and the ratios of Cy3.5 or Tat(FITC) per nanoparticle were calculated assuming 2064 iron atoms per CLIO particle (13). Properties of the nanoparticle are summarized in Table 1. We previously demonstrated that the ability of cells to internalize tat peptide magnetic nanoparticles increases with increasing numbers of peptides per nanoparticle(14). The Tat(FITC)-Cy3.5-CLIO nanoparticle we employed is similar to that of Zhao et al. but differs in the use of two chemically distinct fluorochromes and in the use of a thioether rather than a disulfide linkage. The thioether-linked Tat(FITC)-Cy3.5-CLIO had improved stability between the peptide and nanoparticle when incubated in cell culture media compared to the disulfide-linked nanoparticle (Koch and Josephson, unpublished observations). Cell Culture. HeLa cells were cultured in MEM (minimum essential medium) containing 10% FBS, supplemented with penicillin (100 units/mL)/streptomycin (100 µg/mL), sodium pyruvate (1 mM), and nonessential amino acids (0.1 mM) at 37 °C in a humidified atmosphere

Dual Fluorochrome Tat-nanoparticle in HeLa Cells

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Figure 2. Uptake of Tat(FITC)-Cy3.5-CLIO nanoparticle or Tat(FITC) peptide. (A) Progressive increase in cell associated fluorescence with Tat(FITC)-Cy3.5-CLIO as shown in the FACS profile (frequency distribution of labeled cells). Dotted line is the demarcation between labeled and unlabeled cells. (B) Data from A are plotted as the median fluorescence of labeled cells divided by median unlabeled cells (relative median fluoresecence, RMF). (C and D) Increase in cell fluorescence with Tat(FITC) peptide obtained as in A and B, respectively.

containing 5% CO2. Media, balanced salt solutions, and supplements were from Cellgro (Mediatech Inc, Herndon, VA). HeLa cells were cultured as exponentially growing subconfluent monolayers, except when used in experiments lasting 72 h and more, where cells were allowed to reach confluency. FACS Analysis. Uptake Studies. Exponentially growing HeLa cells were trypsinized and seeded at a density of 105 cells/well in 12 well plates (Becton Dickinson, Bedford, MA). About 18 h later, cells were labeled for 1 h with Tat(FITC)-Cy3.5-CLIO (50 µg Fe/mL), or Tat(FITC) peptide (5 µM) added in complete cell culture medium. Medium was sterile-filtered (0.2 µm, low protein binding, Gelman, Ann Arbor, MI) before addition to cells. After 45, 90, or 180 min, cells were washed 4× with HBSS, trypsinized for 7 min, counted, fixed in 2% paraformaldehyde (Fisher Scientific, reagent grade), and analyzed by flow cytometry on a FacsCalibur (Becton Dickinson, Franklin Lakes, NJ) within 2 h after trypsinization. A total of 10 000 cells per sample was analyzed. The numbers of cells with a fluorescence intensity higher than unlabeled cells was used to calculate percent of labeled cells. The baseline was determined by analyzing unlabeled control cells. RMF (relative median fluorescence) was calculated by dividing the median of the labeled cells by the median of an unlabeled cell population. Retention in Cells. HeLa cells were labeled with Tat(FITC)-Cy3.5-CLIO (50 µg Fe/mL which corresponds to 7.5 µM Tat(FITC) peptide), or Tat(FITC) peptide (5 µM) as described for the uptake studies. After 1 h of labeling, cells were washed 4× with HBSS, and then not immediately trypsinized and analyzed, but incubated in fresh cell culture medium for 3, 24, 48, or 72 h. At respective time points, cells were trypsinized and analyzed as described for uptake studies. Confocal Fluorescence Microscopy. HeLa cells were cultured on Lab-Tek Chambered Coverglass (Nalge Nunc, Naperville, IL) with a density of 3 × 104 cells/cm2. Cells were labeled as described for FACS analysis with

either Tat(FITC)-Cy3.5-CLIO (50 µg/mL Fe) or free Tat(FITC) peptide (5 µM). Cells were observed immediately after labeling, after 24 h and after 72 h. Brightfield and fluorescence images were obtained with a Zeiss PlanNeofluar 40× objective using a Zeiss Axiovert 200 confocal microscope (Zeiss, Jena, Germany) equipped with Argon (488 nm) and HeNe (543 nm) lasers for fluorescence. Exposure time for fluorescence was adjusted at each time point to localize fluorochromes. Background fluorescence was determined by analyzing unlabeled cells. Image acquisition and analysis was done using LSM 5 PASCAL Software. MR Imaging. HeLa cells were seeded in cell culture flasks (175 cm2) at a density of 5 × 104 cells/cm2 and allowed to adhere overnight. Cells were washed with fresh cell culture medium and then incubated for 4 h with sterile filtered Tat(FITC)-Cy3.5-CLIO (200 µg Fe/mL). Cells were trypsinized (5 min) and divided into new cell culture flasks (175 cm2), with each flask containing 2 × 106 cells. Two flasks were harvested immediately, cells of another eight flasks were allowed to adhere and kept in culture for different time points up to 96 h. At each time point, adherent cells were washed 4× with HBSS, trypsinized, transferred to a conical cell culture tube, centrifuged down, and washed three times with HBSS. Finally, cells were resuspended in warm 0.5% agarose to yield a volume of 100 µL and put on ice. The number of cells ranged from 2.5 × 106 cells at start of the experiment to 31 × 106 cells after 7 days, with a doubling time of 24 h at the beginning and then decreasing to 48 h at the end of the experiment. Immobilized cells were then imaged with a 7T MR Scanner (Siemens, Massachusetts General Hospital, Boston, MA). T2 values were from signal intensity data obtained at six echo times (20, 40, 60, 80, 120 ms), with a repetition time of 2 s. Data were fit to the standard exponential transverse relaxation model used previously (15).

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Figure 3. Retention of the Tat(FITC)-Cy3.5-CLIO nanoparticle and Tat(FITC) peptide in HeLa cells. HeLa cells were labeled for 1 h with Tat(FITC)-Cy3.5-CLIO (A and B) or Tat(FITC) peptide (C) and washed. Cells were incubated in fresh cell culture medium for 3, 24, 48, or 72 h, trypsinized, and analyzed by flow cytometry. A relative median fluorescence (RMF) is calculated (median labeled/median unlabeled) and, together with additional time points, shown in Figure 4. RESULTS

Uptake of Tat(FITC)-Cy3.5-CLIO. The uptake of Tat(FITC)-Cy3.5-CLIO was monitored by FACS analysis based on FITC as shown in Figure 2. Figure 2A demonstrates that exposing cells to Tat(FITC)-Cy3.5CLIO results in a uniform labeling of cells and that the amount of cell-associated nanoparticle increases with the time of incubation. Cell labeling was expressed using two parameters, the percent labeled cells and the relative median fluorescence or RMF, obtained by dividing the median fluorescence of labeled cells by the median fluorescence of unlabeled cells. The dotted line indicates a clear division between labeled and unlabeled cells that occurred at the short incubation time of 45 min. RMF and percent of labeled cells are plotted in Figure 2B, which demonstrates that nanoparticle accumulation (RMF) increased at a constant rate over the 180 min incubation period. Also shown are comparable data for the uptake of the tat(FITC), the peptide of the Tat(FITC)-Cy3.5-CLIO nanoparticle, see Figures 2C and 2D. Again labeling was rapid and highly uniform, and cell-associated fluorescence increased over the 180 min incubation period. The Retention of Tat(FITC)-Cy3.5-CLIO in Cells by Fluorescence. We next examined the retention of both FITC fluorescence and Cy3.5 fluorescence of Tat(FITC)-Cy3.5-CLIO in cells, when cells were loaded for 1 h with Tat(FITC)-Cy3.5-CLIO nanoparticle or Tat(FITC) peptide and were then incubated in media for up to 72 h. Representative FACS histograms after cells have been in media for 3 and 72 h are shown in Figures 3AC. Data from the 3 and 72 h time points, together with additional time points, are presented in Figure 4. As shown in Figure 4A, after 72 h cells loaded with Tat(FITC)-Cy3.5-CLIO were still labeled, that is had higher fluorescence than a population of unlabeled cells, by either FITC or Cy3.5 fluorescence. On the other hand, after 72 h, more than 80% of cells labeled with Tat(FITC) peptide at the beginning of the experiment were unlabeled. Figures 4B and 4C show the fluorescence of Tat-

Figure 4. Retention of fluorescence of cells loaded with the Tat(FITC)-Cy3.5-CLIO nanoparticle or the Tat(FITC) peptide. Cells were loaded for 1 h and analyzed by flow cytometry. For all graphs, (1) is the FITC fluorescence from the Tat(FITC) peptide, (2) is the FITC fluorescence from Tat(FITC)-Cy3.5 CLIO, and (9) is the Cy3.5 fluorescence from Tat(FITC)-Cy3.5 CLIO. (A) Percent of labeled cells versus time. (B) Fluorescence per cell as RMF versus time. (C) Total cellular fluorescence (TCF) versus time, where TCF equals RMF times cell number. Experiment is representative for two similar experiments: each data point (A, B) is the median fluorescence intensity of 10 000 cells per sample. Errors bars in (C) represent variations in cell number.

(FITC)-Cy3.5-CLIO or Tat(FITC) peptide loaded cells uncorrected for cell division (B) or corrected for cell division (C). As shown in Figure 4B, the RMF of peptideloaded cells decreased rapidly, while the FITC and Cy3.5 fluorescence from Tat(FITC)-Cy3.5-CLIO-loaded cells decreased similarly and more slowly than peptide-loaded cells. For Figure 4C, total cell fluorescence (TCF) was obtained by multiplying cell number times the RMF. For Tat(FITC)-Cy3.5-CLIO-loaded cells, the loss of TCF was less than the loss of fluorescence per cell (RMF), indicating that a major source of fluorescence loss was the distribution of the labels to daughter cells. A second way of ascertaining the retention of the Tat(FITC)-Cy3.5-CLIO is by determining the T2 spin-spin relaxation times of cells. Cells were loaded with nanoparticle and allowed to proliferate in nanoparticle-free media for up to 144 h, and T2 was determined periodically. With this method, nanoparticles can be distributed between dividing cells, but the entire population of cells is retained and analyzed. As shown in Figure 5, the T2 of cells dcreases from 105 ( 10 ms to 50 ( 5 ms, indicating the internalization of superparamagnetic iron oxide, and did not increase during the 144 h incubation

Dual Fluorochrome Tat-nanoparticle in HeLa Cells

Figure 5. T2 relaxation times of cells labeled with the Tat(FITC)-Cy3.5-CLIO. Cells were labeled (4 h), washed, and incubated in fresh cell culture medium for up to 7 days.

period, indicating retention of the superparamagnetic iron oxide core of the Tat(FITC)-Cy3.5-CLIO nanoparticle. We next examined the intracellular distribution of Tat(FITC)-Cy3.5-CLIO and Tat(FITC) by confocal microscopy as a function of time after loading (Figure 6). Cells were unfixed, to prevent shifts in distribution due to fixation (16), and exposure time was adjusted to correct for the loss of fluorescence (Figure 4B). Nanoparticleloaded cells showed fluorescence in the nucleus and cytoplasm with either FITC or Cy3.5 immediately after labeling or 24 h after labeling. By 72 h after loading, nuclear fluorescence was not observed. Tat(FITC) also showed a broad intracellular distribution after labeling and after 24 h, whereas after 72 h, fluorescence was similar to unlabeled cells. Overlays of FITC and Cy3.5 fluorescence (yellow in Figures 6C, F, and I) showed a similar distribution of FITC and Cy3.5 fluorescence. DISCUSSION

The general design we employed for dual fluorochrome labeled nanoparticles features one fluorochrome directly attached to the cross-linked dextran, Cy3.5, with a second supplied by a FITC-labeled peptide (Figure 1). This allows different peptides to be attached to the same parent nanoparticle with different linkages and valences, and the fate of peptide and nanoparticle can be tracked by fluorescence. Here we use a dual fluorochrome-labeled nanoparticle, Tat(FITC)-Cy3.5-CLIO, with 24 copies of Tat(FITC) attached through a thioether linkage per nanoparticle, and used this nanoparticle to monitor the fate of the Tat(FITC) peptide and nanoparticle (Cy3.5CLIO) after administration to cells. Tat(FITC)-Cy3.5CLIO-loaded cells lost fluorescence from FITC and Cy3.5 with a similar time course (Figure 4), and patterns of intracellular distribution of both fluorochromes (Figure 6) were similar. This suggests that Cy3.5 and FITC remain attached to the nanoparticle coating of crosslinked, polymeric dextran and that this coating was poorly excreted over the time course of our experiments. This result was in contrast to a disulfide-linked Tat(FITC)-Cy3.5-CLIO, made as described (14), where FITC fluorescence was far more rapidly lost than Cy3.5 fluorescence (data not shown). Nanoparticles with disulfide linkage between Tat(FITC) and the Cy3.5-CLIO undergo an intracellular separation of Tat(FITC) and Cy3.5-CLIO, with rapid elimination of the Tat(FITC) peptide and retention of Cy3.5-CLIO. High concentrations of glutathione are believed to create a reducing

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environment in cells, which can break disulfide bonds (17). Similar results with disulfide-linked conjugates have been obtained by others; see for example, ref 18. Tat(FITC) Peptide versus Tat(FITC)-Cy3.5CLIO Nanoparticle Metabolism. Both Tat(FTIC) and Tat(FITC)-CLIO were rapidly internalized, labeling the population of HeLa cells uniformly, with increasing levels of intracellular fluorescence per cell over the 180 min incubation period (Figure 2). While the internalization of the Tat(FITC) and Tat(FITC)-Cy3.5-CLIO were similar, the fates of the peptide and nanoparticle after internalization were quite different. Tat(FITC)-loaded cells showed a sharp decrease in RMF and TCF after 24 h in peptide-free media (Figures 4B and 4C), and by 72 h more than 80% of cells were unlabeled (Figure 4A). With Tat(FITC)-Cy3.5 CLIO, when a correction was made for cell division, cells retained their fluorescence after 24 h in media and lost only about 50% of their FITC or Cy3.5 over the next 48 h. When cells were loaded with Tat(FITC)-Cy3.5-CLIO and incubated in nanoparticlefree media, T2 dropped and did not change over a 144 h incubation period, indicating a general lack of excretion of the superparamagnetic iron oxide core of the nanoparticle. Hence the Tat(FITC) peptide rapidly labeled cells, but fluorescence was rapidly lost, presumably due to the excretion of the peptide or a fragment. The Tat(FITC)-Cy3.5-CLIO also rapidly labeled cells, but the excretion was either slow, based on the retention of fluorescence, or not detectable based on relaxation time measurements. The dominant pathway for nanoparticle elimination was distribution to daughter cells after cell division. The Tat(FITC)-Cy3.5-CLIO nanoparticle is a highly stable nanoparticle because dextran is tightly bound to the surface of the iron oxide by cross-linking, and a reduction-insensitive thioether bond is used to attach the peptide to the nanoparticle. Separation of Cy3.5 and FITC is unlikely, based on the nanoparticle design (Figure 1) that requires a proteolytic cleavage between two highly modified amino acids, the C-terminal cysteine attached to the CLIO nanoparticle, and the penultimate lysine, attached to FITC. Degradation of a major part of the tat peptide may occur without severing the bonds between FITC or Cy3.5 and the coating of cross-linked dextran. The similar retention of the Cy3.5 and FITC fluorescence by cells (Figures 3 and 4) is consistent with this high chemical stability and suggests that both fluorochromes remain attached to the cross-linked coating of dextran after internalization by cells. HeLa cells loaded with Tat(FITC)-Cy3.5-CLIO were unable to digest the superparamagnetic iron oxide sufficiently to cause T2 to increase during our 144 h observation period. When injected intravenously, superparamagnetic iron oxides are phagocytosed, with a drop in the T2 of the liver and spleen (19, 20). Over a period of days, T2 increases back to normal values, due to dissolution of the iron oxide crystal, with the iron joining normal body pools. Our studies suggest that lifetime of superparamagnetic iron oxide in HeLa cells is considerably longer than in phagocytic cells. The retention of T2 depression is a desirable feature that can allow cells to be tracked by MRI in vivo for long periods of time. The ability of tat peptide to deliver a variety of agents of varying sizes and chemical compositions into cells in vitro and in vivo has been explored (1, 21). Particularly intriguing is the potential of the tat peptide to deliver macromolecules such as proteins or enzymes (10, 22, 23) and nanoparticles (9) both into cells and across endo-

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Figure 6. Localization of Tat(FITC)-Cy3.5-CLIO or Tat(FITC) peptide as a function of time by confocal microscopy of unfixed cells. Cells were labeled with Tat(FITC)-Cy3.5-CLIO (A-I) or Tat(FITC) peptide (K-M). FITC fluorescence is in shown in green and Cy3.5 fluorescence in red. The two are superimposed in yellow (C, F, I). Cells were labeled (1 h) analyzed immediately (A-C, K), after 24 h (D-F, L) or after 72 h (G-I, M). Exposure time was adjusted to give similar overall fluorescence intensities at all time points. Arrows indicate perinuclear distribution. Arrowheads indicate nuclear distribution.

thelial and epithelial barriers in vivo. However, a recent study examining radiolabeled tat peptide transport across bladder as an epithelial layer did not find evidence of such transport (24). The loss of cellular fluorescence of Tat(FITC)-Cy3.5-CLIO suggests that the transcellular passage of the nanoparticle, if it occurs, will be a slow and inefficient process compared to internalization. Attempts to study tat-mediated transcellular nanoparticle transport should use analytical methods of very high sensitivity that can detect very stable features of the tat macromolecule or nanoparticle. In the current study we followed the fate of cellular fluorescence for 72 h and internalized superparamagnetic iron oxide over 144 h, a time period during which the fate of a technetium 99mlabeled tat peptide-magnetic nanoparticle would be difficult to observe due to radiochemical decay (t1/2 ) 6 h) (24). On the other hand, a recently developed enzyme immunoassay method for detecting immunoreactive fluorescein, a method that is nonisotopic and which measures difficult to metabolize fluorescein, might provide an accurate view of the magnitude of the transcellular passage of tat-nanoparticles (25). Using the FITC immunoassay the transcellular transport of Tat(FITC)Cy3.5-CLIO using CaCo-2 cell monolayers has been observed to have a lag period time course (Koch et al., manuscript in preparation). Studies of metabolism of Tat(FITC) peptide and Tat(FITC)-Cy3.5-CLIO, such as the current one, can lead to insights regarding the likely rates of tat-mediated, transcellular transport in and through cell layers, and guide the selection of analytical tools to measure such transport.

ACKNOWLEDGMENT

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