International Journal of Pharmaceutics 486 (2015) 88–98
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Novel curcumin-loaded gel-core hyaluosomes with promising burn-wound healing potential: Development, in-vitro appraisal and in-vivo studies Wessam M. El-Refaie a , Yosra S.R. Elnaggar b, * , Magda A. El-Massik a , Ossama Y. Abdallah b a b
Department of Pharmaceutics, Faculty of Pharmacy and Drug Manufacturing, Pharos University in Alexandria, Egypt Department of Pharmaceutics, Faculty of Pharmacy, Alexandria University, Egypt
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 February 2015 Received in revised form 22 March 2015 Accepted 25 March 2015 Available online 27 March 2015
Despite its effectiveness, curcumin (Curc) dermal delivery is handicapped by hydrophobicity, high metabolism and poor skin permeation. In this work, the potential of novel self-assembled nanogels, namely gel-core hyaluosome (GC-HS) to enhance Curc delivery to wound sites, enhance healing rate and decrease scar formation was evaluated. Curc–GC-HS were prepared using film hydration technique and evaluated regarding size, zeta potential (ZP), entrapment efficiency (% EE), and in vitro release. Structure elucidation was performed using light, polarizing and transmission electron microscopy (TEM). In-vivo burn-wound healing potential, skin deposition ability and histological study were evaluated using female Sprague Dawley rats. Curc–GC-HS were compared to conventional transfersomal gel (Curc–T-Pl gel), and other conventional gels. Curc–GC-HS showed nanosize (202.7 0.66 nm), negative ZP (33 2.6 mV) and % EE (96.44 1.29%). TEM revealed discrete vesicles with characteristic bilayer structure. Polarizing microscopy proposed liquid crystalline consistency. Burn-wound healing study showed that Curc–GC-HS was the only system exhibiting marked improvement at day 7 of treatment. At 11th day, Curc–GC-HS treated wounds showed almost normal skin with no scar confirmed by histological analysis. Curc–GC-HS showed five folds higher skin deposition compared to conventional Curc–T-Pl gel. To conclude, novel gelcore hyaluosomes elaborated are promising nanogels able to increase Curc skin penetration and dermal localization while protecting it against degradation. Future perspective encompasses assessing potential of novel nanocarrier for skin cancer therapy. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Curcumin Wound Burn Scar Dermal Vesicles Gel-core liposomes Hyaluronic acid
1. Introduction Skin is repeatedly subjected to wounds either by burns, injuries, or physical trauma. These wounds initiate a set of complicated and synchronized responses for healing and restoration of the skin structure. The healing process is spontaneous and involve different overlapped phases, namely, inflammation (inducing haemostasis and clot formation), fibroplasia and neovascularization, generation of granulation tissue, re-epithelialization, and finally the formation of new extracellular matrix and tissue remodeling (Suguna et al., 2002). In minor wound damages, full restoration of the integrity of tissues structure and barrier function occurs (Yates et al., 2007).
* Corresponding author at: Department of Pharmaceutics, Faculty of Pharmacy, Alexandria University, 1 Khartoum Square, Azarita, Messalla Post Office, P.O. Box 21521, Alexandria, Egypt. Tel.: +20 1147591065; fax: +20 34873273. E-mail address: [email protected] (Y.S.R. Elnaggar). http://dx.doi.org/10.1016/j.ijpharm.2015.03.052 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
However, in severe cases such as large skin injuries, second and third degree burns, several factors are involved and prolong the healing time (Hardwicke et al., 2008). The longer the time for spontaneous healing process, the worse is the obtained result. Edema, inflammation, hypertrophic scars and unsightly pigmentation changes are likely to be associated with such complicated cases. Under these prolonged unfavorable conditions for wound healing, the continuing inflammatory cascade may result in increasing tissue destruction and necrosis rather than healing (Atiyeh and Hayek, 2004). Therefore, inflammation is a common complication in wound healing process. Topical applications of compounds with antioxidative properties using proper materials and antioxidants will be useful against oxidative damage and be helpful to the healing of the wound. Among others, curcumin (Curc) has been reported as a promising wound healing agent when used topically (Moghaddam et al., 2009; Sidhu et al., 1998). It has antioxidant, anti-inflammatory, anti-tumor (apoptosis
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inducing), and anti-angiogenesis effects. Moreover, Curc has received great attention due to its ability to perform multiple actions and to target many cellular pathways (Aggarwal and Sung, 2009). Studies to date have demonstrated better accessibility of Curc to wound sites after topical application compared to its oral administration (Sidhu et al., 1998, 1999). This was attributed to its limited water-solubility and extensive first-pass metabolism (Asai and Miyazawa, 2000; Ravindranath and Chandrasekhara, 1980). With this in mind, different formulations of Curc have been developed to achieve better topical application at the wound site. These include: chitosan–alginate sponges (Dai et al., 2009), polymeric bandages (Mohanty et al., 2012), alginate foams (Hegge et al., 2011), collagen films (Gopinath et al., 2004), and creams (Durgaprasad et al., 2011). Compared to unformulated Curc, it was found that the bioactivity of Curc increased when incorporated into these formulations. However, no significant difference in the wound healing effect was observed between these different formulations. This might be due to the Curc extensive metabolism and rapid elimination regardless the dosage form or the route of administration (Anand et al., 2007; Asai and Miyazawa, 2000; Ravindranath and Chandrasekhara, 1980). Therefore, the infiltration of Curc into cells at the wound site can still potentially be improved especially that studies examining Curc skin delivery never reported its in vivo skin deposition and localization. Several approaches have been investigated for improvement of Curc efficacy (Bisht et al., 2007; Li et al., 2007; Ryu et al., 2006). Most promising are the nanostructured systems that have been recently formulated and investigated for Curc enhanced therapeutic outcomes (Wang et al., 2011). Their higher total surface area and smaller size facilitate the cellular uptake of Curc into cancer cells and reduce their proliferation (Lee et al., 2014, 2015). However, the effect of such nanocarriers in enhancing Curc bioavailability for wound healing applications has not gained considerable attention. Moreover, so far no study has investigated the effect of phospholipid-based vesicular systems on enhancing Curc delivery to wound sites, despite their captivating properties in enhancing drugs skin permeation and dermal localization (Elnaggar et al., 2014). Scar is another common and serious post-wound deformity. Burn scars, because of their clearly visible and stigmatizing appearance, have probably the highest impact on the quality of life compared to other skin diseases (Stella et al., 2008; Van Loey and Van Son, 2003). They have serious physical, functional, and psychological problems. Scars may cause pain, itching; and contractures that may hamper mobility (Van Loey and Van Son, 2003). Deep and severe scars may impair individual’s life as they are often treated with prolonged, painful procedures that in most cases give suboptimal results (Alster and Tanzi, 2003; Bock et al., 2006; Brown et al., 2008; Shaffer et al., 2002). It was reported that the fetus has a faster wound healing progress without leaving scars compared to that occurs in adults (Harrison and Adzick, 1991). Moreover, a more organized collagen was formed unlike that found in adults (Scott Adzick et al., 1985). The peculiar characteristics of the fetal healing were attributed to the HA rich extracellular matrix of the healing tissue (Adzick and Longaker, 1992; Longaker et al., 1991). It was found that the amount of HA was increased rapidly in the extracellular matrix of the healing tissue in both adults and fetus. However, in adults this HA amount significantly reduced to near zero within a week. In the fetus, HA level remains significantly elevated for almost three weeks (Longaker et al., 1991). This may explain the difference in the outcome of wound healing between fetus and adults. HA was examined for its daily topical use in the healing of burn. Topical treatment with 1% HA resulted in shorter healing time of burns under study (29 1.33 days) compared to control (38 2.58 days) (Medeiros et al., 1999). Although, it is
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better than control, the healing time of HA treated group was still considered prolonged. This long time might also result in impaired healing. Recently, the potential of HA as a suitable delivery vehicle to facilitate sustained local release and reduced doses of VN:GF (vitronectin:growth factor) was investigated (Xie et al., 2011). VN: GF was reported to significantly increase re-epithelialization in a porcine deep dermal partial-thickness burn model (Upton et al., 2008). Despite the advantages of HA in wound healing, results obtained from this study (Xie et al., 2011) indicated that, there was no significant difference between the thicknesses of the epidermis treated with VN:GF complexes alone and VN:GF complexes together with HA. Therefore, promising use of HA in burn healing process still needs a step of skin penetration enhancement (El-Refaie et al., 2015). Based on aforementioned background, combining HA and Curc in a tailored nanovesicular system is anticipated to combine their individual privileges for burn-wound healing. In this quest, this work is the first one to present novel HA-based gel-core hyalusomes loaded with Curc (Curc–GC-HS) prepared with simple technology. This system was designed in order to combine positive effect of flexible liposomes in enhancing skin penetration with the reported stability of gel-core liposomes against degradation. In addition, GC-HS utilizes HA as the gelling material with its unique viscoelastic and wound healing properties. The potential of the novel nanocarrier to improve Curc delivery to wound sites, its stability and dermal localization was evaluated. Furthermore, the potential of novel formulation to further enhance the healing rate and reduce scar formation was assessed as well. In vitro characteristics and burn-wound healing potential of the prepared hyaluosomes was compared to conventional transferosomes, conventional Curc pluronic gel, Curc–HA gel and plain HA gel. 2. Materials and methods 2.1. Materials Curcumin was obtained from (Hebei food additive Co., Ltd., China). Hyaluronic acid (HA, MW = 0.8 1.17 10 MDa) was obtained from Shiseido (Japan). Lipoid S100 (phosphatidylcholine from soybean) was kind gift from Lipoid GmbH (Ludwigshafen, Germany). Poloxamer 407 (Kolliphor 407, a sample gift from BASF, Germany). Tween 80 and all other reagents were of analytical grade. 2.2. Preparation of curcumin loaded vesicular gels 2.2.1. Curcumin loaded gel-core hyaluosomes (Curc–GC-HS) Curc–GC-HS were prepared by thin film evaporation technique. Lipoid S100 (PL) together with Tween 80 was dissolved in chloroform. Curc was dissolved in acetone and mixed with the dissolved PL. The solvent was then removed under reduced pressure in a rotary evaporator at 58 C, to obtain a thin film on the flask wall. Evaporation was continued for 2 h after the dry residue appeared, to completely remove all the solvent. Obtained dry lipid films were then hydrated with 1% w/w HA aqueous dispersion. Final Curc concentration in the formulation was 0.15% w/w. The hydrated film was subjected to water bath heating at 58 C for 10 min and vortexing for 2 min; this cycle was repeated four times. The prepared vesicles were then extruded through polycarbonate membranes with pore size of 200 nm at room temperature with the aid of a Liposome Extruder (Model ER-1, Eastern Scientific LLC, USA). The dispersion was hermetically sealed and stored at 4 C (Bragagni et al., 2012). For comparative reasons, Curc free GC-HS were also prepared with the same method except the addition of Curc. Plain and Curc loaded (0.15%) 1% HA aqueous gels were also prepared and used for comparison.
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2.2.2. Curcumin loaded transfersomal pluronic gel (Curc–T-Pl gel) Curc loaded transfersomes (Curc-T) were prepared by the above mentioned procedure using distilled water as a hydrating medium to be used for in vivo comparison. The obtained transfersomal dispersion, containing 0.15% Curc, was then gelled with poloxamer1 407 (Pluronic F-127). Pluronic F-127 (Pl, 25%) was soaked in the obtained Curc-T dispersion and left overnight in the refrigerator then stirred slowly in a closed vessel until homogeneous Curc–T-Pl gel was obtained. Curc–Pl gel was also prepared for comparison by dispersing the drug powder in 25% Pl gel after soaking in water and kept overnight in the refrigerator, and stirred as above till a clear gel containing 0.15% Curc was obtained. 2.3. Particle size, zeta potential and PDI The particle size (PS), polydispersity index (PDI) and zeta potential (ZP) for the prepared Curc–GC-HS were determined by dynamic light scattering (DLS) technique using Zetasizer Nano ZS. (Malvern, Instruments Ltd., Malvern, UK). For measurements, Curc–GC-HS formulations were properly diluted (0.1% v/v) with filtered distilled water to avoid multiscattering phenomena. Measurements were conducted at 25 C. All samples were measured in triplicates and results were represented as mean value SD.
%EE ¼
total drug free drug 100 total drug
2.6. In-vitro release behavior The dialysis method was utilized in order to investigate the release behavior of Curc from Curc–GC-HS, Curc–T-Pl gel, Curc–HA gel, and Curc–Pl gel, respectively (n = 3). Dialysis was carried out through dialysis bag 7 cm long with a molecular mass cut off 12– 14 kDa (VISKING dialysis tubing, SERVA, electrophoresis, Germany) (Elsheikh et al., 2012, 2014). The bags were filled with an amount of the formulation equivalent to 0.3 mg Curc and put in ambered glass bottles containing 10 ml of water: ethanol (1:1). Bottles were kept at 37 C 1 C with 100 rpm shaking throughout the experiment using a shaking water bath. At different time intervals (0.25, 0.5, 1, 2, 3, 4, 6 h), the whole volume of the release medium was withdrawn and replaced with an equal volume of a previously warmed fresh one. The percentage of Curc released was calculated from the cumulative drug amount released at these different time intervals. Drug concentration was determined by measuring UV absorbance at 430 nm. All measurements were performed in triplicates. Data were represented as mean SD. 2.7. In-vivo wound healing
2.4. Morphological characterization and structural elucidation Morphology of the prepared nanovesicles was determined by transmission electron microscopy (TEM). Samples were firstly diluted with distilled water and dropped onto a carbon-coated copper grid and left for 1 min to allow vesicles to adhere on the carbon substrate. The excess dispersion was removed with a filter paper. Negative staining using a 2% phosphotungstic acid solution (w/w) was directly made on the deposit for 45 s. Then the air dried samples were directly examined under the TEM (Freag et al., 2013a,b). Rapid investigation of the effective formation of the vesicles and the absence of un-entrapped drug crystals was performed for undiluted samples by means of a projection microscope fitted camera and photographed at suitable magnification. Moreover, a polarized light microscope (Model CX 31, Olympus, Germany) was used in order to reveal more details on the structure of the prepared systems without any dilutions. 2.5. Determination of entrapment efficiency Percentage entrapment efficiency (% EE) of the prepared Curc– GC-HS and Curc-T (n = 3) was determined by separation of vesicles from the solution after properly diluted, using centrifugation (15.000 rpm for 15 min at 4 C). Supernatant was collected and used for determination of un-entrapped Curc by measuring UV absorbance at 420 nm (Patel et al., 2009). The entrapped drug was calculated by the following equation:
2.7.1. Animals All studies were approved by the Ethics Committee at the Faculty of Pharmacy and Drug Manufacturing, Pharos University in Alexandria. The animals were kept under standard laboratory conditions and veterinary supervision with no restrictions on water and food. 12 female Sprague Dawley rats weighing between 210 and 230 g were randomly separated into two groups each composed of six rats. Rats were kept in individual cages with food and water and were maintained in a 12 h light/12 h dark cycle in a temperature controlled room at 20–25 C. They were given water and standard meals. Just before inflicting the burn, rats were anesthetized using intraperitoneal (IP) thiopental (80–100 mg/kg). The back hair of each rat was shaved with a shaving machine and then the area was depilated with a commercial depilatory cream to obtain a smooth and hairless skin (Jurjus et al., 2007). 2.7.2. Burn-wound model For the formation of the burn-wound, the shaved back skin of anesthetized animals was exposed to hot water. For this procedure, a cylindrical shaped stainless steel cup with radius of 10 mm was placed in hot water (100 C) for 1 min and then on the backs of rats and held for 20 s (Priya et al., 2002). Each rat was subjected to four burns. After the formation of standard, second degree burns; they were covered with cotton and gauze pre-wetted with saline for two days to form a remarkable wound. Fig. 1 shows the steps involved in the formation of skin wounds.
Fig. 1. Steps involved in the formation of skin burn-wound (1: shaved back skin, 2: after the burn, 3: after covering the burn for 2 days with saline wetted cotton).
W.M. El-Refaie et al. / International Journal of Pharmaceutics 486 (2015) 88–98 Table 1 Scale for percentage efficacy index. % Efficacy index values (% EI)
Significance
EI 30% 100% > EI 70% =100%
No improvement Moderate improvement Marked improvement Heal
The rats were divided randomly into two groups each composed of 6 rats. Formulations were examined using six wounds from different rats (n = 6). Formulations were repeatedly applied once daily to the burned areas for 10 days. For the control wounds, they were just cleaned with cotton wetted with normal saline each day. The diameter of the wounds formed were monitored and measured periodically. 2.7.3. Evaluation of the wound healing 2.7.3.1. Measurement of wound size. Progression decrease in the wound size was monitored periodically by measuring the distance between 2 opposite outside edges of the wound margin, using a vernier caliper (Priya et al., 2002). Two measurements approximately 90 from each other were obtained; the largest distance was used as one of the measurements. During the measurements, rats were fixed on a table. Wounds were digitally photographed on 0, 3, 5, 7, and 10 days post wounding with maintaining constant optical zoom. 2.7.3.2. Healing efficacy indices. The wound contraction rate was measured as percentage reduction in the wound size (% efficacy indices (% EI)) at days 2, 4, 7, and 10 from treatment beginning. The % EI of the wound size was calculated by the following equation: %EI ¼
V sp:day V I 100 VI
Vsp.day refers to the diameter (mm) values measured at day 2, 4, 7, and 10, while VI refers to the baseline value measured before treatment. The efficacy indices were then evaluated on a four rank scale specified in Table 1. 2.7.3.3. Morphological examination of scars. Wounds sites were monitored visually for scar formation, redness, and hyper pigmentation all over the study period (10 days) and after end of treatment till day 14 post wounding.
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2.7.3.4. Histological analysis. Full thickness skin biopsies of wounds with surrounding tissues were collected at the 7th and 11th day from wound treatment. Histological assessment was performed only for formulations with highest improvement in wound healing to further compare between them. Specimens were fixed in 10% formaldehyde, embedded in paraffin, cut into 5 mm pieces and stained with hematoxylin and eosin (H&E). Sections were visualized under a light microscope at 10 magnification. Fields were examined regarding the degree of epithelization, presence or absence of inflammatory cells, and the nature of collagen and fibrous tissue (Gong et al., 2013). 2.8. In-vivo skin deposition study The experiment was performed on 5 female Sprague Dawley rats (210–230 g). Rats were divided into two groups; each was composed of two rats. The fifth rat was kept as a control. Hair of the dorsal rats’ skin was removed using a depilatory cream. Using a marker pen, the dorsal skin of each rat was equally divided into six sections. They were exposed to single topical application of weight equivalent to 0.5 mg Curc from Curc–GC-HS, Curc–T-Pl gel, Curc– HA gel, and Curc–Pl gel. Each of the examined formulations was applied to three different sections (n = 3) in each group. In both groups, formulations were applied topically on the rats’ skin and allowed to dry. To avoid leakage from the skin and to get the comparable dose application in all rats, all exposed rats were sedated with IP thiopental (80–100 mg/kg) during the application and kept in a special cage to restrict rats’ movement. Group I rats were sacrificed after 6 h exposure. On the other hand, group II rats were sacrificed after 24 h from dose application. After scarification, the skin was cleaned from formulation residues. For extraction of Curc from collected skin samples 10 ml acetone containing 0.5% sodium lauryl sulfate (SLS) was added to the skin in an ambered glass bottles and kept for shaking over night at room temperature. Obtained dispersions were centrifuged for 10 min. at 10,000 rpm. Control skin samples (collected from the untreated rat) were subjected to the same procedure. Supernatants were withdrawn, and the drug content was determined by measuring UV absorbance at 420 nm. Results were presented as mean of the three readings SD. 2.9. Statistical analysis Statistically significant differences were determined using twotailed and student’s t-test. P < 0.05 was described as the level of significance.
Fig. 2. Morphological examination of vesicles loaded with 0.15% Curc (a) optical micrograph (400) and (b) TEM micrograph of Curc–GC-HS.
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Fig. 3. Morphological examination of vesicles loaded with 0.15% Curc (a) optical micrograph (400) and (b) TEM micrograph of Curc–T-Pl.
3. Results and discussion 3.1. Preparation and characterization of Curc-nanovesicles 3.1.1. Curc–GC-HS In an attempt to improve wound healing characteristics and reduce scar formation, we utilized a novel Curc loaded self assembled HA-based gel-core system. The prepared system yielded vesicles with mean diameter of (202.7 0.66 nm), PDI value of 0.259 0.008, and negative zeta potential of (33.0 2.6 mV) indicating homogenous size distribution with sufficient charge for stabilization. The hydrophobic nature of Curc allows it to become incorporated into the bilayer region of the vesicles with high percentage entrapment efficiency (96.44 1.29%). Morphological examination was performed using optical (Fig. 2a) and transmission electron microscopy (Fig. 2b). Obtained micrographs revealed the presence of well identified vesicles that exist in disperse not aggregated with characteristic clear bicompartmental (bilayer) structure. This might be attributed to the presence of Curc in the outer layer and HA gel in the core of the vesicles of Curc–GC-HS. 3.1.2. Curc–T-Pl gel To investigate the effect of combining HA with Curc in such novel system on wound healing, conventional Curc loaded transfersomes
(Curc-T) were prepared for comparison. Curc-T showed mean vesicles diameter of (320.6 20.7 nm), PDI value of 0.430 0.025, % EE (87.58 3.42) and zeta potential (37.2 3.4 mV). Curc-T optical micrographs (Fig. 3a) revealed the successful formation of vesicles. TEM micrographs (Fig. 3b) also showed well formed vesicles with spherical structure and homogenous size distribution but did not show the clear bilayer structure as in Curc–GC-HS (Fig. 2b). To be suitable for topical application, they were incorporated in 25% Pl gel (Poloxamer1 407). Poloxamers1, particularly Poloxamer1 407, have lately received notable interest as thermo-sensitive hydrogels (Garrastazu Pereira et al., 2013). They are biodegradable, non-toxic, and stable substances that form thermo-sensitive hydrogels in high concentrations. Pl gel was used in a previous study with Curc for wound healing (Kant et al., 2014) and found to have some beneficial effects on healing compared to control. Therefore, Pl (25% w/w) was selected as a gelling agent for Curc-T in the present study. 3.1.3. Structure elucidation Polarizing microscopy was utilized to reveal more details on the structure of the prepared vesicular gel systems (Fig. 4). Unlike Curc–T-Pl gel (Fig. 4b), photomicrographs obtained from Curc–GCHS (Fig. 4a) indicated the formation of a peculiar system that is not a simple gel and not a genuine liquid crystal, but a gel system that incorporates liquid crystals inside. These are commonly lamellar
Fig. 4. Polarizing micrographs of (a) Curc–GC-HS and (b) Curc–T-Pl (400).
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from HA gel compared to other gelling agents (Brown et al., 2001). In addition, Pl is a thermo-sensitive gel that formed a more dense gel structure at the temperature used in the experiment (37 C) which might further reduce Curc release. Comparing vesicular gels with plain gel formulations, it was observed that Curc release from Curc–T-Pl gel was much more retarded than from Curc–Pl gel. This might be due to the vesicles effect that might further slow the release of entrapped drug. On the other hand, in case of HA based systems, unexpected higher release of Curc from GC-HS was observed compared to Curc–HA plain gel. This might be attributed to the presence of Curc as insoluble dispersed large particles in HA gel matrix compared to Curc–GC-HS where Curc occurred in a very finely dispersed molecular state which might enhance its release. 3.3. In-vivo study Fig. 5. In-vitro release behavior of different Curc loaded formulations using dialysis bag method and 1:1 water:ethanol maintained at 37 C as acceptor medium.
structures that are formed around the inner core of the vesicles (Manosroi et al., 2003; Oka et al., 2008). This characteristic structure can make the system formed more stable by reducing the possibility of vesicles aggregation and fusion. 3.2. In-vitro release behavior The aim of this study was to test the ability of the novel designed GC-HS to release their drug load. Fig. 5 shows the drug release behavior of the different Curc loaded formulations namely, Curc suspension in 0.1% sodium lauryl sulphate, Curc in HA gel (Curc-HA), Curc in pluronic gel (Curc-Pl), Curc loaded selfassembled gel-core hyaluosomes (Curc–GC-HS), and Curc loaded transfersomes in pluronic gel (Curc-T-Pl), using dialysis bag method. As shown from the figure, generally all the examined formulations were able to gradually release their drug load by time. They all showed significant (P < 0.05) retardation in the initial release rate compared to Curc suspension. The use of Pl as a gelling agent, either in vesicular systems or plain gels, strongly retarded Curc release when compared to formulations prepared with HA. At 2 h interval, the cumulative release of Curc from Curc– Pl gel and Curc–T-Pl gel was 32.42 1.61 and 24.06 0.8 respectively while from Curc–GC-HS and Curc–HA was 50.32 1.38 and 41.85 1.57 respectively. This might be attributed to the ability of HA to absorb higher amounts of water increasing drug partitioning and release to the acceptor medium. This was in agreement with a previous study that observed higher drug release
As mentioned previously, impairment of wound healing process can be induced by prolonged inflammation and increased oxidative stress. Hence, application of Curc, a well known antioxidant and anti-inflammatory agent, could be important in improving such condition. Moreover, prolonged healing time would result in scar formation. HA was reported to help in reducing scars and improve wound healing. However, it was not sufficiently studied for improving its skin delivery for such application. Therefore, the present study was conducted to evaluate the cutaneous wound healing potential of the topically applied novel Curc–GC-HS that combines the effect of both drugs in addition to the stabilizing effect of the formulation. Rats have been widely utilized in the study of wound healing of different therapies. This was mainly due to their availability, small size, and low cost. In addition, rat skin is similar to that of human in having epidermis, hair follicles, and dermis. Moreover, reepithelization which is the predominant healing mechanism in human skin-is considered the primary response in rats after burns or partial thickness wounds. (Dorsett-Martin and Wysocki, 2008) Therefore, rat burn-wound model was utilized in this study. 3.3.1. Evaluation of wound healing Treatment efficacy of Curc–GC-HS on rats with dermal burnwound was investigated in comparison to other formulations namely Curc–T-Pl gel, Curc–HA gel, Curc–Pl gel, GC-HS, and HA gel. All treated groups showed significant difference in the wound contraction compared to the control group at day 2 of the study period (P < 0.05), Fig. 6. On the other hand, proceeding in the treatment till the end of the 10 days, only Curc–GC-HS followed by Curc–T-Pl and then GC-HS free from Curc showed statistically
Fig. 6. Efficacy indices for the evaluation of wound size reduction of the different groups of rats receiving different treatments.
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Fig. 7. Stages of wound healing of the different therapies.
significant difference compared to the control, Figs. 6 and 7. Comparing these results with those obtained from conventional gels (HA, Curc–Pl, and Curc–HA gels); this might indicate the positive effect of the use of nanostructured vesicular systems in drug delivery either for Curc or HA or both combined. Among the promising formulations, Curc–GC-HS is the only system that demonstrated significant marked improvement at day 7 of treatment and complete heal (% EI = 99.7 0.34%) with minor or no scar at day 10, Fig. 7,Table 2. This might be due to the combined anti inflammatory and anti-oxidant effect of Curc together with the effect of HA with its scar reducing potential in addition of the probability of such novel formulation to increase the dermal localization of their drug load; improving efficacy. GCHS is a novel carrier that combined the effect of elastic vesicles (due to the presence of Tween 80), stable gel core liposomes, and hygroscopic HA. It has been reported that due to its high hygroscopic properties, HA may hydrate the skin creating hydrophilic pathways (Brown and Jones, 2005). The elasticity of the prepared vesicles together with loosening of skin structure by HA could allow the vesicles to penetrate the skin without losing their integrity. Moreover, being core gelled, provided the system with more stability to withstand surrounding environment preventing vesicles damage and early drug leakage (Tiwari et al., 2009). Consequently, Curc might be protected inside the vesicles
that were localized in the skin; accelerating the wound healing process Fig. 8. Evaluating the effect of HA and Curc in conventional gel formulations namely, Curc–Pl gel and HA gel, there was no Table 2 Efficacy indices (% EI) values and their significance for the used formulations at days 7 and 10 of treatment. Formulation
% EI values (significance) Day 7
Day 10
Control
30.15 8.3 (No improvement) 82.39 10.2 (Marked improvement) 40.84 15.9 (Moderate improvement) 28.02 12.39 (No improvement) 28.94 11.4 (No improvement) 63.8 15.49 (Moderate improvement) 30.5 12.5 (No improvement)
52.2 8.62 (Moderate improvement) 99.7 0.34 (Heal) 71.5 11.42 (Marked improvement) 57.28 10.32 (Moderate improvement) 56.5 5.86 (Moderate improvement) 82.19 3.45 (Marked improvement) 53.5 9.43 (Moderate improvement)
Curc–GC-HS GC-HS Curc–HA gel HA gel T-Pl gel Curc–Pl gel
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Fig. 8. A diagram summarizing the mechanism of the novel Curc–GC-HS in enhancing wound healing rate via improving dermal localization of the entrapped Curc in the loosened skin structure.
Fig. 9. Histopathological examination of (a) normal skin (b) treated with Curc–GC-HS at day 7 post wounding and (c) treated with Curc–GC-HS at day 11 post wounding (d) treated with Curc–T-Pl at day 7 post wounding (e) treated with Curc–T-Pl at day 11 post wounding.
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improvement in healing process was observed from both after 7 days and moderate improvement was observed after 10 days (Table 2). Moreover, combination of both drugs in Curc–HA gel did not give any significant improvement compared to each alone (10 days % EI obtained from Curc–Pl gel, HA gel and Curc–HA gel were 53.5 9.43, 56.5 5.86, and 57.28 10.32% respectively, Table 2). This might be due to inability of the drugs to penetrate skin efficiently or to be localized in the skin for prolonged effect. Therefore, they need longer time to give their therapeutic effect which was in agreement with previous studies that observed healing effect for Curc after 14 or 19 days (Chereddy et al., 2013; Gong et al., 2013; Kant et al., 2014) and for HA after 29 days (Medeiros et al., 1999). Hence, the novel self-assembled gel-core hyaluosomes might be a promising carrier for HA and Curc; accelerating the wound healing process with possibility of reducing scar formation. 3.3.2. Histopathological examination For additional confirmation of the quality and maturity of the healed tissues or those with marked improvement namely, Curc loaded self assembled gel-core hyaluosomes (Curc–GC-HS) and Curc loaded transfersomes in pluronic gel (Curc-T-Pl), histological characterization was carried out. Full thickness sections of normal skin and treated wounds, with highest healing potential, were
collected and stained with hematoxylin and eosin (H&E) for microscopical examination of skin layers. The normal skin (Fig. 9a) showed an epidermis layer and areolar woven network of collagen fibers arranged in the dermal layer. On the other hand, Fig. 9b and d reveals histological findings of wounds treated with Curc–GC-HS and Curc–T-Pl gel respectively at 7th day post wounding. Curc–GCHS treated wounds (Fig. 9b) exhibited densely packed collagen fibers with parallel arrangement and high accumulation under well formed thick epithelium. Moreover, numerous collagen bundles were observed in the reticular layer indicating fibroblast tissue formation. In contrast, loose irregular collagen fibers with large dissociating areas in between were observed in Curc–T-Pl gel treated wounds (Fig. 9d). At 11th day post wounding, wounds treated with Curc–GC-HS showed almost normal skin structure with well formed and differentiated epithelium and woven collagen fibers (Fig. 9c). Curc–T-Pl gel treated wounds demonstrated significant increase in the deposition of collagen fibers and hence increase in activity and fibroblast formation (Fig. 9e), indicating that the healing process is incomplete and still active. Worthy mentioning that in all examined treated skin sections no inflammatory cells were detected. These observations indicated the high anti-inflammatory action of Curc and its potential in inducing collagen formation as both examined Curc-loaded vesicular formulations improved wound
Fig. 10. Scar monitoring at day 14 post wounding.
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healing. However, Curc–GC-HS was found to be superior in promoting the formation of the collagen and making re-epithelization in less time compared to Curc–T-Pl gel. This might further prove the combined effect of HA and Curc in addition to the effect of this novel formulation to retain them inside the skin; speeding up the wound healing process with high probability for reducing scar formation. 3.3.3. Scar monitoring As mentioned previously, several steps are involved in wound healing processes as clotting, inflammation, collagen deposition, granulation, spasm of wound, and re-epithelization. Wounds with prolonged healing can be considered most risky for impairment of such process and undue scar formation. Although evaluation of scar development seems necessary, this is still a neglected area, and yet there is no agreement on the best method for scar assessment (Powers et al., 1999). In the current study, visual inspection of scars was performed till 14 days post wounding as a simple method for monitoring of the presence or absence of scars. Scars might be appeared as hypertrophic deformity or impaired pigmentation of the healed wound after epithelialization (Idriss and Maibach, 2009). As shown in Fig. 10 untreated groups were not completely healed and still showing a crust. On the other hand, all treated groups showed almost complete healing and epithelization but with some developed scars. Curc–GC-HS was superior over all formulation as it did not show any hyperpigmentation followed by Curc–T-Pl gel while all others left a scar with different degrees of hyperpigmentation. The greater performance for Curc–GC-HS in wound healing and reducing scar formation might additionally confirm the observed higher rate of collagen development and reepithelization compared to Curc–T-Pl gel and to other formulations. All the previous findings could provide an evidence of the ability of this novel formulation to act as an effective carrier for Curc. It could be able to retain Curc inside the skin and protect it against metabolism; resulting in higher efficacy. This will be examined in the following experiment. 3.3.4. Skin deposition study The aim of this experiment was to examine the ability of the novel GC-HS compared to the other prepared gel formulations to enhance skin penetration of Curc and to protect it inside the skin against metabolism; thus improving its bioavailability and efficacy in wound healing or even in other chronic skin diseases. It should be noted that Curc was selected in this study as it is one of the most poorly skin penetrating compounds in addition to its excessive metabolism, rapid elimination and extreme instability previously observed in different studies (Anand et al., 2007; Asai and Miyazawa, 2000; Ravindranath and Chandrasekhara, 1980; Yang et al., 2007). Moreover, all studies examined Curc skin delivery dealt mainly with its in vitro dialysis or clinical investigations of its effect but never reported its in vivo deposition and localization in the skin. Results obtained from this study indicated that Curc–GCHS was the only formulation that gave detectable amount of Curc deposited in the skin representing 0.83 0.34% of the applied dose after 6 h exposure. After 24 h exposure, Curc–GC-HS showed significantly higher skin deposition of Curc (4.62 1.28%) compared to Curc–T-Pl gel (0.92 0.44%) and no Curc was detected in the skin from plain gel formulations. These results indicated the high potential of the novel GC-HS in protecting loaded Curc against metabolism and increasing its stability and localization inside the skin. This confirmed the ability of the GC-HS for enhancement of Curc bioavailability over other formulations. This may further prove its superior effect in wound healing and suggest its use as a carrier for Curc or any other drug in treatment of other skin diseases.
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4. Conclusion It was concluded that topical application of the novel Curc–GCHS formulation in rat burn-wounds contributed to speed healing, improved histological progress and reduced scar formation. This was attributed to the combined anti inflammatory and antioxidant effect of Curc together with the effect of HA with its scar reducing potential. Furthermore, the potential of the novel GC-HS as an effective carrier for Curc was also proved in the in vivo skin deposition study. It was able to increase skin penetration and dermal localization of Curc; protecting it against degradation and metabolism thus enhancing its bioavailability and therapeutic outcome. This may further prove its superior effect in wound healing and suggest its use as a carrier for Curc or any other drug in treatment of chronic skin diseases. Conflict of interest The authors report no financial or personal conflict of interest. Acknowledgement We would like to express our deep thanks to professor/ Ebtehag El-Ghazawi (Professor of histology and cell biology- Faculty of medicine- Alexandria University) for her help in interpretation of histopathology results. References Adzick, N.S., Longaker, M.T., 1992. Scarless fetal healing therapeutic implications. Ann. Surg. 215, 3–7. Aggarwal, B.B., Sung, B., 2009. Pharmacological basis for the role of curcumin in chronic diseases: an age-old spice with modern targets. Trends Pharmacol. Sci. 30, 85–94. Alster, T.S., Tanzi, E.L., 2003. Hypertrophic scars and keloids. Am. J. Clin. Dermatol. 4, 235–243. Anand, P., Kunnumakkara, A.B., Newman, R.A., Aggarwal, B.B., 2007. Bioavailability of curcumin: problems and promises. Mol. Pharm. 4, 807–818. Asai, A., Miyazawa, T., 2000. Occurrence of orally administered curcuminoid as glucuronide and glucuronide/sulfate conjugates in rat plasma. Life Sci. 67, 2785–2793. Atiyeh, B.S., Hayek, S.N., 2004. An update on management of acute and chronic open wounds: the importance of moist environment in optimal wound healing. Med. Chem. Rev.-Online 1, 111–121. Bisht, S., Feldmann, G., Soni, S., Ravi, R., Karikar, C., Maitra, A., Maitra, A., 2007. Polymeric nanoparticle-encapsulated curcumin (nanocurcumin): a novel strategy for human cancer therapy. J. Nanobiotechnol. 5, 1–18. Bock, O., Schmid-Ott, G., Malewski, P., Mrowietz, U., 2006. Quality of life of patients with keloid and hypertrophic scarring. Arch. Dermatol. Res. 297, 433–438. Bragagni, M., Mennini, N., Maestrelli, F., Cirri, M., Mura, P., 2012. Comparative study of liposomes, transfersomes and ethosomes as carriers for improving topical delivery of celecoxib. Drug. Deliv. 19, 354–361. Brown, B., McKenna, S., Siddhi, K., McGrouther, D., Bayat, A., 2008. The hidden cost of skin scars: quality of life after skin scarring. J. Plast. Reconstr. Aesthet. Surg. 61, 1049–1058. Brown, M., Hanpanitcharoen, M., Martin, G., 2001. An in vitro investigation into the effect of glycosaminoglycans on the skin partitioning and deposition of NSAIDs. Int. J. Pharm. 225, 113–121. Brown, M., Jones, S.A., 2005. Hyaluronic acid: a unique topical vehicle for the localized delivery of drugs to the skin. J. Eur. Acad. Dermatol. Venereol. 19, 308– 318. Chereddy, K.K., Coco, R., Memvanga, P.B., Ucakar, B., des Rieux, A., Vandermeulen, G., Préat, V., 2013. Combined effect of PLGA and curcumin on wound healing activity. J. Control. Release 171, 208–215. Dai, M., Zheng, X., Xu, X., Kong, X., Li, X., Guo, G., Luo, F., Zhao, X., Wei, Y.Q., Qian, Z., 2009. Chitosan-alginate sponge: preparation and application in curcumin delivery for dermal wound healing in rat. Biomed. Res. Int. 2009, 595126. Dorsett-Martin, W.A., Wysocki, A.B., 2008. Rat Models of Skin Wound Healing Sourcebook of Models for Biomedical Research. Springer, pp. 631–638. Durgaprasad, S., Reetesh, R., Hareesh, K., Rajput, R., 2011. Effect of a topical curcumin preparation (BIOCURCUMAX) on burn wound healing in rats. J. Pharm. Biomed. Sci. 8, 1–3. El-Refaie, W.M., Elnaggar, Y.S., El-Massik, M.A., Abdallah, O.Y., 2015. Novel selfassembled, gel-core hyaluosomes for non-invasive management of osteoarthritis: in-vitro optimization ex-vivo and in-vivo permeation. Pharm. Res..
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Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Novel curcumin-loaded gel-core hyaluosomes with promising burn-wound healing potential: Development, in-vitro appraisal and in-vivo studies Wessam M. El-Refaie a , Yosra S.R. Elnaggar b, * , Magda A. El-Massik a , Ossama Y. Abdallah b a b
Department of Pharmaceutics, Faculty of Pharmacy and Drug Manufacturing, Pharos University in Alexandria, Egypt Department of Pharmaceutics, Faculty of Pharmacy, Alexandria University, Egypt
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 February 2015 Received in revised form 22 March 2015 Accepted 25 March 2015 Available online 27 March 2015
Despite its effectiveness, curcumin (Curc) dermal delivery is handicapped by hydrophobicity, high metabolism and poor skin permeation. In this work, the potential of novel self-assembled nanogels, namely gel-core hyaluosome (GC-HS) to enhance Curc delivery to wound sites, enhance healing rate and decrease scar formation was evaluated. Curc–GC-HS were prepared using film hydration technique and evaluated regarding size, zeta potential (ZP), entrapment efficiency (% EE), and in vitro release. Structure elucidation was performed using light, polarizing and transmission electron microscopy (TEM). In-vivo burn-wound healing potential, skin deposition ability and histological study were evaluated using female Sprague Dawley rats. Curc–GC-HS were compared to conventional transfersomal gel (Curc–T-Pl gel), and other conventional gels. Curc–GC-HS showed nanosize (202.7 0.66 nm), negative ZP (33 2.6 mV) and % EE (96.44 1.29%). TEM revealed discrete vesicles with characteristic bilayer structure. Polarizing microscopy proposed liquid crystalline consistency. Burn-wound healing study showed that Curc–GC-HS was the only system exhibiting marked improvement at day 7 of treatment. At 11th day, Curc–GC-HS treated wounds showed almost normal skin with no scar confirmed by histological analysis. Curc–GC-HS showed five folds higher skin deposition compared to conventional Curc–T-Pl gel. To conclude, novel gelcore hyaluosomes elaborated are promising nanogels able to increase Curc skin penetration and dermal localization while protecting it against degradation. Future perspective encompasses assessing potential of novel nanocarrier for skin cancer therapy. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Curcumin Wound Burn Scar Dermal Vesicles Gel-core liposomes Hyaluronic acid
1. Introduction Skin is repeatedly subjected to wounds either by burns, injuries, or physical trauma. These wounds initiate a set of complicated and synchronized responses for healing and restoration of the skin structure. The healing process is spontaneous and involve different overlapped phases, namely, inflammation (inducing haemostasis and clot formation), fibroplasia and neovascularization, generation of granulation tissue, re-epithelialization, and finally the formation of new extracellular matrix and tissue remodeling (Suguna et al., 2002). In minor wound damages, full restoration of the integrity of tissues structure and barrier function occurs (Yates et al., 2007).
* Corresponding author at: Department of Pharmaceutics, Faculty of Pharmacy, Alexandria University, 1 Khartoum Square, Azarita, Messalla Post Office, P.O. Box 21521, Alexandria, Egypt. Tel.: +20 1147591065; fax: +20 34873273. E-mail address: [email protected] (Y.S.R. Elnaggar). http://dx.doi.org/10.1016/j.ijpharm.2015.03.052 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
However, in severe cases such as large skin injuries, second and third degree burns, several factors are involved and prolong the healing time (Hardwicke et al., 2008). The longer the time for spontaneous healing process, the worse is the obtained result. Edema, inflammation, hypertrophic scars and unsightly pigmentation changes are likely to be associated with such complicated cases. Under these prolonged unfavorable conditions for wound healing, the continuing inflammatory cascade may result in increasing tissue destruction and necrosis rather than healing (Atiyeh and Hayek, 2004). Therefore, inflammation is a common complication in wound healing process. Topical applications of compounds with antioxidative properties using proper materials and antioxidants will be useful against oxidative damage and be helpful to the healing of the wound. Among others, curcumin (Curc) has been reported as a promising wound healing agent when used topically (Moghaddam et al., 2009; Sidhu et al., 1998). It has antioxidant, anti-inflammatory, anti-tumor (apoptosis
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inducing), and anti-angiogenesis effects. Moreover, Curc has received great attention due to its ability to perform multiple actions and to target many cellular pathways (Aggarwal and Sung, 2009). Studies to date have demonstrated better accessibility of Curc to wound sites after topical application compared to its oral administration (Sidhu et al., 1998, 1999). This was attributed to its limited water-solubility and extensive first-pass metabolism (Asai and Miyazawa, 2000; Ravindranath and Chandrasekhara, 1980). With this in mind, different formulations of Curc have been developed to achieve better topical application at the wound site. These include: chitosan–alginate sponges (Dai et al., 2009), polymeric bandages (Mohanty et al., 2012), alginate foams (Hegge et al., 2011), collagen films (Gopinath et al., 2004), and creams (Durgaprasad et al., 2011). Compared to unformulated Curc, it was found that the bioactivity of Curc increased when incorporated into these formulations. However, no significant difference in the wound healing effect was observed between these different formulations. This might be due to the Curc extensive metabolism and rapid elimination regardless the dosage form or the route of administration (Anand et al., 2007; Asai and Miyazawa, 2000; Ravindranath and Chandrasekhara, 1980). Therefore, the infiltration of Curc into cells at the wound site can still potentially be improved especially that studies examining Curc skin delivery never reported its in vivo skin deposition and localization. Several approaches have been investigated for improvement of Curc efficacy (Bisht et al., 2007; Li et al., 2007; Ryu et al., 2006). Most promising are the nanostructured systems that have been recently formulated and investigated for Curc enhanced therapeutic outcomes (Wang et al., 2011). Their higher total surface area and smaller size facilitate the cellular uptake of Curc into cancer cells and reduce their proliferation (Lee et al., 2014, 2015). However, the effect of such nanocarriers in enhancing Curc bioavailability for wound healing applications has not gained considerable attention. Moreover, so far no study has investigated the effect of phospholipid-based vesicular systems on enhancing Curc delivery to wound sites, despite their captivating properties in enhancing drugs skin permeation and dermal localization (Elnaggar et al., 2014). Scar is another common and serious post-wound deformity. Burn scars, because of their clearly visible and stigmatizing appearance, have probably the highest impact on the quality of life compared to other skin diseases (Stella et al., 2008; Van Loey and Van Son, 2003). They have serious physical, functional, and psychological problems. Scars may cause pain, itching; and contractures that may hamper mobility (Van Loey and Van Son, 2003). Deep and severe scars may impair individual’s life as they are often treated with prolonged, painful procedures that in most cases give suboptimal results (Alster and Tanzi, 2003; Bock et al., 2006; Brown et al., 2008; Shaffer et al., 2002). It was reported that the fetus has a faster wound healing progress without leaving scars compared to that occurs in adults (Harrison and Adzick, 1991). Moreover, a more organized collagen was formed unlike that found in adults (Scott Adzick et al., 1985). The peculiar characteristics of the fetal healing were attributed to the HA rich extracellular matrix of the healing tissue (Adzick and Longaker, 1992; Longaker et al., 1991). It was found that the amount of HA was increased rapidly in the extracellular matrix of the healing tissue in both adults and fetus. However, in adults this HA amount significantly reduced to near zero within a week. In the fetus, HA level remains significantly elevated for almost three weeks (Longaker et al., 1991). This may explain the difference in the outcome of wound healing between fetus and adults. HA was examined for its daily topical use in the healing of burn. Topical treatment with 1% HA resulted in shorter healing time of burns under study (29 1.33 days) compared to control (38 2.58 days) (Medeiros et al., 1999). Although, it is
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better than control, the healing time of HA treated group was still considered prolonged. This long time might also result in impaired healing. Recently, the potential of HA as a suitable delivery vehicle to facilitate sustained local release and reduced doses of VN:GF (vitronectin:growth factor) was investigated (Xie et al., 2011). VN: GF was reported to significantly increase re-epithelialization in a porcine deep dermal partial-thickness burn model (Upton et al., 2008). Despite the advantages of HA in wound healing, results obtained from this study (Xie et al., 2011) indicated that, there was no significant difference between the thicknesses of the epidermis treated with VN:GF complexes alone and VN:GF complexes together with HA. Therefore, promising use of HA in burn healing process still needs a step of skin penetration enhancement (El-Refaie et al., 2015). Based on aforementioned background, combining HA and Curc in a tailored nanovesicular system is anticipated to combine their individual privileges for burn-wound healing. In this quest, this work is the first one to present novel HA-based gel-core hyalusomes loaded with Curc (Curc–GC-HS) prepared with simple technology. This system was designed in order to combine positive effect of flexible liposomes in enhancing skin penetration with the reported stability of gel-core liposomes against degradation. In addition, GC-HS utilizes HA as the gelling material with its unique viscoelastic and wound healing properties. The potential of the novel nanocarrier to improve Curc delivery to wound sites, its stability and dermal localization was evaluated. Furthermore, the potential of novel formulation to further enhance the healing rate and reduce scar formation was assessed as well. In vitro characteristics and burn-wound healing potential of the prepared hyaluosomes was compared to conventional transferosomes, conventional Curc pluronic gel, Curc–HA gel and plain HA gel. 2. Materials and methods 2.1. Materials Curcumin was obtained from (Hebei food additive Co., Ltd., China). Hyaluronic acid (HA, MW = 0.8 1.17 10 MDa) was obtained from Shiseido (Japan). Lipoid S100 (phosphatidylcholine from soybean) was kind gift from Lipoid GmbH (Ludwigshafen, Germany). Poloxamer 407 (Kolliphor 407, a sample gift from BASF, Germany). Tween 80 and all other reagents were of analytical grade. 2.2. Preparation of curcumin loaded vesicular gels 2.2.1. Curcumin loaded gel-core hyaluosomes (Curc–GC-HS) Curc–GC-HS were prepared by thin film evaporation technique. Lipoid S100 (PL) together with Tween 80 was dissolved in chloroform. Curc was dissolved in acetone and mixed with the dissolved PL. The solvent was then removed under reduced pressure in a rotary evaporator at 58 C, to obtain a thin film on the flask wall. Evaporation was continued for 2 h after the dry residue appeared, to completely remove all the solvent. Obtained dry lipid films were then hydrated with 1% w/w HA aqueous dispersion. Final Curc concentration in the formulation was 0.15% w/w. The hydrated film was subjected to water bath heating at 58 C for 10 min and vortexing for 2 min; this cycle was repeated four times. The prepared vesicles were then extruded through polycarbonate membranes with pore size of 200 nm at room temperature with the aid of a Liposome Extruder (Model ER-1, Eastern Scientific LLC, USA). The dispersion was hermetically sealed and stored at 4 C (Bragagni et al., 2012). For comparative reasons, Curc free GC-HS were also prepared with the same method except the addition of Curc. Plain and Curc loaded (0.15%) 1% HA aqueous gels were also prepared and used for comparison.
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2.2.2. Curcumin loaded transfersomal pluronic gel (Curc–T-Pl gel) Curc loaded transfersomes (Curc-T) were prepared by the above mentioned procedure using distilled water as a hydrating medium to be used for in vivo comparison. The obtained transfersomal dispersion, containing 0.15% Curc, was then gelled with poloxamer1 407 (Pluronic F-127). Pluronic F-127 (Pl, 25%) was soaked in the obtained Curc-T dispersion and left overnight in the refrigerator then stirred slowly in a closed vessel until homogeneous Curc–T-Pl gel was obtained. Curc–Pl gel was also prepared for comparison by dispersing the drug powder in 25% Pl gel after soaking in water and kept overnight in the refrigerator, and stirred as above till a clear gel containing 0.15% Curc was obtained. 2.3. Particle size, zeta potential and PDI The particle size (PS), polydispersity index (PDI) and zeta potential (ZP) for the prepared Curc–GC-HS were determined by dynamic light scattering (DLS) technique using Zetasizer Nano ZS. (Malvern, Instruments Ltd., Malvern, UK). For measurements, Curc–GC-HS formulations were properly diluted (0.1% v/v) with filtered distilled water to avoid multiscattering phenomena. Measurements were conducted at 25 C. All samples were measured in triplicates and results were represented as mean value SD.
%EE ¼
total drug free drug 100 total drug
2.6. In-vitro release behavior The dialysis method was utilized in order to investigate the release behavior of Curc from Curc–GC-HS, Curc–T-Pl gel, Curc–HA gel, and Curc–Pl gel, respectively (n = 3). Dialysis was carried out through dialysis bag 7 cm long with a molecular mass cut off 12– 14 kDa (VISKING dialysis tubing, SERVA, electrophoresis, Germany) (Elsheikh et al., 2012, 2014). The bags were filled with an amount of the formulation equivalent to 0.3 mg Curc and put in ambered glass bottles containing 10 ml of water: ethanol (1:1). Bottles were kept at 37 C 1 C with 100 rpm shaking throughout the experiment using a shaking water bath. At different time intervals (0.25, 0.5, 1, 2, 3, 4, 6 h), the whole volume of the release medium was withdrawn and replaced with an equal volume of a previously warmed fresh one. The percentage of Curc released was calculated from the cumulative drug amount released at these different time intervals. Drug concentration was determined by measuring UV absorbance at 430 nm. All measurements were performed in triplicates. Data were represented as mean SD. 2.7. In-vivo wound healing
2.4. Morphological characterization and structural elucidation Morphology of the prepared nanovesicles was determined by transmission electron microscopy (TEM). Samples were firstly diluted with distilled water and dropped onto a carbon-coated copper grid and left for 1 min to allow vesicles to adhere on the carbon substrate. The excess dispersion was removed with a filter paper. Negative staining using a 2% phosphotungstic acid solution (w/w) was directly made on the deposit for 45 s. Then the air dried samples were directly examined under the TEM (Freag et al., 2013a,b). Rapid investigation of the effective formation of the vesicles and the absence of un-entrapped drug crystals was performed for undiluted samples by means of a projection microscope fitted camera and photographed at suitable magnification. Moreover, a polarized light microscope (Model CX 31, Olympus, Germany) was used in order to reveal more details on the structure of the prepared systems without any dilutions. 2.5. Determination of entrapment efficiency Percentage entrapment efficiency (% EE) of the prepared Curc– GC-HS and Curc-T (n = 3) was determined by separation of vesicles from the solution after properly diluted, using centrifugation (15.000 rpm for 15 min at 4 C). Supernatant was collected and used for determination of un-entrapped Curc by measuring UV absorbance at 420 nm (Patel et al., 2009). The entrapped drug was calculated by the following equation:
2.7.1. Animals All studies were approved by the Ethics Committee at the Faculty of Pharmacy and Drug Manufacturing, Pharos University in Alexandria. The animals were kept under standard laboratory conditions and veterinary supervision with no restrictions on water and food. 12 female Sprague Dawley rats weighing between 210 and 230 g were randomly separated into two groups each composed of six rats. Rats were kept in individual cages with food and water and were maintained in a 12 h light/12 h dark cycle in a temperature controlled room at 20–25 C. They were given water and standard meals. Just before inflicting the burn, rats were anesthetized using intraperitoneal (IP) thiopental (80–100 mg/kg). The back hair of each rat was shaved with a shaving machine and then the area was depilated with a commercial depilatory cream to obtain a smooth and hairless skin (Jurjus et al., 2007). 2.7.2. Burn-wound model For the formation of the burn-wound, the shaved back skin of anesthetized animals was exposed to hot water. For this procedure, a cylindrical shaped stainless steel cup with radius of 10 mm was placed in hot water (100 C) for 1 min and then on the backs of rats and held for 20 s (Priya et al., 2002). Each rat was subjected to four burns. After the formation of standard, second degree burns; they were covered with cotton and gauze pre-wetted with saline for two days to form a remarkable wound. Fig. 1 shows the steps involved in the formation of skin wounds.
Fig. 1. Steps involved in the formation of skin burn-wound (1: shaved back skin, 2: after the burn, 3: after covering the burn for 2 days with saline wetted cotton).
W.M. El-Refaie et al. / International Journal of Pharmaceutics 486 (2015) 88–98 Table 1 Scale for percentage efficacy index. % Efficacy index values (% EI)
Significance
EI 30% 100% > EI 70% =100%
No improvement Moderate improvement Marked improvement Heal
The rats were divided randomly into two groups each composed of 6 rats. Formulations were examined using six wounds from different rats (n = 6). Formulations were repeatedly applied once daily to the burned areas for 10 days. For the control wounds, they were just cleaned with cotton wetted with normal saline each day. The diameter of the wounds formed were monitored and measured periodically. 2.7.3. Evaluation of the wound healing 2.7.3.1. Measurement of wound size. Progression decrease in the wound size was monitored periodically by measuring the distance between 2 opposite outside edges of the wound margin, using a vernier caliper (Priya et al., 2002). Two measurements approximately 90 from each other were obtained; the largest distance was used as one of the measurements. During the measurements, rats were fixed on a table. Wounds were digitally photographed on 0, 3, 5, 7, and 10 days post wounding with maintaining constant optical zoom. 2.7.3.2. Healing efficacy indices. The wound contraction rate was measured as percentage reduction in the wound size (% efficacy indices (% EI)) at days 2, 4, 7, and 10 from treatment beginning. The % EI of the wound size was calculated by the following equation: %EI ¼
V sp:day V I 100 VI
Vsp.day refers to the diameter (mm) values measured at day 2, 4, 7, and 10, while VI refers to the baseline value measured before treatment. The efficacy indices were then evaluated on a four rank scale specified in Table 1. 2.7.3.3. Morphological examination of scars. Wounds sites were monitored visually for scar formation, redness, and hyper pigmentation all over the study period (10 days) and after end of treatment till day 14 post wounding.
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2.7.3.4. Histological analysis. Full thickness skin biopsies of wounds with surrounding tissues were collected at the 7th and 11th day from wound treatment. Histological assessment was performed only for formulations with highest improvement in wound healing to further compare between them. Specimens were fixed in 10% formaldehyde, embedded in paraffin, cut into 5 mm pieces and stained with hematoxylin and eosin (H&E). Sections were visualized under a light microscope at 10 magnification. Fields were examined regarding the degree of epithelization, presence or absence of inflammatory cells, and the nature of collagen and fibrous tissue (Gong et al., 2013). 2.8. In-vivo skin deposition study The experiment was performed on 5 female Sprague Dawley rats (210–230 g). Rats were divided into two groups; each was composed of two rats. The fifth rat was kept as a control. Hair of the dorsal rats’ skin was removed using a depilatory cream. Using a marker pen, the dorsal skin of each rat was equally divided into six sections. They were exposed to single topical application of weight equivalent to 0.5 mg Curc from Curc–GC-HS, Curc–T-Pl gel, Curc– HA gel, and Curc–Pl gel. Each of the examined formulations was applied to three different sections (n = 3) in each group. In both groups, formulations were applied topically on the rats’ skin and allowed to dry. To avoid leakage from the skin and to get the comparable dose application in all rats, all exposed rats were sedated with IP thiopental (80–100 mg/kg) during the application and kept in a special cage to restrict rats’ movement. Group I rats were sacrificed after 6 h exposure. On the other hand, group II rats were sacrificed after 24 h from dose application. After scarification, the skin was cleaned from formulation residues. For extraction of Curc from collected skin samples 10 ml acetone containing 0.5% sodium lauryl sulfate (SLS) was added to the skin in an ambered glass bottles and kept for shaking over night at room temperature. Obtained dispersions were centrifuged for 10 min. at 10,000 rpm. Control skin samples (collected from the untreated rat) were subjected to the same procedure. Supernatants were withdrawn, and the drug content was determined by measuring UV absorbance at 420 nm. Results were presented as mean of the three readings SD. 2.9. Statistical analysis Statistically significant differences were determined using twotailed and student’s t-test. P < 0.05 was described as the level of significance.
Fig. 2. Morphological examination of vesicles loaded with 0.15% Curc (a) optical micrograph (400) and (b) TEM micrograph of Curc–GC-HS.
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Fig. 3. Morphological examination of vesicles loaded with 0.15% Curc (a) optical micrograph (400) and (b) TEM micrograph of Curc–T-Pl.
3. Results and discussion 3.1. Preparation and characterization of Curc-nanovesicles 3.1.1. Curc–GC-HS In an attempt to improve wound healing characteristics and reduce scar formation, we utilized a novel Curc loaded self assembled HA-based gel-core system. The prepared system yielded vesicles with mean diameter of (202.7 0.66 nm), PDI value of 0.259 0.008, and negative zeta potential of (33.0 2.6 mV) indicating homogenous size distribution with sufficient charge for stabilization. The hydrophobic nature of Curc allows it to become incorporated into the bilayer region of the vesicles with high percentage entrapment efficiency (96.44 1.29%). Morphological examination was performed using optical (Fig. 2a) and transmission electron microscopy (Fig. 2b). Obtained micrographs revealed the presence of well identified vesicles that exist in disperse not aggregated with characteristic clear bicompartmental (bilayer) structure. This might be attributed to the presence of Curc in the outer layer and HA gel in the core of the vesicles of Curc–GC-HS. 3.1.2. Curc–T-Pl gel To investigate the effect of combining HA with Curc in such novel system on wound healing, conventional Curc loaded transfersomes
(Curc-T) were prepared for comparison. Curc-T showed mean vesicles diameter of (320.6 20.7 nm), PDI value of 0.430 0.025, % EE (87.58 3.42) and zeta potential (37.2 3.4 mV). Curc-T optical micrographs (Fig. 3a) revealed the successful formation of vesicles. TEM micrographs (Fig. 3b) also showed well formed vesicles with spherical structure and homogenous size distribution but did not show the clear bilayer structure as in Curc–GC-HS (Fig. 2b). To be suitable for topical application, they were incorporated in 25% Pl gel (Poloxamer1 407). Poloxamers1, particularly Poloxamer1 407, have lately received notable interest as thermo-sensitive hydrogels (Garrastazu Pereira et al., 2013). They are biodegradable, non-toxic, and stable substances that form thermo-sensitive hydrogels in high concentrations. Pl gel was used in a previous study with Curc for wound healing (Kant et al., 2014) and found to have some beneficial effects on healing compared to control. Therefore, Pl (25% w/w) was selected as a gelling agent for Curc-T in the present study. 3.1.3. Structure elucidation Polarizing microscopy was utilized to reveal more details on the structure of the prepared vesicular gel systems (Fig. 4). Unlike Curc–T-Pl gel (Fig. 4b), photomicrographs obtained from Curc–GCHS (Fig. 4a) indicated the formation of a peculiar system that is not a simple gel and not a genuine liquid crystal, but a gel system that incorporates liquid crystals inside. These are commonly lamellar
Fig. 4. Polarizing micrographs of (a) Curc–GC-HS and (b) Curc–T-Pl (400).
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from HA gel compared to other gelling agents (Brown et al., 2001). In addition, Pl is a thermo-sensitive gel that formed a more dense gel structure at the temperature used in the experiment (37 C) which might further reduce Curc release. Comparing vesicular gels with plain gel formulations, it was observed that Curc release from Curc–T-Pl gel was much more retarded than from Curc–Pl gel. This might be due to the vesicles effect that might further slow the release of entrapped drug. On the other hand, in case of HA based systems, unexpected higher release of Curc from GC-HS was observed compared to Curc–HA plain gel. This might be attributed to the presence of Curc as insoluble dispersed large particles in HA gel matrix compared to Curc–GC-HS where Curc occurred in a very finely dispersed molecular state which might enhance its release. 3.3. In-vivo study Fig. 5. In-vitro release behavior of different Curc loaded formulations using dialysis bag method and 1:1 water:ethanol maintained at 37 C as acceptor medium.
structures that are formed around the inner core of the vesicles (Manosroi et al., 2003; Oka et al., 2008). This characteristic structure can make the system formed more stable by reducing the possibility of vesicles aggregation and fusion. 3.2. In-vitro release behavior The aim of this study was to test the ability of the novel designed GC-HS to release their drug load. Fig. 5 shows the drug release behavior of the different Curc loaded formulations namely, Curc suspension in 0.1% sodium lauryl sulphate, Curc in HA gel (Curc-HA), Curc in pluronic gel (Curc-Pl), Curc loaded selfassembled gel-core hyaluosomes (Curc–GC-HS), and Curc loaded transfersomes in pluronic gel (Curc-T-Pl), using dialysis bag method. As shown from the figure, generally all the examined formulations were able to gradually release their drug load by time. They all showed significant (P < 0.05) retardation in the initial release rate compared to Curc suspension. The use of Pl as a gelling agent, either in vesicular systems or plain gels, strongly retarded Curc release when compared to formulations prepared with HA. At 2 h interval, the cumulative release of Curc from Curc– Pl gel and Curc–T-Pl gel was 32.42 1.61 and 24.06 0.8 respectively while from Curc–GC-HS and Curc–HA was 50.32 1.38 and 41.85 1.57 respectively. This might be attributed to the ability of HA to absorb higher amounts of water increasing drug partitioning and release to the acceptor medium. This was in agreement with a previous study that observed higher drug release
As mentioned previously, impairment of wound healing process can be induced by prolonged inflammation and increased oxidative stress. Hence, application of Curc, a well known antioxidant and anti-inflammatory agent, could be important in improving such condition. Moreover, prolonged healing time would result in scar formation. HA was reported to help in reducing scars and improve wound healing. However, it was not sufficiently studied for improving its skin delivery for such application. Therefore, the present study was conducted to evaluate the cutaneous wound healing potential of the topically applied novel Curc–GC-HS that combines the effect of both drugs in addition to the stabilizing effect of the formulation. Rats have been widely utilized in the study of wound healing of different therapies. This was mainly due to their availability, small size, and low cost. In addition, rat skin is similar to that of human in having epidermis, hair follicles, and dermis. Moreover, reepithelization which is the predominant healing mechanism in human skin-is considered the primary response in rats after burns or partial thickness wounds. (Dorsett-Martin and Wysocki, 2008) Therefore, rat burn-wound model was utilized in this study. 3.3.1. Evaluation of wound healing Treatment efficacy of Curc–GC-HS on rats with dermal burnwound was investigated in comparison to other formulations namely Curc–T-Pl gel, Curc–HA gel, Curc–Pl gel, GC-HS, and HA gel. All treated groups showed significant difference in the wound contraction compared to the control group at day 2 of the study period (P < 0.05), Fig. 6. On the other hand, proceeding in the treatment till the end of the 10 days, only Curc–GC-HS followed by Curc–T-Pl and then GC-HS free from Curc showed statistically
Fig. 6. Efficacy indices for the evaluation of wound size reduction of the different groups of rats receiving different treatments.
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Fig. 7. Stages of wound healing of the different therapies.
significant difference compared to the control, Figs. 6 and 7. Comparing these results with those obtained from conventional gels (HA, Curc–Pl, and Curc–HA gels); this might indicate the positive effect of the use of nanostructured vesicular systems in drug delivery either for Curc or HA or both combined. Among the promising formulations, Curc–GC-HS is the only system that demonstrated significant marked improvement at day 7 of treatment and complete heal (% EI = 99.7 0.34%) with minor or no scar at day 10, Fig. 7,Table 2. This might be due to the combined anti inflammatory and anti-oxidant effect of Curc together with the effect of HA with its scar reducing potential in addition of the probability of such novel formulation to increase the dermal localization of their drug load; improving efficacy. GCHS is a novel carrier that combined the effect of elastic vesicles (due to the presence of Tween 80), stable gel core liposomes, and hygroscopic HA. It has been reported that due to its high hygroscopic properties, HA may hydrate the skin creating hydrophilic pathways (Brown and Jones, 2005). The elasticity of the prepared vesicles together with loosening of skin structure by HA could allow the vesicles to penetrate the skin without losing their integrity. Moreover, being core gelled, provided the system with more stability to withstand surrounding environment preventing vesicles damage and early drug leakage (Tiwari et al., 2009). Consequently, Curc might be protected inside the vesicles
that were localized in the skin; accelerating the wound healing process Fig. 8. Evaluating the effect of HA and Curc in conventional gel formulations namely, Curc–Pl gel and HA gel, there was no Table 2 Efficacy indices (% EI) values and their significance for the used formulations at days 7 and 10 of treatment. Formulation
% EI values (significance) Day 7
Day 10
Control
30.15 8.3 (No improvement) 82.39 10.2 (Marked improvement) 40.84 15.9 (Moderate improvement) 28.02 12.39 (No improvement) 28.94 11.4 (No improvement) 63.8 15.49 (Moderate improvement) 30.5 12.5 (No improvement)
52.2 8.62 (Moderate improvement) 99.7 0.34 (Heal) 71.5 11.42 (Marked improvement) 57.28 10.32 (Moderate improvement) 56.5 5.86 (Moderate improvement) 82.19 3.45 (Marked improvement) 53.5 9.43 (Moderate improvement)
Curc–GC-HS GC-HS Curc–HA gel HA gel T-Pl gel Curc–Pl gel
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Fig. 8. A diagram summarizing the mechanism of the novel Curc–GC-HS in enhancing wound healing rate via improving dermal localization of the entrapped Curc in the loosened skin structure.
Fig. 9. Histopathological examination of (a) normal skin (b) treated with Curc–GC-HS at day 7 post wounding and (c) treated with Curc–GC-HS at day 11 post wounding (d) treated with Curc–T-Pl at day 7 post wounding (e) treated with Curc–T-Pl at day 11 post wounding.
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improvement in healing process was observed from both after 7 days and moderate improvement was observed after 10 days (Table 2). Moreover, combination of both drugs in Curc–HA gel did not give any significant improvement compared to each alone (10 days % EI obtained from Curc–Pl gel, HA gel and Curc–HA gel were 53.5 9.43, 56.5 5.86, and 57.28 10.32% respectively, Table 2). This might be due to inability of the drugs to penetrate skin efficiently or to be localized in the skin for prolonged effect. Therefore, they need longer time to give their therapeutic effect which was in agreement with previous studies that observed healing effect for Curc after 14 or 19 days (Chereddy et al., 2013; Gong et al., 2013; Kant et al., 2014) and for HA after 29 days (Medeiros et al., 1999). Hence, the novel self-assembled gel-core hyaluosomes might be a promising carrier for HA and Curc; accelerating the wound healing process with possibility of reducing scar formation. 3.3.2. Histopathological examination For additional confirmation of the quality and maturity of the healed tissues or those with marked improvement namely, Curc loaded self assembled gel-core hyaluosomes (Curc–GC-HS) and Curc loaded transfersomes in pluronic gel (Curc-T-Pl), histological characterization was carried out. Full thickness sections of normal skin and treated wounds, with highest healing potential, were
collected and stained with hematoxylin and eosin (H&E) for microscopical examination of skin layers. The normal skin (Fig. 9a) showed an epidermis layer and areolar woven network of collagen fibers arranged in the dermal layer. On the other hand, Fig. 9b and d reveals histological findings of wounds treated with Curc–GC-HS and Curc–T-Pl gel respectively at 7th day post wounding. Curc–GCHS treated wounds (Fig. 9b) exhibited densely packed collagen fibers with parallel arrangement and high accumulation under well formed thick epithelium. Moreover, numerous collagen bundles were observed in the reticular layer indicating fibroblast tissue formation. In contrast, loose irregular collagen fibers with large dissociating areas in between were observed in Curc–T-Pl gel treated wounds (Fig. 9d). At 11th day post wounding, wounds treated with Curc–GC-HS showed almost normal skin structure with well formed and differentiated epithelium and woven collagen fibers (Fig. 9c). Curc–T-Pl gel treated wounds demonstrated significant increase in the deposition of collagen fibers and hence increase in activity and fibroblast formation (Fig. 9e), indicating that the healing process is incomplete and still active. Worthy mentioning that in all examined treated skin sections no inflammatory cells were detected. These observations indicated the high anti-inflammatory action of Curc and its potential in inducing collagen formation as both examined Curc-loaded vesicular formulations improved wound
Fig. 10. Scar monitoring at day 14 post wounding.
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healing. However, Curc–GC-HS was found to be superior in promoting the formation of the collagen and making re-epithelization in less time compared to Curc–T-Pl gel. This might further prove the combined effect of HA and Curc in addition to the effect of this novel formulation to retain them inside the skin; speeding up the wound healing process with high probability for reducing scar formation. 3.3.3. Scar monitoring As mentioned previously, several steps are involved in wound healing processes as clotting, inflammation, collagen deposition, granulation, spasm of wound, and re-epithelization. Wounds with prolonged healing can be considered most risky for impairment of such process and undue scar formation. Although evaluation of scar development seems necessary, this is still a neglected area, and yet there is no agreement on the best method for scar assessment (Powers et al., 1999). In the current study, visual inspection of scars was performed till 14 days post wounding as a simple method for monitoring of the presence or absence of scars. Scars might be appeared as hypertrophic deformity or impaired pigmentation of the healed wound after epithelialization (Idriss and Maibach, 2009). As shown in Fig. 10 untreated groups were not completely healed and still showing a crust. On the other hand, all treated groups showed almost complete healing and epithelization but with some developed scars. Curc–GC-HS was superior over all formulation as it did not show any hyperpigmentation followed by Curc–T-Pl gel while all others left a scar with different degrees of hyperpigmentation. The greater performance for Curc–GC-HS in wound healing and reducing scar formation might additionally confirm the observed higher rate of collagen development and reepithelization compared to Curc–T-Pl gel and to other formulations. All the previous findings could provide an evidence of the ability of this novel formulation to act as an effective carrier for Curc. It could be able to retain Curc inside the skin and protect it against metabolism; resulting in higher efficacy. This will be examined in the following experiment. 3.3.4. Skin deposition study The aim of this experiment was to examine the ability of the novel GC-HS compared to the other prepared gel formulations to enhance skin penetration of Curc and to protect it inside the skin against metabolism; thus improving its bioavailability and efficacy in wound healing or even in other chronic skin diseases. It should be noted that Curc was selected in this study as it is one of the most poorly skin penetrating compounds in addition to its excessive metabolism, rapid elimination and extreme instability previously observed in different studies (Anand et al., 2007; Asai and Miyazawa, 2000; Ravindranath and Chandrasekhara, 1980; Yang et al., 2007). Moreover, all studies examined Curc skin delivery dealt mainly with its in vitro dialysis or clinical investigations of its effect but never reported its in vivo deposition and localization in the skin. Results obtained from this study indicated that Curc–GCHS was the only formulation that gave detectable amount of Curc deposited in the skin representing 0.83 0.34% of the applied dose after 6 h exposure. After 24 h exposure, Curc–GC-HS showed significantly higher skin deposition of Curc (4.62 1.28%) compared to Curc–T-Pl gel (0.92 0.44%) and no Curc was detected in the skin from plain gel formulations. These results indicated the high potential of the novel GC-HS in protecting loaded Curc against metabolism and increasing its stability and localization inside the skin. This confirmed the ability of the GC-HS for enhancement of Curc bioavailability over other formulations. This may further prove its superior effect in wound healing and suggest its use as a carrier for Curc or any other drug in treatment of other skin diseases.
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4. Conclusion It was concluded that topical application of the novel Curc–GCHS formulation in rat burn-wounds contributed to speed healing, improved histological progress and reduced scar formation. This was attributed to the combined anti inflammatory and antioxidant effect of Curc together with the effect of HA with its scar reducing potential. Furthermore, the potential of the novel GC-HS as an effective carrier for Curc was also proved in the in vivo skin deposition study. It was able to increase skin penetration and dermal localization of Curc; protecting it against degradation and metabolism thus enhancing its bioavailability and therapeutic outcome. This may further prove its superior effect in wound healing and suggest its use as a carrier for Curc or any other drug in treatment of chronic skin diseases. Conflict of interest The authors report no financial or personal conflict of interest. Acknowledgement We would like to express our deep thanks to professor/ Ebtehag El-Ghazawi (Professor of histology and cell biology- Faculty of medicine- Alexandria University) for her help in interpretation of histopathology results. References Adzick, N.S., Longaker, M.T., 1992. Scarless fetal healing therapeutic implications. Ann. Surg. 215, 3–7. Aggarwal, B.B., Sung, B., 2009. Pharmacological basis for the role of curcumin in chronic diseases: an age-old spice with modern targets. Trends Pharmacol. Sci. 30, 85–94. Alster, T.S., Tanzi, E.L., 2003. Hypertrophic scars and keloids. Am. J. Clin. Dermatol. 4, 235–243. Anand, P., Kunnumakkara, A.B., Newman, R.A., Aggarwal, B.B., 2007. Bioavailability of curcumin: problems and promises. Mol. Pharm. 4, 807–818. Asai, A., Miyazawa, T., 2000. Occurrence of orally administered curcuminoid as glucuronide and glucuronide/sulfate conjugates in rat plasma. Life Sci. 67, 2785–2793. Atiyeh, B.S., Hayek, S.N., 2004. An update on management of acute and chronic open wounds: the importance of moist environment in optimal wound healing. Med. Chem. Rev.-Online 1, 111–121. Bisht, S., Feldmann, G., Soni, S., Ravi, R., Karikar, C., Maitra, A., Maitra, A., 2007. Polymeric nanoparticle-encapsulated curcumin (nanocurcumin): a novel strategy for human cancer therapy. J. Nanobiotechnol. 5, 1–18. Bock, O., Schmid-Ott, G., Malewski, P., Mrowietz, U., 2006. Quality of life of patients with keloid and hypertrophic scarring. Arch. Dermatol. Res. 297, 433–438. Bragagni, M., Mennini, N., Maestrelli, F., Cirri, M., Mura, P., 2012. Comparative study of liposomes, transfersomes and ethosomes as carriers for improving topical delivery of celecoxib. Drug. Deliv. 19, 354–361. Brown, B., McKenna, S., Siddhi, K., McGrouther, D., Bayat, A., 2008. The hidden cost of skin scars: quality of life after skin scarring. J. Plast. Reconstr. Aesthet. Surg. 61, 1049–1058. Brown, M., Hanpanitcharoen, M., Martin, G., 2001. An in vitro investigation into the effect of glycosaminoglycans on the skin partitioning and deposition of NSAIDs. Int. J. Pharm. 225, 113–121. Brown, M., Jones, S.A., 2005. Hyaluronic acid: a unique topical vehicle for the localized delivery of drugs to the skin. J. Eur. Acad. Dermatol. Venereol. 19, 308– 318. Chereddy, K.K., Coco, R., Memvanga, P.B., Ucakar, B., des Rieux, A., Vandermeulen, G., Préat, V., 2013. Combined effect of PLGA and curcumin on wound healing activity. J. Control. Release 171, 208–215. Dai, M., Zheng, X., Xu, X., Kong, X., Li, X., Guo, G., Luo, F., Zhao, X., Wei, Y.Q., Qian, Z., 2009. Chitosan-alginate sponge: preparation and application in curcumin delivery for dermal wound healing in rat. Biomed. Res. Int. 2009, 595126. Dorsett-Martin, W.A., Wysocki, A.B., 2008. Rat Models of Skin Wound Healing Sourcebook of Models for Biomedical Research. Springer, pp. 631–638. Durgaprasad, S., Reetesh, R., Hareesh, K., Rajput, R., 2011. Effect of a topical curcumin preparation (BIOCURCUMAX) on burn wound healing in rats. J. Pharm. Biomed. Sci. 8, 1–3. El-Refaie, W.M., Elnaggar, Y.S., El-Massik, M.A., Abdallah, O.Y., 2015. Novel selfassembled, gel-core hyaluosomes for non-invasive management of osteoarthritis: in-vitro optimization ex-vivo and in-vivo permeation. Pharm. Res..
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