Targeting Glioma Cells in Vitro with Ascorbate-Conjugated

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Bioconjugate Chem. 2009, 20, 2348–2355

Targeting Glioma Cells in Vitro with Ascorbate-Conjugated Pharmaceutical Nanocarriers Stefano Salmaso,*,†,‡ Juan S. Pappalardo,† Rupa R. Sawant,† Tiziana Musacchio,† Karen Rockwell,† Paolo Caliceti,‡ and Vladimir P. Torchilin† Department of Pharmaceutical Sciences and Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston, Massachusetts 02115, and Department of Pharmaceutical Sciences, University of Padova, via F. Marzolo 5, 35131 Padova, Italy. Received August 19, 2009; Revised Manuscript Received October 16, 2009

6-Ascorbate-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (6-ascorbate-PEG-PE) was synthesized according to a two-step procedure: (1) activation of ascorbic acid with bromine, and (2) synthesis of 6-ascorbatePEG-PE by reacting 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (poly(ethylene glycol))-2000] with an excess of 6-Br-ascorbic acid. The 6-ascorbate-PEG-PE was recovered by precipitation in diethyl ether and purified by gel permeation chromatography. The analysis of the product by 1H NMR and UV-vis spectroscopy confirmed the identity of the conjugate. Liposomes and PEG-PE-based lipid-core micelles were prepared by thin film hydration technique incorporating 6-ascorbate-PEG-PE as targeting moiety. The targeting properties of the ascorbate-decorated nanosystems were tested by fluorescence-activated cell sorting (FACS) analysis and fluorescent microscopy on a panel of tumor cell lines preliminary selected for their ability to express the SVCT2 ascorbate transporter. Cell lines had been selected on the basis of the immunological properties assessed by FACS, which showed that two glioma cell lines, C6 and F98, and fibroblasts NIH/3T3 express plasma membrane-associated SVCT2 transporter for reduced ascorbic acid. Ascorbate-decorated pharmaceutical nanocarriers were endowed with selective targeting properties toward the SVCT2 transporter expressed in glioma cell models. This study shows that SVCT2 transporter for ascorbic acid expressed both in peculiar epithelial cells of the choroid plexus responsible for the filtering of vitamin C into the central nervous system (CNS) and, in some brain tumor cell lines, can be conceivably exploited as a potential target for delivery of drug-loaded pharmaceutical nanocarriers to the brain.

INTRODUCTION In the past decades, a great deal of research has focused on targeting the central nervous system for therapeutic and diagnostic purposes. The goal appeared to be extremely challenging due to the physiological architecture of the biological barriers, namely, the blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier, which have poor permeability and allow for only restricted access to selected molecules. Such barriers limit the permeation to the brain compartment, protecting the nervous systems from toxic compounds, metabolites, viruses, and bacteria. By virtue of these physiologic barriers, most of the targeting strategies for drug delivery to the brain have shown limited efficiency. Central nervous system penetration is favored for molecules having low molecular weight and adequate hydrophilic/ hydrophobic balance (1). Besides unspecific permeation, endothelial cells of the brain capillaries, which constitute the BBB, are provided with selective transport systems for specific nutrients and endogenous biomolecules. Thus, they are responsible for the transport of glucose; neutral, acidic, and basic amino acids; alanine and taurine; some monocarboxylic acids; amines; neuromediators, such as choline; some vitamins; nucleosides; and the peptide transport system for small neurotropic peptides (2, 3). * Corresponding author. Stefano Salmaso, Department of Pharmaceutical Sciences, University of Padua, Via F. Marzolo, 5, 35131 Padova, Italy, phone +39 049 8271602, Fax +39 049 8275366, e-mail: [email protected]. † Northeastern University. ‡ University of Padova.

A limited district of the BBB, namely, the epithelium of the choroid plexus (CP), is considered to be responsible for the maintenance of central nervous system (CNS) homeostasis of few micronutrients, namely, vitamin C and riboflavin (4). It has been found that, in some neurons, ascorbic acid concentration can reach up to 200-fold higher than that measured in the bloodstream (5). Specific physiological mechanisms are operating in order to transfer ascorbic acid from the blood, where its concentration is ∼50 µM, into the cerebrospinal fluid where the concentration is maintained at 200 µM (6). Within the CNS, ascorbic acid plays a pivotal role as a cofactor in a number of different enzymatic activities relating to the neurotransmitters processing and as an antioxidant allowing for neuroprotection. According to H. Tsukaguchi, the reduced form of ascorbic acid is taken up by a mechanism that involves the sodiumdependent-vitamin C transporters SVCT2, whose RNA was detected in the choroid plexus epithelium (7). SVCT2 transporter was found to be expressed by neuroepithelial cells of the choroid plexus and the retinal pigmented epithelium mostly. Despite other mechanisms having been proposed raising options for ascorbic acid penetration mechanism into the brain as the oxidized form, the SVCT2-mediated pathway seems the most likely one, since the oxidized form of vitamin C is undetectable in the blood at physiological conditions and unstable in water (8). Besides, the evidence that ascorbic acid reaches the cerebrospinal fluid through the route of CP and then slowly penetrates the brain substance from the CSF was demonstrated by the intravenous injection of [14C] ascorbic acid (9). How specifically ascorbic acid exits CSF to permeate the brain is still to be investigated, and no extensive data are available in literature so far.

10.1021/bc900369d  2009 American Chemical Society Published on Web 11/23/2009

Targeting Glioma Cells in Vitro

Few in vitro cell models are available for evaluating the mechanism of ascorbic acid uptake mediated by the SVCT2 translocator. SVCT2 transporter expression and function in primary cell culture of astrocytes (10, 11) and in monolayers obtained from the porcine choroid epithelial cells have been investigated (12-14). Many studies concerning SVCT function and targeting for drug delivery have been carried out by using cloned Xenopus oocytes, which were induced to express the desired plasma membrane associated vitamin C translocator (7, 15, 16). Only a limited number of studies are available in the literature on the exploitation of ascorbic acid SVCT transporter as a biological target mediating the brain delivery of therapeutic or diagnostic molecules. Small molecular weight prodrugs were synthesized according to adequate chemical strategies combining ascorbic acid with CNS active molecules and underwent investigation for brain therapeutic activity. It was shown that ascorbic acid endowed such prodrugs with efficient binding features to epithelial-associated SVCT2 translocator (17). Despite the fact that such prodrugs were not found to cross the BBB, they were effective in releasing the drug in the vicinity of the site of action and promote the diffusion through the brain barrier. These results outline the potential of ascorbic acid transporter as a new mediator for drug delivery by using drugloaded systems, which bind the CP epithelium and possibly either translocate through it or release the drug payload that, by virtue of its hydrophilic/hydrophobic balance, can permeate into the brain. So far, the literature is lacking in macromolecular systems aimed at brain targeting, which take advantage of the SVCT2 transporter features, namely, molecular selectivity and physiological disposition. In order to set up a new in vitro cellular model to exploit for brain delivery studies, immunological characterization of a panel of immortalized cell lines was carried out to assess which of them express the SVCT2 transporter. The potential of pharmaceutical nanocarriers, namely, liposomes and lipid-core polymeric micelles, to target such cells via the SVCT transporter was investigated after the nanocarriers were decorated with ascorbate by modifying the 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG2kDa-NH2 with ascorbic acid.

MATERIALS AND METHODS Cell lines, mouse fibroblast NIH/3T3 and melanoma B16-F10, human glioblastoma LN-18 and neuroblastoma SK-N-AS, rat glioma C6 and F98, were purchased from the American Type Culture Collection (Manassas, VA). All cell culture media, RPMI, heat-inactivated fetal bovine serum (FBS), and penicillin/ streptomycin stock solutions were purchased from Cellgro (Herndon, VA). LAB-TEK 4-well cell culture chambers were purchased from Nunc (Rochester, NY). Goat polyclonal antibody anti-SVCT2 transporter (G-19) and donkey antigoat IgG-FITC were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz Biotechnology, CA). Goat antimouse IgG antibody was from ICN Biomedical (Aurora, OH). Egg phosphatidylcholine (PC), cholesterol, 1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[methoxy (poly(ethylene glycol))-2000] (mPEG2kDa-PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(poly(ethylene glycol))2000] (PEPEG2kDa-amine) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamineN-(lissamine rhodamine B sulfonyl) (Rh-PE) were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). Bovine serum albumin and all other chemicals and buffer solution components were from Sigma (St. Louis, MO) and were of analytical grade. Sephadex G25 superfine medium was from GE Healthcare BioSciences Corp. (Piscataway, NJ, USA). Synthesis of 6-Bromodeoxy-ascorbic Acid. The preparation of the 6-bromo derivative of ascorbic acid was performed

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according to the published procedure (18) with some modifications. Two grams of ascorbic acid (11.35 mmol) was added to 2.7 mL of 33% w/v hydrobromic acid in glacial acetic acid (17.18 mmol). The suspension was stirred overnight at room temperature. Afterward, hydrobromic and acetic acid were removed by nitrogen flux and rotary evaporation. Five milliliters of deionized water was added to the syrup, and the mixture was stirred at 60 °C for 30 min. After reduction of the volume by rotary evaporation, the residue was extracted 6 times with 5 mL of ethylacetate, and traces of water were removed by sodium sulfate treatment. The organic solvent was removed under reduced pressure, and the resulting solid product was dissolved in 3 mL of acetonitrile warmed to 70 °C. The solution was cooled to 4 °C to allow crystallization. The white crystals were redissolved and recrystallized according to the same procedure. The final product was recovered by filtration and dried under the vacuum overnight. 6-Bromodeoxy-ascorbic acid dissolved in DMSO-d6 was analyzed by 1H NMR and 13C NMR using a Varian 500 MHz spectrometer. 1H NMR: δ 3.36 (dd, 1H, J ) 10.06, δ 7.14 Hz, BrCCHH), δ 3.60 (dd, 1H, J ) 10.06, 6.56 Hz, BrCCHH), δ 3.96 (t, 1H, J ) 6.67 Hz, BrCH2CHOH), δ 4.83 (broad s, 1H, OCH), δ 5.63 (broad s, 5.63, 1H, BrCH2CHOH), δ 8.42 (s, 1H, OCOCOHCOH), δ 11.66 (s, 1H, OCOCOHCOH). 13C NMR: δ 34.6 (BrC), 68.2 (BrCC), 75.2 (OCH), 152.3 (OCCOH), 118.1 (OCCOHdCOH), 170.3 (OCdO). Mass spectroscopy analysis was carried out by MALDI-TOF using 2,5dihydroxybenzoic acid as a matrix. The negative ion was found to have 237.05 m/z. Synthesis of 6-Ascorbate-PEG-PE. Into a 25 mL roundbottomed flask, 2.8 mL of a 25 mg/mL solution of PE-PEG2kDaamine in chloroform was added (25 µmol, 70 mg). The solvent was removed by rotary evaporation, and the solid residue was dissolved in 2 mL of DMF. Then, 60 mg of 6-bromodeoxyascorbic acid (250 µmol) and 35 µL of triethylamine (250 µmol) were added. The solution was stirred overnight under argon in the dark. The mixture was precipitated by dropwise addition to cold diethyl ether under stirring. The crude precipitate was washed several times with diethyl ether, and the solid was dried under vacuum. The dry powder was dissolved in 2 mL of deionized water, and the derivative was purified by size exclusion chromatography using a column prepacked with Sephadex G25 superfine medium eluted with milli-Q water. Fractions positive both to UV analysis and iodine test were pooled together and lyophilized. Yield was 40 mg (57%). In order to assess the PEG/ascorbate molar ratio in the conjugate, iodine test for PEG (19), UV spectroscopic analysis for ascorbate (20), and Snyder colorimetric test for the residual PEG unconjugated amino groups (21) were carried out on a weighed amount of the purified derivative. 6-Ascorbate-PEGPE dissolved in CDCl3 underwent 1H NMR spectroscopic analysis. 1H NMR: δ 3.73 (d, 1H, OCHCHOHCH2 of ascorbate), δ 3.642 (s, ∼180 H, -[OCH2CH2]n- of PEG), δ 2.308 (m, 4H, OCOCH2 of phospholipid stearate), 1.249 (m, 56 H, -[CH2]n- of phospholipid stearate). Preparation and Characterization of Liposomes and Micelles. Rhodamine-labeled targeted liposomes were prepared by thin film hydration technique. A lipid film was obtained by removing organic solvent from the chloroform solution of 6-ascorbic acid-PEG-PE, egg PC, cholesterol, and Rh-PE in 3/60/30/0.25 molar ratio. The lipid film was suspended in RPMI, pH 7.4, containing 300 µM Tris(2-carboxyethyl)phosphine (TCEP) at a total lipid concentration of 0.435 mg/mL and sonicated in a bath sonicator for 10 min, followed by 11 passages through a mini-extruder equipped with 200 nm pore size polycarbonate filter (Avanti Polar Lipids). Untargeted

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liposomes were prepared according to the same procedure using mPEG2kDa-PE instead of 6-ascorbic acid-PEG-PE. Rhodamine-labeled targeted micelles were prepared by dissolving 6-ascorbic acid-PEG-PE in chloroform. To that solution, 1 mol % of Rh-PE was added. The organic solvent was then removed by rotary evaporation, and the film was rehydrated at a final concentration of 1 mg/mL in RPMI containing TCEP 300 µM. Untargeted micelles were prepared using mPEG2kDaPE according to the same procedure. The micelle and liposome sizes and size distributions were measured by dynamic light scattering (DLS) using a Zeta Plus instrument (Brookhaven Instrument Corporation, Holtsville, NY). The sample suspensions were analyzed after the appropriate dilution required for DLS. For each sample, size distribution measurements were performed six cycles per run. The zeta-potential of liposome and micelle formulations was measured by a Zeta phase Analysis Light scattering (PALS) with an ultrasensitive Zeta Potential Analyzer instrument (Brookhaven Instruments, Holtsville, NY). The micelle and liposome suspensions were diluted properly with a 1 M KCl solution. All zeta-potential measurements were performed in triplicate. Assessment of SVCT2-Expressing Cell Lines by Flow Cytometry. In order to select immortalized cell lines, which express SVCT2 transporter on plasma membrane, cells (NIH/ 3T3, B16-F10, LN-18, SK-N-AS, C6, and F98) were grown in RPMI with 10% FBS in 10 cm tissue culture plates for 72 h. Medium was removed; cells were washed with RPMI and detached by prechilling incubation for 15 min and pipetting. Cells were recovered by centrifugation at 1500 rpm, and 1 million cells were resuspended in 50 µL of PBS containing 3% FBS. Three microliters of a 200 µg/mL goat polyclonal anti SVCT2 transporter (G-19) or goat antimouse IgG antibody was added to each sample and incubated in the dark in an ice-cold bath for 30 min. Cells were then washed once with 1 mL of PBS containing 3% FBS, recovered by centrifugation, resuspended in 50 µL of the same medium, and treated with 3 µL of 200 µg/mL donkey antigoat IgG-FITC for 30 min in the dark in the ice bath. After washing with PBS containing 3% FBS, samples were fixed with 2% paraformaldehyde in PBS and analyzed by FACS (10 000 cells in average count) using BD FACSCalibur flow cytometer and BD CellQuest Pro software (Beckton Dickinson Biosciences, San Jose, CA). Cells were livegated upon acquisition using forward versus side scatter to exclude debris and dead cells. Ascorbate-Mediated Targeting of Pharmaceutical Nanocariers. FACS Analysis. In order to assess the cell-targeting capacity of ascorbate-conjugated nanocarriers, SVCT2-expressing cells incubated with liposomal or micellar formulations were subjected to the FACS analysis. SVCT2-expressing cells (C6, F98) were grown in 10 cm cell culture plates for 48 h. Cells were washed with serum-free medium, detached by prechilling incubation and repetitive pipetting, recovered by centrifugation, and 1 million cells were resuspended in serumfree medium containing fluorescently labeled either targeted or untargeted nanocarriers prepared as described. Samples were incubated at 37 °C in the dark with gentle shaking for 90 min. Afterward, the cell samples were recovered by centrifugation, washed twice with PBS, and gated using forward versus side scatter to exclude debris and dead cells and analyzed (10 000 cells in average count) using BD FACSCalibur flow cytometer and BD CellQuest Pro software. Epi-Fluorescence Microscopy. C6 and F98 cells were investigated using the fluorescence microscopy after incubation with fluorescently labeled targeted liposomes and micelles or untargeted nanocarriers as a reference. After the initial passage in tissue culture flasks, cells were grown in four-well tissue culture

Salmaso et al. Scheme 1

detachable LAB-TEK chambers, at a concentration of 5 × 105 cells per well in RPMI with 10% FBS. After 24 h, the chambers were washed twice with RPMI and then incubated at 37 °C with 0.5 mL of liposome or micelle formulations prepared as described in RPMI. After 1 h incubation, the medium was removed, and the plates were washed with medium three times. Samples were further incubated for 10 min at 37 °C with complete medium containing 0.5 µg/mL of Hoechst 33342 and washed twice with complete medium. Individual slides were covered with coverslips. Cells were observed immediately on a Nikon Eclipse E400 fluorescence microscope equipped with appropriate filters for bright light, Rhodamine and Hoechst detection, and a Nikon N60 camera. For competition studies with ascorbic acid, cells grown on LAB-TEK chambers were washed twice with RPMI and incubated at 37 °C with 0.5 mL of 200 µM ascorbic acid in RPMI for 30 min followed by incubation for an additional 1 h with 6-ascorbate-PEG-PE-containing liposomes (0.464 mg/mL of total lipids). Samples were then washed three times with PBS and mounted with the fluormount medium. Cells were observed immediately by fluorescence microscopy as described, and the fluorescence intensity associated with the cells was quantified by image analysis software (ImageJ).

RESULTS Synthesis and Characterization of 6-Ascorbate-PEG-PE and Ascorbate-Conjugated Nanocarriers. The preparation of the 6-ascorbate-PEG-PE was carried out by a two-step procedure: (1) activation of ascorbic acid with bromine; (2) synthesis of 6-ascorbate-PEG-PE by PE-PEG2kDa-amine reaction with excess of 6-Br-ascorbic acid. The procedure adopted to synthesize the 6-bromodeoxy ascorbic acid allowed, after the crystallization, a 40.5% yield. The 1H NMR spectroscopic analysis showed that hydroxyl groups of ascorbate in positions 2, 3, and 5 are unmodified as confirmed by the corresponding signals at δ 8.42, 11.66, and 5.63, respectively. Mass spectrometry analysis of 6-bromo-ascorbic acid showed a signal at 237.05 m/z, which has been attributed to the monodeprotonated ion [6-bromoscorbate-H+]-1. The reaction of the commercially available PE-PEG2kDa-amine with a 20-fold molar excess of 6-bromo-ascorbate (Scheme 1) allowed for obtaining 6-ascorbate-PEG-PE in 12 h as determined by the disappearance of the primary PEG amino group by ninhydrin assay. The conjugation was carried out in anhydrous conditions in order to avoid water deactivation of the 6-bromoascorbate. The crude product was precipitated in ether and dissolved in water to allow the formation of the micelles. The conjugate was purified by size exclusion chromatography from the unreacted 6-bromo-ascorbate yielding 70% product recovery. UV-vis, iodine, and Snyder colorimetric tests were carried out on a 1



Figure 1. Characterization of SVCT2 expression in different cells. Cells were treated and analyzed by flow cytometry to evaluate expression of SVCT2 transporter. (A,D) C6 cells; (B,E) F98 cells; (C,F) NIH/3T3 cells. Above: cells treated with isotype control. Below: cells treated with goat polyclonal antibody anti SVCT2 transporter.

mg/mL water solution of 6-ascorbate-PEG-PE in order to assess ascorbate, PEG, and free amino groups, respectively. According to the tests, the conjugation yield was 98.9%, i.e., practically complete conjugation of the amino-terminating PEG-phospholipid to ascorbate was achieved by the procedure reported. The chemical identity of the conjugate was confirmed by 1H NMR spectroscopy that showed typical signals of phospholipids at δ 2.308 and 1.249, PEG at δ 3.642, and ascorbate at δ 3.73. The newly synthesized ascorbate-conjugated moiety was incorporated into liposomes or lipid-core PEG-PE micelles. As shown by the dynamic light scattering analysis, the lipid film rehydration techniques and extrusion yielded liposomal formulations of a similar size: 175.1 ( 21.2 nm and 181.9 ( 24.9 nm for untargeted and targeted liposomes, respectively. Micelle size was found to be 13.65 ( 5.1 nm and 21.3 ( 2.4 nm for mPEG2kDa-PE and 6-ascorbate-PEG2kDa-PE micelles, respectively. A slight increase in the micelle size can be ascribed to the additional presence of ascorbate moieties in the outer surface of the micelles. Zeta-potential analysis of the lipid-based nanosystems showed that the presence of the ascorbate on their surface enhances their negative character due to the dissociation of the acid. PEGylated liposomes and PEG-PE-based micelles had zeta-potential of -12.8 ( 3.2 mV and -13.2 ( 2.3 mV, respectively, while ascorbate-conjugated liposomes and micelles showed a slight increase in the negative surface charge with zeta-potentials of -16.4 ( 4.2 mV and -19.2 ( 7.3 mV, respectively. Liposome and micelle sizes and zeta-potentials were unaffected by the presence of the reducing agent TCEP. Selection of SVCT2-Expressing Cell Lines. In order to select a proper cell model suitable for further targeting studies, a panel of immortalized cell lines was tested to identify those expressing SVCT2 translocator on the plasma membrane by using saturating amounts of specific antibodies against the extracellular domain of SVCT2 protein. SK-N-AS (human neuroblastoma), LN18 (human glioblastoma), C6 and F18 (rat gliomas), NIH 3T3 (mouse fibroblasts), and B16-F10 (mouse melanoma) cells were investigated.

Cell lines were grown in an appropriate medium and detached by prechilling incubation and pipeting to prevent surface proteins from being proteolitically degraded by trypsin treatment, which is commonly used to recover cells from culture plastic surfaces. Detached cells were first incubated with goat polyclonal IgG anti SVCT2 transporter, which has selectivity for the detection of SVCT2 of mouse, rat, and human origin as stated by the provider. After washing, cells were incubated with donkey FITC-labeled antigoat antibody as a secondary antibody, and the degree of binding was determined by FACS analysis. Isotype control was performed by incubating cells with goat antimouse IgG in order to detect nonspecific antibody binding on the cell membrane. As shown in Figure 1, 43.9%, 41.1%, and 28.9% of C6, F98, and NIH/3T3 cells stained positive for the SVCT2 expression, respectively (panels D, E and F), while nonspecific immunostaining with the isotype control was about 5% only (panels A, B, and C, respectively). When the FACS analysis was carried out with murine LN18, SK-N-AS, and B16F10 cell lines treated with the monoclonal anti SVCT2 transporter, only a nonspecific weak immunoreactivity was detected, and the cell-associated fluorescence increase accounted for about 5% regardless of cell incubation with goat polyclonal IgG anti SVCT2 transporter or goat antimouse IgG antibody. The FACS experiments demonstrated that such cell lines do not express constitutionally the plasma membrane SVCT2 transporter. Nanocarrier Targeting Studies. FACS Analysis. The ability of ascorbate-conjugated nanocarriers to selectively target SVCT2 transporter-expressing cells was evaluated using FACS analysis after an hour and a half incubation with liposomes or micelles. Ascorbate-free PEGylated liposomes or mPEG2kDa-PE micelles were used as control. Two different cell lines of rat origin, C6 and F98 glioma cell lines, both expressing SVCT2 transporter, were incubated with liposomes containing 3% of 6-ascorbatePEG-PE or with 6-ascorbate-PEG-PE micelles fluorescently labeled with Rh-PE. As depicted in Figure 2, FACS analysis showed that ascorbate conjugation increases C6 cells targeting by 23.2% and 10.1%


Salmaso et al.

Figure 2. FACS analysis of rat C6 cells untreated (A,D), and after 1.5 h treatment with rhodamine-labeled PEG-liposomes (B), ascorbate-PEGliposomes (C), mPEG2kDa-PE micelles (E), and ascorbate-PEG-PE micelles (F).

Figure 3. FACS analysis of rat F98 cells untreated (A,D), and after 1.5 h treatment with rhodamine-labeled PEG-liposomes (B), ascorbate-PEGliposomes (C), mPEG2kDa-PE micelles (E), and ascorbate-PEG-PE micelles (F).

when targeted liposomes and micelles were employed, respectively (pannels C and F), compared to ascorbate-free PEGliposomes and mPEG2kDa-PE micelles controls. Nonspecific cell targeting was shown to account for about 9% and 3% fluorescence increase for PEGylated liposomes and mPEG2kDa-PE micelles (panels B and E, respectively) with respect to untreated cell samples (panels A and D, respectively). Cells were analyzed as a whole without gating (data not shown). Figure 3 reports the results obtained by FACS analysis to evaluate the targeting efficiency of ascorbate-conjugated nanocarriers toward F98 glioma cell line. F98 cells fluorescence

increase was remarkably higher compared to C6 cells when cell samples were treated with ascorbate-decorated liposomes (C) and micelles (F), 42% and 40.1%, respectively. A stronger nonspecific cell-associated fluorescence was also found on the F98 cell line for nontargeted liposome and micelles, 19.1% and 45.2%, respectively (B and E), compared to the C6 cell line, which could be explained by the higher nonspecific uptake activity of these cells. Epifluorescence Microscopy. To additionally evaluate whether the ascorbate can be employed as a targeting moiety to deliver pharmaceutical nanocarriers to SVCT transporter-expressing


Figure 4. Epifluorescent microscopic examination of C6 cell line treated with fluorescently labeled PEG-liposomes (A,B,C) and ascorbate-PEGliposomes (D,E,F). Cell images were acquired in a blue channel for nuclei detection after staining with Hoechst 33342 (panels A and D) and red channel for Rh-PE-labeled liposome detection (panels B and E). Panels C and F are the superimpositions of the two channels. Images were taken at 100× magnification.

cells, C6 cells were also investigated using fluorescence microscopy after the incubation with fluorescently labeled liposomes and micelles. Nuclei were stained by Hoechst 33342 treatment (Figure 4, blue fluorescence on panels A and D). The epifluorescence micrographs obtained clearly support ascorbatePEG-liposome targeting to the investigated glioma cells. In fact, strongly fluorescent red spots associated with the cells were clearly visible (panel E in Figure 4 shows the red fluorescence of targeted liposomes; panel F shows superimposition of blue and red fluorescence). On the contrary, the treatment with nontargeted PEGylated liposomes did not result in any defined fluorescent spots associated to the glioma cells, indicating the lack of targeting toward this cell line (panel B in Figure 4 shows undetactable red fluorescence of PEG-liposomes; panel C shows superimposition of blue and red fluorescence). It is important to note here that PEGylated liposomes are commonly recognized for their stealth properties, which reduce the interaction with biological surfaces. The ascorbate-mediated targeting was investigated also after the treatment of C6 cells with rhodamine-labeled micelles as shown in Figure 5. Panels A and D report nuclei staining by the Hoechst treatment. Panels B and E report red fluorescence of cells treated with untargeted micelles and ascorbate-PEGPE micelles, respectively, while panels C and F are the superimpositions of the two channels. Cells treated with ascorbate-conjugated micelles showed small punctuate red fluorescent spots (panels E and F), which might be due to aggregation of micelles and uptake of the aggregates by C6 glioma cells. Competition studies were performed with epifluorescence microscopy by C6 cells preincubation with free ascorbic acid followed by the treatment with rhodamine-labeled ascorbatePEG-liposomes. The presence of ascorbic acid in the medium dramatically reduced the cell-associated fluorescence as depicted in Figure 6 (panel B) compared to cell samples treated with targeted liposomes only (Figure 6, panel A). The preincubation of cell samples with ascorbic acid followed by the treatment with ascorbate-PEG-liposomes reduced the cell-associated fluorescence induced by targeted nanosystems by 7-fold as determined by the image analysis (Figure 7).

DISCUSSION In the present study, we have investigated the targeting capacity of ascorbic acid moiety to confer selectivity to pharmaceutical


Figure 5. Epifluorescent microscopic examination of C6 cell line treated with fluorescently labeled PEG-PE micelles (A,B,C) and ascorbatePEG-PE micelles (D,E,F). Cell images were acquired in a blue channel for nuclei detection after labeling with Hoechst 33342 (panels A and E) and red channel for rhodamine-labeled micelles detection (panels B and E). Panels C and F are the superimpositions of the two channels. Images were taken at 100× magnification.

Figure 6. Epifluorescent microscopic examination of C6 cells treated with fluorescently labeled ascorbate-PEG-liposomes without (A) and with (B) ascorbic acid preincubation. Images were acquired in a red channel for rhodamine detection. Panels C and D show the bright light acquisition of the same samples. Images were taken at 40× magnification.

Figure 7. Relative fluorescence intensity associated with C6 cells after treatment with rhodamine-labeled ascorbate-PEG-liposomes with or without preincubation with free ascorbic acid.

nanocarriers. A new in vitro cellular model was set up to evaluate the targeting activity of ascorbate-conjugated nanocarriers. Ascorbic acid was activated as a bromo derivative, and the chemical identity of the intermediate was confirmed by 1H NMR


and mass spectroscopy. Activation at the carbon in position 6 of ascorbic acid was chosen according to reported data which showed the biological activity and the selective binding capacity of the 6-deoxy-ascorbic acid derivatives toward the SVCT2 transporter (22). Activated 6-bromodeoxy-ascorbic acid was reacted in anhydrous condition with the primary amino group of commercially available PE-PEG2kDa-amine by using excess of the 6-bromoascorbic acid. At the end, the excess of reactive ascorbic acid was removed by precipitation of the reaction mixture in diethyl ether and size exclusion chromatography that allowed for obtaining a product with high purity. The identity of the conjugated product was investigated by 1H NMR and UV spectroscopy and colorimetric tests for assessing ascorbate and PEG in the conjugate. The tests confirmed high conjugation yield and 1/1 ascorbic acid/PEGPE molar ratio. In order to maintain the conjugated ascorbic acid in the reduced form, which binds selectively SVCT transporter, buffered solutions were kept in the dark and in the presence of TCEP as reducing agent, which allowed the regeneration of traces of oxidized ascorbate eventually formed during the conjugation and purification processes (20). The targeting moiety was incorporated into pharmaceutical nanocarriers, such as liposomes and polymeric lipid-core micelles, by the thin film rehydration method that allowed for obtaining liposomes and micelles with defined dimensions and surface properties. As assessed by photocorrelation spectroscopy, targeted and untargeted liposomes had diameters of about 180 nm and negative zeta potential, which was expectedly increased by the presence of ascorbic acid. Micelle dimensions were found to be in a close range (15-20 nm) regardless of the presence of ascorbate on the surface. Slight increase of the negative zetapotential was found for ascorbate micelles similar to liposomes. In order to set up a cell model to evaluate the targeting performance of ascorbate-decorated nanocarriers, a panel of diverse immortalized cell lines underwent investigation for the expression of SVCT2 transporter by immunostaining and flow cytometry analysis. Keeping in mind the future use of this approach for brain-targeted delivery, we have chosen for investigation a panel of cell lines related to the brain glia (LN18, SK-N-AS, C6, and F98) as well as cells with potential involvement in processing of ascorbic acid (NIH/3T3 and B16-F10) (23, 24). Cells were treated with a goat polyclonal anti SVCT2 trasporter antibody recognizing an extracellular epitope of the protein. Among the cells scanned, two rat glioma cells, C6 and F98, were shown to express plasma membraneassociated SVCT2 transporter. NIH/3T3 cells also gave positive test results. This cell-line phenotypic characterization technique is new with respect to many SVCT transporter expression studies reported in the literature, which were aimed at assessing solely the presence of RNA encoding for such protein, rather than its disposition on the cell surface (25). The partial positivity of the cellular population to the immunological characterization, which results in only partial shift of the cells in Figure 1, can be explained by variation of the ascorbate transporter expression throughout the cell cycle. In light of such immunologic results, it is conceivable that C6 and F98 glioma cells represent a good cancer cell model to analyze a possible use of the SVCT2 transporter for specific targeting of pharmaceutical nanocarriers. Such a model can be complementary to reported primary cell culture (13, 26) and SVCT2-transfected Xenopus oocytes models (22) for investigation of drug delivery systems intended to target SVCT2expressing cells. Besides, such a study introduces the perspective that the SVCT2 transporter can be a good ligand to target the brain through the choroid plexus, where it is selectively expressed, and allow drug delivery systems to reach SVCT2expressing tumor cells.

Salmaso et al.

On the basis of the results originating from the immunological characterization, we have studied the targeting capacity of ascorbate-conjugated liposomes and micelles toward C6 and F98 glioma cells. Flow cytometry analysis of C6 and F98 cells treated with targeted liposomes and micelles labeled with RhPE showed that both nanosystems bind with the target cells much better than nontargeted ascorbate-free nanocarriers used as controls. FACS analysis displayed that untargeted liposomes and micelles associate to a different extent with C6 and F98 cell lines. Nonspecific cell uptake of untargeted liposomes was lower with both cell lines, C6 and F98 (panels B in Figure 2 and 3, respectively), testifying to better “stealth” properties of PEGylated liposomes compared to micelles. Instead, nonspecific cell uptake of mPEG2kDa-PE based micelles is detectable with C6 cells (Figure 2, panel E) but more remarkable in the case of F98 cells (Figure 3, panel E). The different behavior may be ascribed to the diverse physical stability of the two nanosystems. This can allow for membrane fusion of micelles rather than liposomes within the same cell line. On the other hand, the different surface properties of the two cell lines investigated may affect the biological behavior of a given nanosystem. However, the unspecific uptake does not prevail over the effect of ascorbate-mediated cell targeting for both nanosystems. The capacity of ascorbate-conjugated liposomes and micelles to selectively interact with SVCT2-expressing cells was also investigated with glioma C6 cells by fluorescence microscopy. It was found that the cell-associated liposomes (the quantity of cellassociated rhodamine fluorescence) were clearly visible already after 1 h contact with targeted liposomes (Figure 4). Well-defined red spots were found to be coupled to the cells; they may be deposited either on the outer side of the plasma membrane or inside the cytosol undergoing intracellular trafficking. The increase in cellassociated fluorescence observed for ascorbate liposomes compared to nontargeted liposomes may be explained by the ascorbatemediated cellular uptake. Samples treated with nontargeted liposomes do not show red fluorescence (Figure 4, panels B and C). Similar patterns were observed when C6 cells were imaged by fluorescence microscopy after the treatment with ascorbate-targeted and nontargeted PEG-PE micelles (Figure 5). These results clearly demonstrate targeting selectivity of the ascorbate-conjugated nanocarriers toward these cells. In order to confirm the specific addressing of SVCT transporter by ascorbate-conjugated nanocarriers, we have performed the competition study in the presence of an excess of free ascorbic acid. Preincubation of tumor cells with free ascorbic acid strongly reduced the ascorbate-liposome cellular association confirming that the nanosystems selectively target the ascorbate cell membrane transporter. Nanosystems that target solely the ascorbic acid transporter can potentially reach and accumulate in the brain due to local expression of SVCT2, which is limited to choroid plexus epithelial cells, some neurons, tanycytes, astrocites, and the arachnoid membrane (7). The conjugation of the ascorbate moiety to nanocarriers via the carbon in position 6 was shown to preserve its binding recognition for SVCT2 transporter and avoid binding to the glucose (GLUT) transporters, which are targeted by the oxidized ascorbic acid. Furthermore, the eventually oxidized form of conjugated 6-deoxyascorbate cannot bind the GLUT transporters, which are spread over the whole body (22). The targeting of the glucose transporter by ascorbate-conjugated pharmaceutical nanocarriers was unlikely, since the cells were treated with the medium containing 2 g/L of glucose that competes with the oxidized ascorbate if any of its trace is present. According to these considerations, ascorbate moiety can be considered as a potential site-selective targeting agent for brain delivery. As reported in the literature, great efforts have focused on targeting the brain with nanosystems having adequate dimension,


morphology, and surface properties. Systemically administered polymeric nanoparticles were shown to be filtered through fenestrated capillaries of the CP and were rapidly transported into the CSF by absorptive transcytosis (27). Reported nanosystems showed promising performance, which could be ascribed to the tiny dimensions and decoration with a proper BBB transporting vector. Some peculiar features make such colloidal systems potential tools for brain drug delivery and support new perspectives that the CP can be exploited as a physiologic gateway to reach the brain. With this in mind, adequate nanosystems can be designed, which filter off the capillaries and bind to SVCT2 transporters of the choroid ependymal cells (12). At this site, they can either release their payload, which can diffuse into the brain, or undergo transcytosis allowing for further delivery toward the brain. This study has also highlighted that ascorbic acid can be employed as a targeting agent to promote the disposition of drugloaded nanosystems to neoplastic tissues (namely, the gliomas). Our data may facilitate the potential therapeutic use of ascorbateconjugated nanosystems for the treatment of some brainassociated diseases.

ACKNOWLEDGMENT This research was based in part on a concept suggested by Anthony Manganaro.

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Targeting Glioma Cells in Vitro with Ascorbate-Conjugated

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