Quality by design driven development of resveratrol loaded ethosomal hydrogel for improved dermatological benefits via enhanced skin permeation and retention

International Journal of Pharmaceutics 567 (2019) 118448

Contents lists available at ScienceDirect

International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Quality by design driven development of resveratrol loaded ethosomal hydrogel for improved dermatological benefits via enhanced skin permeation and retention

T

Daisy Aroraa,b, , Sanju Nandaa ⁎

a b

Department of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak, Haryana, India Department of Pharmaceutics, I.S.F. College of Pharmacy, Moga, Punjab, India

ARTICLE INFO

ABSTRACT

Keywords: Ethosomes QbD Critical quality attributes Risk assessment analysis Fish bone diagram Factorial design Skin permeation

Resveratrol is a potent anti-oxidant agent and can be used for the effective management of different skin conditions like extrinsic skin ageing, psoriasis, etc. The objective of this research was to develop a dermal delivery system of resveratrol for its improved dermatological benefits for achieving its enhanced skin deposition profile with limited systemic exposure. Resveratrol loaded ethosomal hydrogel was developed and optimized using systematic Quality by Design approach. Firstly, the quality target product profile (QTPP) of ethosomal formulation was defined and critical quality attributes (CQAs) and critical material attributes (CMAs) were screened through risk assessment studies based on fish bone diagram. 32 full factorial design using Design Expert software was employed to optimize the selected CMAs. Concentration of phospholipid (X1) and concentration of ethanol (X2) were selected as independent CMAs. Vesicle size (Y1), entrapment efficiency (Y2), permeation flux (Y3) and drug deposition in dermal layer (Y4) were evaluated as dependant CQAs. Optimized formulation was then evaluated for physicochemical and skin permeation properties. Ethosomal hydrogel was able to significantly enhance the skin permeation parameters and skin deposition of resveratrol in comparison to the conventional cream. The results were highly ratified by CLSM studies in which ethosomal hydrogel was found to be vastly scattered in the deeper skin layers. Thus, there is evidence that systemically developed ethosomal gel can deliver enhanced amounts of bioactives into the skin and it is expected that a number of products for dermal/transdermal applications will be developed in the future based on it.

1. Introduction Largest organ of the human body is skin, presenting a total area of approximately 2 m square. Vital skin function is to provide protection against UV radiation, microbiological invasion, physical/chemical injuries, regulation of the body temperature etc (Degim, 2006). The skin is primarily comprised of three distinctive layers, the epidermis, the dermis and the hypodermis. Each layer exhibits unique cellular makeup and physiological function (Melorose et al., 2011; Menon, 2002). Skin also act as a major site for delivering drugs and bioactives for several topical acute and chronic dermatological conditions such as acne, psoriasis, skin cancer, extrinsic skin ageing and so on. The skin properties play an important role to allow penetration of topically applied drugs or substances into the skin. Drug permeation through the skin includes the diffusion through the intact epidermis and the skin appendages (Negi et al., 2016). The deeper layers of skin i.e. dermis and

⁎

hypodermis are considered as the major site associated with many dermatological disorders. But, the horny layer barrier (i.e. stratum corneum) is the main hurdle and the rate limiting step for the percutaneous absorption of drugs. Overcoming this horny barrier by enhancing the permeation and at the same time, retaining maximum amount of drug in deeper layers, and thus, avoiding systemic absorption to enhance the dermatological benefits via topical route is the need of the hour (Sobanko et al., 2012). Resveratrol (3,4′,5-trans-trihydroxy-stilbene) is a natural polyphenol; a member of stilbene family of phenolic compounds; found in a wide variety of plants such as grapes, nuts and berries (Chedea et al., 2017). Numerous studies in last decade indicated that Resveratrol is an activator of the protein deacetylase sirtuin (SIRT) gene in mitochondria, which is thought to mediate anti-aging, anti-proliferative and anti-inflammatory activity resulting in alteration of gene expression and intonation of numerous metabolic pathways (Ruivo et al., 2015). Thus, it

Corresponding author at: Department of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak 124001, Haryana, India. E-mail address: [email protected] (D. Arora).

https://doi.org/10.1016/j.ijpharm.2019.118448 Received 16 January 2019; Received in revised form 27 May 2019; Accepted 17 June 2019 Available online 18 June 2019 0378-5173/ © 2019 Elsevier B.V. All rights reserved.

International Journal of Pharmaceutics 567 (2019) 118448

D. Arora and S. Nanda

offers several systemic as well as topical biological effects, such as anticancer activity, neuroprotective effect, anti-ageing effect, anti-oxidant activity, anti-inflammatory activity (Pangeni et al., 2014; Yang and Meyskens, 2005). In recent studies, numerous methods and approaches have been employed to improve the dermal flux and skin deposition of resveratrol and related bioactives such as use of iontophoresis (Kushwaha et al., 2016; Yang et al., 2017), electroporation (Tokumoto et al., 2016), microneedles (Petchsangsai et al., 2014), liposomes (Liu et al., 2016), niosomes (Pando et al., 2013), microemulsion (Negi et al., 2016), nanosponges (Ansari et al., 2011), nanosomes (Geusens et al., 2010) etc. But one or other deficiencies are linked with all these like convoluted processing, skin injury, patient incompliance, poor stability and high cost. An alternative delivery strategy used to increase penetration of drug is incorporating the drug into ultra flexible colloidal delivery system such as ethanol containing vesicles i.e. ethosomes. Ethosomal systems are mainly composed of phospholipid, ethanol (10–40%) and water (Ahad et al., 2014). They are unlike the conventional liposomes, which remain confined to the upper layer of the stratum corneum and get accumulated in the skin appendages, with negligible penetration to deeper tissues, due to lack of flexibility. In contrast to this, ethosomes have been reported to enhance permeation of the drug through stratum corneum and subsequent retention in the skin layers (Garg et al., 2016). This is ratified due to presence of ethanol which acts as edge activator and tends to fluidize the highly ordered lipophillic structure of stratum corneum and thus, enhance the permeation flux. This also amends the physicochemical characteristics of vesicles by lowering the phase transition temperature (Tm) of the lipids (Caddeo et al., 2013; Garg et al., 2016). The formulation of such effective and successful vesicular system involves diverse multifunctional excipients and manufacturing steps, to obtain prerequisite quality target product profile (QTPP) (Arora et al., 2016). The conventional system of optimizing drug delivery system basically involves studying the power of one variable at time (OVAT), while keeping others as constant. However by applying this conventional OVAT approach, the explanation of a definite challenging property can be achieved partially, but accomplishment of the best possible composition or process is never assured (Singh et al., 2005). Establishment of “cause-and-effect” relationships using OVAT is not possible and it becomes ineffective when all variables are changed simultaneously. Thus more systematic optimization approaches are need of the hour to avoid such inconsistencies and to produce competent formulation under strictly regulated environment. Quality by Design (QbD) based Design of Experiment (DoE) endows with holistic perception of product and processes to relent the best solution (Singh et al., 2011). QbD is “A systematic approach of development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management” as described in ICH Q8(R2) (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, 2009; Saydam and Takka, 2018). As per ICH Q8 guidelines, defining of Quality target product profile (QTPP), Critical Quality attributes (CQAs), Critical process parameters (CPPs) and Critical material attributes (CMAs) and establishing relationships among them is the initial step for implementing QbD. Several tools of common risk analysis and management are employed such as Fishbone diagrams and failure mode and effect analysis (FMEA) application (ICH, 2005). Design of experiments and multivariate statistical data analysis are indispensable fundamentals of QbD, accepted by recent International Conference of Harmonization Q8 guideline (Saydam and Takka, 2018). In the present investigation, systematic optimization, formulation and evaluation of resveratrol loaded ultra flexible ethanolic vesicles (ethosomes) was conducted using basic tools of QbD i.e. risk assessment analysis and full factorial design. The current study was designed to

determine the influence of concentration of major constituent material variables of vesicles on critical quality attributes which are essential for a stable and efficacious clinical product. 2. Materials and methods 2.1. Materials Pure sample of resveratrol was obtained ex-gratis from Sami labs, Bangalore, India. Phospholipid i.e. Soy phosphatidylcholine (PC) (99% pure), carbopol 934P, Coumarin 6 (C6), stearylamine was purchased from Sigma Aldrich, USA. Ethanol, methanol, propylene glycol and other solvents were procured from Merck Ltd., India. All other chemicals and reagents were obtained from local supplier and of analytical grade. 2.2. Methods 2.2.1. Defining and identifying the QTPP and the CQAs Quality based design (QbD) driven strategy was employed for development of drug formulation. Firstly quality target product profile (QTPP) was defined for formulation of resveratrol loaded ethanol containing vesicles with improved performance and therapeutic benefits (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, 2009). Basic consideration was given for selection of intended use in clinical purpose, route of administration, dosage form, delivery systems, dosage strength(s) and stability. For fulfilling the requirements of defined QTPP, essential CQAs were identified as quality characteristics of flexible vesicles including both formulation and physical attributes. Among various elements, most crucial quality attributes responsible for product performance were selected including vesicle size, drug entrapment efficiency, permeation flux and extent of drug deposition in dermal layer of skin. 2.2.2. Formulation of resveratrol loaded ethosomes Resveratrol loaded ethosomes were developed by reported cold method as described by Sharma et al. (2016) with appropriate modifications as per laboratory setup. Briefly, varying quantities of phospholipid (soya phosphatidylcholine, PC) (20–40 mg/ml), resveratrol (equimolar to PC), stearylamine (2–4 mg/ml) and propylene glycol (1 ml) were dissolved in absolute ethanol (1.5–3.5 ml). To this drug solution, double-distilled water was added in sufficient quantity to make the final volume equal to 10 ml with constant stirring at 1700–2000 rpm for 30 min using magnetic stirrer in a closed vessel at 30 °C. The final preparation was subjected to probe sonication (using Probe sonicator, Lark, USA) for total of 15 min for 3 cycles of 5 min each (15 s on/off cycle). The evenly dispersed ethosomal vesicles were formed. The formulation was then purified to separate free (unentrapped) resveratrol and unstructured materials using ultracentrifugation method. Formulation was then stored in refrigerator for further characterization (Pandey et al., 2015). Conventional liposomes were also prepared for comparative purpose by similar methodology in which PC and cholesterol in 7:3 M ratios (i.e. 35.3 mg/ml PC and 9.1 mg/ml cholesterol) and 125 mg resveratrol were hydrated with 10 ml double-distilled water as above. The liposomal dispersion obtained was processed further as similar to ethosomal vesicles prepared above (Caddeo et al., 2014). 2.2.3. Risk assessment analysis Various quality attributes were screened through risk assessment studies for ethosomes. To perform it, Minitab 18 software (M/s Minitab Inc., Philadelphia, USA) was used to create an Ishikawa fish-bone diagram for development of ethosomes. The main purpose is to look upon a cause-effect relationship among the various material attributes (MAs) and several process parameters (PPs), and expected consequence of 2

International Journal of Pharmaceutics 567 (2019) 118448

D. Arora and S. Nanda

these on the CQAs of the ethosomes. For selection of crucial risk factors having highest effect on selected CQAs, failure mode and effect analysis (FMEA) was performed (Beg et al., 2018; Claycamp et al., 2016). Via extensive literature survey, previous knowledge and brain storming exercise; rank order scores (ranging between 1 and 10) were assigned to the material and process parameters attributes on the basis of severity (S), occurrence (O) and detectability (D) and the risk priority number (RPN) was calculated using the below mentioned equation (Eq. (1)):

RPN = Severity (S )

Occurence (O )

Detectability (D )

employed to formulate the cream (Sharma et al., 2016). 2.2.7. Evaluation of physicochemical characteristics i.e. morphology of vesicles and vesicles loaded hydrogel For morphology (i.e. shape uniformity and lamellarity) transmission electron microscope (H 7500 Hitachi, Japan) was used. Prepared formulations were diluted 100 times with triple distilled water and stained negatively with phosphotungustic acid solution (1%w/v). Microscopic images were taken (Khurana et al., 2013).

(1)

Among the studied material and process attributes, the attributes with high risk scores were selected and studied further for preliminary studies. Various ethosomal formulations were prepared and obtained data for CQAs, i.e., size, percent drug entrapment efficiency, permeation flux and extent of drug deposition in dermal layer of skin was taken for principal component analysis (PCA) for evaluating qualitative and quantitative effects of the studied attributes.

2.2.8. Micromeritic studies Vesicle size, size distribution and zeta potential of vesicles and vesicle loaded hydrogel was determined using zeta sizer (Malvern Instruments, U.K). The formulation was diluted five times with normal saline (0.9% w/v). The number of vesicles per cubic mm was counted microscopically using haemocytometer. The number of vesicles in 80 small squares was counted and average number of vesicles was calculated using the following formula (Eq. (2)) (Sharma et al., 2016):

2.2.4. Statistical optimization of resveratrol loaded ethosomes using full factorial design A two-factor, three-level full factorial design was employed to optimize varied critical material attributes influencing the response variables i.e. critical quality attributes. Concentration of PC (X1) and concentration of ethanol (X2) were selected as independent critical material attributes and varied at three different levels i.e. low, medium and high. Vesicle size (Y1), % entrapment efficiency (Y2), permeation flux J (Y3) and extent of drug deposition in dermal layer of skin SD (Y4) were evaluated as dependant quality attribute. Total 13 batches were prepared including four center points per block. The obtained data was fitted into Design Expert software (Design Expert 11.0.4, Stat-Ease, Minneapolis, MN). Response surface analyses were carried out and contour plots and (3D) response surface plots were constructed to establish the understanding of relationship of variables and its interaction. Effect of different CMAs on CQAs was identified and second-order polynomial models were constructed and fitted into multiple linear regression model. Analysis of variance (ANOVA) was used to validate design (Negi et al., 2016). Constraints for quality attributes were set at target levels and possible formulation composition were determined using checkpoint analysis and desirability approach using Design Expert software. Optimum formulation was selected by a numerical optimization procedure using desirability function (Beg et al., 2018).

Total number of vesicles Total number of vesicles counted in 80 small squares =

dilution factor 4000 Total number of squares counted

(2)

2.2.9. Drug entrapment efficiency Percentage of drug entrapped in ethanolic vesicles and ethosomal gel was determined using the method reported by (Arora et al., 2011). This method includes separation of free drug and unstructured materials via size exclusion chromatography by passing the vesicular formulation through a sephadex G-100 mini column and centrifuged at 2200 × G for 3 min. Vesicles were then ruptured in appropriate volume of chloroform-methanol (2:1 v/v) mixture and analyzed spectrophotometrically at 306 nm. Evaluation of entrapment efficiency was carried out by applying following formula (Eq. (3))

% Entrapment efficiency =

(Amount of drug entrapped ) (Amount of drug added )

100

(3)

2.2.5. Formulation of ethosomal carbopol hydrogel Due to low viscosity and poor skin applicability of ethosomal dispersion, it was incorporated into carbopol hydrogel system. 500 mg Carbopol 934P was dispersed in 50 ml warm distilled water and stirred at 1000 rpm till polymer got homogenously dispersed and then neutralized using 2% triethanolamine solution in sufficient quantity to make the volume 100 ml. It was homogenized till a transparent gel was formed. Optimized ethosomal formulation (Opt-Etho) was then gently levigated in the developed carbopol gel base in the ratio of 5:1, to formulate the resveratrol loaded ethosomal carbopol hydrogel (EthoCBP-Gel). Conventional plain resveratrol carbopol hydrogel (Res-CBPGel) was also prepared for comparative studies containing same amount of resveratrol. For this resveratrol solution in ethanol was prepared and levigated in the developed carbopol gel base in a similar manner. The gel was evaluated visually for checking its color and physical stability as well as for other physical and chemical properties (discussed in further sections) (Garg et al., 2016).

2.2.10. Viscosity, spreadability and pH determination of ethosomal hydrogel Viscosity of the ethosomal hydrogel was determined using Brookfield R/S plus viscometer (Brook-field Engineering Laboratories, Inc, Middleboro, MA). R3-C75 spindle was used and its speed was adjusted at 100 rpm. A small amount of the formulation was applied at the base of viscometer at a temperature of 25 ± 2 °C. A shear rate range from 1 to 100 s−1 was applied. The viscosity was determined from the flow curve obtained at different values of shear rate (Yadav et al., 2016). The spreadability represents the index of ease of application of topical formulation on skin. Plate method was employed to determine the spreadability of gel samples (Nayak et al., 2018). Simply, two acrylic plates were used and 500 mg of ethosomal dispersion incorporated carbopol gel was placed at the centre of the one plate and other was placed above it. Initial diameter of the circle in which gel was spread was calculated. Then, a weight of 500 g was placed on the upper plate for 5 min. The spreading of gel as a function of weight applied was calculated by observing the increase in diameter and spreadability was calculated. pH of the prepared formulation was also measured using a calibrated pH meter (AE Max) (Dhawan and Nanda, 2018).

2.2.6. Formulation of conventional cream of resveratrol Resveratrol loaded conventional cream (Res-Cream) was also formulated for various comparisons. It was composed of 3% resveratrol, 6% span 80, 3% white bees wax, 36% white soft paraffin, 15% liquid paraffin, and quantity sufficient of distilled water. Simple emulsification method of melting and mixing the components with stirring was

2.2.11. Ex vivo skin permeation studies Ex vivo skin permeation study was carried out using freshly excised skin from pig ear (obtained from local slaughter house). Pig skin was used because it is histologically similar to human skin. Stratum corneum of pig pinna, having a thickness of 21–26 μm with average 20 hair follicles per centimeter square area, is quite similar to human forehead 3

International Journal of Pharmaceutics 567 (2019) 118448

D. Arora and S. Nanda

skin having 14–32 hair follicles per centimeter square area. Apart from similarity, its easily availability and wide use in skin permeation studies, makes it most preferable model to be used in the present study (Abd et al., 2016). Shaved dorsal portion of skin was mounted on a static franz diffusion cell having cross-sectional area of 3.14 cm2 and capacity 30 ml. Mixture of ethanol and buffer pH 7.4 in a ratio of 3:7 (v/v) was taken in receptor compartment, maintained at 32 ± 0.5 °C at 100 rpm. Donor compartment was filled with different formulations equivalent to 1.5 mg of drug (ethosomal dispersions, ethosomal carbopol hydrogel (Etho-CBP-Gel), ethanolic solution (Res-Alc-Sol), conventional plain resveratrol containing carbopol hydrogel (Res-CBP-Gel) and conventional cream of Resveratrol (Res-cream)). At predetermined time (1, 2, 4, 6, 8, 10, 12, 18 and 24 h), 0.5 ml samples were withdrawn from the receptor compartment followed by replenishment with fresh media and drug concentration in the sample was analyzed spectrophotometrically. Cumulative amount of drug permeated (Qn) was calculated using following equation (Eq. (4)):

formulations were applied on to the excised and depilated skin of porcine ear (taken from slaughterhouse) and kept at ambient conditions for 24 h. After 24 h, skin was washed with 3 ml of PBS (pH 5.8) and cryo-sectioned using cryostat microtome (Thermo scientific instruments, Microme, Germany). The skin sections of 40 μm were mounted on the glass slides and visualized using CLSM (confocal laser scanning laser microscope) (LSM 510 META, Zeiss, Germany) and photomicrographs were taken (Nayak et al., 2018). 2.2.14. Stability studies Optimized ethosomal formulation and hydrogel were subjected to stability studies to evaluate the effect of different storage conditions. The study was conducted by keeping the formulations at refrigerated condition (4 ± 2 °C) and room temperature (25 ± 2 °C/60 ± 5% RH) for 6 months. The formulations were evaluated for physical as well as chemical stability at periodic time intervals of 0, 1, 3 and 6 months. Physical stability was studied by analyzing vesicle size, zeta potential and pH of formulation. Hydrogel was analyzed for pH, consistency and phase separation. For chemical stability tests, residual drug content in the formulation was investigated at both temperatures for up to 6 months (Negi et al., 2016). The initial drug content was evaluated on the basis of entrapment efficiency of the vesicles and considered as 100%. At periodic time intervals, drug content remaining in the vesicles was evaluated by following the same procedure as reported in previous section for evaluating entrapment efficiency.

n=1

Qn = Cn

Vo

Ci

Vi

(4)

i=1

where, Cn represents the concentration of drug of the receptor compartment at any sampling time, Ci is the drug concentration of the ith sample, and V0 and Vi represent the volumes of the receptor compartment and the sample withdrawn, respectively. Permeation curves were constructed by plotting the cumulative amount of drug permeated through unit area of the skin against time. Permeation rate (flux; J) of drug was calculated from the slope of the regression lines, fitting to the linear portion of the permeability profiles (Negi et al., 2016). Whereas, Permeability coefficient (Kp) was calculated using following equation (Eq. (5)):

Kp =

Jss Co

2.2.15. Statistical analysis Statistical evaluation of results was done using Graph Pad Instat 3 software. All values are given as mean ± S.D. Student’s t-test was used to compare mean values of different groups. Statistical significance was designated as p < 0.05. Multiple comparisons were made using one way analysis of variance (ANOVA) followed by post hoc analysis using Tukey’s test. Statistical significance was considered at p < 0.05.

(5)

where, Co represents the initial drug concentration in the donor cell. Enhancement ratio (Er) was calculated by dividing Jss of the respective formulation by Jss of the control formulation (conventional cream).

3. Results and discussion 3.1. Defining and identifying the QTPP and the CQAs

2.2.12. Skin deposition studies Amount of drug being deposited in dermal layer of skin after permeation studies was also analyzed by method as reported earlier (Negi et al., 2016). After 24 h, the permeation studies were stopped and skin was taken off from the diffusion cell very carefully. Remaining formulation on skin was removed and skin was cleaned and washed with triple distilled water and dried using hair dryer. Then skin was cut into small pieces and drug was extracted with methanol using sonication in bath sonicator for 1 h. The resulting solution was filtered through a membrane (0.45 mm), and filtrate was analyzed for amount of drug deposited in per unit area of skin (SD, µg cm−2).

QTPP of the formulation was defined and identified as per QbD based guidelines. Various quality characteristics were identified (Beg et al., 2018; Lambert, 2010) and enlisted in Table 1 for formulation of resveratrol loaded ethanol containing vesicles with improved performance and therapeutic benefits. Likewise vesicle size, percent drug entrapment efficiency, permeation flux and extent of drug deposition in dermal layer of skin (Skin deposition, SD) were selected as critical quality attributes (CQAs) of the product responsible for product performance are described in Table 2 with their appropriate justification.

2.2.13. Histopathological studies – skin distribution by CLSM The confocal microscopy was performed to confirm the permeation of payloads into the deeper layer of skin after topical application to the skin specimen. Instead of drug, C6 loaded ethosomal dispersion and ethosomal hydrogel were prepared using the same procedure. Conventional hydrogel containing C6 was also prepared. All the

According to preliminary risk assessment analysis, Ishikawa fishbone diagram for development of ethosomes was drawn to look upon a cause-effect relationship among the various material attributes (MAs) and several process parameters (PPs), and expected consequence of these on the CQAs of the ethosomes (Fig. 1). This was then accompanied by FMEA for opting high risk factors by assigning scores and

3.2. Risk assessment analysis

Table 1 Defined QTPP elements for formulation of resveratrol loaded ethosomes. QTPP Elements

Target

Justification(s)

Route of administration Dosage form/delivery systems Dosage type Ex vivo permeation Stability (Physical & chemical)

Topical Ethosomes Rapid release Higher flux and skin retention At least 6 months at various storage temperatures

Recommended route for dermatological benefits Helps in enhanced permeation of bioactives in dermal region Quick release leading to enhanced therapeutic effects Required for achieving higher drug levels in the skin for enhanced benefits To maintain therapeutic potential of the drug during storage period

4

International Journal of Pharmaceutics 567 (2019) 118448

D. Arora and S. Nanda

Table 2 Critical Quality Attributes (CQAs) for Resveratrol loaded ethanolic vesicles and their justifications. CQA

Target

Justification(s)

Vesicle size

In range (100–200 nm)

Percent drug entrapment efficiency

Maximum

Permeation flux

In range (5–7 µg h−1 cm−2)

Extent of drug deposition in dermal layer of skin (Skin deposition, SD)

High

It was considered as highly critical due to its importance in permeation and retention of the bioactives in dermal layer. Smaller size facilities movement inside the layers of skin, but beyond a level it leads to systemic absorption. Higher entrapment efficiency is advantageous for achieving maximal therapeutic response. Hence, it was considered to be highly critical. It was also selected as highly critical quality attribute because better therapeutic efficacy of topical formulation depends on the enhanced flux of formulation into the dermal layer of skin. Too high permeation flux may lead to not requisite systemic delivery of drug. High skin retention is required for topical dermatological benefits in which the target site is located in the dermis region of the skin. Therefore it was also selected as highly critical.

corresponding risk ranking to each of the factors. Rank order scores assigned to the material and process parameters attributes on the basis of severity (S), occurrence (O) and detectability (D) is shown in Table 3. On the basis of risk priority number (RPN), high risk parameters were chosen. These were found to be concentration of phospholipid and concentration of ethanol to be used as they showed RPN scores above 300. While, parameters with medium to low associated risk, were set at optimum levels during the whole study.

It was followed by application of ANOVA to ascertain the statistical significance and the degree of the main effects of each variable and their interactions. The regression model resulted in several 2D graphs (perturbation curves and contour plots) and 3D response surface plots showing the interaction effects of two CMAs on each CQA at one time as shown in Figs. 2–4. ANOVA values (p < 0.05) represented statistical significance and confirmed the adequacy of the model generated. 3.4. Response analysis through polynomial equations

3.3. Statistical optimization using 32 full factorial design

3.4.1. Effect of variables on vesicle size Data was analyzed to fit full second-order quadratic or cubic polynomial equation(s) with added interaction terms to correlate the various studied responses with the examined variables. As depicted in 3Dand 2D-plots (Figs. 2a, 3a and 4a), it is indicated that at lower levels of lipid, increase in the levels of ethanol concentration showed negative influence on vesicle size. On the contrary, increasing the levels of lipid, at constant ethanol concentration, increase in vesicle size was observed (positive influence). Thus, lowest level of lipid while highest level of ethanol concentration, resulted in minimum vesicle size. This can be attributed due to steric stabilization mechanism and edge activation due to high ethanol concentration which resulted in low vesicle size (Dave et al., 2010; Garg et al., 2016). Another reason of reduced vesicle size at higher ethanol concentration may be attributed due to

A two-factor, three-level full factorial design was employed to optimize varied critical material attributes (CMAs) influencing the response variables i.e. critical quality attributes (CQAs) as shown in Table 4. Total 13 batches were prepared including four center points per block. Design matrix created via Design Expert® Software (trial version 11.0.5.0, Stat Ease Inc., Minneapolis, USA) with response data for all experimental runs is shown in table 5. The ranges of Y1, Y2, Y3 and Y4 for all batches were 44–243 nm, 36.14–71.83%, 4.79–7.83 µg h−1 cm−2 and 113.65–323.67 µg cm−2 respectively. The response values were fitted to various models by software and the best fitted model was determined. Design summary, build information and values of R2, SD, and % CV are given in Table 6. It was observed that the best-fitted model was quadratic for most of the responses.

Fig. 1. Ishikawa Fish bone diagram for development of ethosomes. 5

International Journal of Pharmaceutics 567 (2019) 118448

D. Arora and S. Nanda

Table 3 Risk assessment using FMEA for resveratrol loaded ethosomes. S. No.

Name of factor

Severity

Occurrence

Detection

RPN

Impact on CQAs

1 2 3 4 5 6 7 8 9 10

Type of phospholipid Concentration of phospholipid (%) Type of aqueous phase Volume of aqueous phase (ml) Concentration of ethanol (%) Stirring speed (rpm) Stirring Time (min) Type of stirrer Method of preparation Sonication speed and time

6 8 4 4 8 6 4 4 4 3

6 8 4 6 7 5 4 3 3 4

4 6 2 2 7 4 4 2 3 3

144 384 32 48 392 120 64 24 36 36

VS, VS, EE EE VS, VS, VS VS VS VS,

EE EE, J, SD EE, J, SD EE

EE

Risk Ranking: Low risk (0–100), Medium risk (101–300), High risk (301–500). Vesicle size (VS), Entrapment efficiency (EE), Permeation flux (J), Skin deposition (SD).

3.4.2. Effect of variables on entrapment efficiency As depicted by 2D contour plot (Fig. 3b) and perturbation curve (Fig. 1b), percent entrapment efficiency of drug is positively correlated with X1 i.e. ethanol concentration. The quadratic equation indicated that on increasing X1 from low to intermediate levels, % EE increases drastically while, increasing beyond it up to higher levels, extent of increase in % EE was found to be less. This can be attributed to its cosolvent effect, thus aqueous core may entrap high amount of drug, however, higher levels of ethanol concentration results in leakier vesicles. Similar positive influence of lipid concentration on entrapment efficiency was observed due to higher drug carrying capacity of the phosholipids molecules (Jain et al., 2015, 2007). The final mathematical model in terms of coded factors as determined by the Design-Expert software is shown below in Eq. (7) for entrapment efficiency.

Table 4 Variables (CQAs and CMAs) used in full factorial design with coded and actual values of CMAs. CMAs

Coded and Actual Levels Low (−1)

Concentration of Phospholipid (X1) Concentration of ethanol (X2) CQAs Vesicle size (Y1) Entrapment efficiency (Y2) Permeation flux J (µg h−1 cm−2) (Y3) Skin deposition SD (µg cm−2) (Y4)

Medium (0)

20 mg/ml 30 mg/ml 15% v/v 25% v/v Target In range (100–200 nm) Maximum Enhanced but in range Maximum

High (+1) 40 mg/ml 35% v/v

significant reduction in membrane thickness possibly due to the formation of a phase with interpenetrating hydrocarbon chain (Jain et al., 2015). These results were concomitant with previous study which reported that influence of increasing lipids on thickening of matrix structure of vesicles resulted in increased vesicle size. The final mathematical model in terms of coded factors as determined by the DesignExpert software is shown below in Eq. (6) for vesicle size.

Vesicle Size (Y1) = +185.55 + 38.17X1 31.43X12

%EE(Y2) = + 56.94 + 12.83X1 + 5.27X2 +

2.71X12 (7)

3.4.3. Effect of variables on permeation flux Permeation flux was calculated through in vitro skin permeation studies. It was observed that resveratrol ethosomal formulation (Etho 7) had shown maximum flux value, i.e. 7.83 ± 1.12 µg h−1 cm−2 as compared to rigid liposomal formulation (2.78 ± 0.97 µg h−1 cm−2) taken as control. The higher permeability flux of ethosomes as compared to classic liposomes can be attributed to more flexibility of the ethanol containing elastic vesicles and their capacity to maintain vesicle integrity. The most imperative factor allied with elasticity of ethanol containing vesicles is their ability to bypass the lipophillic stratum corneum. Being flexible, size and shape of ethosomes get

60.17X2 + 4.25X1X2 (6)

9.43X22

0.8750X1X2

0.1641X22

The above equation clearly depicted the higher influence of concentration of ethanol with respect to concentration of phospholipid on vesicle size.

Table 5 Response values of experimental runs. Batch No

Etho Etho Etho Etho Etho Etho Etho Etho Etho Etho Etho Etho Etho

1 2 3 4 5* 6 7 8 9 10* 11* 12* 13*

Variable levels in coded form

Response Variables

X1

X2

Y1 (Vesicle size)

Y2 (Entrapment efficiency)

Y3 (Permeation flux)

Y4 (Skin deposition)

−1 0 +1 −1 0 +1 −1 0 +1 0 0 0 0

−1 −1 −1 0 0 0 +1 +1 +1 0 0 0 0

187.7 ± 15.23 216.5 ± 23.12 243.9 ± 27.32 98.6 ± 14.57 188.5 ± 12.51 198.3 ± 21.79 44.6 ± 18.78 124.6 ± 25.43 117.9 ± 31.45 188.5 ± 12.51 188.5 ± 12.51 188.5 ± 12.51 188.5 ± 12.51

36.14 49.63 64.32 41.27 57.31 65.37 47.15 62.75 71.83 57.31 57.31 57.31 57.31

6.80 5.34 4.79 7.21 6.57 5.93 7.83 7.01 6.79 6.57 6.57 6.57 6.57

121.27 294.78 213.78 143.41 323.67 251.83 113.65 204.12 187.23 323.67 323.67 323.67 323.67

± ± ± ± ± ± ± ± ± ± ± ± ±

0.58 1.23 0.87 1.43 1.69 2.35 1.25 0.83 1.22 1.69 1.69 1.69 1.69

± ± ± ± ± ± ± ± ± ± ± ± ±

0.39 0.89 0.90 0.56 1.05 0.45 1.12 0.39 0.65 1.05 1.05 1.05 1.05

± ± ± ± ± ± ± ± ± ± ± ± ±

1.34 2.35 1.44 1.76 0.78 2.78 2.14 1.34 1.89 0.78 0.78 0.78 0.78

**Amounts of drug, SA and PG were kept constant i.e. equimolar to PC, 10% w/w of PC and 10% v/v respectively. All other process variables were also constant. [Values are expressed as mean ± s.d., (n = 3)]. * Formulated in quintuplicate as 4 additional center points per block. 6

International Journal of Pharmaceutics 567 (2019) 118448

D. Arora and S. Nanda

Table 6 Design summary and build information. File Version Study Type Design Type Design Model

11.0.5.0 Response Surface 3 Level Factorial Quadratic

Response

Analysis

R2

Adjusted R2

Predicted R2

Std. Dev.

%CV

Model

R1 R2 R3 R4

Polynomial Polynomial Polynomial Polynomial

0.9656 0.9902 0.9612 0.9648

0.9409 0.9832 0.9482 0.9397

0.8696 0.9142 0.9161 0.8776

13.31 1.29 6.50 20.33

3.98 2.31 2.73 4.39

Quadratic Quadratic 2FI Quadratic

Subtype Runs Blocks

Randomized 13 No Blocks

Fig. 2. Perturbation graph for the effect of CMAs (A = Concentration of phospholipid, B = Concentration of alcohol) on CQAs Y1 (vesicle size), Y2 (drug entrapment efficiency), Y3 (Permeation flux), and Y4 (skin deposition).

modify and these are able to cross the stratum corneum barrier by passing through the small skin pores whose diameter is much smaller than size of vesicle (Ascenso et al., 2015; Sharma et al., 2016). Effects of variables on flux are depicted by the 2D plots (Fig. 2c, 3c) and 3D-plots (Fig. 4c). The results of regression analysis are summarized in Table 6 and the following Eq. (8) was obtained for permeation flux:

Permeation flux(Y3) = + 6.50

thus, aids in achieving rapid permeation of drug through stratum corneum till dermal layer of skin. Another possible reason is high solubilization of lipophillic drug (resveratrol) in ethanol which results in higher permeation flux. The formation of multilamellar vesicles at higher phospholipid concentration is attributed towards the controlled permeation of drug and thus negative influence of phospholipid concentration on permeation flux is observed (Dubey et al., 2007; Garg et al., 2016).

0.7217X1 + 0.7833X2 + 0.2425X1X2 (8)

3.4.4. Effect of variables on skin deposition After regression analysis, following quadratic equation was obtained (Eq. (9))

It was observed that on increasing ethanol concentration, higher values of permeation flux was achieved, while a decrease in flux was observed with increasing lipid concentration. The positive influence of ethanol concentration on flux is might be due to membrane lipids fluidizing property and better skin penetration ability of ethosomes,

SD(Y4) = +318.76 + 45.75X1

20.81X2

4.73X1X2

108.87X12

57.04X22

(9) 7

International Journal of Pharmaceutics 567 (2019) 118448

D. Arora and S. Nanda

Fig. 3. Contour plots for evaluating influence of CMAs on CQAs Y1 (vesicle size), Y2 (drug entrapment efficiency), Y3 (Permeation flux), and Y4 (skin deposition).

achieve the desired goals. CMAs were set to target constraints like vesicle size (Y1) 100–200 nm, % entrapment efficiency (Y2) maximum, permeation flux (Y3) in range and skin retention (Y4) maximum. The optimized formulation was obtained at phospholipid concentration (X1) = 35.3 mg/ml and at concentration of ethanol (X2) = 26% with the corresponding desirability (D) value of 0.845. Predicted values of the responses by software at this factor level combination was found to be as Y1 = 191.089 nm, Y2 = 63.482%, Y3 = 6.211 µg h−1 cm−2 and Y4 = 309.40 µg cm−2, while experimentally observed values at this combination was found to be as Y1 = 196.8 ± 4.19 nm, Y2 = 65.78 ± 3.16%, Y3 = 5.89 ± 0.65 µg h−1 cm−2 and −2 Checkpoint analysis was carried out in Y4 = 299.8 ± 4.21 µg cm which the predicted and experimental values were compared. Results showed high degree of prognostic ability with permissible percentage of prediction error, indicating the accuracy and validity of the design combined with a desirability function for the evaluation and optimization of ethosomal formulation as indicated in Fig. 5.

The 2D (Fig. 2d, 3d) and 3D (Fig. 4d) plots depicted the similar effects of both variables on skin deposition. On increasing the ethanol and phospholipid concentration, skin deposition was found to be increased showing an additive effect of both variables on response. The synergistic amalgamation of phospholipids and ethanol is thought to be accountable for a deeper drug distribution and penetration into the skin layers. Highest value of deposition of drug was achieved at intermediate level, while on further increasing the variables to higher levels, deposition was observed to be decreased. Higher levels of phospholipid concentration results in increased vesicle size thus results in decreased permeability flux as well as skin deposition. Changing ethanol concentration from intermediate to further higher level also results in decrease in deposition. It may be attributed to vesicle disruption at higher ethanol concentration. Thus intermediate levels of both the variables were found to be suitable for optimum skin deposition (Garg et al., 2016). 3.5. Multiple response optimizations through checkpoint analysis and desirability approach

3.6. Evaluation of physicochemical characteristics of optimized ethosomal formulation and corresponding hydrogel

The search for the optimized formulation composition was carried out through post analysis point prediction using the desirability function approach with Design expert software, criterion being one having the maximum desirability value (Ahmed et al., 2016; Sailor et al., 2015). The optimization process was performed by setting the CQAs to

The average vesicle size of optimized formulation was found to be 196.8 ± 4.19 nm. Value of the PDI was found to be 0.221 ± 0.013 indicating a homogeneous population. Liposomal vesicle size was found to be 213.7 ± 6.12 nm with PDI value of 0.276 ± 0.054. The results 8

International Journal of Pharmaceutics 567 (2019) 118448

D. Arora and S. Nanda

Fig. 4. 3D Response surface plots for evaluating influence of CMAs on CQAs Y1 (vesicle size), Y2 (drug entrapment efficiency), Y3 (Permeation flux), and Y4 (skin deposition).

are comparable with ethosomal dispersion. Zeta potential of the optimized ethosomal formulation was found to be +21.31 ± 1.53 mV. The addition of cationic lipid i.e. stearylamine induced the positive charge to the formulation. A number of previous studies have reported the enhanced permeation properties of cationic vesicles as compared to neutral vesicles (Choi et al., 2017; Duangjit et al., 2013; Muzzalupo et al., 2017). Duangjit et al have also studied the effect of cationic vesicles containing meloxicam on its permeation profile and they concluded that cationic transfersomes provided greater meloxicam skin permeation than conventional liposomes and meloxicam suspensions. In another study, Muzzalupo et al. have revealed the expected competence shown by cationic vesicles in interacting with negatively charged surfaces or biomolecules. Thus, it is hypothesized that cationic molecules are attracted towards the negatively charged skin membrane and thus penetrate easily through the stratum corneum. The number of ethanol containing vesicles were also evaluated and found to be optimum. Due to low viscosity and poor skin applicability of ethosomal dispersion, it was incorporated into carbopol hydrogel system. This optimized ethosomal dispersion was incorporated into gel resulted in enhanced viscosity. This is beneficial for improved and easier skin application and also required to retain the consistency as well as retention of formulation within the gel matrix. Leakage of drug may also be

Fig. 5. Contour plot for overall desirability of ethosomes as a function of X1 and X2.

9

International Journal of Pharmaceutics 567 (2019) 118448

D. Arora and S. Nanda

Fig. 7. Permeation profile of different resveratrol formulations. Fig. 6. TEM image of optimized ethosomal hydrogel.

lipid molecules in the polar head group region, thereby, fluidity of stratum corneum is enhanced (Garg et al., 2016). In addition to this, diffusion of the vesicles is also assisted by the phospholipids as they tend to merge well with the skin lipid bilayers and provides better penetration of drug by creating small openings in the stratum corneum. These results are concomitant with previous study (Caddeo et al., 2013; Vitonyte et al., 2017). Thus, combination of edge activator (ethanol) in optimum concentration and high phospholipid content in multilamellar vesicles results in their permeation through stratum corneum and dermal layer in the intact form (Caddeo et al., 2013; Manconi et al., 2011). The permeation flux of resveratrol from ethosomal formulation and its corresponding carbopol hydrogel i.e. Opt-etho and Etho-CBPGel was found to be 5.89 ± 0.87 and 5.52 ± 1.05 µg cm−2 h−1 respectively. The flux of drug from ethanolic solution (Res-Alc-Sol) and Resveratrol hydroethanolic carbopol hydrogel (Res-CBP-Gel) were found to 26.11 ± 2.34 and 19.19 ± 3.56 µg cm−2 h−1 respectively. The results are in accordance with previous study which clearly revealed that the vesicular formulation caused an intermediate permeation by providing a hindrance due to multi lamellar structure of vesicles (Garg et al., 2016; Sharma et al., 2016). This significantly high amount of resveratrol permeated through the skin with conventional hydroethanolic solution and its corresponding gel containing drug depicted the increase systemic exposure of resveratrol and less skin retention. Considering the requirement of the topical formulation, an intermediate permeation flux is required with enhanced skin deposition or retention.

restricted during storage. 1% w/v Carbopol 934P was used due to its versatile properties like non-newtonian pseudoplastic rheological behaviour, good spreadability, viscosity and compliance for topical use (Nayak et al., 2018). Resveratrol loaded ethosomal hydrogel (Etho-CBPGel) was found to be physically stable, free from grittiness and translucent. Integrity of incorporated vesicles was also confirmed through microscopic studies. No evidence of vesicles disruption was found indicating that the vigor involved in hydrogel formulation was well tolerated by the vesicles. The TEM image of optimized ethosomal hydrogel formulation is shown in Fig. 6 revealed that the vesicles has well identified structure and spherical in shape. No loss of drug was observed during gelling procedure as drug content in Etho-CBP-Gel was found to be approximately 100%. Apparent viscosity of ethosomal hydrogel was found to be 35.43 ± 3.22 Pa.s. Rheological studies clearly indicated the shear thinning nature of the systems (Yadav et al., 2016). Viscosity and yield values of the gel were found within the limits representing higher plasticity and smaller fluidity, thus, sufficient rigidity of gel structure is expected (Negi et al., 2016; Yadav et al., 2016). Spreadability of the ethosomal hydrogel (6.72 ± 0.54 cm2) was slightly lesser owing to its increased viscosity than spreadability of plain carbopol hydrogel (7.63 ± 0.31 cm2). pH was found to be 7.4 ± 0.5 confirms the absence of any chances of causing irritation to the skin. Thus, formulation was considered as patient compliant and skin-friendly as desired for any topical formulation. 3.7. Ex vivo drug permeation studies of ethosomal gel formulation

3.8. Skin deposition studies

Ex vivo skin permeation study of different resveratrol formulations i.e. optimized ethosomes (Opt-etho), ethosomal carbopol hydrogel (Etho-CBP-Gel), ethanolic solution (Res-Alc-Sol), Resveratrol hydroethanolic carbopol hydrogel (Res-CBP-Gel) and conventional cream of Resveratrol (Res-cream) was carried out and permeation profiles were compared with each other. Permeation profiles are depicted in Fig. 7 and values of permeation flux, permeability coefficient and enhancement ratio are indicated in Table 7. The permeation rates and permeability coefficient values of resveratrol from the optimized ethosomal formulation and ethosomal hydrogel were 2.18 and 2.04 times higher than the conventional cream formulation as indicated in Table 8 (p < 0.01). This significant enhancement ratio of ethosomal formulation can be depicted by its fluidic characteristics due to the synergistic effects of its constituent excipients like ethanol, propylene glycol and phospholipids. The property of ethanol to fluidize vesicular lipid bilayers as well as stratum corneum lipids mutually provides a greater flexibility to the ethanol containing vesicles for enhanced dermal permeability (Dubey et al., 2007; Jain et al., 2015). A decline in phase transition temperature (Tm) of the stratum corneum is usually observed as ethanol intermingles with its

Skin retention values from the studied formulations are given in Table 8 and Fig. 8. The amount of drug evaluated in receptor compartment as amount permeated is the indicator of transdermal delivery, whereas amount retained in deeper layer of skin, i.e. epidermis and dermis, is the index of topical delivery. The value of drug deposition in skin via ethosomal formulation was observed to be 299.8 ± 20.07 µg cm−2 (62.75 ± 4.21%) and from ethosomal carbopol hydrogel was 308.60 ± 17.30 µg cm−2 (64.6 ± 3.62%). These deposition values are significantly higher (p < 0.001) than the retention achieved by other tested formulations. The drug retained in dermal layer by conventional cream is 4.6 times lesser than that of ethosomal hydrogel. The results can be ascribed due to fusion of lipophillic moieties present in vesicles with the skin and thus forming micro drug reservoirs. Thus, sustained and controlled drug delivery as well as high retention of drug at the target dermal layer of skin is achieved due to these micro drug depots (Jain et al., 2015). The addition of Carbopol 934 into the optimized ethosomal formulation has slightly increased the deposition value as compared to low viscous ethosomal dispersion alone. This slight increase is might be due to the internal 3D structural 10

International Journal of Pharmaceutics 567 (2019) 118448

D. Arora and S. Nanda

Table 7 Permeation parameters of different resveratrol formulations. Formulation Code

Permeation Flux, Jss (µg cm−2 h−1)

Permeability coefficient, Kp (cm h−1) (*10−3)

Enhancement Ratio, Er

Skin retention (%)

Opt-etho Etho-CBP-Gel Res-CBP-Gel Res-Alc-Sol Res-Cream

5.89 ± 0.87 5.52 ± 1.05 19.19 ± 3.56 26.11 ± 2.34 2.70 ± 0.73

3.92 3.68 12.79 17.41 1.80

2.18 2.04 7.10 9.67 –

62.75 ± 4.21 64.60 ± 3.62 15.38 ± 3.17 6.80 ± 2.76 14.05 ± 4.11

[Values are expressed as mean ± s.d., (n = 3)].

network of hydrogel and composite nature of the material. The permeability flux of optimized ethosomes and ethosomal hydrogel were similar without any statistically significant difference (p > 0.05) indicating that addition of carbopol 934 has not caused any interference in optimized permeability of ethosomal vesicles. Conclusively, it was observed that ethosomal hydrogel has shown similar resveratrol permeation and skin deposition profile and also cater the needs of topical application such as easier skin application and high viscosity as compared to ethosomal dispersion. Thus, the results evidently revealed that ethosomal system embedded in carbopol hydrogel is more effective and suitable for enhanced drug delivery into the deeper layers of the skin and consequently may provide improved dermatological benefits. 3.9. Skin distribution by CLSM The CLSM microscopy after application of C6 loaded plain hydrogel, ethosomal dispersion and ethosomal hydrogel was carried out to evaluate the degree of penetration and contributory pathways concerned through the permeation of formulation into the skin. Fig. 9a clearly revealed that plain hydrogel got retained only on upper layer of skin and therefore have not penetrated into the skin adhering prominently to upper layer. However, ethosomal dispersion and ethosomal hydrogel was found to be vastly scattered in the deeper skin layers as shown in Fig. 9b. This was shown by the high intensity of the green fluorescence via ethosomal formulations as compared to the control. This can be attributed to disorganization of lipoid membrane barriers, configuration of channels and passage through skin appendages involving hair follicles, sebaceous glands and associated structures, thus, leading to enhanced penetration ability and better skin deposition potential of ethosomal formulations (Ahmed et al., 2016). The results thus confirmed the appropriateness of the ethosomal formulation as a topical drug delivery system that enhances the permeation and deposition of the associated bioactives through the deeper skin layers.

Fig. 8. Skin deposition of resveratrol from various formulations [Each data represents mean ± S.D. (n = 3). Significance was tested using one way ANOVA and Tukey–Kramer post test. Three asterisks represents p < 0.001 compared to the control Res-Cream].

or change in physical appearance was observed on visual inspection for 6 months. Insignificant changes in vesicle size, zeta potential and residual drug content (p > 0.05) was observed at refrigerated condition. % residual drug content and vesicle size in ethosomal dispersion was found to be 97.43 ± 1.54% and 213.8 ± 5.71 nm (initial vesicle size was 196.8 ± 4.19 nm), p > 0.05 as compared to initial after 6 months as shown in Table 8. In addition to this, developed ethosomal hydrogel formulation was stable enough to maintain pH, consistency and viscosity throughout the test period at refrigerated condition. However, at 25 ± 2 °C an increase in vesicle size was observed. After 3 months it was found to be 254.8 ± 6.79 nm which was observed to be significant (p < 0.05) while, after 6 months it was significantly increased up to 321.9 ± 8.23 nm (p < 0.01). It might be due to vesicular fusion, agglomeration and coalescence of bilayer membrane of vesicles which is clearly revealed by visible signs of physical instability and loss of consistency (Ahad et al., 2013). Whereas, storage at higher temperature, results in decrease of residual drug content i.e. 70.16 ± 2.71% as compared to initial (p < 0.001) over a period of 6 months. Insignificant change in the zeta potential of the vesicular dispersion was observed at all the temperature conditions. This decrease might be attributed due to the degradation and gel-to-liquid transition of lipids bilayers leading to defective membrane packing. Taken as a whole, it

3.10. Stability studies The storage stability of the colloidal carriers is of great concern as it is the principal restraint in the development of clinically acceptable marketed formulations. The storage stability was evaluated by measuring the changes in physical appearance, mean vesicle size, zeta potential, pH and residual drug content after six months storage at the refrigerated condition (4 ± 2 °C) and room temperature (25 ± 2 °C/ 60 ± 5% RH). At refrigerated conditions, no evidence of aggregation

Table 8 Stability Studies of Resveratrol loaded ethosomal gel formulations after 6 months of storage at various conditions. Time

Initial 3 months 6 months

Vesicle Size (nm)

Zeta Potential (mV)

% Residual Drug Content

4 ± 2 °C

25 ± 2 °C

4 ± 2 °C

25 ± 2 °C

4 ± 2 °C

25 ± 2 °C

196.8 ± 4.19 205.4 ± 3.63 213.8 ± 5.71

196.8 ± 4.19 254.8 ± 6.79 321.9 ± 8.23

+21.31 ± 1.53 +20.45 ± 0.87 +20.12 ± 1.19

+21.31 ± 1.53 +20.86 ± 1.88 +19.77 ± 0.76

100 99.65 ± 0.86 97.43 ± 1.54

100 92.12 ± 1.87 70.16 ± 2.71

[Values are expressed as mean ± s.d., (n = 3)].

11

International Journal of Pharmaceutics 567 (2019) 118448

D. Arora and S. Nanda

Fig. 9. CLS micrographs (a) C6 loaded plain hydrogel (b) C6 loaded ethosomal hydrogel.

can be concluded that the ethosomal hydrogel formulations should be stored at lower temperature for maximum stability and minimum drug loss.

Macromol. 67, 99–104. https://doi.org/10.1016/j.ijbiomac.2014.03.011. Ahmed, T.A., El-Say, K.M., Aljaeid, B.M., Fahmy, U.A., Abd-Allah, F.I., 2016. Transdermal glimepiride delivery system based on optimized ethosomal nano-vesicles: preparation, characterization, in vitro, ex vivo and clinical evaluation. Int. J. Pharm. 500, 245–254. https://doi.org/10.1016/j.ijpharm.2016.01.017. Ansari, K.A., Vavia, P.R., Trotta, F., Cavalli, R., 2011. Cyclodextrin-based nanosponges for delivery of resveratrol. in vitro characterisation, stability, cytotoxicity and permeation study. AAPS PharmSciTech 12, 279–286. https://doi.org/10.1208/s12249-0119584-3. Arora, D., Khurana, B., Kumar, M.S., Vyas, S.P., 2011. Oral Immunization against Hepatitis B Virus using. Int. J. Recent Adv. Pharm. Res. 1, 45–51. Arora, D., Khurana, B., Narang, R.K., Nanda, S., 2016. Quality by design (QbD approach for optimization and development of nano drug delivery systems. Trends Drug Deliv. 3, 23–32. Ascenso, A., Raposo, S., Batista, C., Cardoso, P., Mendes, T., Praça, F.G., Bentley, M.V.L.B., Simões, S., 2015. Development, characterization, and skin delivery studies of related ultradeformable vesicles: transfersomes, ethosomes, and transethosomes. Int. J. Nanomed. 10, 5837–5851. https://doi.org/10.2147/IJN.S86186. Beg, S., Saini, S., Bandopadhyay, S., Katare, O.P., Singh, B., 2018. QbD-driven development and evaluation of nanostructured lipid carriers (NLCs) of Olmesartan medoxomil employing multivariate statistical techniques. Drug Dev. Ind. Pharm. 44, 407–420. https://doi.org/10.1080/03639045.2017.1395459. Caddeo, C., Díez-sales, O., Pons, R., Fernàndez-busquets, X., Fadda, A.M., Manconi, M., 2014. Topical anti-inflammatory potential of quercetin in lipid-based nanosystems. In vivo and in vitro evaluation. Pharm. Res. 31, 959–968. https://doi.org/10.1007/ s11095-013-1215-0. Caddeo, C., Diez, O., Valenti, D., Ruiz, A., Maria, A., Manconi, M., 2013. Inhibition of skin inflammation in mice by diclofenac in vesicular carriers: liposomes, ethosomes and PEVs. Int. J. Pharm. 443, 128–136. https://doi.org/10.1016/j.ijpharm.2012.12.041. Chedea, V.S., Vicaş, S.I., Sticozzi, C., Pessina, F., Frosini, M., Maioli, E., Valacchi, G., 2017. Resveratrol: from diet to topical usage. Food Funct. 8, 3879–3892. https://doi. org/10.1039/c7fo01086a. Choi, J.U., Lee, S.W., Pangeni, R., Byun, Y., Yoon, I., 2017. Preparation and in vivo evaluation of cationic elastic liposomes comprising highly skin-permeable growth factors combined with hyaluronic acid for enhanced diabetic wound-healing therapy. Acta Biomater. 57, 197–215. https://doi.org/10.1016/j.actbio.2017.04.034. Claycamp, H.G., Kona, R., Fahmy, R., Hoag, S.W., 2016. Quality-by-design II: application of quantitative risk analysis to the formulation of ciprofloxacin tablets. AAPS PharmSciTech 17, 233–244. https://doi.org/10.1208/s12249-015-0349-2. Dave, V., Kumar, D., Lewis, S., Paliwal, S., 2010. Ethosome for enhanced transdermal drug delivery of aceclofenac. Int. J. Drug Deliv. 2, 81–92. https://doi.org/10.1016/j. rser.2017.10.055. Degim, I.T., 2006. New tools and approaches for predicting skin permeability. Drug Discov. Today 11, 517–523. https://doi.org/10.1016/j.drudis.2006.04.006. Dhawan, S., Nanda, S., 2018. In-vitro estimation of photo-protective potential of pomegranate seed oil and development of a nanoformulation. Curr. Nutr. Food Sci. 14, 1–16. https://doi.org/10.2174/1573401314666180223134235. Duangjit, S., Opanasopit, P., Rojanarata, T., Ngawhirunpat, T., 2013. Evaluation of meloxicam-loaded cationic transfersomes as transdermal drug delivery carriers. AAPS PharmSciTech 14, 133–140. https://doi.org/10.1208/s12249-012-9904-2. Dubey, V., Mishra, D., Dutta, T., Nahar, M., Saraf, D.K., Jain, N.K., 2007. Dermal and transdermal delivery of an anti-psoriatic agent via ethanolic liposomes. J. Control. Release 123, 148–154. https://doi.org/10.1016/j.jconrel.2007.08.005. Garg, B.J., Garg, N.K., Beg, S., Singh, B., Katare, O.P., 2016. Nanosized ethosomes-based hydrogel formulations of methoxsalen for enhanced topical delivery against vitiligo: formulation optimization, in vitro evaluation and preclinical assessment. J. Drug Target. 24, 233–246. https://doi.org/10.3109/1061186X.2015.1070855.

4. Conclusion In this study QbD and DoE concepts were followed for successful development of the ethosomal hydrogel of resveratrol having enhanced dermatological characteristics. The study ratified that the implementation of DoE helped in determining and optimizing influential process and material variables to get desired quality attributes of final product. Intermediate levels of ethanol concentration and phospholipid concentration were found to be the major critical material attributes which significantly influence the vesicle size, entrapment efficiency, drug permeation as well as skin deposition. Supported on regression analysis, it was ratified that to achieve desired dermatological properties of topical formulation, formulation variables can be suitably manipulated. Further, the ethosomal hydrogel were found to be stable and suitable for topical use. Permeation flux and skin deposition was also found to be improved as compared to conventional cream. Thus, it can be concluded that ethosomal hydrogel can be used to develop patient compliant and stable topical product for enhanced dermatological benefits. However, in addition to it, skin irritation and cytotoxicity studies are warranted to assess and for proving its actual clinical acceptance. Acknowledgement Authors are thankful to Sami Labs, Bangalore, India for generously providing the gift samples of Resveratrol. Declaration of Competing Interest The authors declare no conflict of interest. References Abd, E., Yousef, S.A., Pastore, M.N., Telaprolu, K., Mohammed, Y.H., Namjoshi, S., Grice, J.E., Roberts, M.S., 2016. Skin models for the testing of transdermal drugs. Clin. Pharmacol. Adv. Appl. 8, 163–176. https://doi.org/10.2147/CPAA.S64788. Ahad, A., Aqil, M., Kohli, K., Sultana, Y., Mujeeb, M., 2013. Enhanced transdermal delivery of an anti-hypertensive agent via nanoethosomes: statistical optimization, characterization and pharmacokinetic assessment. Int. J. Pharm. 443, 26–38. https:// doi.org/10.1016/j.ijpharm.2013.01.011. Ahad, A., Raish, M., Al-Mohizea, A.M., Al-Jenoobi, F.I., Alam, M.A., 2014. Enhanced antiinflammatory activity of carbopol loaded meloxicam nanoethosomes gel. Int. J. Biol.

12

International Journal of Pharmaceutics 567 (2019) 118448

D. Arora and S. Nanda Geusens, B., Van Gele, M., Braat, S., De Smedt, S.C., Stuart, M.C.A., Prow, T.W., Sanchez, W., Roberts, M.S., Sanders, N.N., Lambert, J., 2010. Flexible nanosomes (SECosomes) enable efficient siRNA delivery in cultured primary skin cells and in the viable epidermis of ex vivo human skin. Adv. Funct. Mater. 20, 4077–4090. https://doi.org/10. 1002/adfm.201000484. ICH, 2005. International conference on harmonization of technical requirements for registration of pharmaceuticals for human use, Quality Risk Management Q9, ICH Harmonised Tripartite Guideline. https://doi.org/10.1371/journal.pone.0150583. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, 2009. Pharmaceutical Development Q8(R2). ICH Harmon. Tripart. Guidel. https://doi.org/10.1111/eff.12061. Jain, S., Patel, N., Madan, P., Lin, S., 2015. Quality by design approach for formulation, evaluation and statistical optimization of diclofenac-loaded ethosomes via transdermal route. Pharm. Dev. Technol. 20, 473–489. https://doi.org/10.3109/ 10837450.2014.882939. Jain, S., Tiwary, A.K., Sapra, B., Jain, N.K., 2007. Formulation and evaluation of ethosomes for transdermal delivery of lamivudine. AAPS PharmSciTech 8, E1–E9. https:// doi.org/10.1208/pt0804111. Khurana, B., Goyal, A.K., Budhiraja, A., Aora, D., Vyas, S.P., 2013. Lipoplexes versus nanoparticles: PDNA/siRNA delivery. Drug Deliv. 20, 57–64. https://doi.org/10. 3109/10717544.2012.752419. Kushwaha, A., Shivakumar, H.N., Murthy, S.N., 2016. Iontophoresis for drug delivery into the nail apparatus: exploring hyponychium as the site of delivery. Drug Dev. Ind. Pharm. 42, 1678–1682. https://doi.org/10.3109/03639045.2016.1165690. Lambert, W.J., 2010. Considerations in developing a target product profile for parenteral pharmaceutical products. AAPS PharmSciTech 11, 1476–1481. https://doi.org/10. 1208/s12249-010-9521-x. Liu, X., Li, Z., Wang, X., Chen, Y., Wu, F., Men, K., Xu, T., Luo, Y., Yang, L., 2016. Novel antimicrobial peptide—modified azithromycin-loaded liposomes against methicillinresistant Staphylococcus aureus. Int. J. Nanomed. 11, 6781–6794. https://doi.org/ 10.2147/IJN.S107107. Manconi, M., Caddeo, C., Sinico, C., Valenti, D., Cristina, M., Biggio, G., Maria, A., 2011. Ex vivo skin delivery of diclofenac by transcutol containing liposomes and suggested mechanism of vesicle – skin interaction. Eur. J. Pharm. Biopharm. 78, 27–35. https:// doi.org/10.1016/j.ejpb.2010.12.010. Melorose, J., Perroy, R., Careas, S., 2011. Nanocosmetics and nanomedicines new approaches for skin care. Statewide Agric. Land Use Baseline 2015. https://doi.org/10. 1007/978-3-642-19792-5. Menon, G.K., 2002. New insights into skin structure: scratching the surface. Adv. Drug Deliv. Rev. 54, S3–S17. https://doi.org/10.1016/S0169-409X(02)00121-7. Muzzalupo, R., Pérez, L., Pinazo, A., Tavano, L., 2017. Pharmaceutical versatility of cationic niosomes derived from amino acid-based surfactants: skin penetration behavior and controlled drug release. Int. J. Pharm. 529, 245–252. https://doi.org/10.1016/j. ijpharm.2017.06.083. Nayak, K., Katiyar, S.S., Kushwah, V., Jain, S., 2018. Coenzyme Q10 and retinaldehyde co-loaded nanostructured lipid carriers for efficacy evaluation in wrinkles. J. Drug Target. 26, 333–344. https://doi.org/10.1080/1061186X.2017.1379527. Negi, P., Singh, B., Sharma, G., Beg, S., Raza, K., Katare, O.P., 2016. Phospholipid microemulsion-based hydrogel for enhanced topical delivery of lidocaine and prilocaine: QbD-based development and evaluation. Drug Deliv. 23, 951–967. https://doi. org/10.3109/10717544.2014.923067. Pandey, V., Golhani, D., Shukla, R., 2015. Ethosomes: Versatile vesicular carriers for efficient transdermal delivery of therapeutic agents. Drug Deliv. 22, 988–1002. https:// doi.org/10.3109/10717544.2014.889777.

Pando, D., Caddeo, C., Manconi, M., Fadda, A.M., Pazos, C., 2013. Nanodesign of olein vesicles for the topical delivery of the antioxidant resveratrol. J. Pharm. Pharmacol. 65, 1158–1167. https://doi.org/10.1111/jphp.12093. Pangeni, R., Sahni, J.K., Ali, J., Sharma, S., Baboota, S., 2014. Resveratrol: review on therapeutic potential and recent advances in drug delivery. Expert Opin. Drug Deliv. 11, 1285–1298. https://doi.org/10.1517/17425247.2014.919253. Petchsangsai, M., Rojanarata, T., Opanasopit, P., Ngawhirunpat, T., 2014. The combination of microneedles with electroporation and sonophoresis to enhance hydrophilic macromolecule skin penetration. Biol. Pharm. Bull. 37, 1373–1382. https://doi.org/ 10.1248/bpb.b14-00321. Ruivo, J., Francisco, C., Oliveira, R., Figueiras, A., 2015. The main potentialities of resveratrol for drug delivery systems. Braz. J. Pharm. Sci. 51, 499–513. https://doi.org/ 10.1590/S1984-82502015000300002. Sailor, G., Seth, A.K., Parmar, G., Chauhan, S., Javia, A., 2015. Formulation and in vitro evaluation of berberine containing liposome optimized by 32 full factorial designs. J. Appl. Pharm. Sci. 5, 23–28. https://doi.org/10.7324/JAPS.2015.50704. Saydam, M., Takka, S., 2018. Development and in vitro evaluation of pH-independent release matrix tablet of weakly acidic drug valsartan using quality by design tools. Drug Dev. Ind. Pharm. 44, 1905–1917. https://doi.org/10.1080/03639045.2018. 1496450. Sharma, G., Goyal, H., Thakur, K., Raza, K., Katare, O.P., 2016. Novel elastic membrane vesicles (EMVs) and ethosomes-mediated effective topical delivery of aceclofenac: a new therapeutic approach for pain and inflammation. Drug Deliv. 23, 3135–3145. https://doi.org/10.3109/10717544.2016.1155244. Singh, B., Dahiya, M., Saharan, V., Ahuja, N., 2005. Optimizing drug delivery systems using systematic “design of experiments”. Part II: retrospect and prospects. Crit. Rev. Ther. Drug Carrier Syst. 22, 27–106. https://doi.org/10.1615/ CritRevTherDrugCarrierSyst.v22.i3.10. Singh, B., Kapil, R., Nandi, M., Ahuja, N., 2011. Developing oral drug delivery systems using formulation by design: vital precepts, retrospect and prospects. Expert Opin. Drug Deliv. 8, 1341–1360. https://doi.org/10.1517/17425247.2011.605120. Sobanko, J.F., Miller, C.J., Alster, T.S., 2012. Topical anesthetics for dermatologic procedures: a review. Dermatologic Surg. 38, 709–721. https://doi.org/10.1111/j.15244725.2011.02271.x. Tokumoto, S., Higo, N., Todo, H., Sugibayashi, K., 2016. Effect of combination of lowfrequency sonophoresis or electroporation with iontophoresis on the mannitol flux or electroosmosis through excised skin. Biol. Pharm. Bull 39, 1206–1210. https://doi. org/10.1248/bpb.b15-00696. Vitonyte, J., Letizia, M., Caddeo, C., Valenti, D., Esteban, J., Usach, I., Nacher, A., Matos, M., Gutiérrez, G., Orrù, G., Fernàndez-busquets, X., Maria, A., Manconi, M., 2017. Bifunctional viscous nanovesicles co-loaded with resveratrol and gallic acid for skin protection against microbial and oxidative injuries. Eur. J. Pharm. Biopharm. 114, 278–287. https://doi.org/10.1016/j.ejpb.2017.02.004. Yadav, N.K., Nanda, S., Sharma, G., Katare, O.P., 2016. Systematically optimized coenzyme q10-loaded novel proniosomal formulation for treatment of photo-induced aging in mice: Characterization, biocompatibility studies, biochemical estimations and anti-aging evaluation. J. Drug Target. 24, 257–271. https://doi.org/10.3109/ 1061186X.2015.1077845. Yang, L., Wu, L., Wu, D., Shi, D., Wang, T., Zhu, X., 2017. Mechanism of transdermal permeation promotion of lipophilic drugs by ethosomes. Int. J. Nanomed. 12, 3357–3364. https://doi.org/10.2147/IJN.S134708. Yang, S., Meyskens, F.L., 2005. Alterations in activating protein 1 composition correlate with phenotypic differentiation changes induced by resveratrol in human melanoma. Mol. Pharmacol. 67, 298–308. https://doi.org/10.1124/mol.104.006023.shown.

13