Brief Article pubs.acs.org/molecularpharmaceutics
Synthesis of Novel Biodegradable Methoxy Poly(ethylene glycol)−Zein Micelles for Effective Delivery of Curcumin Satheesh Podaralla,† Ranjith Averineni, Mohammed Alqahtani, and Omathanu Perumal* Department of Pharmaceutical Sciences, College of Pharmacy, South Dakota State University, Brookings, South Dakota 57007, United States S Supporting Information *
ABSTRACT: Novel biodegradable micelles were synthesized by conjugating methoxy poly(ethylene glycol) (mPEG) to zein, a biodegradable hydrophobic plant protein. The mPEG− zein micelles were in the size range of 95−125 nm with a low CMC (5.5 × 10−2 g/L). The micelles were nonimmunogenic and were stable upon dilution with buffer as well as 10% serum. Curcumin, an anticancer agent with multiple delivery challenges, was encapsulated in mPEG−zein micelles. The micelles significantly enhanced the aqueous solubility (by 1000−2000-fold) and stability (by 6-fold) of curcumin. PEG− zein micelles sustained the release of curcumin up to 24 h in vitro. Curcumin-loaded mPEG−zein micelles showed significantly higher cell uptake than free curcumin in drug-resistant NCI/ADR-RES cancer cells in vitro. Micellar curcumin formulation was more potent than free curcumin in NCI/ADR-RES cancer cells, as evidenced from the 3-fold reduction in IC50 value of curcumin. Overall, this study for the first time reports a natural protein core based polymeric micelle and demonstrates its application for the delivery of hydrophobic anticancer drugs such as curcumin. KEYWORDS: micelles, mPEG−zein micelles, curcumin, nanocarrier, core−shell
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INTRODUCTION Micelles are nanoscopic delivery carriers formed through spontaneous self-assembly of amphiphilic molecules. In an aqueous environment, the micelles form a spherical core−shell architecture where the hydrophobic segment of the amphiphilic molecules forms the core while the hydrophilic segment forms the exterior shell.1 Water insoluble therapeutic and diagnostic agents can be encapsulated inside the hydrophobic core of the micelles.1 Encapsulation of water insoluble agents in micelles offer significant advantages such as improved water solubility, stability, and biodistribution.1−4 Because the size of the micelles are very small (20−200 nm), they can easily extravasate through the leaky vasculature and accumulate in the tumor tissue. This provides a unique advantage to passively target anticancer drugs to the tumor by enhanced permeability and retention (EPR) effect.5 In comparison to other nanocarriers, micelles are much easier to prepare and also provides the flexibility of engineering the core and/or shell to develop active targeting, stimuli responsive, and multifunctional delivery systems.1,4 Many amphiphilic materials are known to form micelles including surfactants and polymers.1−4 Amphiphilic molecules with distinct regions of different affinities toward a given solvent form micelles spontaneously at a defined concentration and temperature. These molecules exist as monomers at low concentrations in the aqueous medium, but as the concentration is increased, aggregation takes place within a very narrow © 2012 American Chemical Society
concentration range. The concentration at which the monomeric amphiphilic molecules form micelles is known as critical micellar concentration (CMC). The formation of micelles is thermodynamically driven by aligning the hydrophobic segments away from the aqueous environment and through van der Waals interactions between the hydrophobic segments in the core.1 Small molecular weight surfactants have high critical micellar concentration (CMC) and lower micellar stability, resulting in dissociation of the micelles upon dilution in vivo.1 Further, the surfactant micelles have low drug loading capacity and show higher toxicity.1 In contrast, polymeric micelles have better micellar stability, higher drug loading efficiency, and lower toxicity.1,2 A variety of amphiphilic copolymers have been used to form micelles, and block copolymers are mainly used in this regard.3,4 Typically, the block copolymers can be synthesized by copolymerizing hydrophobic polymers with hydrophilic polymers. Diblock and triblock copolymers of polyesters and polypeptides have been widely used for preparing micelles.3,4 Alternatively, graft copolymers have also been used to prepare micelles.6 Graft copolymers are branched polymers with a hydrophilic backbone with one or more hydrophobic Received: Revised: Accepted: Published: 2778
December 14, 2011 June 15, 2012 July 6, 2012 July 6, 2012 dx.doi.org/10.1021/mp2006455 | Mol. Pharmaceutics 2012, 9, 2778−2786
Molecular Pharmaceutics
Brief Article
Corporation, USA. Glycine, curcumin, pyrene, dialysis cassettes (cut off ∼10000 Da) were from Pierce, NY, USA. RPMI media (GIBCO), Fetal bovine serum (Atlanta Biologicals Inc., Lawrenceville, GA), and penicillin/streptomycin (GIBCO) were from Fisher Scientific, (New Jersey). Mouse IgG and horseradish peroxidase conjugated to IgG (HRP-Ig G) was purchased from Bethyl laboratories (Montgomery, TX). 3′,3′,5,5′-tetramethylbenzidine (TMB) substrate and all the other chemicals were reagent grade (Sigma, St. Louis MO). PEGylation of Zein. To prepare PEGylated zein, 0.1 g of zein was dissolved in 90% alcohol followed by the addition of 0.1 g of methoxy PEG−succinimidyl succinate. The mixture was incubated for 3 h in a magnetic stirrer (50 rpm) at room temperature. The reaction was stopped by addition of 1 mL of aqueous glycine (1 M) solution to quench the excess PEG ester. The mixture was then diluted with 5 mL of citrate buffer (pH 7.4) and dialyzed (cut off = 10 kDa) against water in a magnetic stirrer (100 rpm) at room temperature for 24 h to remove free PEG and ethanol. The resulting product was then freeze-dried to obtain PEG-zein. Size-Exclusion Chromatography (SEC). To qualitatively confirm the PEGylation of zein, SEC was carried out using a Phenomenex Biosep SEC-S 3000 4.6 mm × 3000 mm column (Phenomenex, Torrance, CA) in a HPLC (Beckman Coulter, Brea, CA, USA) system. The samples were separated using 70% (v/v) ethanol as mobile phase using a flow rate of 0.5 mL/min. The column eluate was monitored at 280 nm. ATR-FTIR Analysis. To further confirm the PEGylation of zein, the ATR-FTIR spectrum of zein, mPEG-ester, and mPEG− zein (5 mg each) were recorded on ZnSe crystal at 2 cm−1 resolution in Nicolet 380 ATR-FTIR spectrophotometer (Thermo Electron Corporation, Madison, WI). Each spectrum was an average of 100 scans. The peak position of functional groups was analyzed using OMNIC software. Proton NMR Analysis. Proton nuclear magnetic resonance (1H NMR) spectroscopy was used to characterize core−shell structure of PEG−zein micelles. The samples (1 mg/mL of zein and PEG−zein) were prepared in D2O and deuterated dimethyl sulfoxide. Samples were subjected to 250 scans in a 400 Hz NMR (Bruker Daltonics Inc., Fremont, CA), and the data was processed using ACD/NMR processor. Determination of Critical Micelle Concentration (CMC). The CMC of mPEG−zein micelles was determined using pyrene as a fluorescent probe. Pyrene solution (12 μg/mL dissolved in acetone) was added to 5 mL glass vials. After complete evaporation of acetone, a series of concentrations of mPEG−zein (0.003 to 5 mg/mL) in 0.01 M phosphate buffered saline (pH 7.4) was added to the mixture, and the final concentration of pyrene was adjusted to 0.12 μg/mL. The mixture was vortexed for 5 min and incubated overnight at 37 °C. The fluorescence spectra of the samples were recorded at the excitation wavelengths of 339 and 334 nm (emission wavelength was fixed at 390 nm). The ratio of absorbance at these two wavelengths (I339/I334) was plotted as a function of increasing logarithmic concentration of mPEG−zein. The concentration of mPEG−zein at which the solution absorbance changed significantly was defined as the CMC. Light Scattering Measurements. The particle size and ζ potential of mPEG−zein micelles was measured by photon correlation spectroscopy using NICOMP 380 ZLS (Particle Sizing Systems, USA). Briefly 2 mg of PEG−zein was dispersed in 10 mL of phosphate buffer (pH 7.4), followed by the measurement of particle size and ζ potential.
polymeric side chains or vice versa. Polyesters and polypeptides are frequently used as the core-forming polymers, while poly(ethylene glycol) (PEG) and polyvinyl pyrrolidone are the most common shell forming hydrophilic polymers.3,4 Lipid core polymeric micelles have also been formed by conjugating PEG to diacyllipids such as phosphatidyl ethanolamine.7 Here for the first time we report a novel protein core polymeric micelle formed using a natural biodegradable water-insoluble protein and a water-soluble polymer. The micelles were prepared by conjugating methoxy poly(ethylene glycol) (mPEG) to zein. Zein is a water-insoluble protein derived from corn and is widely used in food and packaging industry due to its film forming property and ability to provide a moisture barrier.8 It is composed of a high proportion (>50%) of nonpolar amino acids including leucine, proline, alanine, and phenylalanine.9 The molecular weight of zein varies from 22 to 27 kDa and is mainly composed of α-zein.8 Because of its hydrophobicity and biodegradability, zein has been used to prepare microparticles/ nanoparticles for sustained delivery of drugs and nutraceuticals.10−13 However, because of its protein origin and hydrophobicity, the resultant zein micro/nanoparticles can be rapidly taken up by macrophages and may cause immunogenicity.14 Therefore, we hypothesized that PEGylation can prevent opsonization and also provide steric hindrance for enzymatic degradation of zein. Interestingly, as shown here, the mPEG−zein conjugate self-assembled in water to form core−shell micelles. To this end, the main objective of this study was to prepare mPEG−zein micelles, characterize the micelles, test their immunogenicity, and demonstrate their potential as an effective delivery vehicle for curcumin, a water-insoluble anticancer agent. Curcumin is a yellow-colored polyphenol derived from the spice turmeric (Curcuma longa). It has a wide spectrum of therapeutic properties including antioxidant, anti-inflammatory, anticancer, and antimicrobial activities.15,17−19 Curcumin has been widely studied for its chemopreventive and chemotherapeutic effects against various types of human cancers including skin, cervix, lung, prostate, breast, ovarian, bladder, liver, head/ neck, pancreatic, and colorectal cancers as well as leukemias, lymphomas, multiple myeloma, and brain cancer.18 It exerts its anticancer effects by interfering with multiple cell signaling pathways including cell cycle, apoptosis, proliferation, invasion, angiogenesis, metastasis, and inflammation.20 In addition, curcumin is also known to sensitize tumor cells to various chemotherapeutic agents and radiation by downregulating antiapoptotic and multidrug-resistant genes.21 At the same time, curcumin also protects normal tissues from chemotherapy and radiotherapy induced toxicity through its antioxidant effects. Given the diverse anticancer effects of curcumin, it is administered through parenteral and nonparenteral routes.18,22 However, the clinical application of curcumin is limited due to the multiple delivery challenges.22 This includes poor water solubility (Log P = 2.5), chemical instability, poor membrane permeability, metabolic instability, and low bioavailability of curcumin.22 To this end, the goal of this study is to overcome the poor physicochemical properties of curcumin by encapsulating it in mPEG−zein micelles. This feasibility study is aimed at broadly demonstrating the potential of mPEG−zein micelles as a delivery vehicle for curcumin.
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MATERIALS AND METHODS Zein F-6000 (white zein) was purchased from Freeman Industries Inc., (Tuckahole, NY). Methoxy PEG−succinimidyl succinate (1000, 2000, and 5000 Da) was purchased from NOF 2779
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Atomic Force Microscopy (AFM). The particle size and surface morphology of mPEG−zein micelles was analyzed by atomic force microscopy (AFM, Pacific Nanotechnology, Inc., USA). Briefly 2 mg of PEG−zein was dispersed in 20 mL of Milli-Q water and probe sonicated for 1 min. An aliquot (100 μL) of the supernatant was placed on a polyethylene imine-coated coverslip and air-dried. The micelles were imaged in the noncontact mode, and the image was analyzed using Nanorule software (Pacific Nanotechnology, Inc., USA). Transmission Electron microscopy (TEM). The sample was also imaged by TEM (HITACHI H-7000 FA, Japan). Briefly 2 mg of mPEG−zein micelles were dispersed in 20 mL of deionized water and applied to the sample grid. The samples were stained with 1% v/v uranyl acetate, air-dried, and the images were recorded at a magnification of 20 K. Stability of mPEG−Zein Micelles. The change in particle size of mPEG−zein micelles upon dilution in pH 7.4 citrate buffer and 10% fetal bovine serum (FBS) was determined using PCS. The PEG−zein micelles in 10 mM citrate buffer pH 7.4 (2 mg/mL) was diluted 20−500-fold, and the micelle size was determined after 2 h of incubation in citrate buffer at 37 °C. Similarly the micelles were diluted (500-fold) with 10% FBS and kept for 2 h 37 °C before measuring the particle size by PCS. In Vivo Immunogenicity Studies. The immunogenicity of mPEG−zein micelles was determined in mice. All procedures performed in mice were approved by the institutional animal care and use committee. Female BALB/c mice (3− 4 weeks old) were divided into five groups of four animals each. mPEG−zein micelles (100 μg/50 μL) was administered subcutaneously in saline (0.9% w/v sodium chloride) at the first week, and a booster was administered at the fifth week. Blood samples were collected from the retro orbital plexus at the third week after first dose and booster dose, respectively. Saline was used as a negative control. Serum was separated from the blood and diluted 16-fold for determining the IgG levels by sandwich ELISA. Briefly, a 96-well plate was coated with zein (1% w/v in 90% v/v ethanol) and kept for overnight at 4 °C. The wells were washed using buffer (1% Tween 20 in PBS) and blocked with 1% BSA (bovine serum albumin) for 1 h. Then the wells were washed using buffer, followed by incubation with serum (1/16 dilution) for 1 h. After removing the serum, the wells were washed with PBS buffer and incubated with horseradish peroxidase conjugated goat antimouse IgG for 1 h at 37 °C. The washing step was repeated again, and tetramethylbenzidine was added as a substrate followed by incubation for 5 min. The reaction was stopped by the addition of 1 M H2SO4, and the optical density was recorded at 450 nm in a plate reader (SpectromaxM2, micro plate reader, Molecular Devices, Sunnyvale, CA). Preparation of Curcumin Loaded mPEG−Zein Micelles. Curcumin (2 mg; Sigma-Aldrich, USA) and mPEG− zein (100 mg) were dissolved in 20 mL of 90% ethanol, and the mixture was stirred (50 rpm) at 37 °C overnight to allow partitioning of the curcumin into PEG−zein micelles. The mixture was then dialyzed (cut off ∼10000 Da) against deionized water to remove alcohol and free curcumin. The resulting product was then freeze-dried. Determination of Encapsulation Efficiency for Curcumin. Curcumin loaded mPEG−zein micelles (5 mg) was dispersed in 1 mL of Milli-Q water and centrifuged at 5000 rcf for 14 min at 4 °C. An aliquot of the supernatant was diluted with ethanol and used for the determination of free curcumin.
Figure 1. Synthetic scheme for PEGylation of zein. m-PEG-N-hydroxy succinimidyl ester (5 kDa) is conjugated to the free amino group of glutamine in zein. The molecular weight of mPEG−zein is 28.74 kDa as determined from MALDI-TOF.
The pellet was digested with 90% v/v ethanol, and an aliquot was diluted with 90% ethanol to determine the encapsulated curcumin. The curcumin amount was determined by HPLC method.23 Briefly, a C18 column (Waters Corporation, MA, USA) was used and the mobile phase consisted of 60% acetonitrile and 40% citric acid (1% w/v citric acid solution adjusted to pH 3.0 with 50% w/w sodium hydroxide solution). The flow rate was 1.0 mL/min, and the detection wavelength was 420 nm. Standard curve of curcumin (0.031 to 1 μg/mL) in 90% ethanol was generated for the determination of curcumin amount (r2 > 0.99). Free curcumin in the supernatant was subtracted from curcumin in the micelles. The encapsulation efficiency was calculated as percent mg of drug loaded per mg of the mPEG−zein relative to the theoretical loading of curcumin. Encapsulation efficiency was expressed as a mean of three experiments (±SD). Determination of Curcumin Solubility and Stability. The solubility of free and micelle-loaded curcumin was studied by recording the fluorescence spectra using 420 nm as the excitation wavelength. Curcumin (10 μg/mL) was dissolved in methanol and 10%v/v methanol in PBS (pH 7.4), while the curcumin loaded mPEG−zein micelles (equivalent concentration of curcumin) was dispersed in PBS (pH 7.4). For stability studies, the UV absorbance of free curcumin (5 μg/mL in 10% v/v methanol in PBS) and curcumin loaded mPEG−zein micelles were recorded at 420 nm for 24 h under ambient conditions. In Vitro Release Study. Curcumin loaded mPEG−zein micelles (1 mg/mL) was incubated in 1 mL of citrate buffer (pH 7.4 along with 5% tween 80 to maintain the sink conditions and 0.01% butylated hydroxyl anisole as the antioxidant in a centrifuge tube and was maintained at 37 °C in a horizontal shaker water bath at 50 rpm. At each time interval, one vial was removed and was centrifuged at 12000 rcf for 12 min. The pellet was diluted with 90% ethanol, and then the content of curcumin was analyzed using HPLC as described earlier. In Vitro Cell Uptake Studies. To test the cell uptake of curcumin, drug-resistant ovarian cancer cells (NCI/ADR-RES cells) were used. The cells (5000 cells/well) were seeded in a 6-well plate in RPMI medium supplemented with 10% FBS and 1% penicillin−streptomycin. Cells were allowed to attach overnight by incubating at 37 °C under 5% CO2 in an incubator. The next day, the cells were treated with free curcumin (dissolved in 10% v/v DMSO in serum free medium) and equivalent concentration of curcumin loaded PEG−zein micelles (in serum-free medium) for 30−120 min at 37 °C. The cells were washed with PBS, followed by detachment of the cells using trypsin. The cells 2780
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Figure 2. Characterization of mPEG−zein conjugate by (a) FTIR and (b) size-exclusion chromatography.
were resuspended in 1 mL of PBS and used for flow cytometry. In the flow cytometer (FACS, BD biosciences, San Jose, CA), 30000 cells were counted and fluorescence intensity of the cells was detected in FITC channel using a bandpass filter at 530 ± 30 nm. Data were analyzed using CellQuest software, and the mean fluorescence intensity of the cells was measured. In Vitro Cytotoxicity Study. To tests the activity of curcumin, drug-resistant ovarian cancer cells (NCI/ADR-RES cells) were used. The cells (2000 cells/well) were seeded in 96well plates in RPMI medium supplemented with 10% FBS and 1% penicillin−streptomycin. Cells were allowed to attach overnight by incubating at 37 °C under 5% CO2 in an incubator. The next day, the cells were treated with free curcumin (dissolved in 10% v/v DMSO in serum-free medium) and equivalent concentration of curcumin loaded PEG−zein micelles (in serum-free
medium) for 4 days. Starting from second day, the treatment media was replaced with fresh drug-free medium every 48 h and the cell viability was determined on the fifth day by MTT assay. Briefly, the cells were washed three times with PBS and 50 μL of MTT (5 mg/mL in serum-free medium) was added, and cells were incubated for 4 h. After 4 h, 200 μL of DMSO was added and plates were kept on a shaker for 10 min to dissolve formazan crystals. The absorbance was recorded at 570 nm in a plate reader (SpectramaxM2 microplate reader, Molecular Devices, Sunnyvale, CA). The IC50 values were calculated by plotting % cell viability (relative to untreated cells) as a function of curcumin concentration using Graphpad Prism 5 (GraphPad Software Inc., CA, USA) Data Analysis. All the experiments were performed in triplicate unless specified, and the results are expressed as 2781
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Figure 3. Images of PEG−zein micelles by (a) transmission electron microscopy and (b) atomic force microscopy. AFM images from left to right are 2D topography, amplitude, and phase images of a representative sample with a z-scale of 58 nm, 0.75 V, and 64°. The average particle size of 100 particles measured in AFM was 95 ± 10 nm.
mPEG (5 kDa) to zein. It is generally known that PEG binds to SDS micelles and decreases the mobility of the protein in SDSPAGE.24 As a result, the molecular weight of PEGylated protein in SDS-PAGE is usually larger than the actual molecular weight. The conjugation was also confirmed using FTIR (Figure 2a). Amide I and II protein peaks of zein are observed in 1650 and 1500−1540 cm−1, respectively. The NHS ester peak for PEG at 1740 cm−1 disappeared after conjugation with zein. There was a shift in the amide II (1529 cm−1) of zein, but no new peaks were observed as the new amide bonds overlap with the protein amide peaks.25 Further, the conjugate was characterized by size exclusion chromatography (SEC). As can be seen from Figure 2b, PEG−zein conjugate eluted at 7 min and zein eluted at 23 min. On the other hand, PEG eluted at 29 min. The purity of the conjugate is also confirmed from the single peak observed for PEG−zein. Conjugation of PEG to zein resulted in a shift in the melting point of PEG from 55 to 50 °C and reduced the glass transition temperature of zein (Figure 3 in Supporting Information). Characterization of mPEG−Zein Micelles. mPEG−zein micelles were prepared using dialysis process through solvent exchange. mPEG−zein was dissolved in 90% alcohol and dialyzed against deionized water for 24 h. The slow removal of organic solvent favored the formation of mPEG−zein micelles. The micelles were spherical and had a smooth surface as is evident from the transmission electron microscopy and atomic force microscopy (AFM) images in Figure 3. The size of the micelles was measured using photon correlation spectroscopy (PCS) and by AFM (Table 1 and Figure 3). The average size of the micelles found by PCS (123 ± 11 nm) and atomic force microscopy (95 ± 10 nm) were close. The core−shell structure
Table 1. Characteristics of Blank and Curcumin Loaded PEG−Zein Micellesa micelles
particle size (nm)
PDI
ζ potential (mV)
encapsulation efficiency (%)
blank curcumin
95 ± 1.7 124 ± 4
0.21 ± 0.07 0.25 ± 0.03
−7.2 ± 2.5 −7 ± 1.6
NA 95 ± 4
a Each value is a mean of three measurements ± SD; PDI, polydispersity index; NA, not applicable.
mean ± SD. Student t test (Instat, Graph Pad software, CA) was used to compare the different groups, and the results were considered to be significant at p < 0.05.
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RESULTS AND DISCUSSION Characterization of PEGylated Zein. In the present study, PEG−zein was prepared in a single step reaction. The preparation of PEG grafted zein is a simple process compared to the preparation of synthetic block-co-polymers. Zein is composed of 22% of glutamine and aspargine,9 where the amino group in the side chain of these amino acids can be used for PEGylation (Figure 1). Zein is mainly composed of α-zein, and its N-terminal has a glutamine.9 The m-PEG-N-hydroxy succinimidyl ester (5 kDa) was used to form an amide bond with the terminal amino group in zein. The molecular weight of mPEG−zein was determined using SDS-PAGE. Zein showed the bands corresponding to α-zein at 22−23 kDa, while mPEG− zein showed a band at 31 kDa (Figure 1 in Supporting Information). MALDI-TOF analysis showed a peak at 23 kDa for zein and 28.74 kDa for mPEG−zein (Figure 2 in Supporting Information). This corresponds to the conjugation of one 2782
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Figure 4. (a) Proton NMR spectrum of mPEG−zein in deuterated DMSO. Ethylene peak of PEG is observed at 3.6 ppm, while the protein amide of zein peak is observed at 3.4 ppm. In organic solvent, PEG−zein does not form micelles, and hence the signal for both mPEG and zein is observed. (b) Proton NMR spectrum of mPEG− zein in D2O. Because only the hydrophilic shell of the micelles is solvated in D2O, only the ethylene proton signal of mPEG was seen at 3.6 ppm. The zein amide peak was not observed as the hydrophobic core is not soluble in D2O. This confirms the core−shell architecture of mPEG−zein micelles.
Figure 5. (a) Determination of CMC. The ratio of absorbance of pyrene 339 and 334 nm is plotted against logarithmic concentration (g/L) of mPEG−zein. Each data point is a mean of three experiments. The CMC was found to be 0.055 g/L. (b) Particle size of mPEG−zein micelles measured by photon correlation spectroscopy after dilution of the micelles from 20- to 500-fold with citrate buffer (pH 7.4) and kept aside for 2 h at 37 °C. Each data point is a mean of three experiments ± SD.
of mPEG−zein micelles was confirmed by comparing the 1H NMR spectra in dimethyl sulfoxide; (DMSO) and D2O. Because the micelles are not formed in organic solvent (DMSO), both the ethylene protons of PEG and the amide protons of zein were observed (Figure 4a). In contrast, in D2O, only the ethylene protons of PEG were observed while the zein peak disappeared (Figure 4b). The changes in 1H NMR spectra indicate that mPEG−zein assemble into micelles with hydrophobic zein forming the inner core while the hydrophilic mPEG is in the outer shell. The restricted motion of the zein protons in the core resulted in disappearance of zein peak.6 The CMC of mPEG−zein was determined using pyrene as a hydrophobic probe. The fluorescence of pyrene undergoes a significant red-shift from 333 to 338 nm when it is transferred from the aqueous to hydrophobic environment of the micelles (Figure 5a). The CMC for PEG−zein micelle is relatively low (5.5 × 10−2 g/L), indicating the thermodynamic stability of PEG−zein micelles. The CMC is comparable to polypeptide− PEG graft copolymer micelles reported in the literature6 and is at least 5-fold lower than the commercial polyethylene oxide polypropylene oxide block copolymer (Pluronics).26 Low CMC indicates higher stability of the micelles on dilution. As shown in Figure 4b, the size of PEG−zein micelles did not change on 20−500-fold dilution with pH 7.4 buffer. Further the incubation of PEG−zein micelles with 10% serum for 2 h did not significantly change the particle size (123 ± 11 nm in control
Figure 6. Antizein antibody levels after subcutaneous administration of mPEG−zein micelles in mice. The optical density of antizein antibodies in serum was measured after the third week of the first dose and the booster doses respectively. The results are represented as mean ± standard error of mean (n = 4). mPEG−zein micelles did not produce any antizein antibodies, and the values were similar to the saline control (*p > 0.05).
buffer vs 134 ± 42 nm). The CMC and stability of micelles is dependent on the hydrophobicity and molecular weight of the core forming polymer.3,4 The lower CMC can be attributed to the high molecular weight (22−27 kDa) and high proportion (>50%) of nonpolar amino acids in zein.9 One of the concerns with protein polymer is the risk of immunogenicity when 2783
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Figure 8. In vitro release profile of curcumin from mPEG−zein micelles in citrate buffer pH 7.4 containing 5% v/v tween 80 and 0.01% BHT. The release study was conducted for 24 h. Each data point is a mean of three experiments ± SD.
Figure 7. (a) Fluorescence spectra of curcumin (10 μg/mL) in methanol, PBS pH 7.4 (with 10% methanol), and curcumin-loaded PEGylated zein micelles in PBS pH 7.4 (excitation wavelength was 420 nm). The inset shows the curcumin and curcumin loaded PEG− zein micelles in PBS pH 7.4 (10 μg/mL) in the left and right panels respectively. (b) Absorbance of curcumin (10 μg/mL) solution in PBS pH 7.4 as a function of time. (c) Absorbance of curcumin loaded mPEG−zein micelles (curcumin equivalent of 10 μg/mL) solution in PBS pH 7.4 as a function of time.
injected in vivo. PEG−zein was found to be nonimmunogenic and did not elicit any antizein antibodies when injected subcutaneously in mice (Figure 6). Hurtado-López reported that zein microspheres produced antizein antibodies when injected intramuscularly in mice.14 However, no antibodies were produced when the zein microspheres were administered orally to the mice. Given our interest in using mPEG−zein micelles as a drug delivery vehicle for parenteral and topical applications, we tested the immunogenicity by sc injection. However, further studies are required to test the immunogenicity by other delivery routes. Macrophage uptake and immunogenicity is mainly influenced by the particle size and surface hydrophobicity of the particles.27 PEGylation of zein provides a hydrophilic surface and hence can prevent the macrophage uptake and the resultant immune response to zein. PEGylation has been widely used to avoid macrophage uptake and immunogenicity of proteins and nanoparticles.28,29 Further, the smaller size of the micelles may also help in preventing the macrophage uptake and immunogenicity of zein.
Figure 9. (a) In vitro cell uptake of curcumin (dissolved in 10% v/v DMSO) and curcumin loaded mPEG−zein micelles in NCI/ADR-RES drug-resistant human ovarian cancer cells. (b) In vitro cytotoxicity of curcumin (dissolved in 10% v/v DMSO; Curcu-Soln) and curcumin loaded mPEG−zein micelles (Cur-M) in NCI/ADR-RES drug-resistant human ovarian cancer cells. Each data point is a mean of three experiments ± SD. The IC50 for curcumin and curcumin loaded mPEG− zein micelles is 0.104 ± 0.0005 and 0.034 ± 0.0008 μM, respectively.
Curcumin Loaded mPEG−Zein Micelles. To demonstrate the feasibility of using PEG−zein micelles as a drug 2784
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micelles was 3-fold lower than free curcumin (Figure 9b) and is consistent with the significantly higher cell uptake of curcumin micelles Curcumin exerts its anticancer effects by modulating various cell signaling pathways involved in cell apoptosis.20 NCI-ADR-RES is a resistant cell line that overexpresses multidrugresistant P-gp efflux pump.36 The efflux pump reduces the concentration of anticancer agents in the cell, leading to drug resistance. Curcumin is known to modulate the gene expression of multidrug resistance protein in cancer cells, which is advantageous when curcumin is combined with other chemotherapeutic drugs.21 The mPEG−zein micelles can be used for the codelivery of curcumin along with other hydrophobic chemotherapeutic drugs such as doxorubicin or paclitaxel to treat drugresistant cancers. In vivo studies are required to understand the biodistribution and stability of PEG−zein micelles. Future studies will focus on exploring the use of mPEG−zein micelles for curcumin delivery by topical and parenteral routes for treatment of skin cancer and other cancers, respectively.
delivery carrier, curcumin was encapsulated in mPEG−zein micelles. The size of curcumin loaded PEG−zein micelles was comparable to blank micelles (Table 1), and the encapsulation efficiency was 95%. The encapsulation efficiency of curcumin is much higher than other polymeric micelles reported in the literature.30−32 Encapsulation efficiency depends on miscibility of the drug with the hydrophobic polymer.3,4 Therefore, the high encapsulation efficiency can be attributed to the strong hydrophobic interaction between curcumin and zein. The aqueous solubility of curcumin increased by 1000−2000-fold, as evidenced from the significant increase in curcumin fluorescence compared to the free curcumin (Figure 6a). Curcumin has a very poor water solubility (11 ng/mL) and is a major factor that limits its in vivo bioavailability by oral and parenteral routes of drug adminitration.16,33,34 PEG−zein micelles significantly increased the water solubility of curcumin (>10 μg/mL) (Figure 7a). As seen from Figure 6a, the fluorescence of curcumin in mPEG−zein micelles was even higher than the fluorescence of curcumin in 100% methanol and also shifted the peak from 550 to 520 nm. Another major challenge with curcumin is its chemical instability. Curcumin is highly photolabile33,35 but was improved on encapsulation in mPEG−zein micelles. The stability was tested by measuring the curcumin absorbance at 420 nm in phosphate buffer (pH 7.4). As can be seen from Figure 7b, the absorbance of free curcumin decreased rapidly to 50% within 10 min, while the absorbance of curcumin in mPEG−zein micelles decreased gradually and the absorbance was seen up to 6 h (Figure 6b,c). The stability of curcumin was enhanced approximately by 6-fold after encapsulation in mPEG−zein micelles. Further improvement in stability can be achieved by optimizing the formulation and by cross-linking the core or shell of the mPEG−zein micelles. The release of curcumin from mPEG−zein micelles was studied in pH 7.4 in the presence of butylated hydroxyl anisole as an antioxidant. Curcumin release was sustained up to 24 h from mPEG−zein micelles (Figure 8). Further studies in different pH and with enzymes are required to characterize the release of curcumin from PEG−zein micelles. The core or the shell can be modified by cross-linking to further sustain the release of curcumin. Curcumin is rapidly eliminated from the blood by phase I and phase II enzymes, resulting in a short half-life of 30 min.22 Therefore, sustained release of curcumin is important to maintain effective plasma concentrations of curcumin. To test whether the cell uptake and of curcumin can be enhanced by mPEG−zein micelles, we tested the cell uptake in drug-resistant ovarian human cancer cells (NCI/ADR-Res). As can be seen from Figure 9a, the cell uptake of curcumin was significantly enhanced after encapsulation in mPEG−zein micelles. The cell uptake of curcumin micelles was 2−3-fold higher than free curcumin. Free curcumin is taken up by passive diffusion by the cells. The poor aqueous solubility and high lipophilicity of curcumin limits the cell uptake by passive diffusion. This is overcome by encapsulating curcumin in mPEG− zein micelles which leads to higher cell uptake of curcumin. Unlike the free curcumin, the curcumin loaded mPEG−zein micelles can be efficiently taken up endocytosis.1 Endocytosis is an energy-dependent process where macromolecules are transported by vesicles into the cell.1 However, further studies are required to understand the mechanism of cell uptake of PEG-zein micelles. The anticancer effects of free and micelle-encapsulated curcumin was tested by MTT assay in drug-resistant ovarian human cancer cells (NCI/ADR-Res). The IC50 of curcumin
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CONCLUSIONS Overall, the results demonstrate that PEGylated zein forms stable micelles and it can be used to improve the solubility, stability, and cell uptake of hydrophobic drug molecules like curcumin. In addition to being biodegradable, the protein core offers a wide choice of functional groups to conjugate compounds and or modify the core for various applications. Similarly, PEG also offers wide choice for functionalization of the micellar shell for various drug delivery applications. Because both zein and mPEG are biocompatible U.S. FDA approved polymers, mPEG−zein micelles is a safe and promising nanocarrier for hydrophobic drugs like curcumin.
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ASSOCIATED CONTENT
S Supporting Information *
Methods and results of SDS-PAGE, MALDI-TOF, and DSC thermogram. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: 605-688-4745. Fax: 605-688-6232. E-mail: omathanu.
[email protected]. Address: SAV 257, Box 2202C, 1 Admin Lane, South Dakota State University, Brookings, SD 57007. Present Addresses †
Formulations R & D, SRI International, Menlopark, CA 94025, United States. Notes
The authors declare the following competing financial interest(s):Dr. Perumal is an unpaid consultant to Tranzderm Solutions Inc., Brookings, SD. This start-up company has licensed the PEG-zein micelle technology from South Dakota State University.
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ACKNOWLEDGMENTS This work was supported by Governor’s 2010 competitive research seed grant, and grants from the U.S. Small Business Administration and Tranzderm Solutions Inc. Bhimanna Kuppast assisted in the interpretation of NMR spectra. Dr. Linhong Jin from the Central Mass Spectrometry Facility at South Dakota University assisted in MALDI-TOF experiments. 2785
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Molecular Pharmaceutics
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