Article pubs.acs.org/molecularpharmaceutics
Targeted Transferrin-Modified Polymeric Micelles: Enhanced Efficacy in Vitro and in Vivo in Ovarian Carcinoma Rupa R. Sawant,† Aditi M. Jhaveri,† Alexander Koshkaryev,† Lin Zhu,† Farooq Qureshi,‡ and Vladimir P. Torchilin*,† †
Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston, Massachusetts 02115, United States Roche, Global Formulation Research (pRED), Nutley, New Jersey 07110, United States
‡
S Supporting Information *
ABSTRACT: In this study, transferrin (Tf)-modified poly(ethylene glycol)phosphatidylethanolamine (mPEG-PE) micelles loaded with the poorly water-soluble drug, R547 (a potent and selective ATP-competitive cyclin-dependent kinase (CDK) inhibitor), were prepared and evaluated for their targeting efficiency and cytotoxicity in vitro and in vivo to A2780 ovarian carcinoma cells, which overexpress transferrin receptors (TfR). At 10 mM lipid concentration, both Tf-modified and plain micelles solubilized 800 μg of R547. Tf-modified micelles showed enhanced interaction with A2780 ovarian carcinoma cells in vitro. The involvement of TfR in endocytosis of Tfmodified micelles was confirmed by colocalization studies of micelle-treated cells with the endosomal marker Tf-Alexa488. We confirmed endocytosis of micelles in an intact form with micelles loaded with a fluorescent dye and additionally labeled with fluorescent lipid. The in vitro cytotoxicity and in vivo tumor growth inhibition studies in A2780-tumor bearing mice confirmed the enhanced efficacy of Tf-modified R547-loaded micelles compared to free drug solution and to nonmodified micelles. The results of this study demonstrate the potential application of Tf-conjugated polymeric micelles in the treatment of tumors overexpressing TfR. KEYWORDS: polymeric micelles, targeted, transferrin, cancer, drug delivery
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INTRODUCTION Traditional anticancer drug therapy suffers from several drawbacks including poor solubility, a short circulating halflife of the drug, low bioavailability, and most notably systemic toxicities that arise due to the “off-target” effects of chemotherapeutic agents.1−3 Nanocarriers like polymeric micelles have been used successfully to overcome these drawbacks because of a number of favorable properties such as small size, ability to solubilize hydrophobic drugs, good stability, longevity in the systemic circulation, and the ability to accumulate passively in areas with a leaky vasculature via the enhanced permeability and retention (EPR) effect.4,5 The surface of these micelles may be modified with ligands, which can bind to receptors that are preferentially expressed on target cells (also referred to as active targeting), to improve the therapeutic outcome.6,7 A number of ligands have been investigated to date, including antibodies,8,9 aptamers,10,11 peptides,12 sugar moieties,13 transferrin,14,15 and folate residues.16−18 The focus of this research emphasizes the use of polymeric lipid-core micelles made from poly(ethylene glycol)-phosphatidylethanolamine (mPEG-PE). Previous studies from our lab have investigated mPEG-PE micelles for the delivery of poorly soluble drugs like paclitaxel, camptothecin, dequalinium, and photosensitizers such as meso-tetraphenylporphine (TPP).19−22 We have shown that these micelles are capable of accumulating in experimental tumors via the EPR effect23 and, additionally, can be targeted to tumors via attachment of © 2013 American Chemical Society
nucleosome-specific monoclonal antibody 2C5, which recognizes a broad variety of tumor cells through their cell surfacebound nucleosomes.8,19,24 The current study explores the possibility of targeting drug-loaded mPEG-PE micelles to cancer cells using transferrin as a targeting ligand. Transferrin (Tf) (MW 80 kDa) is a serum glycoprotein that helps to transport iron required as a cofactor for DNA synthesis into rapidly growing cells via the transferrin receptor (TfR).25 Because of the rapid rate of proliferation of cancer cells, their demand for iron is much greater than normal cells. This leads to an increased expression of TfR on the surface of cancer cells that can be exploited for the purpose of active targeting of nanocarriers to these cells.26 Tf-conjugated nanocarriers such as liposomes27−30 and polymeric nanoparticles31−33 have been shown to selectively deliver anticancer drugs to tumor cells overexpressing TfR through TfR-mediated endocytosis. However, Tf-modified polymeric micelles have seldom been reported. We have recently reported R547-loaded dual-targeted micelles surface modified with mAb 2C5 and Tf. However, such dual-targeted micelles did not yield the expected increase in efficacy in vivo in A2780-tumor-bearing mice compared to single-ligand-targeted micelles.34 Received: Revised: Accepted: Published: 375
November 5, 2012 October 2, 2013 December 10, 2013 December 10, 2013 dx.doi.org/10.1021/mp300633f | Mol. Pharmaceutics 2014, 11, 375−381
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dialyzed against 1 L of 10 mM PBS, pH 7.4, using cellulose ester membranes with a cut off size of 300 kDa. The amount of Tf in the Tf-PEG3400-PE conjugate was estimated by a bicinchoninic acid (BCA) protein assay with pure holo-Tf as a standard. Both pNP-PEG3400-PE and Tf-PEG3400-PE were characterized by 1H NMR using Varian 400 MHz spectrophotometer. Preparation of Nontargeted and Tf-Targeted Micelles. To incorporate R547 into mPEG2000-PE micelles, we used the lipid film hydration method.38 Briefly, 1 mg of R547 (2 mg/mL in methanol) was mixed with a PEG2000-PE solution in chloroform. Organic solvents were removed by rotary evaporation. The resulting film of the drug/micelle material mixture was freeze-dried to remove any traces of organic solvents. To form micelles, the dried film was hydrated with 10 mM phosphate-buffered saline (PBS), pH 7.4, at room temperature and vortexed vigorously for 5 min. The final lipid concentration in the micelles was 10 mM. Any unincorporated drug was removed by filtration of the micelle suspension through 0.2 μm membrane filters. When required, 1 mol % of the fluorescent probe, Rh-PE, was added to the ingredients during micelle preparation for fluorescent labeling. A hydrophobic fluorescence probe, DiO (2 mol %), was used to represent a hydrophobic drug loaded in the micelle cores and was incorporated into micelles to study the mechanism of cell uptake of the drug-loaded micelles by confocal laser scanning microscopy (CLSM). The postinsertion method was used to modify drug-loaded or empty mPEG2000-PE micelles with Tf-PEG3400-PE conjugate.39,40 The drug loaded or empty mPEG2000-PE micelles were incubated overnight with various amounts of TfPEG3400-PE conjugates (Table I) to form targeted micelles.
R547, a diaminopyrimidine compound, is a potent and selective ATP-competitive cyclin-dependent kinase (CDK) inhibitor, which induces cell-cycle arrest and apoptosis.35,36 R547 has very poor aqueous solubility in physiologically relevant pH conditions. The poor solubility of R547 makes it an ideal candidate for incorporation into mPEG-PE micelles. In this study, we report the development and characterization of R547-loaded, Tf-targeted, mPEG-PE micelles (Figure 1).
Figure 1. Schematic representation of R547-loaded, Tf-targeted PEGPE micelles and their interaction with Tf receptors on cancer cells.
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MATERIALS AND METHODS 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol))-2000] (PEG2000-PE), 1,2-dipalmitoylsn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rh−PE) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were purchased from Avanti Polar Lipids (Alabaster, AL) and used without further purification. pNitrophenylcarbonyl-poly(ethylene glycol)-nitrophenyl carbonate (pNP-PEG3400-pNP) was purchased from Laysan Bio Inc. (Arab, AL). R547 was a gift from Roche (Nutley, NJ). Human holo-Transferrin (Tf) and 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO) were purchased from Sigma (St. Louis, MO). Tf-Alexa488 and Hoechst 33342 were purchased from Invitrogen/Molecular Probes, Inc. (Eugene, OR). Cell Titer Blue cell viability assay reagent was purchased from Promega (Madison, WI). All other chemicals and solvents were purchased as analytical grade reagents and were used without further purification. A2780 cells (human ovarian carcinoma) were purchased from American Type Culture Collection (ATCC) (Manassas, VA). Cell culture media, Dulbecco’s modified Eagle medium (DMEM), penicillin/streptomycin stock solutions, and trypsinEDTA were purchased from Cellgro (Herndon, VA). BD Matrigel was purchased from BD Biosciences (Franklin Lakes, NJ). Synthesis of Tf-PEG3400-PE Conjugate. The TfPEG3400-PE conjugate was prepared by reaction of Tf to the distal tips of mPEG blocks via p-nitrophenylcarbonyl (pNP) groups (using a pNP-PEG3400-PE conjugate). The pNPPEG3400-PE conjugate was synthesized and purified according to a previously established method.37 A solution of mPEG2000-PE in chloroform was mixed with pNP-PEG3400-PE (2 molar excess over Tf), in a small test tube. The molar ratio between mPEG2000-PE and pNP-PEGPE was 4:1. The organic solvents were removed by rotary evaporation followed by freeze-drying. The dried film was hydrated with 5 mM citrate buffered saline, pH 5.5, followed by addition of Tf solution in PBS, pH 7.4. The pH was adjusted to 8.0−8.2 with 100 mM phosphate buffer pH 8.5. The reaction continued overnight at 4 °C. Subsequently, the micelles were
Table I. Typical Micelle Composition for 10 mM Micelle Preparations micelles M 0.25 TM 0.5 TM 1.0 TM
mPEG2000-PE:pNP-PEG3400PE ratio (mol %)
Tf content in Tf-PEG3400-PE conjugate mg/mL
100:0 99.75:0.25
0 1
99.5:0.5 99:1.0
2 4
Characterization of Micelles: Size, drug content and stability of micelles. The micelle size (hydrodynamic diameter) and size distribution were measured by dynamic light scattering (DLS) using a N4 Plus Submicrometer Particle System (Coulter Corporation, Miami, FL, USA). The amount of R547 solubilized in the micelles was determined by reversed phase-HPLC by the method reported earlier.34 The stability of R547-loaded micelles was studied by monitoring the size and drug content during storage at 2−8 °C. The stability of selected R547-loaded micelles was also determined after incubating the micelles at 37 °C for 24h. At various time points micelle size was determined by DLS as described above. For drug content, aliquots of each sample withdrawn were filtered through 0.2 μm membrane filters and then analyzed for drug content by HPLC. Cell Culture. A2780 cells were maintained in DMEM supplemented with 10% FBS, 50 U/mL penicillin and 50 μg/ mL streptomycin. The cell lines were grown in a humidified atmosphere of 5% CO2 at 37 °C. 376
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Table II. Size of empty and R547-loaded formulations micelles M 0.25 TM 0.5 TM 1.0 TM
empty micelle size (nm ± S.D.) 13.2 14.5 15.2 13.2
± ± ± ±
R547-loaded micelle size (nm ± S.D.)
2.5 1.5 2.2 1.3
13.5 15.5 15.0 14.5
± ± ± ±
R547-loaded micelle size (nm ± S.D.) after 1 week storage at 2−8 °C
1.5 2.3 1.2 2.1
12.5 14.7 15.5 15.5
± ± ± ±
2.5 3.3 1.7 2.5
Table III. Drug content of R547-loaded formulations micelles M 0.25 TM 0.5 TM 1.0 TM
drug content of R547-loaded micelle (mg/mL ± S.D.) 0.801 0.815 0.789 0.808
± ± ± ±
drug content of R547-loaded micelle (mg/mL ± S.D.) after 1 week storage at 2−8 °C
0.010 0.008 0.011 0.007
0.792 0.809 0.796 0.815
± ± ± ±
0.015 0.011 0.015 0.009
Figure 2. Effect of ligand (Tf) density and competition with free Tf on micelles to cell interaction as analyzed by flow cytometry. (A) Histogram analysis and (B) mean fluorescence intensity (MFI) expressed as a percent of control cells of interaction of Rh-labeled micelles with A2780 cells. Micelles were modified with increasing amounts of Tf (0.25 to 1 mol %) and incubated with cells for 4 h. A total of 10 000 events were analyzed for each sample and the mean fluorescence intensities (MFI)(FL2) were expressed as a percent of control cells. Legend: untreated cells (black), M treated (green), 0.25TM treated (pink), 0.5TM treated (orange), 1.0TM (blue), 1.0TM+freeTf treated (red). Data represent mean ± S.D. for triplicate samples.
Interaction of Micelles with Cells Using Flow Cytometry and Confocal Microscopy (CLSM). Expression of TfR on A2780 cells was confirmed by flow cytometry (data not shown). A2780 cells were allowed to grow in T75 flasks until about 80% confluent. Trypsin−EDTA was used to detach the cells. For flow cytometry the cells were resuspended in sterile PBS, pH 7.4. Cells (0.5 × 106) were then transferred to centrifuge tubes and incubated with various micelle formulations (0.2 mg/ mL of total lipid) in 0.5 mL of PBS for 4h. For untreated control, cells were treated with 0.5 mL of PBS for 4h. During this incubation period the tubes were shaken intermittently. After incubation, the cells were washed and resuspended in ice cold PBS, pH 7.4. They were analyzed immediately by flow cytometry. The fluorescence of micelle-treated cells was measured in a Becton Dickinson FACScan (Becton), and data analysis was performed using CellQuest software (Becton Dickinson). The red fluorescence was recorded at an emission wavelength of 580 nm (channel FL-2). Cells were gated using forward versus side scatter to exclude debris and dead cells, and a total of 10 000 events were acquired for each sample. In order to demonstrate that the uptake of Tf-modified micelles (TM) occurred through the TfR-endocytic pathway, we showed competitive inhibition of TM uptake with an excess of free human holo-Tf added to the medium at a concentration of 2 mg/mL before the cells were treated with micelles.
For CLSM studies to evaluate the endosomal localization of micelles, A2780 cells grown on glass coverslips to 60−70% confluence were treated with 0.2 mg/mL micelles labeled with Rh-PE for 4 h. Tf-Alexa488 (10 μg/mL) and Hoechst 33342 (1 μg/mL) were added to the cells for 15 min before the micelle treatment was terminated. To check for integrity of micelles during endocytosis, cells were treated with 0.2 mg/mL of Rh-PE and DiO dual-labeled micelles. After incubation, the cells were washed three times with DMEM to remove the unbound micelles and then fixed with 4% paraformaldehyde (15 min at RT). The coverslips were mounted on glass slides with Fluormount-G medium and sealed using a nail lacquer. The slides were observed with a Zeiss LSM 700 inverted confocal microscope (Carl Zeiss Co. Ltd., Jena, Germany) equipped with a 63×, 1.4-numerical aperture plan-apochromat oil-immersion objective. The images were analyzed using the ImageJ 1.42 software (NIH). The quantitative values for Pearson’s correlation coefficient (PCC) and Mander’s overlap coefficient (MOC) were calculated by ImageJ for at least 20 cells from three images obtained from two different experiments. It should be noted that the raw images were utilized as is, and no further image transformation was performed for assessing the cellular colocalization. Cytotoxicity. Cells were plated at a density of 4 × 103 cells per well in 96-well plates (Corning, Inc., NY, USA). They were incubated for 24 h in a humidified atmosphere at 37 °C and 5% CO2. After the incubation, the medium was replaced with free 377
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drug solution, drug-loaded micelles, or empty micelles for 48 h. The free drug solution was prepared from a stock solution (2 mg/mL of R547 in methanol) by diluting the required amount of drug solution to 2 mL with complete media. The micelles were also diluted with complete media. After incubation, each well was washed twice with media, and cell survival was measured using CellTiter-Blue cell viability assay. Tumor Growth Reduction Studies. The experiments were performed on 6−8 week old female nu/nu mice (Charles River Laboratories, Wilmington, MA). A2780 cells (1 ×106) mixed with matrigel (1:1) were injected subcutaneously into the left hind flank and allowed to develop until tumors were approximately 80−130 mm3. The animals were randomized into groups of 5 and injected intravenously through the tail vein with 200 μL of PBS, control formulations, and drug-loaded formulations (equivalent to10 mg/kg of R547) at two day intervals until day 8 (5 total injections). The R547 solution (25% hydroxyproply beta cyclodextrin in an aqueous solution at pH 7) was provided by Roche. All micelle formulations contained 12.5 mM lipid concentration and approximately 1 mg/mL R547. Mice weight and tumors were measured in two dimensions every alternate day using calipers and tumor volumes were calculated as [length × (width)2]/2. Statistical Analysis. The data are expressed as mean ± standard deviation (S.D.). Student’s t test was used to determine the statistical difference between groups. P < 0.05 was considered statistically significant.
Figure 4. Colocalization of DiO (green) and Rh-PE (red) fluorescent markers by confocal microscopy. The nuclei were visualized by staining with Hoechst 33342 (blue). Pearson’s correlation coefficient (PCC) and Mander’s overlap coefficient (MOC) were calculated by ImageJ software for 6 images from two different experiments ± SEM.
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RESULTS AND DISCUSSION Preparation and Characterization of Micelles. Micellar solubilization of R547 (solubility less than 0.001 mg/mL at Figure 5. In vitro cytotoxicity of R547 formulations on A2780 cells after 48 h incubation. Cells (4 ×103) were grown in 96-well plates, and after treatment with various R547 formulations for 48h, the cell viability was determined using the CellTiter-Blue cell viability assay. Data represent mean ± S.D. of three separate determinations. To calculate IC50 GraphPad Prism software was used.
amino groups of Tf. The unreacted Tf was removed by dialysis with PBS, pH 7.4, using cellulose ester membranes with a molecular weight cut off size of 300 kDa. After purification, the amount of Tf-modified conjugate was determined by a BCA protein assay. The reaction efficiency was 60−70% calculated from amount of Tf conjugated to pNP-PEG3400-PE by BCA assay after removal of the free Tf by dialysis. Because we used two molar excess of pNP-PEG3400-PE over Tf, it could be possible that one Tf could react with two or more PEG. However, this did not affect the subsequent micelle formation, as there was no change in size (Table II) and drug content (Table III) of micelles. NMR spectra of both pNP-PEG3400PE and Tf-PEG3400-PE are given in Supporting Information. Based on this estimation, known amounts of Tf-modified PEG3400-PE conjugate were added by the postinsertion method to the drug-loaded or empty mPEG2000-PE micelles and incubated an 4 °C for 24 h. Using this technique, various Tf-targeted micelles were prepared with varying densities of Tf targeting ligand (See Table I). It is important to note that loading of the drug or modification with Tf did not change the size of the micelles significantly (Table II). The average size of the micelles was in the range of 13.2−15.5 nm.
Figure 3. Colocalization of Rh-PE-labeled micelles (red) with the endosomal marker Tf-Alexa488 (green) by confocal microscopy. The nuclei were visualized by staining with Hoechst 33342 (blue). Mean values of Pearson’s correlation coefficient (PCC) and Mander’s overlap coefficient (MOC) were calculated by ImageJ software for five images from two different experiments ± SEM.
neutral pH) increased the solubility of R547 to 0.8 (mg of R547)/mL of the micelle formulation at a10 mM total lipid concentration. The chemistry used for preparation of Tf-PEG3400-PE conjugate is similar to preparation of mAb-PEG3400-PE conjugate successfully developed by us previously without loss of antibody activity. There was no change in size or drug content of the mAb-targeted micelles compared to nonmodified micelles.8,20,24,41 The pNP groups of the pNPPEG3400-PE conjugate reacted rapidly at a pH > 8.0 with 378
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Figure 6. (A) Tumor volumes and (B) percent change in body weight of mice bearing A2780 tumors treated with R547 formulations. A2780 cells (1 ×106) mixed with matrigel (1:1) were injected sc into the flank of female nu/nu mice. Formulations equivalent to 10 mg/kg of R547 were injected via the tail vein every two days, and tumor volumes and body weight were measured every alternate day. Significant differences (p < 0.05) were found between the drug loaded formulations vs their respective control groups as well as between nontargeted and Tf-targeted R547-loaded micelles.
Both R547-loaded micelles and Tf-modified R547-loaded micelles were stable with no signs of drug precipitation when stored at 2−8 °C for 1 week. There was no significant change in size and drug content of micelles during this period (Table II and III). Both R547-loaded micelles and 1% Tf-modified R547loaded micelles did not show any change in size and drug content after incubation at 37 °C for 24h. Cellular Association of Micelles by Confocal Microscopy and Flow Cytometry. The cellular association of Rh-PE labeled empty micelles was evaluated using flow cytometry. The Tf-modified micelles (TM) showed significantly enhanced cellular association compared to nontargeted micelles suggesting the importance of Tf modification (Figure 2). The effect of Tf content on the cellular association was also evaluated. The cellular association of 0.5 mol % and 1 mol % modification (0.5TM and 1.0TM) was higher than with the 0.25 mol % modification (0.25TM). Therefore, we used 1.0TM for further experiments. The involvement of Tf receptors in the micelle endocytosis was confirmed by competitive inhibition studies using free Tf. When cells were preincubated with free Tf, the uptake of TM was blocked (Figure 2B). Tf is internalized into cells via endocytosis through the TfR. An excess of added free Tf blocks these receptors, thus competitively inhibiting the uptake of TM by reducing receptor availability. As a result, we observe a significantly reduced interaction with 1.0TM when cells are pretreated with free Tf versus when they are not. This reduced interaction of 1.0TM is similar to the nontargeted micelles (M). There was no significant change in nontargeted micelles (M) interaction with cells in the presence and absence of free Tf (data not shown). These studies provide a good qualitative insight into the level of intracellular association of R547 when used in different delivery format. Especially the transferring targeted micellar formulation appears to be the preferred choice for better intracellular delivery within tumor cells. More specific quantitative studies can be undertaken as future endeavors in this area for probing the mechanistic aspects on a more quantitative basis to screen the best formulation and/or the targeting ligand approach. Intracellular Localization of Micelles by Confocal Laser Scanning Microscopy (CLSM). Intracellular localization of Rh-PE labeled plain micelles (M) and Tf-micelles (1.0TM) was evaluated by staining the micelle-treated cells with the endosomal marker Tf-Alexa488. TM had significantly
higher localization in the endosomal compartment compared to M (Figure 3). To estimate the integrity of the micelle structure during incubation with cells, A2780 cells were treated with the micelles double-labeled with Rh-PE and DiO. The Rh-PE is incorporated in the lipid layer while the hydrophobic probe DiO is loaded in the core of the micelles. If the micelles are taken up intact during endocytosis, then colocalization of both probes is anticipated during this process. A high level of colocalization for Rh-PE and DiO demonstrated that that the micelles maintained their integrity after 4 h despite their interaction with cells following endocytosis (Figure 4). A relatively higher value for the Pearson correlation coefficient (about a 12-fold increment) and Mander’s overlap coefficient (about a 3-fold increment) for the Tf targeted micelle compared to the non-Tf targeted micelle suggests that the Tf targeting ligand assists in the active targeting of R547, resulting in relatively better cellular internalization. Free Rh-PE and DiO were also used as controls and showed different cellular distribution patterns than when loaded in micelles (see Supporting Information). Increased Cytotoxicity by Tf-Targeted Micelles. Cytotoxicity of the free drug and drug-loaded micelles was evaluated with A2780 cells. Following 48 h incubation, R547loaded TM showed higher toxicity than R547 solution (R547 soln) or R547-loaded M (p < 0.05) as demonstrated by the IC50 values of 0.34 ± 0.100, 0.11 ± 0.010, and 0.064 ± 0.007 μg/mL for R547 solution, R547 M, and R547 TM respectively (Figure 5). Empty M and TM were used as controls and exhibited no cytotoxicity (data not shown). Although R547 by itself is cytotoxic, it is clearly shown in this study that the delivery of R547 using a polymeric micellar nanocarrier enhances the cellular toxicity as demonstrated by a decrease in percent cell viability with an increase in R547 concentration and also very remarkably with the transferrin-targeted polymeric micelles containing R547. For R547 TM, high expression of TfR on the surface of cells enabled a much greater proportion of Tf-targeted micelles to be taken up internalized by the cells relative to their nontargeted counterparts or free drug solution. As a result, more R547 was delivered to cells and this translated to the significantly higher cytotoxicity seen with R547 TM at 48 h. Effect of R547-Loaded Tf-Modified Micelles on the Tumor Growth In Vivo. The treatment with drug-loaded formulations strongly inhibited tumor growth relative to their 379
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ed.; Grobmyer, S.R., Moudgil, B.M., Eds.; Springer Science+Business Media, LLC: New York, 2010; Vol. 624, pp 131−149. (5) Torchilin, V. P. Structure and design of polymeric surfactantbased drug delivery systems. J. Controlled Release 2001, 73 (2−3), 137−172. (6) Timko, B. P.; Whitehead, K.; Gao, W.; Kohane, D. S.; Farokhzad, O.; Anderson, D.; Langer, R. Advances in Drug Delivery. Annu. Rev. Mater. Res. 2011, 41 (1), 1−20. (7) Torchilin, V. P. Targeted polymeric micelles for delivery of poorly soluble drugs. Cell. Mol. Life Sci. 2004, 61 (19−20), 2549− 2559. (8) Torchilin, V. P.; Lukyanov, A. N.; Gao, Z.; PapahadjopoulosSternberg, B. Immunomicelles: targeted pharmaceutical carriers for poorly soluble drugs. Proc. Natl. Acad. Sci. U. S. A. 2003, 100 (10), 6039−6044. (9) Park, J. W.; Kirpotin, D. B.; Hong, K.; Shalaby, R.; Shao, Y.; Nielsen, U. B.; Marks, J. D.; Papahadjopoulos, D.; Benz, C. C. Tumor targeting using anti-her2 immunoliposomes. J. Controlled Release 2001, 74 (1−3), 95−113. (10) Farokhzad, O. C.; Cheng, J.; Teply, B. A.; Sherifi, I.; Jon, S.; Kantoff, P. W.; Richie, J. P.; Langer, R. Targeted nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo. Proc. Natl. Acad. Sci. U. S. A. 2006, 103 (16), 6315−6320. (11) Dhar, S.; Gu, F. X.; Langer, R.; Farokhzad, O. C.; Lippard, S. J. Targeted delivery of cisplatin to prostate cancer cells by aptamer functionalized Pt(IV) prodrug-PLGA-PEG nanoparticles. Proc. Natl. Acad. Sci. U. S. A. 2008, 105 (45), 17356−17361. (12) Hu, Z.; Luo, F.; Pan, Y.; Hou, C.; Ren, L.; Chen, J.; Wang, J.; Zhang, Y. Arg-Gly-Asp (RGD) peptide conjugated poly(lactic acid)poly(ethylene oxide) micelle for targeted drug delivery. J. Biomed. Mater. Res., Part A 2008, 85 (3), 797−807. (13) Nagasaki, Y.; Yasugi, K.; Yamamoto, Y.; Harada, A.; Kataoka, K. Sugar-installed block copolymer micelles: their preparation and specific interaction with lectin molecules. Biomacromolecules 2001, 2 (4), 1067−1070. (14) Ishida, O.; Maruyama, K.; Tanahashi, H.; Iwatsuru, M.; Sasaki, K.; Eriguchi, M.; Yanagie, H. Liposomes bearing polyethyleneglycolcoupled transferrin with intracellular targeting property to the solid tumors in vivo. Pharm. Res. 2001, 18 (7), 1042−1048. (15) Yang, X.; Koh, C. G.; Liu, S.; Pan, X.; Santhanam, R.; Yu, B.; Peng, Y.; Pang, J.; Golan, S.; Talmon, Y.; Jin, Y.; Muthusamy, N.; Byrd, J. C.; Chan, K. K.; Lee, L. J.; Marcucci, G.; Lee, R. J. Transferrin receptor-targeted lipid nanoparticles for delivery of an antisense oligodeoxyribonucleotide against Bcl-2. Mol. Pharmaceutics 2009, 6 (1), 221−230. (16) Gabizon, A.; Horowitz, A. T.; Goren, D.; Tzemach, D.; Mandelbaum-Shavit, F.; Qazen, M. M.; Zalipsky, S. Targeting folate receptor with folate linked to extremities of poly(ethylene glycol)grafted liposomes: in vitro studies. Bioconjugugate Chem. 1999, 10 (2), 289−298. (17) Leamon, C. P.; Weigl, D.; Hendren, R. W. Folate copolymermediated transfection of cultured cells. Bioconjugugate Chem. 1999, 10 (6), 947−957. (18) Park, E. K.; Kim, S. Y.; Lee, S. B.; Lee, Y. M. Folate-conjugated methoxy poly(ethylene glycol)/poly(epsilon-caprolactone) amphiphilic block copolymeric micelles for tumor-targeted drug delivery. J. Controlled Release 2005, 109 (1−3), 158−168. (19) Gao, Z.; Lukyanov, A. N.; Chakilam, A. R.; Torchilin, V. P. PEG-PE/phosphatidylcholine mixed immunomicelles specifically deliver encapsulated taxol to tumor cells of different origin and promote their efficient killing. J. Drug Targeting 2003, 11 (2), 87−92. (20) Sawant, R. R.; Sawant, R. M.; Torchilin, V. P. Mixed PEG-PE/ vitamin E tumor-targeted immunomicelles as carriers for poorly soluble anti-cancer drugs: improved drug solubilization and enhanced in vitro cytotoxicity. Eur. J. Pharm. Biopharm. 2008, 70 (1), 51−57. (21) Mu, L.; Chrastina, A.; Levchenko, T.; Torchilin, V. P. Micelles from Poly(ethylene glycol)-Phosphatidyl Ethanolamine Conjugates (Peg-Pe) as Pharmaceutical Nanocarriers for Poorly Soluble Drug Camptothecin. J. Biomed. Nanotechnol. 2005, 1 (2), 190−195.
control groups at the end of the study at day 10 (p < 0.05) (Figure 6A). There was no significant difference between R547 solution and R547 micelles. Thus, micelles only act as a vehicle for solubilization of the poorly soluble R547. However, Tftargeted R547-loaded micelles (R547 TM) significantly (p < 0.05) inhibited tumor growth compared to R547-loaded micelles (R547 M) at the end of the study at day 10. This can be attributed to the effect active targeting in vivo. The active targeting with transferrin takes advantage of the overexpressed TfR on the cell surface and helps in the cellular uptake and internalization of micelles. This effect results in delivery of higher quantities of R547 to tumors, thus leading to a significantly greater tumor growth inhibition. All formulations were well tolerated as there was no significant change in body weight between the groups (Figure 6B).
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CONCLUSIONS The loading of R547 into mPEG-PE micelles enhanced the solubility of this poorly soluble drug. Modification of these micelles with transferrin improved the cellular uptake efficiency. Both CLSM and flow cytometry confirmed the enhanced uptake of Tf-modified micelles by A2780 ovarian carcinoma cells overexpressing TfR. The results of in vitro cytotoxicity and in vivo tumor growth inhibition studies confirmed the enhanced efficacy of Tf-modified R547 micelles compared to nonmodified micelles. Therefore, Tf-modified micelles might be an effective delivery system for delivery of poorly soluble drugs used for the treatment of TfR-overexpressing carcinomas.
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ASSOCIATED CONTENT
S Supporting Information *
Data providing intrinsic aqueous solubility of R547, NMR spectra of synthesized conjugates, and confocal microscopy of control treatments using free DiO and Rh-PE is provided. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*V. P. Torchilin. Address: Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, 360 Huntington Ave, 140 The Fenway, Room 216, Boston, Massachusetts 02115, United States. E-mail: v.torchilin@neu. edu. Phone: 617-373-3206. Fax 617-373-7509. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research work was funded by a grant from Hoffmann-La Roche Inc., New Jersey. REFERENCES
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