Self-Assembled Penetratin-Deferasirox Micelles as Potential Carriers for Hydrophobic Drug Delivery Dibakar Goswami,1,2 Hector Aguilar Vitorino,1 M. Teresa Machini,3 Breno P. Esp osito1 1

Departamento de Quımica Fundamental, Instituto de Quımica, Universidade de S~ao Paulo, Av. Lineu Prestes 748, 05508000, S~ ao Paulo, Brazil 2

Bio-Organic Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India

3

Departamento de Bioquımica, Instituto de Quımica, Universidade de S~ao Paulo, Av. Lineu Prestes 748, 05508-000, S~ao Paulo, Brazil Received 2 May 2015; accepted 4 May 2015 Published online 13 May 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22672

ABSTRACT:

past two calendar years by emailing the Biopolymers editorial office at [email protected].

There has been a growing interest in the use of micelles with nanofiber geometry as nanocarriers for hydrophobic drugs. Here we show that the conjugate of penetratin, a cell-penetrating peptide (CPP) with blood-brain barrier (BBB) permeability, and deferasirox (DFX), a hydrophobic iron chelator, self-assembles to form micelles at a very low concentration (15 mg/L). The critical micelle concentration (CMC) was determined, and the micelles were used for solubilizing curcumin, a hydrophobic antineurodegenerative drug, for successful delivery across RBE4 cells, a BBB model. Transmission Electron Microscope images of the curcumin-loaded micelles confirmed the formation of nanofibers. These results indicate the potential of C 2015 Wiley CPP-drug conjugates for use as nanocarriers. V

Periodicals, Inc. Biopolymers (Pept Sci) 104: 712–719, 2015. Keywords: penetratin; cell-penetrating peptides; nanofibers; curcumin; neurodegeneration; deferasirox; iron

This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of any preprints from the Additional Supporting Information may be found in the online version of this article Correspondence to: Breno P. Esp osito; e-mail: [email protected]

INTRODUCTION

T

he presence of the Blood-Brain-Barrier (BBB), a tight endothelial junction between the bloodstream and brain parenchyma, prohibits most of the foreign molecules to enter the Central Nervous System.1 Hence one of the most challenging tasks in medicinal chemistry is to find a way to overcome this barrier, and to deliver therapeutic drugs through it. To date, various strategies have been developed for this purpose.2 The most popular and effective approaches are3 (a) invasive, where the BBB is mechanically breached using infusion or microchips; (b) pharmacological, where a known drug is chemically modified to enable its entry through the BBB; and (c) physiological, where the known drug is conjugated to a BBB-permeable moiety. The delivery of therapeutic drugs using nanocarriers, particularly micelles or nanofibers, have also emerged as a promising tool4 because those nanostructures, formed by the selfassembly of a conjugate having suitable amphiphilicity, can contain hydrophobic drugs inside the hydrophobic core, and hence offer more stability and prolonged activity in the systemic circulation. Several polymeric nanocarriers have been designed and tested for this purpose.4b Recently, self-assembly of peptide-drug conjugates has attracted attention as they are able to deliver therapeutic drugs into various cells.5 However, it has been reported that the overall morphology of the core/shell structures of these conjugates has a great effect on their efficiency in chemotherapy.6 It has

C 2015 Wiley Periodicals, Inc. V

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overload, BBB-permeable conjugates of DFX have also been prepared and tested.13 Towards this end, formation of a selfassembled nanostructure may not only improve the BBB permeability and efficacy of DFX, but also it can be useful to solubilize and deliver other hydrophobic drugs across the BBB. Herein, we report the synthesis of a penetratin-DFX conjugate, its self-assembled structure and its in vitro efficacy to deliver hydrophobic molecules across a BBB model (Scheme 1).

MATERIALS AND METHODS Materials

SCHEME 1 Assembly of PEN-DFX and loading of curcumin to the micelles.

been proposed that non-spherical carriers, specially nanofibers have an advantage over the spherical micelles in terms of extended circulating stability, and cell-penetrating ability.7 Hence, micelles with nanofiber geometry have attracted much attention.8 Micelles formed by conjugates of a hydrophobic moiety with cell-penetrating peptides (CPPs) have some inherent advantages for use as nanocarriers. Several polymeric CPP conjugates have been developed to improve the delivery of an encapsulated drug.9,10 CPPs known to cross BBB have also been employed for this purpose. For example, self-assembled micelles from TAT conjugated cholesterol-terminated polyethylene glycol were used to deliver ciprofloxacin across BBB.11a In another study, TAT-modified block copolymer micelles were utilised for brain delivery of coumarin via intranasal administration.11b Inspired by the example of the formation of nanofibers using a 16 amino-acid cationic peptide,5c we envisaged that penetratin, a well-known peptide with BBB permeability,12 can lead to similar self-assembled structures when conjugated to a highly hydrophobic molecule. This peptide has two charged amino acid residues at both terminals. At the pH used this peptide has two charged amino acid residues at both terminals. This favors the formation of nanofibers instead of a cylindrical geometry, because for cylindrical micelles, charged residues in the interfacial region will destabilize the geometry.5c Based on our ongoing research on iron chelators, we chose deferasirox (DFX) as the hydrophobic drug. DFX, a bis-hydroxyl triazole and an orally active tridentate iron chelator, has been recently approved for the treatment of iron overload disorders. However, due to the lower efficacy of DFX in reducing brain iron Biopolymers (Peptide Science)

All Fmoc amino acids and HBTU were purchased from ChemPep Inc. (USA). Fmoc-Lys(Boc)-Wang resin was purchased from Advanced Automated Peptide Protein Technologies (AAPP-TEC, USA). DIC was from Advanced ChemTech (USA), HOBt was from Bachem (USA). DIPEA and ninhydrin were obtained from Applied Biosystems (USA). Piperidine, trifluoroacetic acid (TFA), thioanisole, ethanedithiol (EDT), HEPES, ferrous ammonium sulfate (FAS), calcein and G418 were obtained from Sigma-Aldrich. Phenol was purchased from LabSynth (Brazil). Deferasirox (ExjadeV) was donated by Novartis. All the reagents were of analytical grade and used as received without further purification. The solvents dichloromethane (DCM) and MeOH (analytical grade) were purchased from Merck (Germany), whereas DMSO (analytical grade) was obtained from Sigma-Aldrich. DMF (analytical grade) and acetonitrile (ACN) (chromatographic grade) were purchased from Vetec Fine Chemicals Ltd. (Brazil). Alpha Minimum Eagle Medium 1 : 1 Ham’s F10 Medium (aMEM-F10) was purchased from Vitrocell.HBS (HEPES Buffered Saline; NaCl 150 mM, HEPES 20 mM; pH 7.4) treated with Chelex-100V (Sigma, 1 g/100 mL) was used throughout the experiments. Curcumin (NutsOnline, Brazil) was a gift from Prof. Omar El Seoud (IQUSP) and pyrene (Sigma) was a gift from Dr Mirian Rinaldi (Botanic Institute, S~ao Paulo). R

R

R

Purification of DFX from ExjadeV R

A suspension of finely powdered ExjadeV (500 mg) was suspended in 200 mL aqueous MeOH (50%) and stirred for 30 min. After that, 50 mL of 5M aqueous NaOH was added and further stirred for 3 h. The suspension was then filtered and the filtrate was acidified (pH  2) dropwise with conc. HCl at 08C. A microcrystalline white precipitate of pure DFX appeared, which was filtered and dried under vacuum (380 mg). 1H NMR14 (500 MHz, DMSO-d6) d 6.86-6.88 (m, 1H), 6.99-7.04 (m, 3H), 7.36-7.40 (m, 2H), 7.54-7.57 (m, 3H), 7.978.07 (m, 3H), 10.04 (s, 1H), 10.79 (s, 1H), 13.19 (broad s, 1H).

Synthesis, Purification, and Characterization of Penetratin-DFX Conjugate (Conjugate 1) Penetratin [RQIKIWFQNRRMKWKK] peptide was synthesized manually by solid-phase methodology on Fmoc-Lys(Boc)-Wang resin (0.1 g, 0.56 mmol/g) using the Fmoc/tBu strategy and protocols routinely used by us in the Peptide Chemistry Laboratory of IQ-USP.15The amino acid side-chain protecting groups were trityl (Trt) for Asn and Gln, 2,2,4,6,7-pentamethyl dihydrobenzofuran-5-

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sulfonyl group (Pbf) for Arg, and tert-butyloxycarbonyl (Boc) for Lys and Trp. Fmoc deprotection, in the initial step of peptide assembly on resin and also after each amino acid coupling, was performed for 10 min using a 20% piperidine in DMF solution. Except for Asn and Gln incorporation in the growing peptide-resin, the amino acid couplings were performed by dissolving Fmoc-amino acid, DIC and HOBt (1 : 1 : 1) in a minimal amount of DCM-DMF (1 : 1, v/v), and using 2.5 molar excess relative to the resin aminoacylation level. Couplings of Asn and Gln derivatives employed HBTU/HOBt/DIPEA dissolved in a minimal amount of DMF. The reactions proceeded at room temperature (RT) for 60 min under mechanical shaking. The growing peptidyl-resin was washed (DMF 2 3 1 mL, MeOH 2 3 1 mL, DCM 2 3 1 mL). The efficiencies of Fmoc deprotection and coupling reactions were checked by the ninhydrin test.16 After total peptide assembly on resin, the terminal Fmoc group was removed in 20% piperidine/DMF solution. Conjugation of the -COOH group of DFX with the terminal -NH2 of the resulting resin-peptide was achieved using 2.5 molar excess (relative to the aminoacylation level of the starting resin) of a solution of DFX, DIC, HOBt, and a few drops of DIPEA dissolved in a minimal amount of DCM-DMF 1:1 (v/v). The reaction was complete after 60 min at RT (as indicated by the ninhydrin test). Side-chain protected conjugate was cleaved from the resin and fully deprotected to give the penetratin-DFX conjugate (1) in the mixture of 0.84 mL TFA with 0.06 g phenol, 0.04 mL distilled water, 0.04 mL thioanisole, and 0.02 mL ethanedithiol for 3.5 h at 378C. The crude conjugate was precipitated by addition of dry diethyl ether followed by centrifugation, dissolved in 0.1% aq. TFA, and freeze-dried (to yield 42 mg). Its major component was characterized (by LC-MS) as the desired conjugate 1. Purification of the crude conjugate was accomplished using Waters Model 600E preparative RP-HPLC consisting of a Waters Delta 600 quaternary pump, Waters 2487 dual absorbance UV detector, Rheodyne 3725i-119 manual sample injector, Waters 600 gradient controller, Kipp&Zonen 124 SErecorder and a Vydac C18 preparative column. Elution was monitored at the wavelength of 210 nm. The flow rate was 9.0 mL/min. Eluents were 0.1% TFA/water as solvent A and 60% CH3CN/0.09% TFA as solvent B. For conjugate 1, the following linear gradient was applied: 50% B for 10 min, 50280% B in 60 min. Conjugate 1 was characterized by LC/ESI–MS on a Shimadzu liquid chromatographer (Kyoto, Japan) composed of two LC-20AD pumps, a DGU-20A3 degasser, a CTO-20A column oven, a C18 Shimpack GVP-ODS pre-column, a C18 Shim-pack VP-ODS column and SDP-20AV detector coupled to an AmaZon X electrospray mass spectrometer (Bruker Daltonics, Fahrenheitstrasse, Germany). The software HyStar 3.2 was used in the analysis of the mass spectra obtained. The solvents used for chromatography were: 0.1% TFA/H2O as solvent A and 60% CH3CN/0.09% TFA as solvent B. The linear gradient used was 5 to 95 % B in 30 min. Amino acid analysis of the purified and fully hydrolysed (6M HCl, 1108C, 24h) conjugate was conducted in a DionexVBioLC Chromatography system (Dionex, USA) employing a ion exchange method on a AminoPac PA10 (2.0 3 25 cm) column and an electrochemical detector ED50. R

CMC Determination The critical micelle concentration (CMC) of conjugate 1 was determined17 by measuring fluorescence intensities of the hydrophobic probe pyrene in a solution of 1 in HBS. A methanolic solution of

pyrene (125 lM) was prepared and 50 lL was added to a solution of conjugate 1 at concentrations ranging from 1 to 30 lM, keeping 3 mL as final volume. The solutions were sonicated for 30 min, and the fluorescence emission spectra were recorded in a SPEX FL212 Fluorolog2 spectrofluorometer using a 450 W Xenon lamp as excitation source and two 0.22 m double grating SPEX 1680 monochromators for dispersing the radiation. The entire luminescence setup was fully controlled by a DM3000F spectroscopic computer program and the spectral intensities were automatically corrected for the photomultiplier response. The spectra were recorded at room temperature, using excitation wavelength at 334 nm, excitation slit width at 1 nm, and emission slit width at 6 nm. The intensity ratio of the first peak (I374, 374 nm) and the third peak (I385, 385 nm) of pyrene was plotted against concentration of 1 for the determination of CMC value.

Preparation of Curcumin-Loaded Conjugate 1 Micelles A sample of 1.4 mg of conjugate 1 was dissolved in water and 40 lL of a solution of curcumin in acetone (3.26 mg/mL) was added to it. The mixture was sonicated for 1 h, and thereafter was lyophilised overnight to get a dry powder. A volume of 1 mL HBS was added to the powder, stirred, and centrifuged (3000 rpm for 2 min) to separate the remaining insoluble curcumin. The supernatant solution in HBS was separated, and the solid residue was dissolved in 200 lL DMSO. The absorption intensities at 420 nm were used for quantification of the total and uncapsulated curcumin, against a calibration curve of free curcumin. Curcumin encapsulation efficiency (EE) and loading capacity (LC) were calculated using the following equations:18



 mcurcumin; total 2mcurcumin; free 3100 EEð%Þ5 mcurcumin; total   mcurcumin; total 2mcurcumin; free 3100 LCð%Þ5 ða3f Þ1b

(1) (2)

where a 5 mass of conjugate 1 ; f 5 peptide fraction; b 5 mass of encapsulated curcumin. The spectrophotometric measurements were conducted in a SpectraMax M4 microplate reader (Molecular Devices).

Circular Dichroism Studies Circular dichroism (CD) spectra of aqueous solutions of PEN-DFX and PEN-DFX-Curcumin (20 lM in peptide) were obtained in a Jasco J-815 equipment at 258C, in 10 mm cuvettes at 20 nm/min with background correction. Results are the average of three runs.

Morphology of the Micelles The size and morphology of the curcumin-loaded micelles were determined by transmission electron microscopy (TEM). Three microliters of a 100 mM aqueous solution of curcumin-loaded conjugate 1 was placed on a 300 mesh carbon-coated copper grid, and 3 mL of 2% (w/ w) uranyl acetate solution was added for negative stain. The sample was deposited for 5 min, and excess solution was wiped off by filter paper. The dried sample was observed with a JEOL-JEM 1010 instrument (Japan) operating at 80 kV. The images were analysed using ImageJ 1.37c software.

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allows knowing conjugate concentrations accurately prior to the biological assays and spectroscopic analyses. To the best of our knowledge, this is the first report for the synthesis of a peptide-deferasirox conjugate. By using our protocols and experimental conditions, the synthesis was straightforward and was achieved without any practical surprises. FIGURE 1 Full ESI-MS spectrum and HPLC chromatogram (inset) of Penetratin-DFX conjugate (conjugate 1).

In Vitro Drug Release A sample of 2.4 mg of the micelles formed by lyophilised curcuminloaded conjugate 1 was dissolved in 1 mL of phosphate buffer (pH 7.4) and the resulting solution was incubated at 378C. At predetermined time intervals, it was centrifuged at 3000 rpm for 2 min to sediment the released curcumin, and the supernatant was again incubated. The separated curcumin was redissolved in 200 lL DMSO for spectrophotometric quantification as described above. The release was quantified as

Releaseð%Þ5

  mcurcumin; released mcurcumin; total

CMC Determination The CMC for the self-assembly of the conjugate consisting of a hydrophilic part and a hydrophobic head group to form a micellar structure was determined using a fluorescence method employing the hydrophobic fluorescence probe pyrene. Its solubilisation in the interior hydrophobic core decreases the intensity ratio of I374 to I385, indicating the formation of micelles in solution. As the micelle concentration increased, the ratio decreased sharply. As evident from Figure 2, the CMC of the penetratin-DFX conjugate in water was very low, 6 mM

(3)

Cell Permeation Studies RBE4 cell line was a gift from Prof. Michael Aschner (Albert Einstein College of Medicine, USA). Cells were grown in aMEM/F-10 medium containing 10% FBS, 1% antibiotics and 300 mg/mL G418, and was incubated at 37 8C in a humidified incubator with a 5% CO2 atmosphere. Cells were subcultured three times a week to prevent overcrowding and cell death. In order to conduct the fluorescence microscopy experiments, the cells were trypsinized, transferred (30,000 cells per well) to the wells of a 6-well microplate, and kept for 48 h until complete adherence and confluence. After 48 h, the medium was removed and the cells were washed with warm HBS buffer. Then, the cells were incubated for 30 min with conjugate 1 (100 lM), curcumin (0.1 mg/mL, diluted in HBS from a solution of 3 mg/mL in DMSO), or 100 mM of curcumin-loaded (8.7%) conjugate 1. The images were obtained in an Axiovert 200 (Carl Zeiss, Germany) microscope under 100 3 magnification, recorded on digital camera Canon Power Shot G10, and analyzed using ImageJ 1.37c software.

RESULTS AND DISCUSSION Synthesis, Purification, and Characterization of Penetratin-DFX Conjugate (Conjugate 1) Conjugate 1 was obtained at a 18% yield with purity higher than 95%. After purification, the final targets were evaluated by analytical RP-HPLC analysis and LC-ESI/MS analysis (Figure 1) ([MH]1calcd: 2602.1 g/mol; found: 2602.0 g/mol). Full acid hydrolysis/amino acid analysis of the hydrolyzates showed 38% of peptide content. This information is important, as it Biopolymers (Peptide Science)

FIGURE 2 A: Fluorescent emission spectra of pyrene in presence of increasing concentrations of conjugate 1. B: Plot of ratio of fluorescence peak intensities (I374/I385) as a function of concentration of conjugate 1. (a.u. 5 arbitrary units of fluorescence).

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FIGURE 4 TEM micrograph of curcumin-loaded conjugate 1 nanostructures at a concentration of 100 lM.

Preparation of Curcumin-Loaded Conjugate 1 Micelles

FIGURE 3 A: UV-VIS spectra of 3.3 mg/mL curcumin in DMSO. B: UV-VIS spectra of curcumin-loaded conjugate 1 micelles (8.7%) in water.

To inspect the efficiency of the micelles to solubilize hydrophobic molecules inside the hydrophobic core, and to carry it through BBB, curcumin was chosen as a model hydrophobic drug. Curcumin, a polyphenol, has potential anti-cancer activity.20 Very recently, curcumin has been found to prevent major disabling age-related neurodegenerative disorders,21a and also to restore brain-derived neurotrophic factor levels in mice.21b However, the clinical applications of curcumin are restricted due to its low water solubility, and low bioavailability. Until now, several micellar nanocarriers have been tested for enhancing its bioavailability.20,22

( 15 mg/L). This low CMC of conjugate 1 can be attributed to the high hydrophobicity of DFX, and makes it beneficial for use in very low concentrations.

Circular Dichroism Studies In aqueous medium, penetratin presents a CD spectrum characteristic of random coil, with a strong negative peak at 190– 200 nm and a slight positive peak at 230 nm.19 The conjugation of penetratin to DFX, or the load of curcumin to PENDFX, did not alter this feature as the CD spectra did not display signs of secondary structures such as a-helixes or b-sheets (Supporting Information Figure S1), indicating that even though the DFX hydrophobic moiety favours proximity of the peptide molecules, the high polarity of the medium prevents organization. FIGURE 5 In vitro release profile of curcumin encapsulated in conjugate 1 micelles. Results are the average of two separated experiments.

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FIGURE 6 Permeation of conjugate 1 (A0), curcumin (B0), and curcumin-loaded conjugate 1 micelles (C0) in RBE4 cells. The histograms show the cell population against fluorescence density for cells treated with curcumin (B1, xc 5 8.90 6 0.98 f.u./pixel2), and curcumin-loaded conjugate 1 micelles (C1, xc 5 15.81 6 1.83 f.u./pixel2). Fluorescence values were corrected for background. (f.u. 5 fluorescence units).

In our study, curcumin was loaded into the micelle using a method described in the experimental section. The UV-VIS spectra of pure curcumin and the curcumin-loaded conjugate 1 micelles are shown in Figure 3. The drug loading was found to be 8.7% with an encapsulation efficiency of 59.1%. This loading efficiency might be further improved by optimizing the loading parameters; here, we used curcumin-loaded micelles with 8.7% loading to determine its efficacy to permeate the BBB. Biopolymers (Peptide Science)

Morphology of the Micelles A typical TEM image of the curcumin-loaded micelles (Figure 4) shows them to have average diameter of 4 nm and length of 17 nm. According to previous reports,5c the diameter indicates formation of nanofibers rather than cyllindrical micelles. The morphology of the micelle is highly dependent on the competition between the intra-/inter-peptide hydrogen bonding and

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the shielding of the hydrophobic DFX from the aqueous environment. The formation of nanofibers clearly indicates that the stabilisation arising from hydrogen bonding supersedes the shielding of the hydrophobic DFX. The presence of charged amino acid residues (arginine and lysine) also favours the formation of nanofiber geometry.

In Vitro Drug Release The drug release profile (Figure 5) at 378C showed that the encapsulated curcumin was released slowly (up to 65%) over a period of 60 h, indicating that the curcumin molecules were well encapsulated within the micelle core. Within the initial phase (10–20 h) curcumin is adsorbed in the surface of the carrier material quickly released.23a It is important to notice that besides diffusion other mechanisms may be involved at the same time in curcumin release, such as swelling/erosion and degradation of the micelles.23b Other peptide-based micelles display similar release kinetics to PEN-DFX.23c However, the effect of micelle composition markedly affects release. Poly(ethyleneglycol)-based micelles for example slow curcumin release, with 50–70% after incubation for 9 days.23a,23d Chitosan-based micelles released 70% of curcumin after 5 days of incubation.23e These differences indicate that the careful choice of micelle composition may tune the release kinetics.

Cell Permeation Studies RBE4 cell line is a well-accepted in vitro model for the BBB, and has been used extensively to investigate the transendothelial export of various molecules.24 The permeability of the curcumin-loaded micelles to RBE4 cells was studied via fluorescence microscopy, and was compared with that of curcumin. As shown in Figure 6, the curcumin-loaded penetratinDFX micelles could penetrate RBE4 cells within 30 min of incubation. As expected, the cells treated with conjugate 1 solution did not show any fluorescence. However, when compared to curcumin-treated cells, xc (the cell population with maximum fluorescence) for the cells treated with curcuminloaded conjugate 1 micelles was significantly higher, indicating a substantial increase in the bioavailability of curcumin. In both cases the fluorescence densities were corrected after subtracting an average background density from those of the cells. This increased permeation of curcumin in a BBB model can be attributed exclusively to the presence of penetratin, a CPP with known ability to translocate the cellular membranes by interacting with the lipids.25 So far, several formulations of curcumin, including encapsulation into nanostructures,20,22 have been developed. However, very few of these formulations are

actually capable of permeating through BBB.26 Considering the beneficial effects of curcumin for neurodegenerative diseases, we believe that its encapsulation into micelles of a CPP-drug conjugate can be effective in translocating curcumin across BBB. Obviously, these micelles can also be a vehicle for the increased brain permeability of DFX. Considering that brain iron overload is at the basis of several neurodegenerative processes such as Parkinson or Alzheimer Diseases,27 coadministration of curcumin and DFX might have a synergic effect on the treatment of these illnesses. Finally, these micelles can further be utilized as nanocarriers in general, translocating hydrophobic antibiotics across the BBB. Our findings on the self-assembly of a CPP-drug conjugate may open various pathways for delivering otherwise nonpermeable drugs into the brain.

CONCLUSIONS Conjugation of DFX to penetratin leads to self-assembly into nanofibers, which can be used to encapsulate hydrophobic drugs like curcumin. The enhanced loading of curcumin into RBE4 cells indicates that PEN-DFX could be an effective nanocarrier for drugs used in neurodegenerative diseases. In vivo tests are required to verify this possibility. DG receives a post-doctoral fellowship from FAPESP (2011/ 18958-0). FAPESP and CNPq funded these investigations. The authors thank Mr Latif Khan, Mr Alfredo Duarte, Mr Jesus Alvarado and Dr. Cleber Wanderlei Liria for discussions and technical assistance.

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Self-assembled penetratin-deferasirox micelles as potential carriers for hydrophobic drug delivery.

There has been a growing interest in the use of micelles with nanofiber geometry as nanocarriers for hydrophobic drugs. Here we show that the conjugat...
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