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Improved bioavailability of raloxifene hydrochloride using limonene containing transdermal nano-sized vesicles
Journal of Drug Delivery Science and Technology 52 (2019) 468–476
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Improved bioavailability of raloxifene hydrochloride using limonene containing transdermal nano-sized vesicles
T
Ayesha Waheeda, Mohd Aqila,∗, Abdul Ahadb, Syed Sarim Imamc, Thasleem Moolakkadatha, Zeenat Iqbala, Asgar Alia a
Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard (Deemed University), M. B. Road, New Delhi, 110062, India Department of Pharmaceutics, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh, 11451, Saudi Arabia c School of Pharmacy, Glocal University, Saharanpur, U.P, India b
ARTICLE INFO
ABSTRACT
Keywords: Limonene Penetration enhancer containing vesicles Raloxifene hydrochloride Transfersomes
The present investigation was carried out with the aim of optimization of raloxifene hydrochloride-entrapped, limonene containing transfersomes for transdermal delivery. Several formulations were prepared by varying independent variables such as Phospholipid, sodium cholate and limonene while vesicles size, entrapment efficiency and polydispersity index were the dependent variables for Box-Behnken Design. Further, bioavailability assessment of raloxifene in Wistar rats was evaluated after transdermal application and compared with its oral formulation. It was observed that the optimized raloxifene loaded transfersomes formulation showed vesicle size of 107.8 nm, polydispersity index of 0.252 and entrapment efficiency of 90.2% which is close to the predicted values of 114.45 nm, 0.251 and 82.82% respectively generated by the Design Expert software. The TEM image shows the outline and the core of the well identified sealed spherical structure. Confocal laser microscopy study demonstrated that the prepared transfersomes formulation was capable to increase the permeation of probe dye into deeper layer of rat's skin in comparison to the control dye solution. The transdermal flux presented by the transfersomes gel formulation was found to be 13.20 μg/cm2/h whereas the control gel showed the transdermal flux of 2.35 μg/cm2/h across rat skin. Further the in vivo pharmacokinetic study revealed that the relative bioavailability following application of transfersomes gel in Wistar rats was found be increased by 2.71 times as compared to the oral suspension of raloxifene. It was concluded that the prepared transfersomes formulation was found to be a potentially useful drug carrier for the enhancement of raloxifene bioavailability in rats.
1. Introduction Raloxifene hydrochloride (RLX) is regarded as a BCS class II drug, it is promptly absorbed from the GI tract and face extensive first-pass metabolism and about sixty percent of a dosage is absorbed; nevertheless, its absolute bioavailability is merely two percent [1]. Transdermal delivery is of one the methods to improve bioavailability of several class of drug particularly for BCS class II drugs. Past decades have witnessed significantly increased interest in the exploration of new transdermal techniques to enhance drug permeation into or through the skin [2]. Transfersomes help the drug to pass across the stratum corneum unlike the conventional liposomes, edge activators incorporate into the lipid bilayer increasing the resulting vesicle elasticity [3,4]. Due to the elasticity, transfersomes vesicles are capable to squeeze via networks one-tenth the diameter of the vesicles and permitting them to penetrate across the stratum corneum via intercellular
∗
route [5]. Previously RLX -loaded transfersomes were prepared by Mahmood et al., 2014. The best chosen formulation exhibited vesicles size of 134 nm with an EE of 91% and flux of 6.5 μg/cm2/h [6]. In another study, Mahmood et al., 2018 prepared RLX-loaded ethosomes formulations. The final formulation presented flux of 22.14 ± 0.83 μg/ cm2/h. Authors concluded that the animal study showed 157% times increased in relative bioavailability of RLX-ethosomes formulation when compared to oral control formulation in rats [7]. In another study, Joshi et al., 2018 prepared RLX -transfersomes formulations. Authors concluded that the RLX-transfersomes suspension exhibited better release than RLX-transfersomes loaded cream, gel and patch formulations [8]. In present study, we have utilized the combined effect of transfersomes (flexible drug carrier) and terpene (natural penetration enhancer). Terpenes are acquired from natural sources, they are less toxic
Corresponding author. E-mail addresses: [email protected] (M. Aqil), [email protected], [email protected] (A. Ahad).
https://doi.org/10.1016/j.jddst.2019.05.019 Received 5 March 2019; Received in revised form 28 April 2019; Accepted 7 May 2019 Available online 08 May 2019 1773-2247/ © 2019 Elsevier B.V. All rights reserved.
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and are efficient penetration enhancers for both lipophilic and hydrophilic drugs; the FDA included them in generally regarded as safe (GRAS) category [9,10]. Limonene is a hydrocarbon lipophilic terpene and it fluidizes or disturbs the integrity of the stratum corneum for improved the passage of drugs across the skin [11,12]. Earlier reports are available that reflects the use of limonene as an encouraging permeation enhancer for the delivery of drugs via transdermal route [12–15]. Hence, the purpose of this study was to examine the effect of phospholipid 90G (X1, −1 = 40 mg, 0 = 60 mg and +1 = 80 mg), sodium cholate (X2, −1 = 6 mg, 0 = 7.5 mg and +1 = 9 mg), and limonene (X3, −1 = 15 μl, 0 = 20 μl and +1 = 25 μl) on the vesicles size, EE and PDI of RLX loaded transfersomes. Further, in vitro skin permeation, CLSM, skin irritation and bioavailability assessment of RLX loaded formulation were investigated.
The EE was calculated using following equation EE (%) = (Total drug − drug detected only in supernatant) / Total drug × 100 2.6. Transmission electron microscopy The vesicles surface morphology of the transfersomes was evaluated by “transmission electron microscopy” [19]. The sample was placed on copper grids then “negatively stained by phosphotungstic acid (1%)” then permitted to air dried and then visualized by microscope at “an accelerating voltage of 100 kV”. 2.7. Confocal laser scanning microscopy (CLSM) For CLSM study excised rat skin was treated with rhodamine loaded optimized transfersomes formulation for 24 h and compared it rat's skin treated with plain rhodamine solution. After 24 h, the excised rat skin was clean with distilled water and glass slides were prepared and visualized by confocal microscope with an “argon laser beam with excitation at 540 nm” and “emission at 625 nm”.
2. Materials and methods 2.1. Materials RLX and Sodium cholate was a generous gift from Kusum Healthcare Pvt. Ltd., Delhi and Thomas Baker, Mumbai respectively. Phospholipon 90G was a generous gift from Lipoid, Germany. Limonene was purchased from Sigma-Aldrich, USA. All other reagents are of analytical grade and HPLC grade.
2.8. Preparation of RLX loaded transfersomes gel The final RLX transfersomes preparation was transformed to a gel preparation by dissolving 1% Carbopol 934 in water with continuous stirring. After complete dispersal of Carbopol into water, the dispersion was allowed for nightlong to complete swelling. After this, triethanolamine and bezalkonium chloride was added to obtain a homogenous dispersion of gel. Lastly, the final RLX-transfersomes formulation was added to the gel base by gentle stirring to get the final gel preparation [21].
2.2. Preparation of RLX-entrapped limonene-containing transfersomes Transfersomes are prepared by thin layer evaporation procedure [16–18]. Briefly, round bottomed flask having mixture of phospholipid, RLX and sodium cholate previously dissolved in methanol/chloroform (1:1 v/v) was attached with “rotary evaporator” maintained under reduced pressure and allows the organic solvent to evaporate. Later the thin dried lipid film so formed on the inner wall of round bottomed flask was rehydrated for 30 min at a rotation of 60 rpm with phosphate buffered pH 7.4. At last, the dispersion having large vesicles was probe sonicated at 4 °C for 10 min (gap of 5 min between each cycle) to produced transfersomes.
2.9. Evaluation of RLX loaded transfersomes gel 2.9.1. Homogeneity, pH, spreadability, extrudability and drug content The developed gel was placed in clear and transparent container and examined visually for visual aspects and existence of any masses formation and the gel pH was assessed by pH meter. Method for the evaluation of gel spreadability and extrudability are well explained by other researchers [22,23]. Further, the drug content was found out by dissolving the gel (1 g) in methanol (100 ml) then sonicated for the extraction of drug in methanol. Subsequently the final mixture was filtered, diluted and examined by means of UV spectrophotometer.
2.3. Experimental design for optimization of transfersomes A Box-Behnken design (3-factor, 3-level) was used for the preparation of transfersomes and the Design Expert (version 11.0) software generated 15 different formulations by varying the composition of phospholipid 90G (X1, −1 = 40 mg, 0 = 60 mg and +1 = 80 mg), sodium cholate (X2, −1 = 6 mg, 0 = 7.5 mg and +1 = 9 mg), and limonene (X3, −1 = 15 μl, 0 = 20 μl and +1 = 25 μl) and the formulations were characterized based on the dependent responses like vesicles size (Y1), polydispersity index (PDI, Y2), and entrapment efficiency (EE, Y3).
2.9.2. Texture analysis The gel was analyzed in texture analyzer (Stable Micro Systems, Exponent Lite, Vienna, U.K.) with a back extruding probe of 30 mm diameter. Various parameters like firmness, cohesiveness, consistency and index of viscosity were investigated.
2.4. Vesicle size, size distribution and zeta potential measurements
2.9.3. In vitro drug release study For the evaluation of drug release, the transfersomes gel was kept in activated dialysis bag installed in dissolution chamber. The dissolution chamber was filled with 200 ml of PBS, pH 7.4 and isopropyl alcohol (70:30 v/v), the medium was agitated with magnetically stirred at 600 rpm, and the medium temperature was kept at 37 ± 0.5 °C. The Aliquots of 2 ml was pipetted out at each interval and refilled with fresh vehicle. The results found were fit into “drug release kinetic models”.
For analysis, RLX loaded transfersomes samples were diluted hundred times with Milli-Q water and the vesicles size and size distribution were evaluated by “Zetasizer Nano ZS (Malvern Instruments Ltd, Worcestershire, UK)” at 25 ± 1 °C and at a scattering angle of 90°. Zeta potential of samples was also evaluated by same instrument at 25 ± 1 °C. 2.5. Determination of EE (%)
2.9.4. In vitro skin permeation study In this study the prepared rat's skin specimen was fixed on Franz vertical cell (surface area 6.33 cm2). The receiver cell was made full with PBS (pH 7.4) and isopropyl alcohol (15 ml, 70:30 v/v) which was agitated with magnetic bead at 600 rpm and the temperature of vehicle
Separation of un-entrapped drug from the prepared transfersomes was carried out by centrifugation method [19]. Transfersomes formulation was centrifuged at 25000 rpm for 1 h and the supernatant was removed and analyzed by UV- spectrophotometer at 287 nm λmax [20]. 469
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“hematoxylin and eosin” and examined for any histopathological changes using optical microscopy equipped with photographic arrangement [27].
Table 1 Observed response in Box–Behnken design for the optimization of RLX loaded transfersomes formulations. Formulation code
RLX RLX RLX RLX RLX RLX RLX RLX RLX RLX RLX RLX RLX RLX RLX
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Independent variables
Dependent variables
X1
X2
X3
Y1
Y2
Y3
60.00 40.00 40.00 40.00 60.00 80.00 60.00 40.00 60.00 80.00 60.00 60.00 80.00 60.00 80.00
7.50 7.50 6.00 9.00 6.00 7.50 7.50 7.50 9.00 9.00 7.50 9.00 6.00 6.00 7.50
20.00 15.00 20.00 20.00 15.00 25.00 20.00 25.00 25.00 20.00 20.00 15.00 20.00 25.00 15.00
136.2 108.27 198.32 92.12 162.7 151.5 131.8 121.92 156.52 148.3 138.8 106.2 199.6 235.6 134.1
85.11 65.25 61.12 72.41 82.42 88.72 86.08 76.6 93.16 90.91 88.54 93.14 85.28 84.31 86.37
0.221 0.356 0.371 0.299 0.315 0.316 0.219 0.292 0.253 0.391 0.215 0.371 0.303 0.305 0.301
3. Results and discussion 3.1. Optimization of RLX-entrapped limonene-containing transfersomes The 15 formulations generated by the design expert software were developed and their outcomes are demonstrated in Table 1. Model summary analysis presented that the quadratic model was obtained to be the most well conformed model for the chosen three responses (Table 2). The consequences of independent variables on Y1, Y2 and Y3 (responses) are revealed in Fig. 1. 3.2. Response Y1: effect of independent variables on vesicles size The vesicles size of various RLX loaded formulations were presented in Table 1. The least vesicles size (92.12 nm) was observed for formulation F4 while the maximum vesicles size of was obtained as 235.6 nm for formulation F14 (Table 1). It was noted that presence of phospholipid in formulation showed positive effect on vesicles size. For example, formulation F2 having 40 mg of phospholipid presented vesicles size of 108.27 nm while the formulation F15 having maximum concentration of phospholipid (80 mg) showed vesicles size of 134.1 nm. Similar results was obtained with formulation F8 having 40 mg phospholipid showing vesicles size of 121.92 nm while the formulation F6 having 80 mg of phospholipid presented vesicles size of 151.5 nm. On contrary sodium cholate showed negative impact on vesicles size. Formulation 3 possessing sodium cholate 6 mg showing vesicles size of 198.32 nm while the formulation 4 having 9 mg sodium cholate showed vesicles size of 92.12 nm. Similar results were also observed for formulation 5 (sodium cholate 6 mg) depicted vesicles size of 162.7 nm although the formulation 12 (sodium cholate 9 mg) showed the vesicles size of 106.2 nm (Table 1). On the other hand, presence of limonene in the transfersomes also exhibited positive effect of size of vesicles. It was noticed that the vesicles size of transfersomes was augmented on raising the limonene concentration in the formulation. For example, formulation F2 having limonene 15 μl presented vesicles size of 108.27 nm, while the formulation F8 having limonene 25 μl showed vesicles size of 121.92 nm. Similar results were also observed for formulation 5 (limonene 15 μl) presented vesicles size of 162.7 nm while the formulation 14 (limonene 25 μl) showed the vesicles size of 235.6 nm.
X1, Phospholipid (mg); X2, Sodium cholate (mg); X3, Limonene (μl); Y1, Vesicles size (nm); Y2, EE (%); Y3, PDI.
was maintained at 37 ± 0.5 °C. Transfersomes gel containing 1 mg of RLX was placed on the skin surface and 1 ml sample was pipetted out at 0, 0.5, 1, 2, 4, 6, 12, and 24 h from receiver cell and refilled with fresh vehicle [24]. Subsequently the withdrawn samples were examined for RLX content by UV- spectrophotometers. 2.9.5. In vivo pharmacokinetic study Wistar rats (200–250 g) were obtained after the study was sanctioned by “Jamia Hamdard Animal Ethics Committee (JHAEC, Registration No. 173/GO/Re/S/2000/CPCSEA), New Delhi, India” (Project Proposal no. 1417)”. Rats were separated into groups; Group 1 served as normal control, Group 2 received oral RLX suspension and Group 3 received RLX loaded transfersomes gel. After treatment, blood samples were withdrawn at 0, 2, 4, 8, 12, 24 and 48 h then the plasma was collected and examined for RLX content by HPLC method [25]. 2.9.6. Skin irritation study The rats were separated into 3 groups (n = 5). Group I was a control, Group II received 0.8% v/v aqueous solution of formalin as a standard irritant (Positive control). Group III was treated with optimized transfersomes gel formulation. Abdominal hairs of rats were clipped by electric clipper about 24 h before the application of test formulations. Fresh formulation and formalin solution were applied on back side of rats and rats were observed at 1 h, 24 h, 48 h, 72 h and on 7th day for any scores of edema and erythema after removal of formulations [26].
3.3. Response Y2: effect of independent variables on EE The minimum EE of 61.12% was found with formulation F3 while the formulation F9 presented maximum EE of 93.16%. The presence of phospholipid produced a positive effect on the EE, it was observed in 3D-response graph (Fig. 1) that on increasing in phospholipid, the EE of RLX in transfersomes also increased.
2.9.7. Skin histopathology study For histopathology study, rat's skin specimen from RLX loaded transfersomes gel treated and control skin areas were stained with
Table 2 Summary of results of regression analysis for responses Y1, Y2, and Y3 for fitting to quadratic model. Quadratic model
R2
Adjusted R2
Predicted R2
S.D.
% C.V.
Adequate precision
Response Y1 0.9453 0.8468 0.1411 15.15 10.23 10.647 Response Y2 0.9728 0.9239 0.6307 2.67 3.23 14.843 0.9730 0.9244 0.5739 0.015 5.12 13.016 Response Y3 Regression equation of the fitted quadratic model 2 2 Vesicle size = +135.60 + 14.11 × X1 −36.64 × X2 +19.28 × C +13.73 × X1 × X2 +0.94 × X1 × X3 −5.64 × X2 × X3 −6.16 × X1 +30.15 × X2 −0.49 × X32 EE = +86.58 + 9.49 × X1 +4.56 × X2 +1.95 × X3 −1.41 × X1 × X2 −2.25 × X1 × X3 −0.47 × X2 × X3 −9.08 × X12 −0.062 × X22 +1.74 X32 PDI = +0.22–0.00087–004 × X1 +0.0025–003 × X2 −0.022 × X3 +0.040 × X1 × X2 +0.020 × X1 × X3 −0.027 × X2 × X3 +0.064 × X12 +0.059 × X22 +0.034 X32
X1, Phospholipid (mg); X2, Sodium cholate (mg); X3, Limonene (μl); Y1, Vesicles size (nm); Y2, EE (%); Y3, PDI; CV, coefficient of variation; SD, standard deviation. 470
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Fig. 1. 3D-response surface plot showing effect of independent variables on (A) vesicles size (B) EE and (C) PDI.
Formulation F3 (phospholipid 40 mg) showed the EE of 61.12% while the formulation F13 (phospholipid 80 mg) presented EE of 85.28%. Likewise, formulation F2 (phospholipid 40 mg) showed EE of 65.25% while the formulation F15 having phospholipid 80 mg presented EE of 86.37% (Table 1). Comparable results was also observed for formulation F8 (phospholipid 40 mg; EE 76.60%) and formulation F6 (phospholipid 80 mg; EE 88.72%). Similarly, sodium cholate also showed positive effect of EE, increase in EE was observed on increasing sodium cholate. Formulation F3 having 6 mg sodium cholate presented EE of 61.12% while formulation F4 having 9 mg sodium cholate presented EE of 72.41%. Similar results were observed for formulation F5 (sodium cholate 6 mg) presented EE of 82.42% and formulation F12 (sodium cholate 9 mg) presented EE of 93.14% (Table 1). Limonene also displayed a convinced consequence on EE; it was detected that on raising the limonene concentration the EE of RLX also increased in vesicles. For example, F2 (limonene 15 μl) showed EE of 65.25% while the F8 (limonene 25 μl) showed EE of 76.60%. Likewise, formulation F5 (limonene 15 μl) presented EE of 82.42% while formulation F14 (limonene 25 μl) presented EE of 84.31% (Table 1).
Formulation F3 (sodium cholate 6 mg) exhibited PDI of 0.371 while the formulation F4 (sodium cholate 9 mg) exhibited PDI of 0.299. Limonene also exhibited negative impact on PDI; it was found that on raising the limonene concentration the PDI values reduced. Formulation F2 (limonene 15 μl) presented PDI of 0.356 and formulation F8 (limonene 25 μl) presented PDI of 0.292 (Table 1). The point prediction optimization method of Design Expert software generated the optimized formulation based on the above revealed outcomes. The formulation with phospholipid (43.75 mg), sodium cholate (8.78 mg) and limonene (25 μl) was found to fulfill requisites of an optimized transfersomes formulation. The optimized formulation showed vesicle size of 107.8 nm (Fig. 2A) with PDI and EE of 0.252 and 90.2% which is close to the predicted values (vesicles size of 114.45, EE of 82.82% and PDI of 0.251) generated by the Design Expert software. The optimized formulation exhibited zeta potential value of −20.9 mV (Fig. 2B). It was reported that negatively charged vesicles formulations substantially improved skin permeation of drugs via transdermal route [19]. 3.5. Transmission electron microscopy
3.4. Response Y3: effect of independent variables on PDI
The transmission electron micrograph of RLX loaded transfersomes formulation is shown in Fig. 2C. The image displays the outline and the core of the well identified sealed spherical structure.
The least PDI of 0.215 was observed for formulation F11 and the maximum PDI of 0.391 was observed for formulation F10 (Table 1). It was noticed that sodium cholate delivered negative impact on PDI. 471
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Fig. 2. Figure showing (A) vesicles size distribution curve and (B) zeta potential and (C) transmission electron micrograph of optimized RLX loaded transfersomes formulation.
3.6. CLSM
3.7. Evaluation of transfersomes gel
CLSM micrographs presented in Fig. 3, it can be clearly seen that Rhodamine-B plain solution did not facilitate the probe diffusion into the deeper layer of the skin (Fig. 3A). The dye remains confined only to the superior layer of the skin. Instead, Rhodamine-B loaded transfersomes was well penetrated and disseminated in the skin with high fluorescence intensity into the deeper layer of the skin (Fig. 3B). Hence it is suggested that the prepared transfersomes formulation was capable to increase the permeation of probe into deeper layer of skin in relatively large quantity. This indicates that prepared transfersomes could act as an important drug carrier in enhancing drug absorption across skin.
3.7.1. Homogeneity, pH, spreadability, extrudability and drug content The prepared transfersomes gel formulation demonstrated a pleasant, smooth homogeneous appearance and was free from presence of any gritty particles. The pH of prepared gel formulation was found to be 6.81 ± 0.23, which are regarded as tolerable, to circumvent the possibility of irritation on application to the skin. The prepared gel formulation demonstrated the spreadability and extrudability of 6.89 ± 0.378 g cm/sec and 161.27 ± 2.74 g respectively. The drug content in the gel formulation was found to be 98.13 ± 1.13%. 3.7.2. Texture analysis The transfersomes gel formulation was evaluated for texture 472
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Fig. 3. Confocal laser scanning micrographs of (A) rat skin treated with Rhodamin B solution and (B) rat skin treated with optimized transfersomes formulation.
analysis and the parameters such as firmness, cohesiveness, consistency and index of viscosity were evaluated. It was noted that the optimized transfersomes formulation loaded gel formulation presented firmness, cohesiveness, consistency and index of viscosity of 566.43 g, −419.58 g, 4473.81 g s, and −1554.47 g s respectively. The report of texture analysis obtained from software was presented in Fig. 4. The results exhibited good texture of gel which represents it as an ideal gel for dermal uses.
3.7.4. Skin permeation study The RLX loaded transfersomes gel was subjected to permeation study through rat skin for the analysis of flux and it was compared with control gel containing drug. The transdermal flux presented by the transfersomes gel formulation was found to be 13.20 μg/cm2/h whereas the control gel showed the transdermal flux of 2.35 μg/cm2/h across rat skin. The developed transfersomes gel formulation presented enhancement ratio of 5.61 in comparison to control formulation.
3.7.3. In vitro drug release The “% cumulative drug release” of the RLX loaded transfersomes gel was 78.31% in the dissolution medium. Fig. 5 demonstrated that the maximum value of R2 was detected for the Higuchi matrix (R2 = 0.9991), after that R2 = 0.9663 for Korsmeyer–Peppas, R2 = 0.9590 for Zero order and R2 = 0.9589 for First-order. In addition, the value of n (release exponent) was < 0.5; it implies that the transfersomes gel formulation follows Fickian diffusion release mechanism.
3.7.5. In vivo pharmacokinetic study The Tmax value of RLX was substantially increased after transdermal application in comparison to its oral administration (Fig. 6). It was observed that the Tmax following transfersomes gel application on Wistar rats was found to be 8.0 ± 0.26 h while the Tmax following an oral administration of RLX suspension was found to be 4.0 ± 0.15 h. On contrary, Cmax of RLX was decreased in Wistar rats following transfersomes gel as compared to the values presented by the control group (Fig. 6). The Cmax of 176.42 ± 32.52 ng/ml was observed after
Fig. 4. Texture analysis report of RLX loaded transfersomes gel formulation. 473
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Fig. 5. The plots of the kinetics of drug release from the RLX transfersomes gel through dialysis membrane, fitted to (A) zero order, (B) first order, (C) Higuchi, and (D) Peppas' mathematical kinetics model.
bioavailability from developed transfersomes gel as compared to oral suspension could be due to the nano sized flexible vesicles. Nano sized vesicles are able to permeate easily into the stratum corneum and could enhanced the transport of drug across skin. 3.7.6. Skin irritation study The skin irritation score in control group's animals was found to be 0. The standard irritant (formalin) group presented skin irritation score of 2.63 ± 0.49. Rats treated with transfersomes gel presented the skin irritation scores of 0.20 ± 0.06 (Table 4). Hence it was determined that the RLX loaded transfersomes gel could be considered to be nonirritant and nontoxic for transdermal drug delivery. Fig. 6. Plasma concentration profiles of RLX after transdermal application of transfersomes gel and oral RLX suspension in Wistar rats.
3.7.7. Histopathological study The histopathological study of the rat skin section of normal untreated rat skin (Fig. 7A) and rat skin sample after transfersomes gel treatment (Fig. 7B) did not reveal any change in the state of the skin. The photomicrograph of control clearly shows the intact epidermis and viable dermis, however following transfersomes gel treatment there was no obvious changes was observed. Consequently, it was determined that the transfersomes gel was found to be a tolerable nano-carrier system for the transdermal RLX delivery.
Table 3 Pharmacokinetic parameters of RLX in Wistar rats after administration of RLX oral suspension and application of RLX loaded transfersomes gel. Parameters
RLX suspension
RLX loaded transfersomes gel
Tmax (h) Cmax (ng/ml) AUC0→t (ng.h/ml) Elimination rate constant (Kel) Relative Bioavailability
4.0 ± 0.15 176.42 ± 32.52 1764.44 ± 76.82 0.061762 ± 0.003
8.0 ± 0.26 151.68 ± 25.16 4781.82 ± 124.65 0.001126 ± 0.0002
–
2.71
Table 4 Mean skin irritation scores for control, positive control and transfersomes gel treated group.
Wistar rats was treated with transfersomes gel while Cmax value of 151.68 ± 25.16 ng/ml was observed when rats were treated with an oral formulation of RLX (Table 3). The AUC0→t of oral RLX formulation and RLX transfersomes gel was found to be 1764.44 ng h/ml and 4781.82 ng h/ml respectively. The relative bioavailability following transfersomes gel in Wistar rats was found be increased by 2.71 times as compared to the oral suspension of RLX (Table 3). The increased in RLX 474
Rat no.
Control
Positive control
Transfersomes gel
1 2 3 4 5 Average skin irritation score
00 00 00 00 00 00
3.14 2.14 3.00 2.00 2.86 2.63 ± 0.49
0.12 0.19 0.21 0.30 0.17 0.20 ± 0.06
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Fig. 7. Photomicrograph of section of (A) untreated rat skin and (B) optimized RLX loaded transfersomes gel formulation treated rat skin (10x).
4. Conclusion In the current study, RLX loaded transfersomes were prepared and optimized by Box–Behnken design with success. Best formulation showed nano-sized range vesicles with EE of 90%. Confocal study had shown improved permeation of dye loaded transfersomes across rat's skin as compared to control dye solution. In addition pharmacokinetic study presented improved bioavailability of RLX in Wistar rats after transdermal application as compared to its control oral formulation.
[5] [6] [7] [8]
Conflicts of interest
[9]
None.
[10]
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Improved bioavailability of raloxifene hydrochloride using limonene containing transdermal nano-sized vesicles
T
Ayesha Waheeda, Mohd Aqila,∗, Abdul Ahadb, Syed Sarim Imamc, Thasleem Moolakkadatha, Zeenat Iqbala, Asgar Alia a
Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard (Deemed University), M. B. Road, New Delhi, 110062, India Department of Pharmaceutics, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh, 11451, Saudi Arabia c School of Pharmacy, Glocal University, Saharanpur, U.P, India b
ARTICLE INFO
ABSTRACT
Keywords: Limonene Penetration enhancer containing vesicles Raloxifene hydrochloride Transfersomes
The present investigation was carried out with the aim of optimization of raloxifene hydrochloride-entrapped, limonene containing transfersomes for transdermal delivery. Several formulations were prepared by varying independent variables such as Phospholipid, sodium cholate and limonene while vesicles size, entrapment efficiency and polydispersity index were the dependent variables for Box-Behnken Design. Further, bioavailability assessment of raloxifene in Wistar rats was evaluated after transdermal application and compared with its oral formulation. It was observed that the optimized raloxifene loaded transfersomes formulation showed vesicle size of 107.8 nm, polydispersity index of 0.252 and entrapment efficiency of 90.2% which is close to the predicted values of 114.45 nm, 0.251 and 82.82% respectively generated by the Design Expert software. The TEM image shows the outline and the core of the well identified sealed spherical structure. Confocal laser microscopy study demonstrated that the prepared transfersomes formulation was capable to increase the permeation of probe dye into deeper layer of rat's skin in comparison to the control dye solution. The transdermal flux presented by the transfersomes gel formulation was found to be 13.20 μg/cm2/h whereas the control gel showed the transdermal flux of 2.35 μg/cm2/h across rat skin. Further the in vivo pharmacokinetic study revealed that the relative bioavailability following application of transfersomes gel in Wistar rats was found be increased by 2.71 times as compared to the oral suspension of raloxifene. It was concluded that the prepared transfersomes formulation was found to be a potentially useful drug carrier for the enhancement of raloxifene bioavailability in rats.
1. Introduction Raloxifene hydrochloride (RLX) is regarded as a BCS class II drug, it is promptly absorbed from the GI tract and face extensive first-pass metabolism and about sixty percent of a dosage is absorbed; nevertheless, its absolute bioavailability is merely two percent [1]. Transdermal delivery is of one the methods to improve bioavailability of several class of drug particularly for BCS class II drugs. Past decades have witnessed significantly increased interest in the exploration of new transdermal techniques to enhance drug permeation into or through the skin [2]. Transfersomes help the drug to pass across the stratum corneum unlike the conventional liposomes, edge activators incorporate into the lipid bilayer increasing the resulting vesicle elasticity [3,4]. Due to the elasticity, transfersomes vesicles are capable to squeeze via networks one-tenth the diameter of the vesicles and permitting them to penetrate across the stratum corneum via intercellular
∗
route [5]. Previously RLX -loaded transfersomes were prepared by Mahmood et al., 2014. The best chosen formulation exhibited vesicles size of 134 nm with an EE of 91% and flux of 6.5 μg/cm2/h [6]. In another study, Mahmood et al., 2018 prepared RLX-loaded ethosomes formulations. The final formulation presented flux of 22.14 ± 0.83 μg/ cm2/h. Authors concluded that the animal study showed 157% times increased in relative bioavailability of RLX-ethosomes formulation when compared to oral control formulation in rats [7]. In another study, Joshi et al., 2018 prepared RLX -transfersomes formulations. Authors concluded that the RLX-transfersomes suspension exhibited better release than RLX-transfersomes loaded cream, gel and patch formulations [8]. In present study, we have utilized the combined effect of transfersomes (flexible drug carrier) and terpene (natural penetration enhancer). Terpenes are acquired from natural sources, they are less toxic
Corresponding author. E-mail addresses: [email protected] (M. Aqil), [email protected], [email protected] (A. Ahad).
https://doi.org/10.1016/j.jddst.2019.05.019 Received 5 March 2019; Received in revised form 28 April 2019; Accepted 7 May 2019 Available online 08 May 2019 1773-2247/ © 2019 Elsevier B.V. All rights reserved.
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and are efficient penetration enhancers for both lipophilic and hydrophilic drugs; the FDA included them in generally regarded as safe (GRAS) category [9,10]. Limonene is a hydrocarbon lipophilic terpene and it fluidizes or disturbs the integrity of the stratum corneum for improved the passage of drugs across the skin [11,12]. Earlier reports are available that reflects the use of limonene as an encouraging permeation enhancer for the delivery of drugs via transdermal route [12–15]. Hence, the purpose of this study was to examine the effect of phospholipid 90G (X1, −1 = 40 mg, 0 = 60 mg and +1 = 80 mg), sodium cholate (X2, −1 = 6 mg, 0 = 7.5 mg and +1 = 9 mg), and limonene (X3, −1 = 15 μl, 0 = 20 μl and +1 = 25 μl) on the vesicles size, EE and PDI of RLX loaded transfersomes. Further, in vitro skin permeation, CLSM, skin irritation and bioavailability assessment of RLX loaded formulation were investigated.
The EE was calculated using following equation EE (%) = (Total drug − drug detected only in supernatant) / Total drug × 100 2.6. Transmission electron microscopy The vesicles surface morphology of the transfersomes was evaluated by “transmission electron microscopy” [19]. The sample was placed on copper grids then “negatively stained by phosphotungstic acid (1%)” then permitted to air dried and then visualized by microscope at “an accelerating voltage of 100 kV”. 2.7. Confocal laser scanning microscopy (CLSM) For CLSM study excised rat skin was treated with rhodamine loaded optimized transfersomes formulation for 24 h and compared it rat's skin treated with plain rhodamine solution. After 24 h, the excised rat skin was clean with distilled water and glass slides were prepared and visualized by confocal microscope with an “argon laser beam with excitation at 540 nm” and “emission at 625 nm”.
2. Materials and methods 2.1. Materials RLX and Sodium cholate was a generous gift from Kusum Healthcare Pvt. Ltd., Delhi and Thomas Baker, Mumbai respectively. Phospholipon 90G was a generous gift from Lipoid, Germany. Limonene was purchased from Sigma-Aldrich, USA. All other reagents are of analytical grade and HPLC grade.
2.8. Preparation of RLX loaded transfersomes gel The final RLX transfersomes preparation was transformed to a gel preparation by dissolving 1% Carbopol 934 in water with continuous stirring. After complete dispersal of Carbopol into water, the dispersion was allowed for nightlong to complete swelling. After this, triethanolamine and bezalkonium chloride was added to obtain a homogenous dispersion of gel. Lastly, the final RLX-transfersomes formulation was added to the gel base by gentle stirring to get the final gel preparation [21].
2.2. Preparation of RLX-entrapped limonene-containing transfersomes Transfersomes are prepared by thin layer evaporation procedure [16–18]. Briefly, round bottomed flask having mixture of phospholipid, RLX and sodium cholate previously dissolved in methanol/chloroform (1:1 v/v) was attached with “rotary evaporator” maintained under reduced pressure and allows the organic solvent to evaporate. Later the thin dried lipid film so formed on the inner wall of round bottomed flask was rehydrated for 30 min at a rotation of 60 rpm with phosphate buffered pH 7.4. At last, the dispersion having large vesicles was probe sonicated at 4 °C for 10 min (gap of 5 min between each cycle) to produced transfersomes.
2.9. Evaluation of RLX loaded transfersomes gel 2.9.1. Homogeneity, pH, spreadability, extrudability and drug content The developed gel was placed in clear and transparent container and examined visually for visual aspects and existence of any masses formation and the gel pH was assessed by pH meter. Method for the evaluation of gel spreadability and extrudability are well explained by other researchers [22,23]. Further, the drug content was found out by dissolving the gel (1 g) in methanol (100 ml) then sonicated for the extraction of drug in methanol. Subsequently the final mixture was filtered, diluted and examined by means of UV spectrophotometer.
2.3. Experimental design for optimization of transfersomes A Box-Behnken design (3-factor, 3-level) was used for the preparation of transfersomes and the Design Expert (version 11.0) software generated 15 different formulations by varying the composition of phospholipid 90G (X1, −1 = 40 mg, 0 = 60 mg and +1 = 80 mg), sodium cholate (X2, −1 = 6 mg, 0 = 7.5 mg and +1 = 9 mg), and limonene (X3, −1 = 15 μl, 0 = 20 μl and +1 = 25 μl) and the formulations were characterized based on the dependent responses like vesicles size (Y1), polydispersity index (PDI, Y2), and entrapment efficiency (EE, Y3).
2.9.2. Texture analysis The gel was analyzed in texture analyzer (Stable Micro Systems, Exponent Lite, Vienna, U.K.) with a back extruding probe of 30 mm diameter. Various parameters like firmness, cohesiveness, consistency and index of viscosity were investigated.
2.4. Vesicle size, size distribution and zeta potential measurements
2.9.3. In vitro drug release study For the evaluation of drug release, the transfersomes gel was kept in activated dialysis bag installed in dissolution chamber. The dissolution chamber was filled with 200 ml of PBS, pH 7.4 and isopropyl alcohol (70:30 v/v), the medium was agitated with magnetically stirred at 600 rpm, and the medium temperature was kept at 37 ± 0.5 °C. The Aliquots of 2 ml was pipetted out at each interval and refilled with fresh vehicle. The results found were fit into “drug release kinetic models”.
For analysis, RLX loaded transfersomes samples were diluted hundred times with Milli-Q water and the vesicles size and size distribution were evaluated by “Zetasizer Nano ZS (Malvern Instruments Ltd, Worcestershire, UK)” at 25 ± 1 °C and at a scattering angle of 90°. Zeta potential of samples was also evaluated by same instrument at 25 ± 1 °C. 2.5. Determination of EE (%)
2.9.4. In vitro skin permeation study In this study the prepared rat's skin specimen was fixed on Franz vertical cell (surface area 6.33 cm2). The receiver cell was made full with PBS (pH 7.4) and isopropyl alcohol (15 ml, 70:30 v/v) which was agitated with magnetic bead at 600 rpm and the temperature of vehicle
Separation of un-entrapped drug from the prepared transfersomes was carried out by centrifugation method [19]. Transfersomes formulation was centrifuged at 25000 rpm for 1 h and the supernatant was removed and analyzed by UV- spectrophotometer at 287 nm λmax [20]. 469
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“hematoxylin and eosin” and examined for any histopathological changes using optical microscopy equipped with photographic arrangement [27].
Table 1 Observed response in Box–Behnken design for the optimization of RLX loaded transfersomes formulations. Formulation code
RLX RLX RLX RLX RLX RLX RLX RLX RLX RLX RLX RLX RLX RLX RLX
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Independent variables
Dependent variables
X1
X2
X3
Y1
Y2
Y3
60.00 40.00 40.00 40.00 60.00 80.00 60.00 40.00 60.00 80.00 60.00 60.00 80.00 60.00 80.00
7.50 7.50 6.00 9.00 6.00 7.50 7.50 7.50 9.00 9.00 7.50 9.00 6.00 6.00 7.50
20.00 15.00 20.00 20.00 15.00 25.00 20.00 25.00 25.00 20.00 20.00 15.00 20.00 25.00 15.00
136.2 108.27 198.32 92.12 162.7 151.5 131.8 121.92 156.52 148.3 138.8 106.2 199.6 235.6 134.1
85.11 65.25 61.12 72.41 82.42 88.72 86.08 76.6 93.16 90.91 88.54 93.14 85.28 84.31 86.37
0.221 0.356 0.371 0.299 0.315 0.316 0.219 0.292 0.253 0.391 0.215 0.371 0.303 0.305 0.301
3. Results and discussion 3.1. Optimization of RLX-entrapped limonene-containing transfersomes The 15 formulations generated by the design expert software were developed and their outcomes are demonstrated in Table 1. Model summary analysis presented that the quadratic model was obtained to be the most well conformed model for the chosen three responses (Table 2). The consequences of independent variables on Y1, Y2 and Y3 (responses) are revealed in Fig. 1. 3.2. Response Y1: effect of independent variables on vesicles size The vesicles size of various RLX loaded formulations were presented in Table 1. The least vesicles size (92.12 nm) was observed for formulation F4 while the maximum vesicles size of was obtained as 235.6 nm for formulation F14 (Table 1). It was noted that presence of phospholipid in formulation showed positive effect on vesicles size. For example, formulation F2 having 40 mg of phospholipid presented vesicles size of 108.27 nm while the formulation F15 having maximum concentration of phospholipid (80 mg) showed vesicles size of 134.1 nm. Similar results was obtained with formulation F8 having 40 mg phospholipid showing vesicles size of 121.92 nm while the formulation F6 having 80 mg of phospholipid presented vesicles size of 151.5 nm. On contrary sodium cholate showed negative impact on vesicles size. Formulation 3 possessing sodium cholate 6 mg showing vesicles size of 198.32 nm while the formulation 4 having 9 mg sodium cholate showed vesicles size of 92.12 nm. Similar results were also observed for formulation 5 (sodium cholate 6 mg) depicted vesicles size of 162.7 nm although the formulation 12 (sodium cholate 9 mg) showed the vesicles size of 106.2 nm (Table 1). On the other hand, presence of limonene in the transfersomes also exhibited positive effect of size of vesicles. It was noticed that the vesicles size of transfersomes was augmented on raising the limonene concentration in the formulation. For example, formulation F2 having limonene 15 μl presented vesicles size of 108.27 nm, while the formulation F8 having limonene 25 μl showed vesicles size of 121.92 nm. Similar results were also observed for formulation 5 (limonene 15 μl) presented vesicles size of 162.7 nm while the formulation 14 (limonene 25 μl) showed the vesicles size of 235.6 nm.
X1, Phospholipid (mg); X2, Sodium cholate (mg); X3, Limonene (μl); Y1, Vesicles size (nm); Y2, EE (%); Y3, PDI.
was maintained at 37 ± 0.5 °C. Transfersomes gel containing 1 mg of RLX was placed on the skin surface and 1 ml sample was pipetted out at 0, 0.5, 1, 2, 4, 6, 12, and 24 h from receiver cell and refilled with fresh vehicle [24]. Subsequently the withdrawn samples were examined for RLX content by UV- spectrophotometers. 2.9.5. In vivo pharmacokinetic study Wistar rats (200–250 g) were obtained after the study was sanctioned by “Jamia Hamdard Animal Ethics Committee (JHAEC, Registration No. 173/GO/Re/S/2000/CPCSEA), New Delhi, India” (Project Proposal no. 1417)”. Rats were separated into groups; Group 1 served as normal control, Group 2 received oral RLX suspension and Group 3 received RLX loaded transfersomes gel. After treatment, blood samples were withdrawn at 0, 2, 4, 8, 12, 24 and 48 h then the plasma was collected and examined for RLX content by HPLC method [25]. 2.9.6. Skin irritation study The rats were separated into 3 groups (n = 5). Group I was a control, Group II received 0.8% v/v aqueous solution of formalin as a standard irritant (Positive control). Group III was treated with optimized transfersomes gel formulation. Abdominal hairs of rats were clipped by electric clipper about 24 h before the application of test formulations. Fresh formulation and formalin solution were applied on back side of rats and rats were observed at 1 h, 24 h, 48 h, 72 h and on 7th day for any scores of edema and erythema after removal of formulations [26].
3.3. Response Y2: effect of independent variables on EE The minimum EE of 61.12% was found with formulation F3 while the formulation F9 presented maximum EE of 93.16%. The presence of phospholipid produced a positive effect on the EE, it was observed in 3D-response graph (Fig. 1) that on increasing in phospholipid, the EE of RLX in transfersomes also increased.
2.9.7. Skin histopathology study For histopathology study, rat's skin specimen from RLX loaded transfersomes gel treated and control skin areas were stained with
Table 2 Summary of results of regression analysis for responses Y1, Y2, and Y3 for fitting to quadratic model. Quadratic model
R2
Adjusted R2
Predicted R2
S.D.
% C.V.
Adequate precision
Response Y1 0.9453 0.8468 0.1411 15.15 10.23 10.647 Response Y2 0.9728 0.9239 0.6307 2.67 3.23 14.843 0.9730 0.9244 0.5739 0.015 5.12 13.016 Response Y3 Regression equation of the fitted quadratic model 2 2 Vesicle size = +135.60 + 14.11 × X1 −36.64 × X2 +19.28 × C +13.73 × X1 × X2 +0.94 × X1 × X3 −5.64 × X2 × X3 −6.16 × X1 +30.15 × X2 −0.49 × X32 EE = +86.58 + 9.49 × X1 +4.56 × X2 +1.95 × X3 −1.41 × X1 × X2 −2.25 × X1 × X3 −0.47 × X2 × X3 −9.08 × X12 −0.062 × X22 +1.74 X32 PDI = +0.22–0.00087–004 × X1 +0.0025–003 × X2 −0.022 × X3 +0.040 × X1 × X2 +0.020 × X1 × X3 −0.027 × X2 × X3 +0.064 × X12 +0.059 × X22 +0.034 X32
X1, Phospholipid (mg); X2, Sodium cholate (mg); X3, Limonene (μl); Y1, Vesicles size (nm); Y2, EE (%); Y3, PDI; CV, coefficient of variation; SD, standard deviation. 470
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Fig. 1. 3D-response surface plot showing effect of independent variables on (A) vesicles size (B) EE and (C) PDI.
Formulation F3 (phospholipid 40 mg) showed the EE of 61.12% while the formulation F13 (phospholipid 80 mg) presented EE of 85.28%. Likewise, formulation F2 (phospholipid 40 mg) showed EE of 65.25% while the formulation F15 having phospholipid 80 mg presented EE of 86.37% (Table 1). Comparable results was also observed for formulation F8 (phospholipid 40 mg; EE 76.60%) and formulation F6 (phospholipid 80 mg; EE 88.72%). Similarly, sodium cholate also showed positive effect of EE, increase in EE was observed on increasing sodium cholate. Formulation F3 having 6 mg sodium cholate presented EE of 61.12% while formulation F4 having 9 mg sodium cholate presented EE of 72.41%. Similar results were observed for formulation F5 (sodium cholate 6 mg) presented EE of 82.42% and formulation F12 (sodium cholate 9 mg) presented EE of 93.14% (Table 1). Limonene also displayed a convinced consequence on EE; it was detected that on raising the limonene concentration the EE of RLX also increased in vesicles. For example, F2 (limonene 15 μl) showed EE of 65.25% while the F8 (limonene 25 μl) showed EE of 76.60%. Likewise, formulation F5 (limonene 15 μl) presented EE of 82.42% while formulation F14 (limonene 25 μl) presented EE of 84.31% (Table 1).
Formulation F3 (sodium cholate 6 mg) exhibited PDI of 0.371 while the formulation F4 (sodium cholate 9 mg) exhibited PDI of 0.299. Limonene also exhibited negative impact on PDI; it was found that on raising the limonene concentration the PDI values reduced. Formulation F2 (limonene 15 μl) presented PDI of 0.356 and formulation F8 (limonene 25 μl) presented PDI of 0.292 (Table 1). The point prediction optimization method of Design Expert software generated the optimized formulation based on the above revealed outcomes. The formulation with phospholipid (43.75 mg), sodium cholate (8.78 mg) and limonene (25 μl) was found to fulfill requisites of an optimized transfersomes formulation. The optimized formulation showed vesicle size of 107.8 nm (Fig. 2A) with PDI and EE of 0.252 and 90.2% which is close to the predicted values (vesicles size of 114.45, EE of 82.82% and PDI of 0.251) generated by the Design Expert software. The optimized formulation exhibited zeta potential value of −20.9 mV (Fig. 2B). It was reported that negatively charged vesicles formulations substantially improved skin permeation of drugs via transdermal route [19]. 3.5. Transmission electron microscopy
3.4. Response Y3: effect of independent variables on PDI
The transmission electron micrograph of RLX loaded transfersomes formulation is shown in Fig. 2C. The image displays the outline and the core of the well identified sealed spherical structure.
The least PDI of 0.215 was observed for formulation F11 and the maximum PDI of 0.391 was observed for formulation F10 (Table 1). It was noticed that sodium cholate delivered negative impact on PDI. 471
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Fig. 2. Figure showing (A) vesicles size distribution curve and (B) zeta potential and (C) transmission electron micrograph of optimized RLX loaded transfersomes formulation.
3.6. CLSM
3.7. Evaluation of transfersomes gel
CLSM micrographs presented in Fig. 3, it can be clearly seen that Rhodamine-B plain solution did not facilitate the probe diffusion into the deeper layer of the skin (Fig. 3A). The dye remains confined only to the superior layer of the skin. Instead, Rhodamine-B loaded transfersomes was well penetrated and disseminated in the skin with high fluorescence intensity into the deeper layer of the skin (Fig. 3B). Hence it is suggested that the prepared transfersomes formulation was capable to increase the permeation of probe into deeper layer of skin in relatively large quantity. This indicates that prepared transfersomes could act as an important drug carrier in enhancing drug absorption across skin.
3.7.1. Homogeneity, pH, spreadability, extrudability and drug content The prepared transfersomes gel formulation demonstrated a pleasant, smooth homogeneous appearance and was free from presence of any gritty particles. The pH of prepared gel formulation was found to be 6.81 ± 0.23, which are regarded as tolerable, to circumvent the possibility of irritation on application to the skin. The prepared gel formulation demonstrated the spreadability and extrudability of 6.89 ± 0.378 g cm/sec and 161.27 ± 2.74 g respectively. The drug content in the gel formulation was found to be 98.13 ± 1.13%. 3.7.2. Texture analysis The transfersomes gel formulation was evaluated for texture 472
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Fig. 3. Confocal laser scanning micrographs of (A) rat skin treated with Rhodamin B solution and (B) rat skin treated with optimized transfersomes formulation.
analysis and the parameters such as firmness, cohesiveness, consistency and index of viscosity were evaluated. It was noted that the optimized transfersomes formulation loaded gel formulation presented firmness, cohesiveness, consistency and index of viscosity of 566.43 g, −419.58 g, 4473.81 g s, and −1554.47 g s respectively. The report of texture analysis obtained from software was presented in Fig. 4. The results exhibited good texture of gel which represents it as an ideal gel for dermal uses.
3.7.4. Skin permeation study The RLX loaded transfersomes gel was subjected to permeation study through rat skin for the analysis of flux and it was compared with control gel containing drug. The transdermal flux presented by the transfersomes gel formulation was found to be 13.20 μg/cm2/h whereas the control gel showed the transdermal flux of 2.35 μg/cm2/h across rat skin. The developed transfersomes gel formulation presented enhancement ratio of 5.61 in comparison to control formulation.
3.7.3. In vitro drug release The “% cumulative drug release” of the RLX loaded transfersomes gel was 78.31% in the dissolution medium. Fig. 5 demonstrated that the maximum value of R2 was detected for the Higuchi matrix (R2 = 0.9991), after that R2 = 0.9663 for Korsmeyer–Peppas, R2 = 0.9590 for Zero order and R2 = 0.9589 for First-order. In addition, the value of n (release exponent) was < 0.5; it implies that the transfersomes gel formulation follows Fickian diffusion release mechanism.
3.7.5. In vivo pharmacokinetic study The Tmax value of RLX was substantially increased after transdermal application in comparison to its oral administration (Fig. 6). It was observed that the Tmax following transfersomes gel application on Wistar rats was found to be 8.0 ± 0.26 h while the Tmax following an oral administration of RLX suspension was found to be 4.0 ± 0.15 h. On contrary, Cmax of RLX was decreased in Wistar rats following transfersomes gel as compared to the values presented by the control group (Fig. 6). The Cmax of 176.42 ± 32.52 ng/ml was observed after
Fig. 4. Texture analysis report of RLX loaded transfersomes gel formulation. 473
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Fig. 5. The plots of the kinetics of drug release from the RLX transfersomes gel through dialysis membrane, fitted to (A) zero order, (B) first order, (C) Higuchi, and (D) Peppas' mathematical kinetics model.
bioavailability from developed transfersomes gel as compared to oral suspension could be due to the nano sized flexible vesicles. Nano sized vesicles are able to permeate easily into the stratum corneum and could enhanced the transport of drug across skin. 3.7.6. Skin irritation study The skin irritation score in control group's animals was found to be 0. The standard irritant (formalin) group presented skin irritation score of 2.63 ± 0.49. Rats treated with transfersomes gel presented the skin irritation scores of 0.20 ± 0.06 (Table 4). Hence it was determined that the RLX loaded transfersomes gel could be considered to be nonirritant and nontoxic for transdermal drug delivery. Fig. 6. Plasma concentration profiles of RLX after transdermal application of transfersomes gel and oral RLX suspension in Wistar rats.
3.7.7. Histopathological study The histopathological study of the rat skin section of normal untreated rat skin (Fig. 7A) and rat skin sample after transfersomes gel treatment (Fig. 7B) did not reveal any change in the state of the skin. The photomicrograph of control clearly shows the intact epidermis and viable dermis, however following transfersomes gel treatment there was no obvious changes was observed. Consequently, it was determined that the transfersomes gel was found to be a tolerable nano-carrier system for the transdermal RLX delivery.
Table 3 Pharmacokinetic parameters of RLX in Wistar rats after administration of RLX oral suspension and application of RLX loaded transfersomes gel. Parameters
RLX suspension
RLX loaded transfersomes gel
Tmax (h) Cmax (ng/ml) AUC0→t (ng.h/ml) Elimination rate constant (Kel) Relative Bioavailability
4.0 ± 0.15 176.42 ± 32.52 1764.44 ± 76.82 0.061762 ± 0.003
8.0 ± 0.26 151.68 ± 25.16 4781.82 ± 124.65 0.001126 ± 0.0002
–
2.71
Table 4 Mean skin irritation scores for control, positive control and transfersomes gel treated group.
Wistar rats was treated with transfersomes gel while Cmax value of 151.68 ± 25.16 ng/ml was observed when rats were treated with an oral formulation of RLX (Table 3). The AUC0→t of oral RLX formulation and RLX transfersomes gel was found to be 1764.44 ng h/ml and 4781.82 ng h/ml respectively. The relative bioavailability following transfersomes gel in Wistar rats was found be increased by 2.71 times as compared to the oral suspension of RLX (Table 3). The increased in RLX 474
Rat no.
Control
Positive control
Transfersomes gel
1 2 3 4 5 Average skin irritation score
00 00 00 00 00 00
3.14 2.14 3.00 2.00 2.86 2.63 ± 0.49
0.12 0.19 0.21 0.30 0.17 0.20 ± 0.06
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Fig. 7. Photomicrograph of section of (A) untreated rat skin and (B) optimized RLX loaded transfersomes gel formulation treated rat skin (10x).
4. Conclusion In the current study, RLX loaded transfersomes were prepared and optimized by Box–Behnken design with success. Best formulation showed nano-sized range vesicles with EE of 90%. Confocal study had shown improved permeation of dye loaded transfersomes across rat's skin as compared to control dye solution. In addition pharmacokinetic study presented improved bioavailability of RLX in Wistar rats after transdermal application as compared to its control oral formulation.
[5] [6] [7] [8]
Conflicts of interest
[9]
None.
[10]
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[11]
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