Specific Association of Thiamine-Coated Gadolinium Nanoparticles

Thiamine-coated nanoparticle association with THTR1 and ... The nanoparticle cell association was ..... Each data point represents the mean ( SD (n ) ...
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Bioconjugate Chem. 2003, 14, 404−411

Specific Association of Thiamine-Coated Gadolinium Nanoparticles with Human Breast Cancer Cells Expressing Thiamine Transporters Moses O. Oyewumi,† Shuqian Liu,‡ Jeffrey A. Moscow,‡ and Russell J. Mumper*,† Division of Pharmaceutical Sciences, College of Pharmacy, and Department of Pediatrics, Chandler Medical Center, University of Kentucky, Lexington, Kentucky 40536-0082. Received January 2, 2003; Revised Manuscript Received January 28, 2003

Thiamine (vitamin B1) was investigated as a tumor-specific ligand for gadolinium nanoparticles. Solid nanoparticles containing gadolinium hexanedione (1.5 mg/mL) were engineered from oil-in-water microemulsion templates and coated with thiamine ligands. Thiamine ligands were synthesized by conjugating thiamine to either distearoylphosphatidylethanolamine (DSPE) or fluorescein via a poly(ethylene glycol) (PEG) spacer (Mw 3350). The efficiency of thiamine ligand attachment to nanoparticles was evaluated using gel permeation chromatography (GPC). Cell association studies were carried using a methotrexate-resistant breast cancer cell line, MTXRZR75, transfected with thiamine transporter genes (THTR1 and THTR2). Thiamine-coated nanoparticle association with THTR1 and THTR2 cells was significantly greater than that with control breast cancer cells (MTXRZR75 transfected with the empty expression vector pREP4) (p < 0.01; t-test). The nanoparticle cell association was significantly dependent on the extent of thiamine ligand coating on nanoparticles, expression of thiamine transporters in cells, temperature of incubation, and the concentration of competitive inhibitor (free thiamine). Further studies are warranted to assess the potential of the engineered thiaminecoated gadolinium (Gd) nanoparticles in neutron capture therapy of tumors.

INTRODUCTION

The application of nanoparticles as drug delivery systems has received increasing attention due to the opportunities of achieving drug targeting and controlled release (Cho et al., 2001). As such, nanoparticles have been widely applied in the delivery of drugs, genes, and vaccines to specific cells and tissues of interest with potential reduction of toxicity as well as increased therapeutic effects (Allen et al., 1995; Kreuter, 1995). Various types of targeting ligands that have been employed in cell specific nanoparticle formulations include antibodies, peptides, and vitamins (Vyas et al., 2001; Wang and Low, 1998). The utilization of vitamins in targeted delivery has been based on the fact that all eukaryotic cells have specific uptake mechanisms for essential vitamins such as folic acid, vitamin B12, and biotin (Russell-Jones et al., 1999). In particular, many studies carried out on folate receptors indicated that folate-conjugated macromolecules can be specifically taken up by folate receptor-bearing tumor cells (Lee and Low, 1995; Atkinson et al., 2001). Based on the previous work with folate, the present study has been designed to investigate the feasibility of engineering thiaminecoated nanoparticles and to determine the extent to which the presence of thiamine coating on nanoparticles could confer specificity to cells that overexpress thiamine transporters. To our knowledge, no previous work has * Corresponding author: Russell J. Mumper, Ph.D., Assistant Professor of Pharmaceutical Sciences, Assistant Director, Center for Pharmaceutical Science and Technology, College of Pharmacy, University of Kentucky, 907 Rose St., Lexington, KY 40536-0082. Tel: (859) 257-2300 ext. 258, Fax: (859) 323-5985, E-mail: [email protected]. † Division of Pharmaceutical Sciences. ‡ Department of Pediatrics.

been done on the feasibility of thiamine as a cell specific ligand. Thiamine is a member of the vitamin B family and is a water soluble micronutrient that is essential for normal cell function, growth, and development. Like other vitamin B family members, such as folic acid, thiamine has specified transport mechanisms in all eukaryotic cells (Said et al., 1999; Zhao et al., 2002). Earlier studies using gene mapping techniques have identified the genes for thiamine transporters THTR1 and THTR2 (Rajgoal et al., 2001; Lo and Wang, 2002). In its coenzyme form, thiamine pyrophosphate plays a critical role in carbohydrate metabolism (Said et al., 2001; Singleton and Martin, 2001). Specifically, thiamine pyrophosphate participates in the carboxylation of pyruvic and R-ketoglutamic acids as well as in the utilization of pentose. Thiamine deficiency in humans leads to a variety of clinical abnormalities such as cardiovascular and neurological disorders (Seligmann et al., 2001; Lee et al., 1998). In addition, the importance of thiamine in the biosynthesis of many cell constituents, including neurotransmitters and nucleic acid precursors, has been shown to be the basis for the observed increased thiamine utilization in tumor cells (Singleton and Martin, 2001). Additional studies conducted in animals and humans showed that tumor growth might be related to the depletion of tissue thiamine stores, apparently because of the increased thiamine utilization (Lee et al., 1998; Weng et al., 1999; Cascante et al., 2000). Further studies carried out using radiolabeled glucose indicated that tumor cells rely heavily on nonoxidative transketolase (TK) pathway for ribose synthesis to build nucleic acids (Comin-Aduix et al., 2001). As such, antithiamine compounds have been employed in several tumor models to inhibit nucleic acid synthesis and tumor proliferation (Boros et al., 1997).

10.1021/bc0340013 CCC: $25.00 © 2003 American Chemical Society Published on Web 02/27/2003

Thiamine-Coated Gadolinium Nanoparticles

Therefore, in the present study, gadolinium nanoparticles coated with thiamine were engineered, and selective cell association was evaluated using cells that overexpress thiamine transporters. We earlier reported on the engineering of nanoparticles from oil-in-water microemulsion templates (Oyewumi and Mumper, 2002a; Oyewumi and Mumper, 2002b). Nanoparticle formulations contained a final concentration of gadolinium hexanedione (GdH) of 1.5 mg/mL. Gadolinium is a potential anticancer agent for neutron capture therapy (NCT) (Shih and Brugger, 1992). To obtain thiaminecoated nanoparticles, thiamine ligands synthesized to contain either DSPE or fluorescein groups were added to preformed nanoparticle suspensions. Cell specificity of the engineered thiamine-coated nanoparticles was assessed on the basis of the rate and extent of cell association with human breast cancer cells transfected with thiamine transporter genes THTR1 and THTR2. Cells transfected with the empty expression vector pREP4 were used as controls in all experiments. The feasibility of engineering thiamine-coated gadolinium nanoparticles was evaluated as well as the potential of achieving cell specific association due to the thiamine coating. EXPERIMENTAL PROCEDURES

Materials. Emulsifying wax was obtained from Spectrum Chemicals (New Brunswick, NJ), and polyoxyl 20 stearyl ether (Brij 78) was purchased from Uniqema (Wilmington, DE). Distearoylphosphatidylethanolamine (DSPE)-PEG-NHS and fluorescein (FL)-PEG-NHS were purchased from Shearwater Polymers (Huntsville, AL). Thiamine hydrochloride was obtained from Aldrich Chemicals (Milwaukee, WI). Sephadex G-75, Sepharose CL-4B, and potassium ferricyanide were purchased from Sigma Chemicals (St. Louis, MO). Fluorescein-DOPE was purchased from Avanti Polar Lipids (Alabaster, AL). Gadolinium hexanedione was synthesized as previously described (Oyewumi and Mumper, 2002b). Preparation of Nanoparticles from Microemulsion Templates. Solid nanoparticles were engineered from oil-in-water microemulsion templates using emulsifying wax as the oil phase (matrix material). Briefly, 2 mg of emulsifying wax accurately weighed into a glass vial was melted together with 1.5 mg of gadolinium hexanedione (GdH) on a hotplate. Various volumes of polyoxyl 20 stearyl ether (Brij 78; 100 mM) were added to the melted mixture at 55 °C under magnetic stirring. Water (0.22 µm filtered) was added to make the final volume of 1 mL. The formation of an oil-in-water microemulsion was verified by the clarity of the mixture and by photon correlation spectroscopy (PCS) using a Coulter N4 Plus Submicron Particle Sizer at 55 °C. Using optimal microemulsion templates, nanoparticles were prepared by simply cooling the stirring microemulsions to room temperature. The properties and characteristics of nanoparticles cured in each preparation were investigated as described below. Synthesis of Thiamine Ligands. Thiamine ligands were synthesized by chemically linking thiamine to either DSPE or fluorescein via a PEG spacer (Mw 3350). The procedure was based on the modified method of Jayamani and Low (1992). Briefly, 2.0 mg of thiamine was reacted at room temperature for 2 h with 10 mg of either DSPEPEG-NHS or Fluorescein-PEG-NHS in 1 mL of phosphate buffered saline (pH 7.4). Afterward, unreacted thiamine was removed from reaction mixture by dialysis in deionized water since free (unreacted) thiamine will

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interfere in cell targeting. The conjugated thiamine was purified further by passing the reaction mixture through a gel permeation column (2.5 cm × 5.0 cm; Sephadex G-75) with phosphate buffer, pH 5.0 as the mobile phase. Thiamine conjugated to either DSPE or fluorescein was eluted at the void volume of the GPC column as determined by a thiochrome assay. The procedure of the thiochrome assay involved oxidizing thiamine in each GPC fraction to thiochrome and subsequently measuring fluorescence intensity at 365 nm (excitation) and 445 nm (emission). The efficiency of thiamine conjugation to either DSPE-PEG or Fluorescein-PEG was 40% and 18%, respectively. The conjugate was verified by IR spectroscopy. Addition of Thiamine Ligands to Nanoparticle Preparations. Using a stock aqueous solution of thiamine ligand (0.5 mg/mL), various amounts of thiamine ligand were added to cured nanoparticle suspensions at 25 °C. The mixture was then gently stirred for 4 h at 25 °C. The efficiency of thiamine attachment/adsorption was assessed by gel permeation chromatography (GPC) elution profiles using a Sepharose CL-4B column. Briefly, 80 µL of thiamine-coated nanoparticle suspensions were passed down the Sepharose CL-4B column (1.5 cm × 8 cm) using deionized water (0.22 µm filtered) as the mobile phase. The elution of thiamine-coated nanoparticles and free thiamine ligand in all the GPC fractions was detected by laser light scattering counts per second (CPS) and the thiochrome assay method as mentioned above. In a separate study, the GPC elution profiles of control nanoparticles (without thiamine) and free thiamine ligand were obtained to serve as references. Based on the GPC elution profiles, the efficiency of thiamine ligand coating was calculated as the percentage of the ratio of the area under thiamine-coated nanoparticle profiles to the area under the total elution profiles. Calculation of the concentration of thiamine and total number of thiamine molecules used in coating nanoparticles was based on the coating efficiency data. Photon Correlation Spectroscopy (PCS). The particle sizes of thiamine-coated nanoparticles were determined using an N4 Plus Submicron Particle Sizer at 20 °C by scattering light at angle 90° for 180 s (Beckman Coulter Corporation, Miami, FL). Prior to particle size measurements, the nanoparticles were diluted (1:10 v/v) with water (0.22 µm filtered), to ensure that the light scattering signal as indicated by the particle counts per second (CPS) was within the sensitivity range of the instrument. Gel Permeation Chromatography (GPC). To obtain the GPC elution profiles of nanoparticles, 80 µL of nanoparticle suspensions was passed down a Sephadex G-75 column (1.5 cm × 8 cm) using deionized water (0.22 µm filtered) as the mobile phase. The elution of thiaminecoated nanoparticles was detected by laser light scattering counts per second and thiochrome assay using fluorescence spectroscopy (Hitachi Model F-2000). Transmission Electron Microscopy (TEM). The size and morphology of nanoparticles were observed using a JEOL Electron Microscope in the Imaging Facility Unit of the University of Kentucky. A carbon-coated 200-mesh copper specimen grid was glow-discharged for 1.5 min. One drop of nanoparticle suspension was deposited on the grid and allowed to stand for 1.5 min after which any excess fluid was removed with filter paper. The grid was later stained with 1 drop of 1% uranyl acetate (0.22 µm filtered) for 30 s, and any excess stain was removed. The grids were allowed to dry for an additional 10 min before examination under the electron microscope.

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Transfection and Expression of THTR1 and THTR2 Genes in Human Breast Cancer Cells. The transfection and expression of THTR1 and THTR2 thiamine transporter genes in breast cancer cells (MTXRZR75 cells) was previously described (Liu et al., 2003). Briefly, the characteristics of THTR1 and THTR2 were defined in the host cell line of MTXRZR75 cells because these cells have no detectable specific reduced folate carrier activity (Dixon et al., 1991; Dixon et al., 1994) which can transport thiamine pyrophosphate (Zhao et al., 2002). First, the THTR1 cDNA was cloned by RT-PCR using skeletal muscle RNA as a template, with primers which span the putative open reading frame: THTR1-U1 5′CAGTTGGCGGAGGAGGAGAAGGAAG-3′ and at the 3′ end THTR1-L2 5′-AAGGTATTAGTCAAGTGGCTGCTGT3′. For THTR2, the cDNA was cloned by RT-PCR using placenta RNA as a template, with the primers which span the putative open reading frame. The primers used were: THTR2-U1 5′-GGG GTA CCT AGT GAG CGA TTT GGT GAA CAG AC-3′ and THTR2-L2 5′-CCG CTC GAG TAT GCC ACC CAT CTC AAA ATC TTT-3′. Unique restriction enzyme sites for directional cloning into the multiple cloning site of the episomal expression vector were added to the 5′ ends of these primers. MTXRZR75 cells were transfected with pREP4/THTR1, pREP4/ THTR2, or pREP4 alone using LipofectAMINE reagent (Invitrogen, Carlsbad CA). Quantitative RT-PCR for measuring RNA levels was performed by using a Roche LightCycler, which uses real time fluorescence detection for quantitative measurement of PCR products. The quantitative measurement of each gene in each cell line was normalized to the relative amount of actin RNA in each cell line, as a control for equivalent cDNA loading in each sample. Thiamine uptake studies were performed in a manner similar to previous studies of MTX uptake (Moscow et al., 1995). [3H]-Labeled thiamine was obtained from American Radiolabeled Chemicals (St. Louis, MO), and its purity was assayed by the manufacturer. For the initial thiamine uptake studies, cells were plated at a density of 1 × 105 in 6-well Linbro dishes in medium containing 10% fetal bovine serum. After 48-72 h of growth, cells were washed three times in transport medium (125 mM NaCl, 4.8 mM KCl, 5.6 mM D-glucose, 1.2 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, and 25 mM HEPES, pH 7.4) without [3H]-labeled thiamine and then exposed to 20 nM [3H]-labeled thiamine in transport medium. At specified intervals, the transport medium was aspirated, and the plates immersed in three successive washes of ice-cold 1X Dulbecco’s phosphate-buffered saline containing Mg2+ and Ca2+ (D-PBS). The cells were then solubilized by overnight incubation in 0.2 N NaOH and neutralized with 0.2 N HCl, and the radioactivity was determined by liquid scintillation counting as previously described (Moscow et al., 1997). Protein concentrations were determined by the Bradford assay according to manufacturer’s instructions (BioRad) using a spectrophotometer (Beckman). Nonspecific binding was determined by exposure of cells to transport medium for less than 5 s and was subtracted from measured values to indicate specific uptake. Cell Culture. Cells were maintained and grown at 37 °C in Improved MEM Zinc Option (IMEM) (Gibco BRL, Gaitherburg, MD) supplemented with 10% fetal bovine serum (FBS) and 250 µg/mL hygromycin B. Medium was changed into Improved MEM Zinc Option (IMEM) without thiamine (Gibco BRL, Gaitherburg, MD) before the cell association studies of thiamine-coated nanoparticles.

Oyewumi et al.

Figure 1. Chemical structures of thiamine ligands synthesized by chemically linking thiamine to either distearoylphosphatidylethanolamine (DSPE) or fluorescein (FL) via a PEG spacer: (A) thiamine-PEG-fluorescein, (B) thiamine-PEG-DSPE.

Nanoparticle Cell Association Studies. The cells were seeded in 24-well plates at a density of 105 cells/ mL in 1 mL of medium and incubated at 37 °C in a humidified 5% CO2/air incubator for 24 h. To determine the effect of nanoparticle concentration on cell association, various dilutions of thiamine-coated nanoparticles or control nanoparticles (without thiamine) in cell growth medium were made in the concentration range of 20 to 360 µg/mL. After replacing the medium in each well with nanoparticles suspended in medium, the plates were incubated at 37 °C or 4 °C for various times. Prior to the cell association study at 4 °C, a 10 min equilibration time was allowed for nanoparticle suspensions and cells. In a separate experiment, nanoparticle suspensions (180 µg/ mL) were incubated in the presence of various concentrations of free thiamine to serve as a competitive inhibitor. At the end of the incubation time, nanoparticle suspensions were removed from the wells, and the cell monolayers were rinsed three times with cold PBS and lysed using 1x lysis buffer (Promega). Cell associated fluorescence was measured using a fluorometer (Hitachi Model F-2000). Fluorescein-DOPE was used as a fluorescence marker in the studies. Initial experiments showed that fluorescein-DOPE (0.25%) could be entrapped in nanoparticles with ∼100% efficiency. The amount of fluorescent-labeled nanoparticles associated with cells was measured by quantifying the intensity of internalized fluorescence at 521 nm (emission) with excitation at 497 nm. The percentage of nanoparticle cell association was calculated from the ratio of the observed cell associated fluorescence to the total fluorescence added to the cells. Additional control experiments were carried out to compare the total cell protein content of cells incubated with various concentrations of thiamine-coated nanoparticles ranging from 0 to 360 µg/mL. The total cell protein content in the cell lysate from each well was determined using the Coomassie Plus assay protocol (Pierce, Rockford, IL). RESULTS AND DISCUSSION

Synthesis of Thiamine Ligands. The chemical structures of the thiamine ligands are shown in Figure 1. The

Thiamine-Coated Gadolinium Nanoparticles

thiamine ligands were synthesized based on the nucleophilic attack of the hydroxyl group of thiamine on the electrophilic group of either DSPE-PEG-NHS or FLPEG-NHS. The purpose of the synthesis was to chemically link thiamine to hydrophobic groups such as DSPE or fluorescein via a PEG spacer. The presence of the hydrophobic groups on the thiamine ligand was particularly necessary to ensure attachment to nanoparticles. Since the thiamine ligand was added to nanoparticle suspension by physical mixing, adsorption/insertion of the ligand on nanoparticles was facilitated by the presence of hydrophobic anchors. In addition, the PEG spacer theoretically facilitated cell recognition of the thiamine ligand on nanoparticles. Previous studies have demonstrated the importance of PEG spacers in targeted delivery systems (Lee and Low, 1995; Goren et al., 2000). Engineering Thiamine-Coated Nanoparticles. The nanoparticle engineering process was based on the formation of oil-in-water microemulsions that were prepared at 55 °C, which upon cooling in one vessel resulted in the production of nanoparticles. Emulsifying wax was used as nanoparticle matrix material and Brij 78 (polyoxyl 20 stearyl ether) as the surfactant. The procedure of engineering nanoparticles from oil-in-water microemulsion templates was reported earlier (Oyewumi and Mumper, 2002a). Emulsifying wax is a nonionic wax comprised of cetostearyl alcohol and a polyethylene derivative of a fatty acid in a molar ratio of about 20:1. GdH was incorporated in nanoparticles by the addition to the oil phase (matrix materials) of the microemulsion templates. GdH is a gadolinium compound, synthesized by the complexation of Gd3+ with 2,4-hexanedione (Oyewumi and Mumper, 2002b). In addition to having a relatively higher weight percentage of Gd compared to other complexes of Gd (such as Gd-DTPA, Gd-DOTA, Gd-EDTA), GdH possesses other suitable properties such as lower melting point (55 °C) and increased hydrophobicity that could facilitate entrapment in nanoparticles. The final nanoparticle preparation was made with emulsifying wax (2 mg/mL) containing 1.5 mg of GdH using 3.0 mM Brij 78 as the surfactant. The choice and concentration of Brij 78 was based on results from earlier studies (Oyewumi and Mumper, 2002a). Solid nanoparticles containing GdH were engineered using the ternary system: melted oil, surfactant, and water. To obtain thiamine-coated nanoparticles, various concentrations of thiamine ligand were added to nanoparticle suspensions at 25 °C. The mixture of nanoparticles and ligand was gently stirred at room temperature to ensure complete adsorption/insertion of thiamine ligand on the nanoparticles. A TEM micrograph of thiamine-coated nanoparticles is shown in Figure 2. The engineered nanoparticles had a uniform particle size of about 100 nm. Although the addition of increasing amounts of thiamine ligand (0.15% w/w to 2.5% w/w) resulted in a slight increase in nanoparticle size, the average nanoparticle size was maintained below 100 nm (Figure 3). Further, the polydispersity index ranged from 0.2 to 0.4 with the addition of thiamine ligand at concentrations below 2.5% w/w, indicating uniformity of the coated nanoparticles. In particular, the nanoparticle size of about 100 nm is considered to be suitable for cell targeting since several other groups using liposomes and other macromolecules have shown that particles with small size (less than 400 nm and ideally less than 150 nm) are more efficient in cell targeting than larger particles (Desai et al., 1996; Jain, 1997). The extent of thiamine ligand attachment to nanoparticles was determined by gel permeation chromatography

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Figure 2. Transmission electron micrograph (TEM) showing the size and morphology of thiamine-coated emulsifying wax (2 mg/mL) nanoparticles containing gadolinium hexanedione (1.5 mg).

Figure 3. The average nanoparticle size after the addition of various concentrations of thiamine-PEG-DSPE (0-2.5% w/w). The particle size of each sample (after GPC purification) was determined by laser light scattering at 25 °C. Each value represents the mean ( SD (n ) 3).

(GPC) based on the elution of free thiamine and thiaminecoated nanoparticles. Thiamine elution as free thiamine ligand or thiamine ligand attached to nanoparticles was monitored by the thiochrome assay. To calculate the coating efficiency of the ligand, both the elution of control nanoparticles (without thiamine) and free thiamine ligand were obtained and used as references. GPC elution of nanoparticles occurred at fraction 3 (void volume) as

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Oyewumi et al.

Figure 5. Association of thiamine-coated nanoparticle (THNP) with THTR1, THTR2, and pREP4 cells as a function of nanoparticle concentrations. Cell-associated fluorescence after 15 min was measured at 497 nm (excitation) and 521 nm (emission) using fluorescein-DOPE as the marker. Each data point represents the mean ( SD (n ) 4).

Figure 4. (A) Gel permeation chromatography (GPC) elution profiles demonstrating that the elution of free thiamine ligand (0) was separated from that of thiamine-coated nanoparticles (9). Thiamine-coated nanoparticles were obtained by adding 0.2% w/w thiamine-PEG-DSPE to nanoparticle suspensions at 25 °C. The elution of thiamine from GPC column (Sepharose CL-4B) was detected. The amount of thiamine in each GPC fraction was determined by fluorescence intensity at 445 nm (emission) and 365 nm (excitation) using a thiochrome assay. (B) Efficiency of thiamine ligand coating on nanoparticles (NP). Various concentrations (0-1.2% w/w) of either thiamine-PEGDSPE (9) or thiamine-PEG-fluorescein (0) were added to nanoparticle suspensions at 25 °C. Each value represents the mean ( SD (n ) 3).

monitored by laser light scattering counts per second (CPS) while the elution of free thiamine ligand occurred later than fraction 3 (Figure 4A). The coating efficiencies of various concentrations of thiamine-PEG-DSPE and thiamine-PEG-FL are shown in Figure 4B. At thiamine ligand concentrations below 0.2% w/w, comparable results were obtained for the two ligands indicating high coating efficiencies. The observed decrease in thiamine ligand coating efficiency at high concentrations was, however, pronounced with thiamine-PEG-FL. The difference in the trend observed for the two ligands indicates that hydrophobic groups on the ligand are necessary for optimal ligand attachment to the nanoparticles. Compared to DSPE, fluorescein is more hydrophilic due to the many hydroxyl groups that most likely participated in hydrogen bonding with the water continuous phase. Nanoparticle Cell Association Studies. MTXRZR75 cells transfected with THTR1 and THTR2 genes were referred to as simply THTR1 and THTR2 cells, respectively, while the same cell line transfected with the empty expression vector (pREP4) was referred to as pREP4 cells. The presence of THTR1 and THTR2 trangenes in MTXRZR75 cells was confirmed by PCR reactions. Also, the MTXRZR75 cells transfected with the cDNAs for THTR1 and THTR2 showed an increase in initial thiamine uptake relative to the control cell line. At 4 min,

uptake of radiolabeled thiamine at an extracellular concentration of 20 nM was 47% greater in the THTR1transfected cells than control cells, and 133% greater in the THTR2-transfected cells than in the control cells. Additional initial studies were carried out using thiaminecoated nanoparticles. After incubation with cells, the total protein in all the wells was measured and did not show any significant difference between different concentrations of nanoparticles (0-360 µg/mL) (p > 0.05; t-test) (data not shown). It was also observed that thiaminecoated nanoparticles were stable (based on nanoparticle size and thiamine coating efficiency) when incubated at 37 °C (for 120 min) in either fetal bovine serum (10%) or PBS (pH 7.4). Results obtained on thiamine-coated nanoparticle cell association as a function of nanoparticle concentration are shown in Figure 5. The extent of cell association of thiamine-coated nanoparticles was viewed as a reflection of thiamine transport activity present in all the cells. In comparison to pREP4 cells, nanoparticle cell association was greater with THTR1 and THTR2 cells most likely due to the higher level of expression of thiamine transport genes in these cells. For instance, at a nanoparticle concentration of 180 µg/mL, the concentration of nanoparticles in association with THTR1 and THTR2 cells was 90% and 185% higher than with control cells (pREP4), respectively (Figure 5). The enhanced nanoparticle association in transfected cells was expressed further by calculating the number of nanoparticles (∼100 nm diameter) that associated with THTR1, THTR2, and control cells. Using the data in Figure 5 (at a nanoparticle concentration of 180 µg/mL), the number of nanoparticles that associated with a single THTR1, THTR2, and pREP4 cell was calculated. The analysis estimated that a single THTR1 and THTR2 cell had approximately 30 000 to 70 000 more nanoparticles associated than a single pREP4 cell at the same test condition. Further experiments were carried out to demonstrate the involvement of thiamine transporter genes in nanoparticle cell association. The effects of various concentrations of thiamine ligand used in coating nanoparticles were studied. As shown in Figure 6, at concentrations of thiamine ligand from 0.1 to 0.6% w/w, nanoparticle cell association with THTR1 and THTR2 cells was significantly greater (p < 0.01; t-test) than with control cells (pREP4), probably due to the less expression of thiamine transporters in control cells. However, at concentrations of thiamine

Thiamine-Coated Gadolinium Nanoparticles

Figure 6. The effect of thiamine ligand concentration added to nanoparticle formulations on cell association. Results were expressed as percentage of the ratio of cell-associated fluorescence and total fluorescence added to cells. Cell-associated fluorescence was measured at 521 nm (emission) and 497 nm (excitation) after 15 min incubation of thiamine-coated nanoparticles (THNP) (180 µg/mL) with either THTR1, THTR2, and pREP4 cells. Values represent mean ( SD (n ) 4).

ligand above 1% w/w, nanoparticle association with THTR1- and THTR2-transfected cells decreased significantly to such a level that was comparable to pREP4 cells (p < 0.11; t-test). This decrease in nanoparticle association with increased concentration of thiamine ligand can be attributed to the expected increase in the amount of free thiamine ligand in nanoparticle preparations. Since the nanoparticle preparations were applied in the study without GPC purification, the free thiamine ligand (unattached to nanoparticles) most likely competed with thiamine-coated nanoparticles for binding to the transporters. Subsequent experiments were carried out using nanoparticles coated with 0.2% w/w thiamine ligand containing 10 µM of thiamine (as determined by thiochrome assay). The incubation time of 15 min was used in all the cases. Experiments conducted at various incubation time intervals (ranging from 5 to 60 min), showed that nanoparticle cell association (at 37 °C) occurred rapidly within 5 min and resulted in a plateau at 15 min (data not shown). The involvement of thiamine transporters in nanoparticle cell association was investigated further by using thiamine-coated nanoparticles and control nanoparticles (without thiamine ligand). As shown in Figure 7, association of thiamine-coated nanoparticles with THTR1- and THTR2-transfected cells was observed to be significantly higher than control nanoparticles (p < 0.05). The possibility of thiamine transporters mediating nanoparticle cell association was reflected from the results obtained in pREP4 cells. Compared with THTR1- and THTR2-transfected cells, thiaminecoated nanoparticle association occurred to a lower extent in pREP4 cells and the effect was comparable to control nanoparticles (p > 0.11; t-test). In contrast to the data obtained at 37 °C, studies conducted at 4 °C showed reduction (p < 0.005; t-test) in association of thiaminecoated nanoparticles in all the cells (Figure 7). The reduction in nanoparticle cell association could be due to the expected reduced activity of thiamine transporter at 4 °C. Earlier studies have shown that ligand binding, association and endocytic activities are suppressed significantly at 4 °C (Shikata et al., 2002). As such, cell association at 4 °C will most likely be due to simple adhesion of nanoparticles to the cell surfaces. Since, adhesion alone did not fully account for the extent of cell association observed at 37 °C, the trend thus suggests

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Figure 7. Cell association of (180 µg/mL) of thiamine-coated nanoparticles (THNP) or control nanoparticles (CTRNP) after incubation at 37 °C or 4 °C for 15 min with THTR1, THTR2, and pREP4 cells. Control nanoparticles (CTRNP) had no thiamine ligand coating. After the incubation, cell-associated fluorescence was measured at 521 nm (emission) and 497 nm (excitation). Each data point represents the mean ( SD (n ) 4).

Figure 8. The effect of coincubation of free thiamine on thiamine-coated nanoparticles (THNP) (180 µg/mL). Cell-associated fluorescence after incubation at 37 °C for 15 min was measured at 521 nm (emission) and 497 nm (excitation). Each value represents the mean ( SD (n ) 3).

the involvement of thiamine transporters in nanoparticle cell association. The specificity of nanoparticle association to the transporters was investigated by coincubation of free thiamine with thiamine-coated nanoparticles. The result showed that free thiamine inhibited cell association of thiamine-coated nanoparticles (Figure 8). The competitive effect of free thiamine was most evident in THTR1- and THTR2-transfected cells and at the highest concentration of free thiamine studied. For instance, coincubation of 100 mM free thiamine resulted in reduction of nanoparticle cell association to 31%, 15%, and 72% of its initial value (0 mM free thiamine) in THTR1, THTR2, and control cells, respectively, indicating that the reduction of nanoparticle cell association due to free thiamine competition was more pronounced in THTR1and THTR2-transfected cells. In fact, at concentrations of free thiamine that were 10-fold to 100-fold higher than the concentration of thiamine ligand on nanoparticles, significant reduction of nanoparticle association was observed only with THTR1- and THTR2-transfected cells (p < 0.005, t-test), and not with the control cells (p > 0.2; t-test) (Figure 8). The studies demonstrated higher specificity of thiamine-coated nanoparticle association with THTR1- and THTR2-transfected cells compared with the control cells and dependence on the extent of expression of thiamine transporters genes in these cells.

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The specific mechanism of nanoparticle cell association (involving either binding to the transporters alone and/ or cell uptake) is yet to be investigated. Regardless of the mechanism of nanoparticle cell association, achieving tumor cell specificity of thiamine-coated gadolinium nanoparticles may potentially be employed in Gd neutron capture therapy (NCT) of tumors. GdNCT is a potential cancer therapy that utilizes a stable, nonradioactive Gd-157 nuclide delivered to tumor cells which upon irradiation by thermal or epithermal neutrons produces localized cytotoxic radiations. The tumor killing effect of GdNCT is due to the emission of prompt gamma rays followed by a series of low-energy conversion and Auger electrons. A favorable characteristic of GdNCT is that the cytotoxic emissions are deposited at long ranges within the target tumor site (Allen et al., 1989); as such, the location of Gd within the target tumor cell is not critical to the therapeutic effectiveness. In general, thiamine-coated Gd nanoparticles have been engineered and characterized. The potential of using thiamine as a cell specific ligand has been investigated. Results showed that nanoparticle cell specific association was dependent on the extent of expression of thiamine transporters genes. Additional studies are warranted to determine the extent of expression of thiamine transporters in various human tumors compared to normal tissues and to assess the potential of the thiamine-coated Gd nanoparticles in neutron capture therapy. LITERATURE CITED (1) Allen, B. J., McGregor, B. J., and Martin, R. F. (1989) Neutron capture therapy with gadolinium-157. Stahlenther Onkol. 165, 156-158. (2) Allen, T. M., Brandeis, E., Hansen, C. B., Kao, G. Y., and Zaplipsky, S. (1995) A new strategy for attachment of antibodies to sterically stabilized liposomes resulting in efficient targeting to cancer cells. Biochim. Biophys. Acta 1237, 99-108. (3) Atkinson, S. F., Bettinger, T., Seymour, L. W., Behr, J., and Ward, C. (2001) Conjugation of folate via gelonin carbohydrate residues retains ribosomal-inactivating properties of the toxin and permits targeting to folate receptor positive cells. J. Biol. Chem. 276, 27930-27935. (4) Boros, L. G., Puigjaner, J., Cascante, M., Lee, W. N. P., Brandes, J. L., Bassilian, S., Yusurf, F. I., Williams, R. D., Muscarella, P., Melvin, W. S., and Schirmer, W. J. (1997) Oxythiamine and dehydroepiandrosterone inhibit the nonoxidative synthesis of ribose and cancer cell proliferation. Cancer Res. 57, 4242-4248. (5) Cascante, M., Centelles, J. J. Veech, R. L., Lee, W. N., and Boros, L. G. (2000) Role of thiamine (vitamin B-1) and transketolase in tumor cell proliferation. Nutr. Cancer 2, 150-154. (6) Cho, C. S., Cho, K. Y., Park, I. K., Kim, S. H., Sasagawa, T., Uchiyama, M., and Akaike, T. (2001) Receptor-mediated delivery of trans-retinoic acid to hepatocyte using poly (Llactic acid) nanoparticles coated with galactose-carrying polystyrene. J. Controlled Release 77, 7-15. (7) Comin-Aduix, B. Boren, J. Martonez, S. Moro, C., Centelles. J. J. Trebukhina, R., Petushok, N., Lee, W. P., Boros, L. G., and Cascante, M. (2001) The effect of thiamine supplementation on tumour proliferation. A metabolic control analysis study. Eur. J. Biochem. 268, 4177-4182. (8) Desai, M. P., Labhasetwar, V., Amidon, G. L., and Levy, R. J. (1996) Gastrointestinal uptake of biodegradable microparticles in Caco-2 cells is size dependent. Pharm. Res. 14, 15681573. (9) Dixon, K. H., Trepel, J. B., Eng, S. C., and Cowan, K. H. (1991) Folate transport and the modulation of antifolate sensitivity in a methotrexate-resistant human breast cancer cell line. Cancer Commun. 3, 357-365.

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