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Nanocarrier with Self-Antioxidative Property for Stabilizing and Delivering Ascorbyl Palmitate into Skin
Nanocarrier with Self-Antioxidative Property for Stabilizing and Delivering Ascorbyl Palmitate into Skin SIRINAPA JANESIRISAKULE, TARIT SINTHUSAKE, SUPASON WANICHWECHARUNGRUANG Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Received 25 March 2013; revised 20 May 2013; accepted 29 May 2013 Published online 17 June 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23641 ABSTRACT: The concept of a nanocarrier with a self-antioxidative property to deliver and stabilize a labile drug while at the same time providing a free radical scavenging activity is demonstrated. Curcumin was grafted onto a poly(vinyl alcohol) [PV(OH)] chain, and the nanocarriers fabricated from the obtained curcumin-grafted PV(OH) polymer [CUR-PV(OH)] showed a good free radical scavenging activity. Ascorbyl palmitate (AP) could be effectively loaded into the CUR-PV(OH) at 29% by weight. The CUR-PV(OH)-encapsulated AP was 77% more stable than the free (unencapsulated) AP, and 47% more stable than AP encapsulated in the control nanocarrier with no antioxidative property [cinnamoyl-grafted PV(OH); CIN-PV(OH)]. Although coencapsulation of curcumin and AP into CIN-PV(OH) showed some improvement on the AP stability, AP was more stable when encapsulated in CUR-PV(OH). Compared with the free AP, encapsulated AP within the CUR-PV(OH) nanocarriers showed not only a better penetration into pig skin dermis via hair follicle pathway followed by the release and diffusion of the AP, but also a greater AP stability after skin application. Although a proof of principle is shown for CUR-PV(OH) and AP, it is likely that other carriers of the same principal could be designed and applied to different oxidation-sensitive drugs. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2770–2779, 2013 Keywords: curcumin; antioxidant; ascorbyl palmitate; skin delivery; polymeric drug carrier; nanoparticles; transdermal drug delivery; skin; stability
INTRODUCTION The main function of drug carriers is to deliver their payload (drug molecules) to the desired target sites, reducing nontarget (systemic) exposure and increasing the exposure concentrations and/or duration per administered dose at the target site(s). To achieve these aims, various properties have been introduced into carriers besides the inherited physical barrier, such as the ability to bind to specific proteins on the target cells, stealth characteristics to avoid the host’s immune system, and the ability to release drugs under specific conditions, at specific pH or temperature values. In most encapsulated drug systems, the carrier materials are the major content as compared with the payload drug content, and so it would be logical to, if possible, make the carrier material Additional Supporting Information may be found in the online version of this article. Supporting Information Correspondence to: Supason Wanichwecharungruang (Telephone: +662-2187634; Fax: +662-2541309; E-mail: psupason@ chula.ac.th) Journal of Pharmaceutical Sciences, Vol. 102, 2770–2779 (2013) © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association
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possess a therapeutic function or biological activity. So far, only a few bioactive carriers have been reported, which include liposomes made from ascorbyl palmitate (AP),1 nanocarriers with UV absorptive properties,2,3 delivery systems made from L-ascorbic (vitamin C) derivatives,4,5 and carriers with anticancer activity.6 Furthermore, UV absorptive carriers have been shown to slowly release fragrance molecules and at the same time act as an UV screening material for the skin.3 Carriers with anticancer activity that showed good antitumor activity on their own were reported to work synergistically with their payload to combat the drug resistant cancerous cells.6 There are many labile bioactive molecules that need a better technology to keep their chemical structure unchanged during storage and/or during their transport to the required target site in the body. As the most common cause of the deterioration of active molecules is oxidation, usually, through free radical mechanisms initiated by reactive oxygen species, we expect that a carrier system with an integral free radical scavenging activity will be able to lessen such deterioration and so increase the stability of their
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otherwise oxidation-sensitive payloads. As a result, a nanocarrier with an antioxidative property was fabricated and is reported here. Vitamin C has long been used in pharmaceutical and cosmetic products. Its biological activities are based on its function as a cofactor for a number of enzymes, most notably the hydroxylases involved in collagen synthesis,7,8 and as a nonenzymatic watersoluble antioxidant. Vitamin C can efficiently protect biomolecules against oxidative degradation through its ability to scavenge and reduce reactive oxidizing molecules and free radicals.9 Although the in vitro dose-dependent pro-oxidant activity of vitamin C has been reported previously,10–12 recent studies have indicated no adverse effects of vitamin C on normal cells.12,13 The use of vitamin C in skin care is still popular, not only for its antiageing effect but also for its antitanning effect among Asian women,14,15 because the compound can also inhibit melanin synthesis.16,17 However, vitamin C is unstable and can be easily oxidized under aerobic conditions, the rate of which is increased with increasing heat or UV light levels.18 Many derivatives of vitamin C have been introduced to reduce the ease of its oxidation, including various esters, such as AP and retinyl ascorbate,19,20 bis(Lascorbic acid-3,3 )phosphate, and L-ascorbic acid 2phosphate.21 Among the many vitamin C derivatives, AP has been popularly used as an antioxidant additive in food, pharmaceutical, medical, and cosmetic products because of its improved stability and better skin penetration as compared with vitamin C.22 The successful encapsulation of AP into microemulsions,23 bilayer vesicles,1,22 polymeric nanoparticles,24 and solid lipid nanoparticles that resulted in some retardation of AP degradation,22 has been reported. Here, we used AP as an example to show that its degradation can be delayed when encapsulated in a nanocarrier system with an antioxidative property. In addition, the nanocarriers were designed to possess an optimal size so they could be entrapped in the hair follicular shunts and act as reservoirs of AP for the sustained release into the skin tissue. To make a nanocarrier with an antioxidative property, we attached the natural dietary antioxidant curcumin onto poly(vinyl alcohol) [PV(OH)], a nontoxic, biocompatible, and biodegradable hydrophilic polymer, so as to impart not only an amphiphilic character, but also an antioxidative property to the polymer. The obtained curcumin-grafted PV(OH) polymer [CURPV(OH)] was then fabricated into a nanocarrier and its free radical scavenging activity was evaluated. Next, AP was encapsulated into the CUR-PV(OH) nanocarrier and the stability of the encapsulated AP was evaluated. To answer whether the antioxidative CUR-PV(OH) nanocarrier would give better AP protection than one with no antioxidative property (control nanocarrier), AP was also encapsulated DOI 10.1002/jps
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into cinnamoyl-grafted PV(OH) [CIN-PV(OH)] and its stability was compared with that encapsulated in CUR-PV(OH). Finally, the release of AP from the CUR-PV(OH) nanocarriers, ex vivo penetration into pig skin, and stability of the AP after skin application, were all investigated.
MATERIALS AND METHODS Materials Ascorbyl palmitate was obtained from Roche (Basle, Switzerland). PV(OH) (MW 124,000–186,000 and 87%–89% deacetylated), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and glutaric anhydride were obtained from Sigma–Aldrich Chemical Company (Steinheim, Germany). Cinnamoyl chloride and curcumin were obtained from Acros Organics (Geel, Belgium). Synthesis of the Antioxidative Nanocarrier: Poly(Vinyl Alcohol-Co-Vinyl Glutarycurcumin), CUR-PV(OH) (Scheme 1) A mixture of 3.0 g curcumin, 0.6 g glutaric anhydride, and 4–5 drops pyridine in 60 mL dry dimethylformamide (DMF) was refluxed for 4 h and then the glutarylcurcumin formed was purified on a silica gel column, using a 20%–50% (v/v) ethyl acetate gradient in hexane as the mobile phase. The obtained glutarylcurcumin (2.5 g) in 20 mL of dry DMF was then added to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (0.05 g), followed by PV(OH) (0.09 g) and 1-hydroxybenzotriazole (0.06 g), and the mixture was kept at 0◦ C for 24 h under a nitrogen (N2 ) atmosphere. Subsequently, the reaction mixture was dialyzed against 30% (v/v) methanol in distilled water using a regenerated cellulose tubular membrane (CeluSep T4 dialysis tube; MWCO 12,000–14,000; Membrane Filtration Products, Seguin, Texas). Dry particles were obtained by freeze-drying the aqueous suspension. The product was characterized by 1 H NMR (Varian Mercury Spectrometer, 400.00 MHz; Varian Company, Palo Alto, California), Fourier transform infrared spectroscopy (FT-IR; Impact 410 Nicolet Fourier transform Infrared spectrophotometer; Nicolet Instruments Technologies, Inc., Madison, Wisconsin), UV–Vis spectrophotometry (UV, 2500 UV–Vis spectrophotometer; Shimadzu Corporation, Kyoto, Japan), and differential scanning calorimetry (DSC; Mettler DSC 822, Mettler Toledo, Columbus, Ohio) analyses. The DSC analysis was performed at a heating and cooling rate of 10◦ C/min under a N2 flow of 20 mL/min.
Curcumin-Grafted PV(OH) Polymer (CUR-PV(OH)) White solid. Degree of substitution (DS): 0.059. Tg : 81.4◦ C. FT-IR (KBr, cm−1 ) 3302 (O H str), JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 8, AUGUST 2013
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Scheme 1.
Synthesis of CUR-PV(OH) and CIN-PV(OH).
2919 (C H str), 1732 (C O str), 1626 (C C str), 1417 (CH2 bend), and 1250, 1121, and 1092 (C O str). 1 H NMR (CDCl3 , 400 MHz, *, ppm): 7.65 (O C CHCH Ar PV(OH)), d, J = 16 Hz, 1H), 7.55 (O C CHCH Ar, d, J= 16 Hz, 1H), 7.30–6.85 (Ar H, 6H), 6.8 (O C CH CHAr PV(OH), d, J = 16 Hz, 1H), 6.7 [(O C CH CHAr), d, J = 16 Hz, 1H], 5.70–5.90 ppm (keto–enol proton of curcumin moieties), 4.75, 4.55, 4.45 [ CH OH of PV(OH) backbone], 3.83 [CHOH and CHOC(O)CH3 of PV(OH) backbone, and OCH3 ], and 3.0–1.0 [br, CH3 CO and CH CH2 CH of PV(OH) backbone]. UV– Visible spectroscopy (DMSO): λmax at 412 nm. Synthesis of the Control (No Antioxidative Property) Nanocarrier: Poly(Vinyl Alcohol-Co-Vinyl Cinnamate), CIN-PV(OH) (Scheme 1) Cinnamoyl-grafted PV(OH) was prepared as previously described.25 In brief, 1.19 g PV(OH) was dissolved in heated anhydrous DMF. Then, pyridine (2.18 mL) was added and the obtained clear solution was poured into a round bottom flask containing 4.0 g freshly prepared cinnamoyl chloride. The mixture was stirred at 80◦ C–90◦ C for 1–2 h before the product was precipitated with 1.0% (w/v) aqueous Na2 CO3 , JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 8, AUGUST 2013
washed with water and dried under vacuum to a constant weight.
Cinnamoyl-Grafted PV(OH) Polymer (CIN-PV(OH)) Pale yellow solid. DS: 0.17. Tg : 71◦ C. FT-IR (KBr, cm−1 ): 3472 (OH), 3063, 3023 ( C H, str), 2929 ( C H str), 1720 (C O, str), 1636, 1575 (C C, str), 1441 ( CH2 bend) and 1312, 1248, and 1174 (C O str). 1 H NMR (400 MHz, DMSO-d6 , *, ppm): 8.17 (br, Ar CH), 7.65 (br, Ar H), 7.41 (br, Ar H), 6.58 (br, CHCOOR), 5.5–4.1 (br, OH), 4.0–3.5 (br, CHOH and CHOCOCH3 ), and 2.30–0.80 [br, CH3 CO and CH CH2 CH of PV(OH) backbone]. UV–Vis (DMSO) λmax , nm (g, M−1 cm−1 , monomeric unit−1 ): 282 (2544). Encapsulation
AP-Loaded CUR-PV(OH) Curcumin-grafted PV(OH) polymer (30 mg) was dissolved in DMF (10 mL) and then 15 mg of AP in 10 mL of DMF was added, mixed, and dialyzed against water. The resultant suspension of AP-loaded CUR-PV(OH) nanocarriers was collected, the volume adjusted to 20 mL with water, and aliquots DOI 10.1002/jps
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of the suspension were subjected to analysis by scanning electron microscopy (SEM; JEM-6400; Jeol, Ltd., Tokyo, Japan), transmission electron microscopy (TEM; JEM-2100; Jeol, Ltd.), and dynamic light scattering (DLS). The remaining suspension was freezedried to obtain the dry AP-loaded CUR-PV(OH) nanocarriers.
AP-Loaded CIN-PV(OH) The AP-loaded-CIN-PV(OH) nanocarriers were prepared as described for AP-loaded CUR-PV(OH) above, except that CUR-PV(OH) was replaced with an equal molar amount (same number of monomeric units) of CIN-PV(OH).
AP + Curcumin-Loaded CIN-PV(OH) Ascorbyl palmitate and curcumin were coloaded into the CIN-PV(OH) nanocarriers by dissolving CIN-PV(OH) (30 mg), AP (30 mg), and curcumin (60 mg) in DMF (20 mL) and then dialyzing the obtained mixture against water. Encapsulation Efficiency and Loading Level The amount of AP loaded into each type of nanocarrier was analyzed using high-performance liquid chromatography (HPLC). The suspension of the AP-loaded nanocarriers was centrifugally filtered using a 10,000-Da MWCO centrifugal-filter (Amicon Ultra-15; Millipore, County Cork, Ireland), and the solid product was very quickly rinsed with methanol to remove the AP attached to the outside of the nanocarriers and around the filter. The washed liquid was combined with the supernatant obtained from the dialysis process during the carrier preparation, and was analyzed for AP content by HPLC. The residual amounts of AP in the filtrate and the wash were then subtracted from the amount of AP initially used, to obtain the amount of encapsulated AP. From that, the encapsulation efficiency (EE) and loading level were calculated according to Eqs. 1 and 2, respectively: EE(%) =
weight of encapsulated AP × 100 , weight of AP used
Loading(%) =
(1)
weight of encapsulated AP × 100 . (2) weight of polymer used
Quantitation of AP by HPLC High-performance liquid chromatography was performed on a ThermoFinnigan P4000 (pump), connected to a UV–Vis detector (UV6000LP) using a C18 reversed-phase column (100 × 4.6 mm2 Hypersil ODS; Agilent Technologies, Santa Clara, California). The mobile phase was a 15:2:3 (v/v/v) mixture of methanol–acetonitrile–0.02 M phosphate buffer DOI 10.1002/jps
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pH 2.5, at a flow rate of 1.5 mL/min. Detection was performed at 254 nm, and the sample injection volume was 20 :L. Stability of the Encapsulated AP The stability of the four different AP samples, AP encapsulated in either (a) CUR-PV(OH) or (b) CIN-PV(OH) nanocarriers, (c) AP coencapsulated with curcumin in the CIN-PV(OH) nanocarrier, and (d) unencapsulated AP, was evaluated on the freezedried samples. The freeze-dried samples were kept at 30◦ C for 45 days under a light proof condition. At various time intervals, a few milligrams of each sample was withdrawn and analyzed for its AP content. The free AP sample was directly quantified for the level of AP by HPLC, whereas the three encapsulated AP samples were first subjected to extraction by soaking in methanol under a light proof and N2 atmosphere, then centrifugally filtered from the extracted polymer material, and the obtained methanol extract (filtrate) then analyzed for AP content by HPLC. DPPH Scavenging Activity The DPPH radical scavenging activity was used to measure the free radical scavenging activity as previously described.26 Briefly, 195 :L of 100 :M DPPH solution (in ethanol) and 5 :L of the test material (performed as serial dilutions) were mixed in a 96-well plate, and the absorbance was measured at 515 nm after incubation in the dark for 30 min at room temperature. The level of residual DPPH-free radicals was determined from the absorbance. Samples included aqueous suspensions of CUR-PV(OH) and CIN-PV(OH), plus the 10% (v/v) aqueous ethanol solution of free (unencapsulated) curcumin. Butylated hydroxytoluene (1.0 mM) and the solvent only were used as the 100% and 0% radical scavenging controls, respectively. Assays were performed in triplicate wells, and the data are expressed as the mean of these, in terms of the percent ratio of the level of residual to initial DPPH radicals.
Ex Vivo Pig Skin Penetration Fresh porcine ear skin from a one-month-old White Large piglet was obtained from Manoch Farm (Phetchaboon, Thailand), and was kept at 4◦ C from excision immediately after death to its use, which was within 8 h of execution. To evaluate the ability of the AP-loaded CUR-PV(OH) nanocarriers to penetrate into the pig skin, 10 :L of an aqueous suspension of AP-loaded CUR-PV(OH) (0.705 mg AP/mL) was applied onto a 1.0 × 2.0 cm2 skin piece to give a final AP skin coverage of 3.52 :g/cm2 . The skin piece was massaged for 2 min to induce the movement of the hair and then left at room temperature for 30 min before being subjected JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 8, AUGUST 2013
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to confocal fluorescence laser microscopy [CFLM; Nikon, Tokyo, Japan Digital Eclipse C1-Si, Plan Apochromat VC 100×, Diode Laser (405 nm; Melles Griot, Carlsbad, California), a Nikon TE2000-U microscope, a 32-channel-PMT-spectral-detector, and C1plus/C1si EZ-C1 Version 3.80m control software]. The obtained spectral output signal of each pixel was then unmixed into the respective CUR-PV(OH), AP, and skin/ hair autofluorescence components using chemometric analysis (image algorithms). Images indicating the location of the CUR-PV(OH), AP, and skin tissue and hair were then constructed using the obtained resolved signals. Stability of AP after Skin Application Thirty microliter of an aqueous suspension of APloaded CUR-PV(OH) nanocarriers (2.2 mg AP/mL) was applied onto a 2.5 × 2.5 cm2 piece of the pig ear skin to give a final AP skin coverage of 10.56 :g cm−2 . The skin piece was massaged for 2 min, left at room temperature for 3 h, then homogenized with 10 mL ethyl acetate under a N2 atmosphere for 2 min followed by ultrasonic vibration for 5 min. The mixture was then centrifuged at 21130 g for 10 min under a N2 atmosphere. The supernatant was harvested and the residue was extracted again with 5 mL ethyl acetate. Water (5 mL) was then added to the combined supernatants and the mixture was saturated with N2 and vortexed. The ethyl acetate layer was collected, dried under a N2 flow, and subjected to AP quantitation by HPLC. Similar experiments were carried out with free AP in place of the AP-loaded CUR-PV(OH) nanocarriers. Five replicate experiments were performed.
ous suspension indicated spherical particles with an average anhydrous diameter of 232.5 ± 29.44 nm. The hydrodynamic diameter of the CUR-PV(OH) nanocarriers, as determined by DLS, was 269.8 ± 19.45 nm (poly dispersity index (PDI) of 0.6). At similar curcumin moiety concentrations, the DPPH-free radical scavenging activity of CUR-PV(OH) was 48% of that of the free curcumin Synthesis of the Control (No Antioxidant Activity) CIN-PV(OH) Nanocarrier The cinnamoyl groups were successfully grafted onto the PV(OH) backbone (Scheme 1), to give CIN-PV(OH) with a DS of 0.17, as verified through the NMR spectrum (SI; Fig. S4). SEM and TEM images of the dry CIN-PV(OH) sample showed spherical particles with diameters of 269.8 ± 19.43 and 259.4 ± 16.92 nm, respectively. The aqueous suspension gave a similar hydrodynamic diameter of 270.5 ± 18.74 nm, as determined by DLS. The obtained nanocarrier suspension showed no free radical scavenging activity when tested by the DPPH scavenging assay. Encapsulation The AP loading percentages are shown in Table 1. DLS analyses of the AP-loaded CUR-PV(OH), AP-loaded CIN-PV(OH), and AP + curcumin coloaded CIN-PV(OH) nanocarriers gave hydrodynamic diameters of 278.2 ± 14.21, 290.5 ± 16.51, and 306.2 ± 18.66 nm, respectively (Fig. 1), whereas the SEM and/or TEM images indicated a spherical morphology with an average anhydrous diameter of 264.6 ± 18.27, 283.3 ± 20.02, and 302.4 ± 15.16 nm, respectively, suggesting very little swelling in water. AP Stability
RESULTS Synthesis of the Antioxidant Containing CUR-PV(OH) Nanocarrier Glutarylcurcumin was successfully synthesized from curcumin and glutaric anhydride and purified by column chromatography [see Nuclear Magnetic Resonance (NMR) spectrum in Supporting Information (SI), Fig. S1]. Glutarylcurcumin was successfully grafted onto PV(OH) with a DS of 0.059 (SI; Figs. S2 and S3). The CUR-PV(OH) dispersed well in water. SEM and TEM images of the dry CUR-PV(OH) aqueTable 1.
Samples were kept dry at 30◦ C under a light proof condition for 45 days and the amount of AP in the samples was periodically evaluated (Fig. 2). The AP encapsulated in CUR-PV(OH) was 47% and 77% more stable than the AP encapsulated in the control CIN-PV(OH) and free AP, respectively.
Ex Vivo Pig Skin Penetration The fluorescent spectra of AP and are shown in Figure 3a. AP-loaded nanocarriers were massaged into the and the images of the fluorescent
CUR-PV(OH) CUR-PV(OH) pig ear skin, signals from
Loading Percentages and Encapsulation Efficiency (EE) of the Three Particles
Samples AP-loaded CUR-PV(OH) (AP + curcumin) coloaded CIN-PV(OH) AP-loaded CIN-PV(OH)
Curcumin Loading (%, w/w)a
AP Loading (%, w/w)a
EE of AP (%)a
Mole Ratio of AP: Curcumin
– 19.0 ± 0.21 –
29.0 ± 0.32 19.4 ± 0.23 27.3 ± 0.52
80.9 ± 0.16 62.5 ± 0.11 75.9 ± 0.28
1.16:1 0.91:1 –
Data are shown as the mean ± 1 SD and are derived from three independent samples.
a
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HPLC analysis. The content of AP in the skin was 71 ± 14% and 18 ± 6% of the original amount applied onto the skin for the AP-loaded CUR-PV(OH) and free AP, respectively.
DISCUSSION Synthesis of the CUR-PV(OH) Nanocarrier
Figure 1. (a) Scanning electron microscopy image of the AP-loaded CUR-PV(OH) nanocarrier. (b–d) TEM images of the (b) AP-loaded CUR-PV(OH), (c) AP + curcumin coloaded CIN-PV(OH), and (d) AP-loaded CIN-PV(OH) nanocarriers.
Figure 2. Stability of dry free AP (unencapsulated AP), or AP encapsulated in different nanocarriers, after storage at 30◦ C under a light proof condition with normal air exposure. Data are shown as the mean ± 1 SD, derived from four independent repeats. The Mann–Whitney U test indicated a significant difference among the four samples at P ≤ 0.05 during 15–45 days.
CUR-PV(OH) and AP in the pig ear skin were taken after 2 h and are shown in Figures 3b and 3c, respectively. Stability of AP after Skin Application The AP-loaded CUR-PV(OH) and the free AP were separately applied onto freshly excised pig ear skin and left for 3 h at 37◦ C. The AP in the skin tissue was then recovered by solvent extraction followed by DOI 10.1002/jps
To fabricate a nanocarrier with an antioxidative property, PV(OH) was chosen as the backbone hydrophilic polymer because of not only its well-known safety but also its abundance of hydroxyl moieties for chemical derivatization. Curcumin was first derivatized into glutarylcurcumin by reacting curcumin with glutaric anhydride and purifying the monofunctionalized product from other species (bifunctionalized product, curcumin, and glutaric acid) through silica column chromatography (SI; Fig. S1). Then, the obtained glutarylcurcumin was grafted onto PV(OH) via ester bond formation using 1-ethyl- 3(3-dimethyl-aminopropyl)carbodiimide (EDCI)as the coupling agent and the unattached glutarylcurcumin was subsequently dialyzed out from the CUR-PV(OH) product. Successful grafting was confirmed through the 1 H NMR spectrum of the product (SI; Fig. S2). The DS of curcumin (0.059) was deduced from the ratio between the integration of peaks at 1.2–1.8 ppm [ CH CH2 CH of PV(OH) backbone] and the peaks at 6.5–7.0 ppm (Ar CHCH C O and one aromatic proton of the curcumin moieties). The UV–Vis absorption spectrum of CUR-PV(OH) showed a maximum absorbance at 422 nm, a blueshift from that of free curcumin (429 nm) (SI; Fig. S3). The blueshift indicates a lower degree of conjugation in the curcumin core structure resulting from the replacement of the hydroxyl group on the aromatic ring with an acyl group. With the attached hydrophobic curcumin moieties on the hydrophilic PV(OH) backbone, the CUR-PV(OH) became amphiphilic. Therefore, it was likely that as the CUR-PV(OH) self-assembled into spherical nanocarriers during the solvent displacement process (DMF was displaced with water), the hydrophobic curcumin moieties would locate at the core, away from water with their hydrophilic hydroxyl groups located at the nanocarrier’s surface, in full contact with the water medium. Curcumin-grafted PV(OH) polymer dispersed well in water, with a hydrodynamic diameter, as determined by DLS, of 269.8 ± 19.45 nm and a PDI of 0.6, indicating mid-range polydispersity). SEM and TEM images showed a spherical morphology with a slightly smaller average anhydrous diameter of 232 ± 29.44 nm, suggesting only minimal swelling in the aqueous environment. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 8, AUGUST 2013
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Figure 3. Confocal fluorescence laser microscopy images showing skin penetration by the AP-loaded CUR-PV(OH) nanocarriers. (a) Fluorescent spectra of CUR-PV(OH) and AP; (b, C) fluorescent images of the skin tissue at 2 h after the material was applied to the stratum corneum and showing (b) the CUR-PV(OH) fluorescent signal and (c) the AP signals.
At similar curcumin moiety concentrations, the DPPH-free radical scavenging activity of CUR-PV(OH) was 48% of that of the free curcumin. The free radical scavenging activity of curcumin is a result of both the highly conjugated phenolic and the keto–enol functionalities in the curcumin structure.27 Thus, by derivatizing only one phenolic group, each grafted curcumin still possessed one phenolic and one conjugated keto–enol moiety with which to scavenge free radicals. Nevertheless, the steric effect of the polymer around the grafted curcumin moieties probably partially hindered their free radical scavenging activity, and this would explain why the grafted curcumin moiety possessed only half (rather than close to 66% if the activity of the conjugated keto–enol group is assumed for simplicity to equate to that of the phenolic groups) the activity of the ungrafted curcumin. Synthesis of the CIN-PV(OH) Nanocarrier As the CUR-PV(OH) nanocarriers still possessed free radical scavenging activity (∼48% of that of the free curcumin), it was expected that they could provide an extra defense against oxidative degradation for their payload. To test this notion, a similar nanocarrier but without an antioxidative activity was required. For this reason, CIN-PV(OH) was synthesized. The hydrophobic cinnamoyl groups were grafted onto the PV(OH) backbone through esterification of the carboxylic functionality of cinnamic acid and the hydroxyl moieties on the PV(OH) (Scheme 1). Successful grafting was verified by the 1 H NMR spectrum of the CIN-PV(OH) (SI; Fig. S4), with a DS of 0.17 being obtained from the ratio of the integrated peaks at 6.2–8.4 ppm ( COCHCHAr H) against those at 0.8–2.3 ppm ( CH CH2 CH and CH3 CO of PV(OH) backbone).21 The obtained CINPV(OH) could self-assemble into stable spherical nanocarriers during the displacement of DMF with water. SEM and TEM images of the anhydrous samples indicated a spherical morphology with diameters of 269.8 ± 19.43 and 259.4 ± 16.92 nm, respectively, which was similar to the hydrodynamic diameter of JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 8, AUGUST 2013
the particles (270 ± 18.74 nm) obtained by DLS analysis. Thus, we obtained a CIN-PV(OH) nanocarrier with no free radical scavenging activity (confirmed by DPPH scavenging assay) and a similar low level of swelling in an aqueous environment to be used as control carrier. Encapsulation Encapsulation of AP into the CUR-PV(OH) or CIN-PV(OH) nanocarriers was performed by allowing self-assembly of the CUR-PV(OH) or CIN-PV(OH) polymer in the presence of AP (or AP and curcumin for the coencapsulation in the CIN-PV(OH)) during the displacement of DMF with water. It was speculated that during the self-assembly of CUR-PV(OH) or CIN-PV(OH), the hydrophobic AP molecules would automatically locate themselves at the hydrophobic cores of the forming polymeric nanocarriers, to have minimal interaction with the hydrophilic water. The AP loading percentage and EE (Table 1) were determined as outlined in the Material and Methods section. The loading value of AP in CUR-PV(OH) nanocarriers was very close to that in the control CIN-PV(OH) ones. However, in the AP plus curcumin coloaded CIN-PV(OH), the AP loading level obtained was significantly lower. This could be explained through the fact that the coloaded curcumin probably took up a significant space in each CIN-PV(OH) nanocarrier, reducing the space available to load the AP. Accordingly, the EE of AP was also significantly lower in the AP plus curcumin coloaded CIN-PV(OH). The higher AP loading capacity and EE of the CUR-PV(OH) nanocarrier, compared with the CIN-PV(OH) one that was coloaded with AP and curcumin, indicated another advantage of using the antioxidative CUR-PV(OH) nanocarrier systems. In other words, using the CUR-PV(OH) nanocarrier not only gave better protection to the AP than coloading the AP with curcumin into the CIN-PV(OH) one, but also allowed a higher EE and loading capacity to be obtained. SEM and TEM analyses revealed that the AP-loaded CUR-PV(OH), AP-loaded DOI 10.1002/jps
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CIN-PV(OH), and the AP plus curcumin coloaded CIN-PV(OH) nanocarriers were all spherical with an average dry diameter of 264.6 ± 18.27, 283.3 ± 20.02, and 302.4 ± 15.16 nm, respectively (Fig. 1). These agree with the hydrodynamic diameters obtained from DLS analysis of 278.2 ± 14.21, 290.5 ± 16.51, and 306.2 ± 18.66, respectively, confirming the minimal swelling in water.
In Vitro AP Stability The AP-loaded CUR-PV(OH), AP-loaded CIN-PV(OH), and the AP plus curcumin coloaded CIN-PV(OH) nanocarriers, along with free AP, were kept dry at 30◦ C under a light proof condition for 45 days and the amount of AP in the samples was periodically evaluated. The results (Fig. 2) indicated that the free AP degraded much quicker than all three encapsulated APs, with the residual free AP level decreasing to <50%, <25%, <10%, and 0% after 6, 10, 20, and 45 days, respectively. Of the encapsulated APs, AP in the CUR-PV(OH) nanocarriers degraded the slowest with approximately 80% residual AP after 45 days, followed by the AP that was coloaded with curcumin in CIN-PV(OH), with AP loaded in CIN-PV(OH) being the least stable. This supported that curcumin could help retard the degradation of AP, and that the protection was more effective when the curcumin molecules were covalently grafted onto the polymeric PV(OH) wall than when they were coloaded into the CIN-PV(OH) nanocarrier to essentially the same level. In fact, it should be noted here again that the grafted curcumin in the CUR-PV(OH) nanocarrier contained only one phenolic group per molecule, whereas the coloaded curcumin in the CIN-PV(OH) nanocarrier contained two phenolic groups per molecule and so theoretically had 50% more free radical scavenging activity than that of the grafted curcumin (if we assume for simplicity that the conjugated keto–enol group is as effective as the phenolic groups). Indeed, as reported above, at a similar concentration of curcumin moieties, the DPPH-free radical scavenging activity of the CUR-PV(OH) was 48% of the value of the free curcumin. Nevertheless, the grafted curcumin in the CUR-PV(OH) was more effective in protecting the encapsulated AP than the coloaded curcumin in the CIN-PV(OH) nanocarrier at corresponding AP: curcumin molar ratios (Table 1). The reason for this lies in the arrangement of the curcumin moieties. Curcumin moieties along the nanocarrier wall material acted as a protective barrier, scavenging the invading-free radicals and so effectively protecting the encapsulated AP inside the nanocarrier (Fig. 4). This resulted in an improved stability of the AP in the CUR-PV(OH) nanocarriers, despite the reduced radical scavenging activity of the DOI 10.1002/jps
Figure 4. Representative models of (left) the AP-loaded control nanocarriers, (middle) AP + curumin coloaded control nanocarriers, and (right) AP-loaded CUR-PV(OH) nanocarriers.
grafted curcumin compared with that of the original curcumin.
Ex Vivo Pig Skin Penetration The AP-loaded-CUR-PV(OH) nanocarriers were massaged into the pig ear skin and subsequently subjected to analysis by CFLM. It should be noted here that the massage was carried out to induce the hair movement that would enhance the moving of the carriers along the hair shaft, step by step down the hair cuticle into the hair follicles by the gear pump mechanism.28 This is reasonable given the fact that skin creams are generally applied by repeated massaging into the skin. Because the fluorescent spectrum of AP is different from that of the CUR-PV(OH) nanocarriers (Fig. 3a), the fluorescent signal from AP was resolved from the signal from CUR-PV(OH) using chemometric analysis (image algorithms). In a similar way, the autofluorescence from the tissue components was removed. As a result, the obtained spectral output signal of each pixel was unmixed into CUR-PV(OH), AP, and skin–hair autofluorescence components, yielding separate images for the locations of the CUR-PV(OH) and AP. The fluorescent signals from the CUR-PV(OH) and AP could be observed clearly along the hair follicles (Figs. 3b and 3c, respectively), indicating that the AP-loaded CUR-PV(OH) nanocarriers could be trapped in the hair follicle shunts. As the location of the AP fluorescent signal did not always match the location of the CUR-PV(OH) signal, it was likely that during the 2 h period, some of the AP had already been released and diffused away from the nanocarriers. Thus, the AP-loaded CUR-PV(OH) nanocarriers could be trapped in the hair follicles and release some of the loaded AP that then diffused into the tissue surrounding the hair follicles. Accumulation of the AP-loaded nanocarriers at the hair follicles should offer a good AP reservoir for the skin. As demonstrated earlier, the AP inside the CUR-PV(OH) nanocarriers is quite stable and so this AP reservoir will slowly supply the skin with undegraded AP. It should be noted here that the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 8, AUGUST 2013
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entrapment of drug-loaded nanocarriers in hair follicles has been reported previously.29 However, here we also showed evidence of AP release from those entrapped nanocarriers. In fact, the fluorescent signal of AP was detected at a depth of more than 200 :m from the stratum corneum, and so some of the AP was in the dermal layer of the pig ear skin. Stability of AP After Skin Application As reported above, when encapsulated in the CUR-PV(OH) nanocarrier, the stability of AP was improved significantly when tested in vitro. We then speculated that the improved AP stability would hold even when the materials were applied onto the skin. Accordingly, the AP-loaded CUR-PV(OH) nanocarriers and the free AP were separately applied onto freshly excised pig ear skin as above and left for 3 h at 37◦ C. The AP in the skin tissue was then recovered by solvent extraction followed by HPLC analysis. The content of AP in the skin was 71% and 18% of the original amount applied onto the skin for the AP-loaded CUR-PV(OH) nanocarriers and free AP, respectively. It was obvious that the antioxidative CUR-PV(OH) nanocarriers could effectively increase the AP stability even when applied on the skin. This was likely to result from the free radical protection afforded by the antioxidative nanocarrier and the localization of some of the AP-loaded nanocarriers inside the hair follicles. The nanocarriers were physically stable even when dry (SEM analysis) and so the antioxidative nanocarriers could effectively protect their payload efficiently even after skin application. Localization of the nanocarriers inside the hair follicles would automatically decrease the air and light exposure of the materials and thus might also have contributed to the slower AP oxidation compared with the materials sitting on the skin fully exposed to such environmental oxidative factors.
CONCLUSIONS The concept of a nanocarrier with a self-antioxidative property, to stabilize and deliver a labile drug, was demonstrated through the stabilizing and delivery of the labile AP into the pig skin using a nanocarrier fabricated from CUR-PV(OH). The CUR-PV(OH) nanocarriers possessed a free radical scavenging activity, as tested by the DPPH assay. Loading of AP into the CUR-PV(OH) resulted in nanocarriers with an average dry diameter of 232 ± 29.4 nm and a slightly larger hydrodynamic size of 269.8 ± 19.4 nm (PDI of 0.6), with an AP loading of 29% (w/w) and an EE of 81%. The AP encapsulated in CUR-PV(OH) showed the highest storage stability compared with that coencapsulated with curcumin in CIN-PV(OH) and especially compared with the AP encapsulated alone in CIN-PV(OH) and the free AP. CFLM analysis indiJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 8, AUGUST 2013
cated that the AP-loaded CUR-PV(OH) nanocarriers could penetrate pig skin via the hair follicle pathway and release AP to subsequently diffuse into the dermis.
ACKNOWLEDGMENTS The authors thank the Thailand Research Fund (RDG5650009), the Advanced Material Cluster at Chulalongkorn University and the higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission (CU-56-AM02), for financial support.
REFERENCES 1. Gopinath D, Ravi D, Rao BR, Apte SS, Renuka D, Rambhau D. 2004. Ascorbyl palmitate vesicles (Aspasomes): Formation, characterization and applications. Int J Pharm 271:95–11. 2. Kidsaneepoiboon P, Wanichwecharungruang SP, Chooppawa T, Deephum R, Panyathanmaporn T. 2011. Organic–inorganic hybrid polysilsesquioxane nanospheres as UVA/UVB absorber and fragrance carrier. J Mater Chem 21:7922–7930. 3. Anumansirikul N, Wittayasuporn M, Klinubol P, Tachaprutinun A, Wanichwecharungruang SP. 2008. UV-screening chitosan nanocontainers: Increasing the photostability of encapsulated materials and controlled release. Nanotechnology 19:205101. 4. Zhuang D, Nie J, Yang J. 2011. Poly(ascorbyl acrylate)s: Synthesis and evaluation of their redox polymerization ability in the presence of hydrogen peroxide. J Polym Sci Pol Chem 49:1999–2007. 5. Wang Y, Yang J. 2011. Synthesis and self-assembly of novel amphiphilic copolymers poly(lactic acid)-block-poly(ascorbyl acrylate). J Polym Sci, Part A: Polym Chem 49:3988–3996. 6. Amornwachirabodee K, Chiablaem K, Wacharasindhu S, Lirdprapamongkol K, Svasti J, Vchirawongkwin V, Wanichwecharungruang SP. 2012. Paclitaxel delivery using carrier made from curcumin derivative: Synergism between carrier and the loaded drug for effective cancer treatment. J Pharm Sci 101:3779–3786. 7. Traber MG, Stevens JF. 2011. Vitamins C and E: Beneficial effects from a mechanistic perspective. Free Radic Biol Med 51:1000–1013. 8. Phillips CL, Combs SB, Pinnell SR. 1994. Effects of ascorbic acid on proliferation and collagen synthesis in relation to the donor age of human dermal fibroblasts. J Invest Dermatol 103:228–232. 9. Colven RM, Pinnell SR. 1996. Topical vitamin C in aging. Clin Dermatol 14(2):227–234. 10. Podmore ID, Griffiths HR, Herbert KE, Mistry N, Mistry P, Lunec J. 1998. Vitamin C exhibits pro-oxidant properties. Nature 392:559–559. 11. Carr A, Frei B. 1999. Does vitamin C act as a pro-oxidant under physiological conditions? FASEB J 13:1007–1024. 12. Putchala MC, Ramani P, Sherlin HJ, Premkumar P, Natesan A. 2013. Ascorbic acid and its pro-oxidant activity as a therapy for tumours of oral cavity—A systematic review. Arch Oral Biol 58:563–574. 13. Chen Q, Espey MG, Sun AY, Pooput C, Kirk KL, Krishna MC, Khosh DB, Drisko J, Levine M. 2008. Pharmacologic doses of ascorbate act as a prooxidant and decrease growth of aggressive tumor xenografts in mice. Proc Natl Acad Sci USA 105:11105-11109. DOI 10.1002/jps
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14. Ohshima H, Mizukoshi K, Oyobikawa M, Matsumoto K, Takiwaki H, Kanto H, Itoh M. 2009. Effects of vitamin C on dark circles of the lower eyelids: Quantitative evaluation using image analysis and echogram. Skin Res Technol 15:214–217. 15. Zussman J, Ahdout J, Kim J. 2010. Vitamins and photoaging: Do scientific data support their use? J Am Acad Dermatol 63:507–525. 16. Lerner AB, Fitzpatrick TB. 1950. Biochemistry of melanin formation. Physiol Rev 30:91–126. 17. Ros JR, Rodriguez-Lopez JN, Garcia-Canovas F. 1993. Effect of L-ascorbic acid on the monophenolase activity of tyrosinase. Biochem J 295:309–312. 18. Austria R, Semenzato A, Bettero A. 1997. Stability of vitamin C derivatives in solution and topical formulations. J Pharm Biomed Anal 15:795–801. 19. Song QX, Wei DZ, Zhou WY, Xu WQ, Yang SL. 2004. Enzymatic synthesis and antioxidant properties of L-ascorbyl oleate and L-ascorbyl linoleate. Biotechnol Lett 26:1777–1780. 20. Abdulmajed K, Heard CM. 2004. Topical delivery of retinyl ascorbate co-drug: 1. Synthesis, penetration into and permeation across human skin. Int J Pharm 280:113–124. 21. Nomura H, Ishiguro T, Morimoto S. 1969. Studies on Lascorbic acid derivatives. 3. Bis(L-ascorbic acid-3,3’)phosphate and L-ascorbic acid 2-phosphate. Chem Pharm Bull (Tokyo) 17:387–393. ˇ 22. Kristl J, Volk B, Gaˇsperlin M, Sentjurc M, Jurkoviˇc P. 2003. Effect of colloidal carriers on ascorbyl palmitate stability. Eur J Pharm Sci 19:181–189.
DOI 10.1002/jps
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ˇ 23. Spiclin P, Gaˇsperlin M, Kmetec V. 2001. Stability of ascorbyl palmitate in topical microemulsions. Int J Pharm 222:271– 279. 24. Yoksan R, Jirawutthiwongchai J, Arpo K. 2010. Encapsulation of ascorbyl palmitate in chitosan nanoparticles by oil-inwater emulsion and ionic gelation processes. Colloids Surf, B 76:292–297. 25. Luadthong C, Tachaprutinun A, Wanichwecharungruang SP. 2008. Synthesis and characterization of micro/nanoparticles of poly(vinyl alcohol-co-vinyl cinnamate) derivatives. Eur Polym J 44:1285–1295. 26. Suwannateep N, Banlunara W, Wanichwecharungruang SP, Chiablaem K, Lirdprapamongkol K, Svasti J. 2011. Mucoadhesive curcumin nanospheres: Biological activity, adhesion to stomach mucosa and release of curcumin into the circulation. J Control Release 151:176–182. 27. Lin L, Lee KH. 2006. Structure-activity relationships of curcumin and its analogs with different biological activities. In Studies in natural products chemistry; Atta-ur R, Ed. Elsevier, London, UK pp 785–812. 28. Lademann J, Richter H, Teichmann A, Otberg N, BlumePeytavi U, Luengo J, Weiß B, Schaefer UF, Lehr CM, Wepf R, Sterry W. 2007. Nanoparticles—An efficient carrier for drug delivery into the hair follicles. Eur J Pharm Biopharm 66:159–164. 29. Knorr F, Lademann J, Patzelt A, Sterry W, Blume-Peytavi U, Vogt A. 2009. Follicular transport route—Research progress and future perspectives. Eur J Pharm Biopharm 71:173–180.
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INTRODUCTION The main function of drug carriers is to deliver their payload (drug molecules) to the desired target sites, reducing nontarget (systemic) exposure and increasing the exposure concentrations and/or duration per administered dose at the target site(s). To achieve these aims, various properties have been introduced into carriers besides the inherited physical barrier, such as the ability to bind to specific proteins on the target cells, stealth characteristics to avoid the host’s immune system, and the ability to release drugs under specific conditions, at specific pH or temperature values. In most encapsulated drug systems, the carrier materials are the major content as compared with the payload drug content, and so it would be logical to, if possible, make the carrier material Additional Supporting Information may be found in the online version of this article. Supporting Information Correspondence to: Supason Wanichwecharungruang (Telephone: +662-2187634; Fax: +662-2541309; E-mail: psupason@ chula.ac.th) Journal of Pharmaceutical Sciences, Vol. 102, 2770–2779 (2013) © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association
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possess a therapeutic function or biological activity. So far, only a few bioactive carriers have been reported, which include liposomes made from ascorbyl palmitate (AP),1 nanocarriers with UV absorptive properties,2,3 delivery systems made from L-ascorbic (vitamin C) derivatives,4,5 and carriers with anticancer activity.6 Furthermore, UV absorptive carriers have been shown to slowly release fragrance molecules and at the same time act as an UV screening material for the skin.3 Carriers with anticancer activity that showed good antitumor activity on their own were reported to work synergistically with their payload to combat the drug resistant cancerous cells.6 There are many labile bioactive molecules that need a better technology to keep their chemical structure unchanged during storage and/or during their transport to the required target site in the body. As the most common cause of the deterioration of active molecules is oxidation, usually, through free radical mechanisms initiated by reactive oxygen species, we expect that a carrier system with an integral free radical scavenging activity will be able to lessen such deterioration and so increase the stability of their
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otherwise oxidation-sensitive payloads. As a result, a nanocarrier with an antioxidative property was fabricated and is reported here. Vitamin C has long been used in pharmaceutical and cosmetic products. Its biological activities are based on its function as a cofactor for a number of enzymes, most notably the hydroxylases involved in collagen synthesis,7,8 and as a nonenzymatic watersoluble antioxidant. Vitamin C can efficiently protect biomolecules against oxidative degradation through its ability to scavenge and reduce reactive oxidizing molecules and free radicals.9 Although the in vitro dose-dependent pro-oxidant activity of vitamin C has been reported previously,10–12 recent studies have indicated no adverse effects of vitamin C on normal cells.12,13 The use of vitamin C in skin care is still popular, not only for its antiageing effect but also for its antitanning effect among Asian women,14,15 because the compound can also inhibit melanin synthesis.16,17 However, vitamin C is unstable and can be easily oxidized under aerobic conditions, the rate of which is increased with increasing heat or UV light levels.18 Many derivatives of vitamin C have been introduced to reduce the ease of its oxidation, including various esters, such as AP and retinyl ascorbate,19,20 bis(Lascorbic acid-3,3 )phosphate, and L-ascorbic acid 2phosphate.21 Among the many vitamin C derivatives, AP has been popularly used as an antioxidant additive in food, pharmaceutical, medical, and cosmetic products because of its improved stability and better skin penetration as compared with vitamin C.22 The successful encapsulation of AP into microemulsions,23 bilayer vesicles,1,22 polymeric nanoparticles,24 and solid lipid nanoparticles that resulted in some retardation of AP degradation,22 has been reported. Here, we used AP as an example to show that its degradation can be delayed when encapsulated in a nanocarrier system with an antioxidative property. In addition, the nanocarriers were designed to possess an optimal size so they could be entrapped in the hair follicular shunts and act as reservoirs of AP for the sustained release into the skin tissue. To make a nanocarrier with an antioxidative property, we attached the natural dietary antioxidant curcumin onto poly(vinyl alcohol) [PV(OH)], a nontoxic, biocompatible, and biodegradable hydrophilic polymer, so as to impart not only an amphiphilic character, but also an antioxidative property to the polymer. The obtained curcumin-grafted PV(OH) polymer [CURPV(OH)] was then fabricated into a nanocarrier and its free radical scavenging activity was evaluated. Next, AP was encapsulated into the CUR-PV(OH) nanocarrier and the stability of the encapsulated AP was evaluated. To answer whether the antioxidative CUR-PV(OH) nanocarrier would give better AP protection than one with no antioxidative property (control nanocarrier), AP was also encapsulated DOI 10.1002/jps
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into cinnamoyl-grafted PV(OH) [CIN-PV(OH)] and its stability was compared with that encapsulated in CUR-PV(OH). Finally, the release of AP from the CUR-PV(OH) nanocarriers, ex vivo penetration into pig skin, and stability of the AP after skin application, were all investigated.
MATERIALS AND METHODS Materials Ascorbyl palmitate was obtained from Roche (Basle, Switzerland). PV(OH) (MW 124,000–186,000 and 87%–89% deacetylated), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and glutaric anhydride were obtained from Sigma–Aldrich Chemical Company (Steinheim, Germany). Cinnamoyl chloride and curcumin were obtained from Acros Organics (Geel, Belgium). Synthesis of the Antioxidative Nanocarrier: Poly(Vinyl Alcohol-Co-Vinyl Glutarycurcumin), CUR-PV(OH) (Scheme 1) A mixture of 3.0 g curcumin, 0.6 g glutaric anhydride, and 4–5 drops pyridine in 60 mL dry dimethylformamide (DMF) was refluxed for 4 h and then the glutarylcurcumin formed was purified on a silica gel column, using a 20%–50% (v/v) ethyl acetate gradient in hexane as the mobile phase. The obtained glutarylcurcumin (2.5 g) in 20 mL of dry DMF was then added to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (0.05 g), followed by PV(OH) (0.09 g) and 1-hydroxybenzotriazole (0.06 g), and the mixture was kept at 0◦ C for 24 h under a nitrogen (N2 ) atmosphere. Subsequently, the reaction mixture was dialyzed against 30% (v/v) methanol in distilled water using a regenerated cellulose tubular membrane (CeluSep T4 dialysis tube; MWCO 12,000–14,000; Membrane Filtration Products, Seguin, Texas). Dry particles were obtained by freeze-drying the aqueous suspension. The product was characterized by 1 H NMR (Varian Mercury Spectrometer, 400.00 MHz; Varian Company, Palo Alto, California), Fourier transform infrared spectroscopy (FT-IR; Impact 410 Nicolet Fourier transform Infrared spectrophotometer; Nicolet Instruments Technologies, Inc., Madison, Wisconsin), UV–Vis spectrophotometry (UV, 2500 UV–Vis spectrophotometer; Shimadzu Corporation, Kyoto, Japan), and differential scanning calorimetry (DSC; Mettler DSC 822, Mettler Toledo, Columbus, Ohio) analyses. The DSC analysis was performed at a heating and cooling rate of 10◦ C/min under a N2 flow of 20 mL/min.
Curcumin-Grafted PV(OH) Polymer (CUR-PV(OH)) White solid. Degree of substitution (DS): 0.059. Tg : 81.4◦ C. FT-IR (KBr, cm−1 ) 3302 (O H str), JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 8, AUGUST 2013
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Scheme 1.
Synthesis of CUR-PV(OH) and CIN-PV(OH).
2919 (C H str), 1732 (C O str), 1626 (C C str), 1417 (CH2 bend), and 1250, 1121, and 1092 (C O str). 1 H NMR (CDCl3 , 400 MHz, *, ppm): 7.65 (O C CHCH Ar PV(OH)), d, J = 16 Hz, 1H), 7.55 (O C CHCH Ar, d, J= 16 Hz, 1H), 7.30–6.85 (Ar H, 6H), 6.8 (O C CH CHAr PV(OH), d, J = 16 Hz, 1H), 6.7 [(O C CH CHAr), d, J = 16 Hz, 1H], 5.70–5.90 ppm (keto–enol proton of curcumin moieties), 4.75, 4.55, 4.45 [ CH OH of PV(OH) backbone], 3.83 [CHOH and CHOC(O)CH3 of PV(OH) backbone, and OCH3 ], and 3.0–1.0 [br, CH3 CO and CH CH2 CH of PV(OH) backbone]. UV– Visible spectroscopy (DMSO): λmax at 412 nm. Synthesis of the Control (No Antioxidative Property) Nanocarrier: Poly(Vinyl Alcohol-Co-Vinyl Cinnamate), CIN-PV(OH) (Scheme 1) Cinnamoyl-grafted PV(OH) was prepared as previously described.25 In brief, 1.19 g PV(OH) was dissolved in heated anhydrous DMF. Then, pyridine (2.18 mL) was added and the obtained clear solution was poured into a round bottom flask containing 4.0 g freshly prepared cinnamoyl chloride. The mixture was stirred at 80◦ C–90◦ C for 1–2 h before the product was precipitated with 1.0% (w/v) aqueous Na2 CO3 , JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 8, AUGUST 2013
washed with water and dried under vacuum to a constant weight.
Cinnamoyl-Grafted PV(OH) Polymer (CIN-PV(OH)) Pale yellow solid. DS: 0.17. Tg : 71◦ C. FT-IR (KBr, cm−1 ): 3472 (OH), 3063, 3023 ( C H, str), 2929 ( C H str), 1720 (C O, str), 1636, 1575 (C C, str), 1441 ( CH2 bend) and 1312, 1248, and 1174 (C O str). 1 H NMR (400 MHz, DMSO-d6 , *, ppm): 8.17 (br, Ar CH), 7.65 (br, Ar H), 7.41 (br, Ar H), 6.58 (br, CHCOOR), 5.5–4.1 (br, OH), 4.0–3.5 (br, CHOH and CHOCOCH3 ), and 2.30–0.80 [br, CH3 CO and CH CH2 CH of PV(OH) backbone]. UV–Vis (DMSO) λmax , nm (g, M−1 cm−1 , monomeric unit−1 ): 282 (2544). Encapsulation
AP-Loaded CUR-PV(OH) Curcumin-grafted PV(OH) polymer (30 mg) was dissolved in DMF (10 mL) and then 15 mg of AP in 10 mL of DMF was added, mixed, and dialyzed against water. The resultant suspension of AP-loaded CUR-PV(OH) nanocarriers was collected, the volume adjusted to 20 mL with water, and aliquots DOI 10.1002/jps
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of the suspension were subjected to analysis by scanning electron microscopy (SEM; JEM-6400; Jeol, Ltd., Tokyo, Japan), transmission electron microscopy (TEM; JEM-2100; Jeol, Ltd.), and dynamic light scattering (DLS). The remaining suspension was freezedried to obtain the dry AP-loaded CUR-PV(OH) nanocarriers.
AP-Loaded CIN-PV(OH) The AP-loaded-CIN-PV(OH) nanocarriers were prepared as described for AP-loaded CUR-PV(OH) above, except that CUR-PV(OH) was replaced with an equal molar amount (same number of monomeric units) of CIN-PV(OH).
AP + Curcumin-Loaded CIN-PV(OH) Ascorbyl palmitate and curcumin were coloaded into the CIN-PV(OH) nanocarriers by dissolving CIN-PV(OH) (30 mg), AP (30 mg), and curcumin (60 mg) in DMF (20 mL) and then dialyzing the obtained mixture against water. Encapsulation Efficiency and Loading Level The amount of AP loaded into each type of nanocarrier was analyzed using high-performance liquid chromatography (HPLC). The suspension of the AP-loaded nanocarriers was centrifugally filtered using a 10,000-Da MWCO centrifugal-filter (Amicon Ultra-15; Millipore, County Cork, Ireland), and the solid product was very quickly rinsed with methanol to remove the AP attached to the outside of the nanocarriers and around the filter. The washed liquid was combined with the supernatant obtained from the dialysis process during the carrier preparation, and was analyzed for AP content by HPLC. The residual amounts of AP in the filtrate and the wash were then subtracted from the amount of AP initially used, to obtain the amount of encapsulated AP. From that, the encapsulation efficiency (EE) and loading level were calculated according to Eqs. 1 and 2, respectively: EE(%) =
weight of encapsulated AP × 100 , weight of AP used
Loading(%) =
(1)
weight of encapsulated AP × 100 . (2) weight of polymer used
Quantitation of AP by HPLC High-performance liquid chromatography was performed on a ThermoFinnigan P4000 (pump), connected to a UV–Vis detector (UV6000LP) using a C18 reversed-phase column (100 × 4.6 mm2 Hypersil ODS; Agilent Technologies, Santa Clara, California). The mobile phase was a 15:2:3 (v/v/v) mixture of methanol–acetonitrile–0.02 M phosphate buffer DOI 10.1002/jps
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pH 2.5, at a flow rate of 1.5 mL/min. Detection was performed at 254 nm, and the sample injection volume was 20 :L. Stability of the Encapsulated AP The stability of the four different AP samples, AP encapsulated in either (a) CUR-PV(OH) or (b) CIN-PV(OH) nanocarriers, (c) AP coencapsulated with curcumin in the CIN-PV(OH) nanocarrier, and (d) unencapsulated AP, was evaluated on the freezedried samples. The freeze-dried samples were kept at 30◦ C for 45 days under a light proof condition. At various time intervals, a few milligrams of each sample was withdrawn and analyzed for its AP content. The free AP sample was directly quantified for the level of AP by HPLC, whereas the three encapsulated AP samples were first subjected to extraction by soaking in methanol under a light proof and N2 atmosphere, then centrifugally filtered from the extracted polymer material, and the obtained methanol extract (filtrate) then analyzed for AP content by HPLC. DPPH Scavenging Activity The DPPH radical scavenging activity was used to measure the free radical scavenging activity as previously described.26 Briefly, 195 :L of 100 :M DPPH solution (in ethanol) and 5 :L of the test material (performed as serial dilutions) were mixed in a 96-well plate, and the absorbance was measured at 515 nm after incubation in the dark for 30 min at room temperature. The level of residual DPPH-free radicals was determined from the absorbance. Samples included aqueous suspensions of CUR-PV(OH) and CIN-PV(OH), plus the 10% (v/v) aqueous ethanol solution of free (unencapsulated) curcumin. Butylated hydroxytoluene (1.0 mM) and the solvent only were used as the 100% and 0% radical scavenging controls, respectively. Assays were performed in triplicate wells, and the data are expressed as the mean of these, in terms of the percent ratio of the level of residual to initial DPPH radicals.
Ex Vivo Pig Skin Penetration Fresh porcine ear skin from a one-month-old White Large piglet was obtained from Manoch Farm (Phetchaboon, Thailand), and was kept at 4◦ C from excision immediately after death to its use, which was within 8 h of execution. To evaluate the ability of the AP-loaded CUR-PV(OH) nanocarriers to penetrate into the pig skin, 10 :L of an aqueous suspension of AP-loaded CUR-PV(OH) (0.705 mg AP/mL) was applied onto a 1.0 × 2.0 cm2 skin piece to give a final AP skin coverage of 3.52 :g/cm2 . The skin piece was massaged for 2 min to induce the movement of the hair and then left at room temperature for 30 min before being subjected JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 8, AUGUST 2013
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to confocal fluorescence laser microscopy [CFLM; Nikon, Tokyo, Japan Digital Eclipse C1-Si, Plan Apochromat VC 100×, Diode Laser (405 nm; Melles Griot, Carlsbad, California), a Nikon TE2000-U microscope, a 32-channel-PMT-spectral-detector, and C1plus/C1si EZ-C1 Version 3.80m control software]. The obtained spectral output signal of each pixel was then unmixed into the respective CUR-PV(OH), AP, and skin/ hair autofluorescence components using chemometric analysis (image algorithms). Images indicating the location of the CUR-PV(OH), AP, and skin tissue and hair were then constructed using the obtained resolved signals. Stability of AP after Skin Application Thirty microliter of an aqueous suspension of APloaded CUR-PV(OH) nanocarriers (2.2 mg AP/mL) was applied onto a 2.5 × 2.5 cm2 piece of the pig ear skin to give a final AP skin coverage of 10.56 :g cm−2 . The skin piece was massaged for 2 min, left at room temperature for 3 h, then homogenized with 10 mL ethyl acetate under a N2 atmosphere for 2 min followed by ultrasonic vibration for 5 min. The mixture was then centrifuged at 21130 g for 10 min under a N2 atmosphere. The supernatant was harvested and the residue was extracted again with 5 mL ethyl acetate. Water (5 mL) was then added to the combined supernatants and the mixture was saturated with N2 and vortexed. The ethyl acetate layer was collected, dried under a N2 flow, and subjected to AP quantitation by HPLC. Similar experiments were carried out with free AP in place of the AP-loaded CUR-PV(OH) nanocarriers. Five replicate experiments were performed.
ous suspension indicated spherical particles with an average anhydrous diameter of 232.5 ± 29.44 nm. The hydrodynamic diameter of the CUR-PV(OH) nanocarriers, as determined by DLS, was 269.8 ± 19.45 nm (poly dispersity index (PDI) of 0.6). At similar curcumin moiety concentrations, the DPPH-free radical scavenging activity of CUR-PV(OH) was 48% of that of the free curcumin Synthesis of the Control (No Antioxidant Activity) CIN-PV(OH) Nanocarrier The cinnamoyl groups were successfully grafted onto the PV(OH) backbone (Scheme 1), to give CIN-PV(OH) with a DS of 0.17, as verified through the NMR spectrum (SI; Fig. S4). SEM and TEM images of the dry CIN-PV(OH) sample showed spherical particles with diameters of 269.8 ± 19.43 and 259.4 ± 16.92 nm, respectively. The aqueous suspension gave a similar hydrodynamic diameter of 270.5 ± 18.74 nm, as determined by DLS. The obtained nanocarrier suspension showed no free radical scavenging activity when tested by the DPPH scavenging assay. Encapsulation The AP loading percentages are shown in Table 1. DLS analyses of the AP-loaded CUR-PV(OH), AP-loaded CIN-PV(OH), and AP + curcumin coloaded CIN-PV(OH) nanocarriers gave hydrodynamic diameters of 278.2 ± 14.21, 290.5 ± 16.51, and 306.2 ± 18.66 nm, respectively (Fig. 1), whereas the SEM and/or TEM images indicated a spherical morphology with an average anhydrous diameter of 264.6 ± 18.27, 283.3 ± 20.02, and 302.4 ± 15.16 nm, respectively, suggesting very little swelling in water. AP Stability
RESULTS Synthesis of the Antioxidant Containing CUR-PV(OH) Nanocarrier Glutarylcurcumin was successfully synthesized from curcumin and glutaric anhydride and purified by column chromatography [see Nuclear Magnetic Resonance (NMR) spectrum in Supporting Information (SI), Fig. S1]. Glutarylcurcumin was successfully grafted onto PV(OH) with a DS of 0.059 (SI; Figs. S2 and S3). The CUR-PV(OH) dispersed well in water. SEM and TEM images of the dry CUR-PV(OH) aqueTable 1.
Samples were kept dry at 30◦ C under a light proof condition for 45 days and the amount of AP in the samples was periodically evaluated (Fig. 2). The AP encapsulated in CUR-PV(OH) was 47% and 77% more stable than the AP encapsulated in the control CIN-PV(OH) and free AP, respectively.
Ex Vivo Pig Skin Penetration The fluorescent spectra of AP and are shown in Figure 3a. AP-loaded nanocarriers were massaged into the and the images of the fluorescent
CUR-PV(OH) CUR-PV(OH) pig ear skin, signals from
Loading Percentages and Encapsulation Efficiency (EE) of the Three Particles
Samples AP-loaded CUR-PV(OH) (AP + curcumin) coloaded CIN-PV(OH) AP-loaded CIN-PV(OH)
Curcumin Loading (%, w/w)a
AP Loading (%, w/w)a
EE of AP (%)a
Mole Ratio of AP: Curcumin
– 19.0 ± 0.21 –
29.0 ± 0.32 19.4 ± 0.23 27.3 ± 0.52
80.9 ± 0.16 62.5 ± 0.11 75.9 ± 0.28
1.16:1 0.91:1 –
Data are shown as the mean ± 1 SD and are derived from three independent samples.
a
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HPLC analysis. The content of AP in the skin was 71 ± 14% and 18 ± 6% of the original amount applied onto the skin for the AP-loaded CUR-PV(OH) and free AP, respectively.
DISCUSSION Synthesis of the CUR-PV(OH) Nanocarrier
Figure 1. (a) Scanning electron microscopy image of the AP-loaded CUR-PV(OH) nanocarrier. (b–d) TEM images of the (b) AP-loaded CUR-PV(OH), (c) AP + curcumin coloaded CIN-PV(OH), and (d) AP-loaded CIN-PV(OH) nanocarriers.
Figure 2. Stability of dry free AP (unencapsulated AP), or AP encapsulated in different nanocarriers, after storage at 30◦ C under a light proof condition with normal air exposure. Data are shown as the mean ± 1 SD, derived from four independent repeats. The Mann–Whitney U test indicated a significant difference among the four samples at P ≤ 0.05 during 15–45 days.
CUR-PV(OH) and AP in the pig ear skin were taken after 2 h and are shown in Figures 3b and 3c, respectively. Stability of AP after Skin Application The AP-loaded CUR-PV(OH) and the free AP were separately applied onto freshly excised pig ear skin and left for 3 h at 37◦ C. The AP in the skin tissue was then recovered by solvent extraction followed by DOI 10.1002/jps
To fabricate a nanocarrier with an antioxidative property, PV(OH) was chosen as the backbone hydrophilic polymer because of not only its well-known safety but also its abundance of hydroxyl moieties for chemical derivatization. Curcumin was first derivatized into glutarylcurcumin by reacting curcumin with glutaric anhydride and purifying the monofunctionalized product from other species (bifunctionalized product, curcumin, and glutaric acid) through silica column chromatography (SI; Fig. S1). Then, the obtained glutarylcurcumin was grafted onto PV(OH) via ester bond formation using 1-ethyl- 3(3-dimethyl-aminopropyl)carbodiimide (EDCI)as the coupling agent and the unattached glutarylcurcumin was subsequently dialyzed out from the CUR-PV(OH) product. Successful grafting was confirmed through the 1 H NMR spectrum of the product (SI; Fig. S2). The DS of curcumin (0.059) was deduced from the ratio between the integration of peaks at 1.2–1.8 ppm [ CH CH2 CH of PV(OH) backbone] and the peaks at 6.5–7.0 ppm (Ar CHCH C O and one aromatic proton of the curcumin moieties). The UV–Vis absorption spectrum of CUR-PV(OH) showed a maximum absorbance at 422 nm, a blueshift from that of free curcumin (429 nm) (SI; Fig. S3). The blueshift indicates a lower degree of conjugation in the curcumin core structure resulting from the replacement of the hydroxyl group on the aromatic ring with an acyl group. With the attached hydrophobic curcumin moieties on the hydrophilic PV(OH) backbone, the CUR-PV(OH) became amphiphilic. Therefore, it was likely that as the CUR-PV(OH) self-assembled into spherical nanocarriers during the solvent displacement process (DMF was displaced with water), the hydrophobic curcumin moieties would locate at the core, away from water with their hydrophilic hydroxyl groups located at the nanocarrier’s surface, in full contact with the water medium. Curcumin-grafted PV(OH) polymer dispersed well in water, with a hydrodynamic diameter, as determined by DLS, of 269.8 ± 19.45 nm and a PDI of 0.6, indicating mid-range polydispersity). SEM and TEM images showed a spherical morphology with a slightly smaller average anhydrous diameter of 232 ± 29.44 nm, suggesting only minimal swelling in the aqueous environment. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 8, AUGUST 2013
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Figure 3. Confocal fluorescence laser microscopy images showing skin penetration by the AP-loaded CUR-PV(OH) nanocarriers. (a) Fluorescent spectra of CUR-PV(OH) and AP; (b, C) fluorescent images of the skin tissue at 2 h after the material was applied to the stratum corneum and showing (b) the CUR-PV(OH) fluorescent signal and (c) the AP signals.
At similar curcumin moiety concentrations, the DPPH-free radical scavenging activity of CUR-PV(OH) was 48% of that of the free curcumin. The free radical scavenging activity of curcumin is a result of both the highly conjugated phenolic and the keto–enol functionalities in the curcumin structure.27 Thus, by derivatizing only one phenolic group, each grafted curcumin still possessed one phenolic and one conjugated keto–enol moiety with which to scavenge free radicals. Nevertheless, the steric effect of the polymer around the grafted curcumin moieties probably partially hindered their free radical scavenging activity, and this would explain why the grafted curcumin moiety possessed only half (rather than close to 66% if the activity of the conjugated keto–enol group is assumed for simplicity to equate to that of the phenolic groups) the activity of the ungrafted curcumin. Synthesis of the CIN-PV(OH) Nanocarrier As the CUR-PV(OH) nanocarriers still possessed free radical scavenging activity (∼48% of that of the free curcumin), it was expected that they could provide an extra defense against oxidative degradation for their payload. To test this notion, a similar nanocarrier but without an antioxidative activity was required. For this reason, CIN-PV(OH) was synthesized. The hydrophobic cinnamoyl groups were grafted onto the PV(OH) backbone through esterification of the carboxylic functionality of cinnamic acid and the hydroxyl moieties on the PV(OH) (Scheme 1). Successful grafting was verified by the 1 H NMR spectrum of the CIN-PV(OH) (SI; Fig. S4), with a DS of 0.17 being obtained from the ratio of the integrated peaks at 6.2–8.4 ppm ( COCHCHAr H) against those at 0.8–2.3 ppm ( CH CH2 CH and CH3 CO of PV(OH) backbone).21 The obtained CINPV(OH) could self-assemble into stable spherical nanocarriers during the displacement of DMF with water. SEM and TEM images of the anhydrous samples indicated a spherical morphology with diameters of 269.8 ± 19.43 and 259.4 ± 16.92 nm, respectively, which was similar to the hydrodynamic diameter of JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 8, AUGUST 2013
the particles (270 ± 18.74 nm) obtained by DLS analysis. Thus, we obtained a CIN-PV(OH) nanocarrier with no free radical scavenging activity (confirmed by DPPH scavenging assay) and a similar low level of swelling in an aqueous environment to be used as control carrier. Encapsulation Encapsulation of AP into the CUR-PV(OH) or CIN-PV(OH) nanocarriers was performed by allowing self-assembly of the CUR-PV(OH) or CIN-PV(OH) polymer in the presence of AP (or AP and curcumin for the coencapsulation in the CIN-PV(OH)) during the displacement of DMF with water. It was speculated that during the self-assembly of CUR-PV(OH) or CIN-PV(OH), the hydrophobic AP molecules would automatically locate themselves at the hydrophobic cores of the forming polymeric nanocarriers, to have minimal interaction with the hydrophilic water. The AP loading percentage and EE (Table 1) were determined as outlined in the Material and Methods section. The loading value of AP in CUR-PV(OH) nanocarriers was very close to that in the control CIN-PV(OH) ones. However, in the AP plus curcumin coloaded CIN-PV(OH), the AP loading level obtained was significantly lower. This could be explained through the fact that the coloaded curcumin probably took up a significant space in each CIN-PV(OH) nanocarrier, reducing the space available to load the AP. Accordingly, the EE of AP was also significantly lower in the AP plus curcumin coloaded CIN-PV(OH). The higher AP loading capacity and EE of the CUR-PV(OH) nanocarrier, compared with the CIN-PV(OH) one that was coloaded with AP and curcumin, indicated another advantage of using the antioxidative CUR-PV(OH) nanocarrier systems. In other words, using the CUR-PV(OH) nanocarrier not only gave better protection to the AP than coloading the AP with curcumin into the CIN-PV(OH) one, but also allowed a higher EE and loading capacity to be obtained. SEM and TEM analyses revealed that the AP-loaded CUR-PV(OH), AP-loaded DOI 10.1002/jps
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CIN-PV(OH), and the AP plus curcumin coloaded CIN-PV(OH) nanocarriers were all spherical with an average dry diameter of 264.6 ± 18.27, 283.3 ± 20.02, and 302.4 ± 15.16 nm, respectively (Fig. 1). These agree with the hydrodynamic diameters obtained from DLS analysis of 278.2 ± 14.21, 290.5 ± 16.51, and 306.2 ± 18.66, respectively, confirming the minimal swelling in water.
In Vitro AP Stability The AP-loaded CUR-PV(OH), AP-loaded CIN-PV(OH), and the AP plus curcumin coloaded CIN-PV(OH) nanocarriers, along with free AP, were kept dry at 30◦ C under a light proof condition for 45 days and the amount of AP in the samples was periodically evaluated. The results (Fig. 2) indicated that the free AP degraded much quicker than all three encapsulated APs, with the residual free AP level decreasing to <50%, <25%, <10%, and 0% after 6, 10, 20, and 45 days, respectively. Of the encapsulated APs, AP in the CUR-PV(OH) nanocarriers degraded the slowest with approximately 80% residual AP after 45 days, followed by the AP that was coloaded with curcumin in CIN-PV(OH), with AP loaded in CIN-PV(OH) being the least stable. This supported that curcumin could help retard the degradation of AP, and that the protection was more effective when the curcumin molecules were covalently grafted onto the polymeric PV(OH) wall than when they were coloaded into the CIN-PV(OH) nanocarrier to essentially the same level. In fact, it should be noted here again that the grafted curcumin in the CUR-PV(OH) nanocarrier contained only one phenolic group per molecule, whereas the coloaded curcumin in the CIN-PV(OH) nanocarrier contained two phenolic groups per molecule and so theoretically had 50% more free radical scavenging activity than that of the grafted curcumin (if we assume for simplicity that the conjugated keto–enol group is as effective as the phenolic groups). Indeed, as reported above, at a similar concentration of curcumin moieties, the DPPH-free radical scavenging activity of the CUR-PV(OH) was 48% of the value of the free curcumin. Nevertheless, the grafted curcumin in the CUR-PV(OH) was more effective in protecting the encapsulated AP than the coloaded curcumin in the CIN-PV(OH) nanocarrier at corresponding AP: curcumin molar ratios (Table 1). The reason for this lies in the arrangement of the curcumin moieties. Curcumin moieties along the nanocarrier wall material acted as a protective barrier, scavenging the invading-free radicals and so effectively protecting the encapsulated AP inside the nanocarrier (Fig. 4). This resulted in an improved stability of the AP in the CUR-PV(OH) nanocarriers, despite the reduced radical scavenging activity of the DOI 10.1002/jps
Figure 4. Representative models of (left) the AP-loaded control nanocarriers, (middle) AP + curumin coloaded control nanocarriers, and (right) AP-loaded CUR-PV(OH) nanocarriers.
grafted curcumin compared with that of the original curcumin.
Ex Vivo Pig Skin Penetration The AP-loaded-CUR-PV(OH) nanocarriers were massaged into the pig ear skin and subsequently subjected to analysis by CFLM. It should be noted here that the massage was carried out to induce the hair movement that would enhance the moving of the carriers along the hair shaft, step by step down the hair cuticle into the hair follicles by the gear pump mechanism.28 This is reasonable given the fact that skin creams are generally applied by repeated massaging into the skin. Because the fluorescent spectrum of AP is different from that of the CUR-PV(OH) nanocarriers (Fig. 3a), the fluorescent signal from AP was resolved from the signal from CUR-PV(OH) using chemometric analysis (image algorithms). In a similar way, the autofluorescence from the tissue components was removed. As a result, the obtained spectral output signal of each pixel was unmixed into CUR-PV(OH), AP, and skin–hair autofluorescence components, yielding separate images for the locations of the CUR-PV(OH) and AP. The fluorescent signals from the CUR-PV(OH) and AP could be observed clearly along the hair follicles (Figs. 3b and 3c, respectively), indicating that the AP-loaded CUR-PV(OH) nanocarriers could be trapped in the hair follicle shunts. As the location of the AP fluorescent signal did not always match the location of the CUR-PV(OH) signal, it was likely that during the 2 h period, some of the AP had already been released and diffused away from the nanocarriers. Thus, the AP-loaded CUR-PV(OH) nanocarriers could be trapped in the hair follicles and release some of the loaded AP that then diffused into the tissue surrounding the hair follicles. Accumulation of the AP-loaded nanocarriers at the hair follicles should offer a good AP reservoir for the skin. As demonstrated earlier, the AP inside the CUR-PV(OH) nanocarriers is quite stable and so this AP reservoir will slowly supply the skin with undegraded AP. It should be noted here that the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 8, AUGUST 2013
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entrapment of drug-loaded nanocarriers in hair follicles has been reported previously.29 However, here we also showed evidence of AP release from those entrapped nanocarriers. In fact, the fluorescent signal of AP was detected at a depth of more than 200 :m from the stratum corneum, and so some of the AP was in the dermal layer of the pig ear skin. Stability of AP After Skin Application As reported above, when encapsulated in the CUR-PV(OH) nanocarrier, the stability of AP was improved significantly when tested in vitro. We then speculated that the improved AP stability would hold even when the materials were applied onto the skin. Accordingly, the AP-loaded CUR-PV(OH) nanocarriers and the free AP were separately applied onto freshly excised pig ear skin as above and left for 3 h at 37◦ C. The AP in the skin tissue was then recovered by solvent extraction followed by HPLC analysis. The content of AP in the skin was 71% and 18% of the original amount applied onto the skin for the AP-loaded CUR-PV(OH) nanocarriers and free AP, respectively. It was obvious that the antioxidative CUR-PV(OH) nanocarriers could effectively increase the AP stability even when applied on the skin. This was likely to result from the free radical protection afforded by the antioxidative nanocarrier and the localization of some of the AP-loaded nanocarriers inside the hair follicles. The nanocarriers were physically stable even when dry (SEM analysis) and so the antioxidative nanocarriers could effectively protect their payload efficiently even after skin application. Localization of the nanocarriers inside the hair follicles would automatically decrease the air and light exposure of the materials and thus might also have contributed to the slower AP oxidation compared with the materials sitting on the skin fully exposed to such environmental oxidative factors.
CONCLUSIONS The concept of a nanocarrier with a self-antioxidative property, to stabilize and deliver a labile drug, was demonstrated through the stabilizing and delivery of the labile AP into the pig skin using a nanocarrier fabricated from CUR-PV(OH). The CUR-PV(OH) nanocarriers possessed a free radical scavenging activity, as tested by the DPPH assay. Loading of AP into the CUR-PV(OH) resulted in nanocarriers with an average dry diameter of 232 ± 29.4 nm and a slightly larger hydrodynamic size of 269.8 ± 19.4 nm (PDI of 0.6), with an AP loading of 29% (w/w) and an EE of 81%. The AP encapsulated in CUR-PV(OH) showed the highest storage stability compared with that coencapsulated with curcumin in CIN-PV(OH) and especially compared with the AP encapsulated alone in CIN-PV(OH) and the free AP. CFLM analysis indiJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 8, AUGUST 2013
cated that the AP-loaded CUR-PV(OH) nanocarriers could penetrate pig skin via the hair follicle pathway and release AP to subsequently diffuse into the dermis.
ACKNOWLEDGMENTS The authors thank the Thailand Research Fund (RDG5650009), the Advanced Material Cluster at Chulalongkorn University and the higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission (CU-56-AM02), for financial support.
REFERENCES 1. Gopinath D, Ravi D, Rao BR, Apte SS, Renuka D, Rambhau D. 2004. Ascorbyl palmitate vesicles (Aspasomes): Formation, characterization and applications. Int J Pharm 271:95–11. 2. Kidsaneepoiboon P, Wanichwecharungruang SP, Chooppawa T, Deephum R, Panyathanmaporn T. 2011. Organic–inorganic hybrid polysilsesquioxane nanospheres as UVA/UVB absorber and fragrance carrier. J Mater Chem 21:7922–7930. 3. Anumansirikul N, Wittayasuporn M, Klinubol P, Tachaprutinun A, Wanichwecharungruang SP. 2008. UV-screening chitosan nanocontainers: Increasing the photostability of encapsulated materials and controlled release. Nanotechnology 19:205101. 4. Zhuang D, Nie J, Yang J. 2011. Poly(ascorbyl acrylate)s: Synthesis and evaluation of their redox polymerization ability in the presence of hydrogen peroxide. J Polym Sci Pol Chem 49:1999–2007. 5. Wang Y, Yang J. 2011. Synthesis and self-assembly of novel amphiphilic copolymers poly(lactic acid)-block-poly(ascorbyl acrylate). J Polym Sci, Part A: Polym Chem 49:3988–3996. 6. Amornwachirabodee K, Chiablaem K, Wacharasindhu S, Lirdprapamongkol K, Svasti J, Vchirawongkwin V, Wanichwecharungruang SP. 2012. Paclitaxel delivery using carrier made from curcumin derivative: Synergism between carrier and the loaded drug for effective cancer treatment. J Pharm Sci 101:3779–3786. 7. Traber MG, Stevens JF. 2011. Vitamins C and E: Beneficial effects from a mechanistic perspective. Free Radic Biol Med 51:1000–1013. 8. Phillips CL, Combs SB, Pinnell SR. 1994. Effects of ascorbic acid on proliferation and collagen synthesis in relation to the donor age of human dermal fibroblasts. J Invest Dermatol 103:228–232. 9. Colven RM, Pinnell SR. 1996. Topical vitamin C in aging. Clin Dermatol 14(2):227–234. 10. Podmore ID, Griffiths HR, Herbert KE, Mistry N, Mistry P, Lunec J. 1998. Vitamin C exhibits pro-oxidant properties. Nature 392:559–559. 11. Carr A, Frei B. 1999. Does vitamin C act as a pro-oxidant under physiological conditions? FASEB J 13:1007–1024. 12. Putchala MC, Ramani P, Sherlin HJ, Premkumar P, Natesan A. 2013. Ascorbic acid and its pro-oxidant activity as a therapy for tumours of oral cavity—A systematic review. Arch Oral Biol 58:563–574. 13. Chen Q, Espey MG, Sun AY, Pooput C, Kirk KL, Krishna MC, Khosh DB, Drisko J, Levine M. 2008. Pharmacologic doses of ascorbate act as a prooxidant and decrease growth of aggressive tumor xenografts in mice. Proc Natl Acad Sci USA 105:11105-11109. DOI 10.1002/jps
NANOCARRIER WITH SELF-ANTIOXIDATIVE PROPERTY
14. Ohshima H, Mizukoshi K, Oyobikawa M, Matsumoto K, Takiwaki H, Kanto H, Itoh M. 2009. Effects of vitamin C on dark circles of the lower eyelids: Quantitative evaluation using image analysis and echogram. Skin Res Technol 15:214–217. 15. Zussman J, Ahdout J, Kim J. 2010. Vitamins and photoaging: Do scientific data support their use? J Am Acad Dermatol 63:507–525. 16. Lerner AB, Fitzpatrick TB. 1950. Biochemistry of melanin formation. Physiol Rev 30:91–126. 17. Ros JR, Rodriguez-Lopez JN, Garcia-Canovas F. 1993. Effect of L-ascorbic acid on the monophenolase activity of tyrosinase. Biochem J 295:309–312. 18. Austria R, Semenzato A, Bettero A. 1997. Stability of vitamin C derivatives in solution and topical formulations. J Pharm Biomed Anal 15:795–801. 19. Song QX, Wei DZ, Zhou WY, Xu WQ, Yang SL. 2004. Enzymatic synthesis and antioxidant properties of L-ascorbyl oleate and L-ascorbyl linoleate. Biotechnol Lett 26:1777–1780. 20. Abdulmajed K, Heard CM. 2004. Topical delivery of retinyl ascorbate co-drug: 1. Synthesis, penetration into and permeation across human skin. Int J Pharm 280:113–124. 21. Nomura H, Ishiguro T, Morimoto S. 1969. Studies on Lascorbic acid derivatives. 3. Bis(L-ascorbic acid-3,3’)phosphate and L-ascorbic acid 2-phosphate. Chem Pharm Bull (Tokyo) 17:387–393. ˇ 22. Kristl J, Volk B, Gaˇsperlin M, Sentjurc M, Jurkoviˇc P. 2003. Effect of colloidal carriers on ascorbyl palmitate stability. Eur J Pharm Sci 19:181–189.
DOI 10.1002/jps
2779
ˇ 23. Spiclin P, Gaˇsperlin M, Kmetec V. 2001. Stability of ascorbyl palmitate in topical microemulsions. Int J Pharm 222:271– 279. 24. Yoksan R, Jirawutthiwongchai J, Arpo K. 2010. Encapsulation of ascorbyl palmitate in chitosan nanoparticles by oil-inwater emulsion and ionic gelation processes. Colloids Surf, B 76:292–297. 25. Luadthong C, Tachaprutinun A, Wanichwecharungruang SP. 2008. Synthesis and characterization of micro/nanoparticles of poly(vinyl alcohol-co-vinyl cinnamate) derivatives. Eur Polym J 44:1285–1295. 26. Suwannateep N, Banlunara W, Wanichwecharungruang SP, Chiablaem K, Lirdprapamongkol K, Svasti J. 2011. Mucoadhesive curcumin nanospheres: Biological activity, adhesion to stomach mucosa and release of curcumin into the circulation. J Control Release 151:176–182. 27. Lin L, Lee KH. 2006. Structure-activity relationships of curcumin and its analogs with different biological activities. In Studies in natural products chemistry; Atta-ur R, Ed. Elsevier, London, UK pp 785–812. 28. Lademann J, Richter H, Teichmann A, Otberg N, BlumePeytavi U, Luengo J, Weiß B, Schaefer UF, Lehr CM, Wepf R, Sterry W. 2007. Nanoparticles—An efficient carrier for drug delivery into the hair follicles. Eur J Pharm Biopharm 66:159–164. 29. Knorr F, Lademann J, Patzelt A, Sterry W, Blume-Peytavi U, Vogt A. 2009. Follicular transport route—Research progress and future perspectives. Eur J Pharm Biopharm 71:173–180.
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