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Targeting Tumor Cells through Chitosan-Folate Modified Microcapsules Loaded with Camptothecin Alice Galbiati,† Claudio Tabolacci,† Blasco Morozzo Della Rocca,† Palma Mattioli,† Simone Beninati,† Gaio Paradossi,‡ and Alessandro Desideri*,† †
Department of Biology Department of Chemical Sciences and Technologies University of Rome Tor Vergata, Via della Ricerca Scientifica, Rome 00133, Italy
‡
bS Supporting Information ABSTRACT: Poly(vinyl alcohol) microcapsules have been tailored as carriers to deliver camptothecin, an anticancer drug poorly soluble in water. The capsules have been reacted with a chitosanfolate complex in order to selectively target cancer cells overexpressing the folic acid receptor. Microcapsules decorated with the chitosanfolate complex have been characterized in their uptake and release of camptothecin, following the absorption band at λ = 370 nm diagnostic of the drug molecule. The selectivity of chitosanfolate microcapsules in targeting cancer cells has been demonstrated by fluorescence microscopy using HeLa cells, overexpressing the folate receptor and NIH3t3 fibroblasts as a negative control. The chitosanfolate microcapsules loaded with camptothecin significantly reduce the proliferation of HeLa tumor cells, while they have a negligible effect on fibroblasts. This work demonstrates that the chitosanfolate microcapsules represent a promising system to selectively target hydrophobic drugs, such as camptothecin, to tumor cells.
’ INTRODUCTION Camptothecin (CPT) and its derivatives are chemical poisons that specifically target topoisomerase I, forming a stable ternary complex with the enzyme and DNA, inhibiting the unknotting process that is associated with replication and transcription.13 These drugs are used in the treatment of ovarian carcinoma4 (the fifth leading cause of cancer deaths in women) and in the treatment of small-cell lung cancer (SCLC) recurrent disease, which is among the most lethal malignancies.5,6 Unfortunately, such chemical agents display a high degree of toxicity also against normal tissues that show enhanced proliferation rates, such as the bone marrow, gastrointestinal tract, and hair follicles.7,8 Several systems have been devised to better deliver CPT, as those based on lipid nanoparticles,9,10 dendrimers,11 solid lipid nanoparticles,12 and perfluorocarbon nanoemulsions,13 but the main problem remains the selectivity toward tumor cells. Various approaches have been used to selectively target drugs against the tumor1416 such as the use of monoclonal antibodies,17 or systems able to sense the changes in temperature and redox signals,18 pH,19 or membrane potential between normal cells and cancer cells, and similar strategies have been recently applied for the transport of CPT.20,21 A promising strategy concerns the use of ligands specific for membrane receptors differentially expressed on pathological cells.22,23 Folate receptors (FR) are vastly overexpressed in a wide variety of human tumors, including ovarian, endometrial, colorectal, breast, r 2011 American Chemical Society
lung, renal cell carcinomas, brain metastases derived from epithelial cancers, and neuroendocrine carcinomas, but rarely are found on normal cell surface . Folate (FA) has been conjugated to many polymers, such as polyethylenimine,24 poly(L-lysine),25 poly(ethylene glycol), and chitosan,26 to give complexes that can be internalized by cells through a FR-mediated endocytic mechanism.27 Among the various polymers, chitosan displays important useful properties being biodegradable and biocompatible since it can be degraded by lysozymes into N-acetyl-glucosamine, that is subsequently excreted as carbon dioxide via the glycoprotein synthetic pathway.26 For this reason, chitosan has been widely used for different pharmaceutical and medical applications28 and has also been used for gene complexation.2932 The delivery systems made by folate-conjugated polymers increase either the intracellular uptake of proteins or the transfection rate of DNA compared with unmodified polymer delivery systems.33,34 Poly(vinyl alcohol) (PVA) is a water-soluble and injectable polymer, which can be used as the basis for the construction of various structures. PVA has been recently used to produce microbubbles, air-filled biocompatible spherical structures that can be used as novel polymer based ultrasound contrast agents Received: December 3, 2010 Revised: April 28, 2011 Published: May 06, 2011 1066
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Figure 1. General scheme for the synthesis of chitosanfolate modified microcapsules (MC-Chi-FA). The chitosanfolate (Chi-FA) derivative is obtained by reacting chitosan and folate in DMSO using EDC to activate the COOH folate groups. Chi-FA is linked to MC using a reductive amination procedure.
(UCA),35 as well as other already commercially available enhancers for echographic investigations.36 In the presence of ethanol, the air present in PVA microbubbles is replaced by the solvent, giving rise to microcapsules (MC), that retain the spherical shape, with a mean diameter ranging from 3 to 5 μm depending on the preparations.37 They are versatile and biocompatible microstructures whose surface has free OH groups and reactive aldehydes that can be used to functionalize them with different molecules.37 In this work, folic acid has been bound on a flexible arm of chitosan, which has then been conjugated to the PVA microcapsules (Figure 1), in order to be specifically directed against tumor cells of epithelial origin such as the HeLa cells. The loading and release of the antitumor drug CPT has been investigated, in both concentration and rate, for chitosan and chitosan-folate modified and unmodified microcapsules, and the specific interaction with tumor cells has been assayed by fluorescence microscopy. The results indicate that the chitosanfolate modified microcapsules specifically target tumor cells and strongly reduce their proliferation, while having a negligible effect on control cells, suggesting that they can represent an interesting system to be taken in consideration toward applications of targeted delivery.
’ MATERIALS AND METHODS Materials. All solvents and reagents were of analytical grade and were used as received. Poly vinyl alcohol (PVA) was obtained from Sigma Aldrich. Number average molecular weight determined by membrane osmometry was 30 000 (5000 g/mol).
Weight average molecular weight, determined by static light scattering, was 70 000 (10 000 g/mol). Low molecular weight (50 kDa) chitosan, 7585% deacetylated, rhodamine-β-isothiocyanate, (S)-(þ)-camptothecin, 95% HPLC, Triton X-100, folic acid, and dimethyl sulfoxide (DMSO) were Sigma products. Sodium periodate, and inorganic acids and bases, used for telechelic PVA preparation and microballoon synthesis and modifications, were RPE products from Carlo Erba. Cell culture media were obtained from Gibco. Alexa-488-conjugated phalloidin was obtained from Molecular Probes. Instruments. High-resolution 1H NMR spectra were recorded on a Digital NMR Spectrospin AVANCE 300, Bruker. Chitosan and chitosanfolate 3 mg/mL are dissolved in D2O with 3% v/v CD3COOD solution, using tetramethylsilane (TMS) as the internal standard. The release kinetics of the drug were performed using Perkin-Elmer Lambda 2 UV/vis double beam spectrophotometer. Fluorescence microscopy images were obtained with the DeltaVision system (Applied Precision, Washington, USA) equipped with an Olympus inverted microscope IX 70. The image workstation includes SoftWoRx software for digital image acquisition, deconvolution, and optical sectioning. Synthesis of PVA-Coated Microbubbles and Conversion into Microcapsules. Synthesis of telechelic PVA was previously described.38,39 Stable (air-filled) PVA-coated microbubbles were prepared by cross-linking telechelic PVA at the water/air interface. Vigorous stirring at room temperature for 2 h of 2% telechelic PVA aqueous solution (200 mL) by an Ultra-Turrax T-25 at 8000 rpm equipped with a Teflon coated tip generated a fine foam of telechelic PVA acting both as colloidal stabilizer and as air bubble coating agent. The cross-linking reaction was carried 1067
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Bioconjugate Chemistry out at room temperature. Floating microbubbles were separated from solid debris and extensively dialyzed against Milli-Q water. The aqueous suspensions of microbubbles were obtained and used for microcapsule preparation. PVA microbubbles were converted into solvent-filled microcapsules by equilibrating an aqueous suspension in ethanol 70% solution. The microcapsule suspension was then extensively washed by a series of centrifugations at 1000g to remove excess ethanol. Chemical Modifications. Rhodamination of PVA-MC. Suspensions of 1 mg/mL of MC were added with 10 μL of rhodamine-β-isothiocyanate40 solution 2.3 mg/mL in DMSO and stirred for 1 h. The rhodamine excess was removed by at least 5 centrifugation steps at 1000g for 10 min, changing the external medium. Synthesis of Chi-Rod. Chitosan 0.5% (w/v) was suspended in 100 mL of acetic acid 3% (v/v) solution. 50 mg of rhodamine-βisothiocyanate was then added and stirring maintained for 48 h in the dark at 4 °C. The solution was adjusted to pH 9 with NaOH, and unreacted rhodamine-β-isothiocyanate was removed carrying out at least 5 centrifugation steps at 1000g for 10 min. The synthesized compound was extensively dialyzed against Milli-Q-water. Synthesis of Chi-FA and Chi-FA-Rod. 1.2 mmol of EDC (N(3-dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride) were added to 3 mmol of FA dissolved in 50 mL of DMSO, and left to react for 1 h in the dark at room temperature. Then, 50 mL of a 1% (w/w) chitosan or chitosanrhodamine in 20 mM acetate buffer pH 4.7 was added to the reaction. After 16 h, the solution was brought to pH 9 with NaOH, and the compound purified by centrifugation followed by 3 days of dialysis against acetate buffer and extensive dialysis against Milli-Q water using a 12 kDa cutoff dialysis membrane. The compound was then freezedried. Preparation of Chitosan Functionalized Microcapsules. Chitosan was conjugated to microcapsules by reductive amination. Chi or Chi-FA was dissolved in a sodium acetate 0.2 M, acetic acid 0.3 M buffer at pH 4.5 to a concentration of 0.15% (w/v). A 10 mL sample of 1 mg/mL microcapsule aqueous suspension was added with 4 μL of Chi or Chi-FA solution at room temperature. The pH was carefully adjusted to 5.0 with acetate buffer, following the addition of Na(CN)BH3. The resulting suspension was stirred for 1 day at room temperature. The microcapsule suspension was then extensively dialyzed and washed to remove low molecular weight reaction products and unreacted chitosan. Adsorption of the CPT on MC or MC-Chi-FA and Evaluation of Drug Release Kinetics. Suspensions of 1 mg/mL of MC or MC-Chi-FA were added with CPT in DMSO at a final concentration of 100 μM and stirred for 4 days in the dark. Then, three washes were performed by centrifugation at 1000g to remove the drug that was not adsorbed. The concentration of drug in the washing is calculated by spectrophotometer following the absorption at λ = 370 nm. The percentage of CPT adsorbed on microcapsules was indirectly estimated by subtracting the total concentration of drug present in the wash to the total drug used for the adsorption. A suspension of MC o MC-Chi-FA 1 mg/mL (20 mL), previously adsorbed with CPT 100 μM, was resuspended in Dulbecco’s modified Eagle’s medium (D-MEM) without phenol red, with or without 10% fetal calf serum (FCS), and subdivided into 20 aliquots of 1 mL. The MC was kept in an incubator shaking at 140 rpm, 37 °C. At different times, the samples were
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centrifuged for 5 min at 2000g and the supernatant was read spectrophotometrically at λ = 370 nm. Absorption values were fitted with the equation A = Amax[1 exp(Kt)] to estimate the time constant of the kinetics (K1) and maximum release Amax. Cell Culture and Proliferation Assay. HeLa cells were cultured in RPMI-1640 and NIH3t3 cells were cultured in D-MEM, supplemented with 10% fetal calf serum (FCS), 200 mM glutamine, 100 U/mL penicillin, and 0.1 mg/mL streptomycin, and maintained in humidified atmosphere of 5% CO2 at 37 °C. Cells were harvested twice a week and used at about 80% confluence. For proliferation studies, cells were detached with trypsin/ EDTA for 5 min and centrifuged. Cells were resuspended in RPMI-1640 medium without folate and seeded in 96-well culture dishes (2.5 103 cells per well) and cells were left to grow for 48 h. After this time, medium was refreshed and cells were incubated for additional 48 h with MC, MC-Chi, and MC-Chi-FA (either adsorbed with CPT or not). At the end of incubation, medium was refreshed again, and after 48 h, the percentage of cell proliferation was evaluated by the sulforhodamine assay,41 calculating the values as a percentage of the control, considered to be 100%. Proliferation assays were also carried out by cell counting using a Neubauer modified chamber. In this case, cells are seeded in 12-well culture dishes at 7.5 103 cells per well. This protocol was established in order to discriminate between the antiproliferative effect due to the diffusion of CPT released from the microcaplules in the media and the one due to the direct cellmicrocapsule interaction. Microcapsule Localization. For fluorescence studies, cells were grown in Lab-Tek chamber slides (Nunc Inc.) in RPMI1640 medium without folate and treated with MC as described previously. After 48 h of incubation, cells were washed, fixed with 3.8% paraformaldehyde solution in phosphate buffered saline, and permeated by using 0.1% Triton X-100. Actin filaments were labeled by using Alexa-488-conjugated phalloidin according to the manufacturer’s instructions. Fluorescent signals were detected and photographed using a Delta Vision 3.0 microscope. To assess the direct involvement of folate mediated interaction, localization experiments have been carried out also in the presence of free folate. After 48 h of culture in folate-free medium, MC-Chi-FA were given to cells in either folate-free or folate-supplemented medium (final concentration 4 mg/L). Cells were fixed and stained after the 2 day treatment and localization assays were performed as described.
’ RESULTS AND DISCUSSION ChitosanFolate Complex Formation and Functionalization of Microcapsules. Chitosanfolate has been synthesized
using published protocols42 by reacting the NH2 group of chitosan with the COOH groups of folate (Figure 1). The γcarboxylate group is the most reactive one, although the Rcarboxylate is also available for conjugation. The reaction has been verified through 1H NMR spectroscopy that is not able to distinguish between the two contributions. The 1H NMR spectrum of chitosan (Figure 2a) shows a series of peaks corresponding to protons linked to the C3, C4, and C2 carbon atoms of the sugar and to the methyl protons, falling at 3.90, 3.77, 3.18, and 2.07 ppm, respectively. The degree of chitosan deacetylation, obtained from the ratio of the integrals of the methyl peak over the C2 one, is found to be 85%. The NMR proton spectrum of chitosanfolate (Figure 2b), when
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Figure 2. NMR spectra of chitosan and of the chitosanfolate derivative. (a) 1H-NMR spectrum of chitosan solution in D2O with 3% (v/v) CD3COOD; (b) 1H-NMR spectrum of 3 mg/mL of the chitosanfolate adduct in D2O with 3% (v/v) CD3COOD. Inset: Chemical structure of the chitosanfolate complex. In gray, the numbering of carbons of chitosan; and in black, the numbering of folate (see text for peak assignments).
Figure 3. Fluorescence microscopy of decorated MC, adsorbed with CPT. Fluorescence microscope images of MC labeled with chitosan rhodaminefolate, and absorbed with camptothecin. Left panel shows the rhodamine signal (red) on MC, the middle one the CPT signal (blue), and the right one the overlay of the two signals (violet).
compared to chitosan, shows additional peaks at 8.73, 7.66, and 6.83 ppm due to the folate aromatic rings and one at 2.40 ppm due to the protons linked to C22 (see inset of Figure 2 for the labeling of the different groups). The ratio between the integral of one of the folate aromatic peaks and the chitosan C2 peak at 3.14 ppm allows us to quantify the percentage of folate substitution on the chitosan NH2 groups that is found to be 5%. On the basis of nominal molecular weight, this percentage corresponds to 15 folates per chitosan. Chitosan (Chi) and chitosanfolate (ChiFA) have been also functionalized with rhodamine (Rod)43 to give the Chi-Rod and Chi-FA-Rod complex. All the synthesized complexes have been then conjugated to microcapsules (MC) using the reductive amination procedure in the presence of Na(CN)BH3,35,37 to yield MC-Chi, MC-Chi-FA, and their rhodaminated counterparts MC-Chi-Rod and MC-Chi-FA-Rod.
The success of decoration and the ability of the modified MC’s to incorporate CPT has been verified by incubating the microcapsules with 100 μM CPT for four days and observing their fluorescence microscopy images. Figure 3, representing the MCChi-FA-Rod system, shows that the red signal coming from rhodamine (λex = 540 nm, λem = 625 nm, left panel) colocalizes with the blue signal originating from CPT (λex = 370 nm, λem = 420 nm, middle panel), since they have an identical shape. The colocalization is confirmed by the overlay of the two images (right panel), that gives spheres characterized by a violet color. To our knowledge, this is the first time that CPT adsorbance on PVA MC is reported. CPT Adsorption and Release. The adsorption of the drug has been carried out incubating the MC or MC-Chi-FA with camptothecin for 4 days with continuous stirring. The microcapsules have then been extensively washed in water and the concentration of drug bound on the MC’s has been indirectly estimated quantifying the drug present in the washes by absorption spectroscopy measurements at λ = 370 nm, corresponding to the maximum in the camptothecin absorption spectrum. Considering an extinction coefficient ε = 17 670 (M cm)1 for CPT at λ = 370 nm, it has been evaluated that MC and MC-ChiFA adsorb about 68% and 82% of the external drug concentration, respectively. When resuspended in D-MEM at 37 °C, the maximum of released drug, in the case of MC, is obtained after one hour, while in the case of MC-Chi and MC-Chi-FA, it is obtained after about 5 h. In detail, fitting the data, reported as percentage of release normalized to maximum released drug, gives rise to a rate of release of 2.4 ( 0.2 h1 for MC, 0.68 ( 0.02 1069
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Bioconjugate Chemistry h1 for MC-Chi, and 0.52 ( 0.02 h1 for MC-Chi-FA (Figure S1). Figure 4 shows the release kinetics of CPT from MC, MCChi and MC-Chi-FA at 37 °C in the culture medium supplemented with 10% serum. The release properties of the microcapsules are influenced by the presence of serum in the medium; in fact, the release rates, obtained by fitting the data as described in the Methods section, correspond to 1.4 ( 0.2 h1 for MC, 1.4
Figure 4. Drug release kinetics from MC. CPT release kinetics in D-MEM medium supplemented with 10% FCS at 37 °C, for nude microcapsules (MC, circles), chitosan microcapsules (MC-Chi, squares), and chitosanfolate microcapsules (MC-Chi-FA, triangles).
Figure 5. Localization of MC on the cells. Fluorescence microscopy image of HeLa tumor cells (up) and NIH3t3 fibroblast cells (down), treated for 48 h with MC (left), MC-chitosan (middle), and MCchitosan-folate (right). The cells have been cultured in folate-free medium.
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( 0.3 h1 for MC-Chi, and 2.7 ( 0.8 h1 for MC-Chi-FA, values larger than those obtained in the absence of serum. Interaction of MC with Different Cell Lines. The ability of MC and MC-Chi-FA to interact with cells has been tested incubating the capsules with two different cell types: HeLa cells, that overexpress the FR, and NIH3t3 fibroblasts, not expressing the receptor on their surface, as a negative control. The cells have been incubated for two days with 100 μg/mL of MC, MC-Chi, and MC-Chi-FA, all labeled with rhodamine-β-isothiocyanate on PVA, and then they have been extensively washed, stained, and imaged. Representative images from these experiments are shown in Figure 5. Control cells, fibroblasts, show no uptake of MC, independent of their functionalization, while HeLa cells exhibit a folate-dependent interaction. In our experiments, the unfunctionalized MC have never been found to interact with HeLa cells, while a small number of adhesions has been occasionally found for MC-Chi, likely due to a fusogenic activity of chitosan. In marked contrast, MC-Chi-FA frequently interact with the surface of HeLa cells and sometimes are also internalized, as reported in Figure S2 showing a large number of microcapsules bound to cells. The internalization of MC-Chi-FA by HeLa cells can be appreciated through a series of optical sections above and below the focal plane of the cell, such as the ones reported in Figure S3, where a representative sequence from outside to inside the cell is shown. A further hint to the nature of folate-mediated interaction is given by competition experiments, in which functionalized MC have been administered to cells together with folate in the medium. The effect of competition is striking, as in this case the uptake of MC-Chi-FA is substantially suppressed (Figure S2). Effect of MC on Cell Proliferation. The impact of MCs on healthy and cancer cells has been followed, incubating cells in the presence of MC, MC-Chi, and MC-Chi-FA, either previously adsorbed with CPT or not. For drug adsorption, the microcapsules have been incubated with 100 μM CPT, and the amount of drug adsorbed was quantified. The quantity of MC used for the cell treatment is different and depends on the MC functionalization. For each experiment, we have taken care to have a concentration of adsorbed CPT equal to the IC50 of the specific cell line. In detail, a CPT concentration of 40 nM, corresponding to the IC50 value for the HeLa cells,44 and one of 12 μM, corresponding to the IC50 for the NIH3t3,45 have been used. The cells have been incubated with the microcapsules for 48 h and
Figure 6. Effect of the decorated MC on cell lines. Proliferation of NIH3t3 fibroblast (left) and HeLa cells (right), treated for 48 h with MC, MCchitosan (MC-Chi), and MC-chitosan-folate (MC-Chi-FA), with or without CPT adsorption. The treatment was removed and substituted with fresh medium, and then the proliferation was assayed after 48 h. Asterisks mark statistical significance (p < 0.01). 1070
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Bioconjugate Chemistry allowed to grow on fresh medium for additional 48 h before being assayed on their proliferation (Figure 6). This method has been established to allow the discrimination of the effect due to the diffusion of the drug lost from the capsules in the media from the one due to a direct capsulescell interaction. Evaluation of cell proliferation after 48 h treatment indicates that the nude MC, previously incubated with CPT, has the same effect on HeLa viability as the chitosanfolate functionalized ones (Figure S4). Analysis of antiproliferative effect after media change and incubation for two additional days highlights the mortality due to a direct microcapsulecell interaction (Figure 6). In the absence of CPT, a difference in proliferation no larger than 10% has been found on both cell lines for any kind of incubated MC. The antiproliferative effect of the microcapsules is slightly larger in the HeLa than in the control NIH3t3 cells for the MC-Chi system, a phenomenon that is not easy to interpret, although a more intense interaction of chitosan with HeLa cells could account for it. The most striking difference is observed upon incubation with the MC-Chi-FA. In this latter case, in fact less than 20% cells survive in the case of HeLa cells, while more than 80% of NIH3t3 cells are still active. The same results were obtained with the alternative method of cell counting, described in the Methods section, for which control cell numbers were approximately 2 105 and 3 105 per well, for HeLa and NIH3t3, respectively. This result confirms that the overexpression of the folate receptor permits a very efficient and selective targeting of HeLa cells from the MC-Chi-FA.
’ CONCLUSION In this work, PVA microcapsules have been functionalized with chitosan or chitosanfolate and have been characterized for their ability to absorb and release CPT. The presence of chitosan or chitosanfolate on MC leads to greater absorption of the drug, likely because of an increased surface area of the functionalized microstructures, that can then accommodate a greater amount of drug. Notably, MC-Chi-FA are able to bind and to be internalized by HeLa cells overexpressing the folate receptor, while no interaction is observed with NIH3t3 fibroblasts used as negative control (Figures 5 and S5). MC-Chi are found to occasionally adhere to both cell lines, likely because of the fusogenic properties of chitosan, due to the positive charges that allow electrostatic interactions with the negatively charged cell surface.32,46 The results of cellular localization fully match proliferation experiments; in fact, MC-Chi-FA, interacting with HeLa cells, impact their proliferation while not perturbing the growth of NIH3t3 cells (Figure 6). These results indicate that microcapsules modified with chitosan folate are a promising system to target camptothecin in tumors of epithelial origin since the presence of folate allows a selective recognition of HeLa tumor cells. Other studies will be performed on different cell lines also using functionalized microbubbles in the effort to design multivalent therapeutic agents that combine detectability (e.g., contrast agents for imaging techniques) with tissue specific drug delivery. ’ ASSOCIATED CONTENT
bS
Supporting Information. Drug release kinetics in D-MEM, folate dependent localization, MC-Chi-Fa interaction,
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and uptake. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Prof. Alessandro Desideri, Department of Biology, University of Rome Tor Vergata, Via della Ricerca Scientifica, Rome, Italy, Tel. þ39 06 72594326, Fax þ39 06 2022798. E-mail: desideri@ uniroma2.it.
’ ACKNOWLEDGMENT A.D. acknowledges support by the AIRC project number 10121 ’ ABBREVIATIONS: FA, folic acid; FR, folate receptor; PVA, poly(vinyl alcohol); MC, microcapsules; MC-Chi, chitosan conjugated MC; MC-Chi-FA, chitosan-folate conjugated MC; MC-Chi-Rod, chitosan-rhodamine conjugated MC; CPT, camptothecin ’ REFERENCES (1) Hsiang, Y. H., and Liu, L. F. (1988) Identification of mammalian DNA topoisomerase I as an intracellular target of the anticancer drug camptothecin. Cancer. Res. 48, 1722–1726. (2) Ulukan, H., and Swaan, P. W. (2002) Camptothecins: a review of their chemotherapeutic potential. Drugs 62, 2039–2057. (3) Hede, M. S., Petersen, R. L., Frøhlich, R. F., Kr€uger, D., Andersen, F. F., Andersen, A. H., and Knudsen, B. R. (2007) Resolution of Holliday junction substrates by human topoisomerase I. J. Mol. Biol. 365, 1076–1092. (4) Peng, L. H., Chen, X. Y., and Wu, T. X. (2008) Topotecan for ovarian cancer. Cochrane Database Syst. Rev. 2, CD005589. (5) Sandler, A. B. (2001) Irinotecan in small-cell lung cancer: the US experience. Oncology 15, 11–12. (6) Bruzzese, F., Rocco, M., Castelli, S., Di Gennaro, E., Desideri, A., and Budillon, A. (2009) Synergistic antitumor effect between vorinostat and topotecan in small cell lung cancer cells is mediated by generation of reactive oxygen species and DNA damage-induced apoptosis. Mol. Cancer Ther. 8, 3075–87. (7) Mross, K., Richly, H., Schleucher, N., Korfee, S., Tewes, M., Scheulen, M. E., Seeber, S., Beinert, T., Schweigert, M., Sauer, U., Unger, C., Behringer, D., Brendel, E., Haase, C. G., Voliotis, D., and Strumberg, D. (2004) A phase I clinical and pharmacokinetic study of the camptothecin glycoconjugate, BAY 383441, as a daily infusion in patients with advanced solid tumors. Ann. Oncol. 15, 1284–1294. (8) Pizzolato, J. F., and Saltz, L. B. (2003) The camptothecins. Lancet 361, 2235–2242. (9) Huang, Z. R., Hua, S. C., Yang, Y. L., and Fang, J. Y. (2008) Development and evaluation of lipid nanoparticles for camptothecin delivery: a comparison of solid lipid nanoparticles, nanostructured lipid carriers, and lipid emulsion. Pharmacol. Sin. 29, 1094–1102. (10) Koo, O. M., Rubinstein, I., and Onyuksel, H. (2005) Camptothecin in sterically stabilized phospholipid micelles: a novel nanomedicine. Nanomedicine 1, 77–84. (11) Venditto, V. J., Allred, K., Allred, C. D., and Simanek, E. E. (2009) Intercepting the synthesis of triazine dendrimers with nucleophilic pharmacophores: a general strategy toward drug delivery vehicles. Chem. Commun. (Camb.) 37, 5541–5542. (12) Yang, S., Zhu, J., Lu, Y., Liang, B., and Yang, C. (1999) Body distribution of camptothecin solid lipid nanoparticles after oral administration. Pharm. Res. 16, 751–757. (13) Fang, J. Y., Hung, C. F., Hua, S. C., and Hwang, T. L. (2009) Acoustically active perfluorocarbon nanoemulsions as drug delivery 1071
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