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Formulation and optimization of nanotransfersomes using experimental design technique for accentuated transdermal delivery of valsartan
POTENTIAL CLINICAL RELEVANCE Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 237 – 249
Research Article
nanomedjournal.com
Formulation and optimization of nanotransfersomes using experimental design technique for accentuated transdermal delivery of valsartan Abdul Ahad, MPharm, PhD, Mohammed Aqil, MPharm, PhD⁎, Kanchan Kohli, MPharm, PhD, Yasmin Sultana, MPharm, PhD, Mohammed Mujeeb, MPharm, PhD, Asgar Ali, MPharm, PhD Faculty of Pharmacy, Hamdard University, New Delhi, India Received 8 November 2010; accepted 4 June 2011
Abstract The purpose of this work was to develop and statistically optimize nanotransfersomes for enhanced transdermal of valsartan vis-àvis traditional liposomes. Nanotransfersomes bearing valsartan were prepared by conventional rotary evaporation method and characterized for various parameters including entrapment efficiency, vesicles shape, size, size distribution, and skin permeation. In vivo antihypertensive activity conducted on Wistar rats was also taken as a measure of performance of nanotransfersomes and liposomes. Nanotransfersomes proved significantly superior in terms of amount of drug permeated in the skin, with an enhancement ratio of 33.97 ± 1.25 when compared to rigid liposomes. This was further confirmed through a confocal laser scanning microscopy study. Nanotransfersomes showed better antihypertensive activity in comparison to liposomes by virtue of better permeation through Wistar rat skin. Finally, it could be concluded that the nanotransfersomes accentuates the transdermal flux of valsartan and could be used as a carrier for effective transdermal delivery of valsartan. From the Clinical Editor: In this paper, the authors discuss the development and optimization of nanotransfersomes for enhanced transdermal of valsartan and demonstrate accentuated transdermal compared to standard preparations. © 2012 Elsevier Inc. All rights reserved. Key words: Box-Behnken design; Elastic liposomes; Hypertension; Transfersomes; Valsartan
The greatest obstacle for transdermal delivery is the barrier property of the stratum corneum (SC).1,2 Many approaches have been used to breach the skin barrier; for example, the use of lipid vesicles to modulate the SC is gaining interest. The first articles to report on the effectiveness of vesicles for skin delivery were published in the early 1980s. Most groups concluded that liposomes did not act as transport systems.3 Conventional liposomes have been generally reported to remain confined to the upper layer of the SC and to accumulate in the skin appendages, with minimal penetration to deeper tissues, because of their large size and lack of flexibility.4,5 Further intensive research over the past two decades led to the introduction and development of a new class of lipid vesicles, the ultradeformable (elastic or ultraflexible) liposomes that have been termed Transfersomes (IDEA AG, Munich, Germany). Several studies have reported that TransferAbdul Ahad thanks the Council for Scientific and Industrial Research (CSIR), India (File No. 09/591 (0084)/2009-EMR-I), for providing financial assistance in the form of a senior research fellowship. ⁎Corresponding author: Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University, New Delhi 110 062, India. E-mail address: [email protected] (M. Aqil).
somes were able to improve in vitro skin delivery of various drugs6 and to penetrate intact skin in vivo, transferring therapeutic amounts of drugs with efficiency comparable with subcutaneous administration.7-12 The key factors that confer ultradeformability to the liposomes have been considered to be edge activators (EAs), the special surfactants incorporated into Transfersomes (e.g., sodium cholate or sodium deoxycholate, SDC). Because Transfersomes are composed of surfactant, they have better rheology and hydration properties, which are responsible for their superior skin penetration ability. Less deformable vesicles, including traditional liposomes, are confined to the skin surface where they dehydrate completely and fuse, so they have less penetration power than Transfersomes. Transfersomes are optimized in this respect, thus attaining maximum flexibility, and hence they can take full advantage of the transepidermal osmotic gradient (water concentration gradient).13 The aim of the present study was to optimize the nanovesicles formulations (nanotransfersomes) for enhanced skin delivery of a model drug valsartan, a lipophilic antihypertensive drug having low oral bioavailability of about 25%. It has low molecular weight (435.5) and melting point (116–117°C) with a log partition coefficient of 4.5 and a mean biological half-life of 7.5 hours; there are no reports of skin irritation attributed to valsartan.
1549-9634/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2011.06.004 Please cite this article as: A. Ahad, M. Aqil, K. Kohli, Y. Sultana, M. Mujeeb, A. Ali, Formulation and optimization of nanotransfersomes using experimental design technique for accentuated transdermal delivery of vals.... Nanomedicine: NBM 2012;8:237-249, doi:10.1016/j.nano.2011.06.004
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Valsartan has been previously identified as a promising candidate for transdermal drug delivery.14-16
Table 1 Variables in Box-Behnken design for preparation of valsartan nanotransfersomes Factor
Methods Materials Valsartan was received as a gratis sample from Ranbaxy Research Laboratories Ltd. (Gurgaon, India). Phospholipon 90G (PL90G) was received as a gift sample from Phospholipid GmbH (Nattermannallee, Germany). Water for high-performance liquid chromatography (HPLC) was purchased from Thomas Baker Chemicals Ltd. (Mumbai, India). SDC and cholesterol were purchased from Spectrochem Pvt Ltd. (Mumbai, India). Rhodamine Red-X 1,2 dihexadecanoyl-sn-glycero-3-phosphoethanolamine trimethylammonium salt (RR) was purchased from Molecular Probes (Eugene, Oregon). Absolute ethanol was purchased from Merck (Darmstadt, Germany). All other chemicals used were of reagent grade and were used as received. Double-distilled water was used for all experiments. Animals Albino Wistar rats (6-8 weeks old, 100-125 g) were supplied by Central Animal House of Hamdard University and kept under standard laboratory conditions in 12 hours light/dark cycle at 25 ± 2°C. Animals were nourished with a pellet diet (Lipton India Ltd., Bangalore, India) and water ad libitum. The animals were received after the study was duly approved by the University Animal Ethics Committee, and Committee for the Purpose of Control and Supervision on Experiments on Animals (CPSCEA), government of India. Wistar rats were euthanized with prolonged ether anesthesia and the abdominal skin of each rat was excised. Hairs on the skin of animals (thickness ∼0.6 mm) were removed with electrical clippers, subcutaneous tissues were surgically removed, and the dermis side was wiped with isopropyl alcohol to remove residual adhering fat. The skin was washed with phosphate buffered saline, wrapped in aluminum foil, and stored in a deep freezer at −20°C until further use (used within 2 weeks of preparation). On the day of the experiment, skin was brought to room temperature (22°C) and skin samples were mounted over the diffusion cells in such a way that the SC side faced the donor compartment whereas the dermis faced the receiver compartment. Preparation of nanotransfersomes and liposomes using the factorial designs Valsartan transfersomal formulations (VTFs) were prepared by conventional thin-layer evaporation technique13,17 using factorial design, A four-factor three-level Box-Behnken design was employed to study the effect of independent variables on dependent variables as shown in Table 1. Twenty-nine formulations were prepared according to the experimental design shown in Table 2. The entrapment efficiency (EE %), vesicle size, and transdermal flux obtained from the skin permeation study of nanotransfersomes bearing valsartan are presented in Table 2. The valsartan liposomes formulation (VLF; ratio of PL90G to cholesterol, 85:15) that were used as a control in the present study were prepared by the rotary evaporation sonication method as
Independent variables X1 = Phospholipon 90G (mg) X2 = Sodium deoxycholate (mg) X3 = Valsartan (mg) X4 = Sonication time (minutes) Dependent variables Y1 = EE % Y2 = Vesicles size (nm) Y3 = Flux (μg/cm2/hr)
Level used, actual (coded) Low (−1)
Medium (0)
High (+1)
75 5 40 15
85 15 60 25
95 25 80 35
described above. Similarly, RR (0.03% w/v)-loaded transfersomes and liposomes were prepared for confocal laser microscopy. Experimental design A four-factor, three-level Box-Behnken design was used to explore the quadratic response surfaces and for constructing a second-order polynomial models using Design Expert (Version 7.1.6; Stat-Ease Inc., Minneapolis, Minnesota). A design matrix comprising 29 experimental runs was constructed, for which the nonlinear computer-generated quadratic model is defined as: Y = b0 + b1 X1 + b2 X2 + b3 X3 + b12 X1 X2 + b13 X1 X3 + b23 X2 X3 + b11 X12 + b22 X22 + b33 X32 where Y is the measured response associated with each factor level combination; b0 is constant; b1, b2, b3 are linear coefficients, b12, b13, b23 are interaction coefficients between the three factors, b11, b22, b33 are quadratic coefficients computed from the observed experimental values of Y from experimental runs; and X1, X2, and X3 are the coded levels of independent variables. The terms X1X2 and X1 2 (i = 1, 2, or 3) represent the interaction and quadratic terms, respectively.18 The independent variables selected were the amount of the phospholipids 90G (X1), SDC (X2), valsartan (X3), and sonication time (X4). The dependent variables were EE % (Y1), vesicle size (Y2), and flux (Y3), with constraints applied on the formulation of nanotransfersomes. The concentration range of independent variables under study is shown in Table 1 along with their low, medium, and high levels, which were selected based on the results from preliminary experimentation. The concentration range of phospholipids 90G (X1), SDC (X2), valsartan (X3), and sonication time (X4) used to prepare the 29 formulations and the respective observed responses are given in Table 2. Vesicles shape, size, and size distribution Nanotransfersomes vesicles were visualized by using a Morgagni 268D (Fei Electron Optics, Eindhoven, the Netherlands) transmission electron microscope; digital micrograph and soft imaging viewer software were used to perform the image capture and analysis.19 The vesicles size and size distribution were determined by dynamic light scattering method, using a
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Table 2 Observed response in Box-Behnken design for valsartan transfersomal formulation (VTF) Formulation code
VTF1 VTF2 VTF3 VTF4⁎ VTF5 VTF6⁎ VTF7 VTF8 VTF9⁎ VTF10 VTF11 VTF12 VTF13 VTF14 VTF15 VTF16 VTF17 VTF18 VTF19 VTF20 VTF21 VTF22 VTF23 VTF24 VTF25 VTF26 VTF27 VTF28⁎ VTF29⁎
Independent variables
Dependent variables
X1 (mg)
X2 (mg)
X3 (mg)
X4 (minutes)
Y1 (%) (mean ± SD)
Y2 (nm) (mean ± SD)
Y3 (μg/cm2/hr) (mean ± SD)
0 −1 −1 0 −1 0 +1 0 0 −1 +1 0 +1 0 0 0 0 −1 0 +1 0 +1 +1 −1 0 0 0 0 0
−1 0 −1 0 0 0 0 +1 0 0 0 +1 0 −1 −1 0 0 0 0 +1 0 −1 0 0 −1 +1 +1 0 0
−1 0 0 0 +1 0 0 0 0 0 0 0 +1 0 +1 +1 −1 0 +1 0 −1 0 −1 −1 0 −1 +1 0 0
0 0 0 0 0 0 −1 +1 0 −1 +1 −1 0 −1 0 0 +1 +1 +1 0 −1 0 0 0 +1 0 0 0 0
76.24 ± 4.23 69.76 ± 3.20 63.94 ± 3.65 88.62 ± 6.36 62.73 ± 5.35 87.56 ± 6.42 93.56 ± 5.45 79.46 ± 4.78 89.74 ± 6.47 70.59 ± 5.07 91.84 ± 8.12 82.82 ± 4.54 77.44 ± 3.74 78.45 ± 5.30 73.23 ± 4.21 77.53 ± 3.52 80.36 ± 5.04 75.23 ± 3.65 75.23 ± 4.32 81.62 ± 5.20 81.33 ± 5.77 90.98 ± 7.53 92.34 ± 7.09 65.75 ± 6.12 79.88 ± 6.22 77.45 ± 4.27 72.55 ± 4.52 86.74 ± 5.11 87.21 ± 4.75
158 ± 8 75 ± 4 120 ± 8 135 ± 10 90 ± 4 132 ± 7 176 ± 9 90 ± 5 138 ± 6 115 ± 5 155 ± 9 125 ± 7 160 ± 10 170 ± 8 162 ± 10 145 ± 7 110 ± 5 72 ± 4 115 ± 6 150 ± 8 139 ± 7 190 ± 12 163 ± 9 85 ± 5 138 ± 7 105 ± 6 115 ± 6 131 ± 7 133 ± 7
525.63 ± 25.47 210.71 ± 15.41 298.89 ± 25.85 620.06 ± 42.63 286.21 ± 22.42 625.32 ± 45.89 419.53 ± 32.28 470.26 ± 40.78 618.44 ± 49.05 255.74 ± 22.44 471.25 ± 32.74 425.26 ± 29.84 448.26 ± 37.54 490.32 ± 32.08 500.84 ± 25.75 531.02 ± 28.63 552.86 ± 24.70 304.04 ± 20.81 600.35 ± 42.78 411.83 ± 29.63 541.94 ± 32.74 425.76 ± 27.89 432.63 ± 32.74 312.05 ± 19.74 520.86 ± 30.44 445.64 ± 29.32 487.34 ± 29.85 626.57 ± 37.19 617.32 ± 32.25
X1, Phospholipon 90G (mg); X2, sodium deoxycholate (mg); X3, valsartan (mg); X4, sonication time (minutes); Y1, EE % ; Y2, = vesicle size (nm) ; Y3, flux (μg/cm2/hr). ⁎ Indicates the center point of the design.
computerized inspection system (Zetasizer, HAS 3000; Malvern Instruments, Malvern, United Kingdom).20 Entrapment efficiency EE %, expressed as a percentage of the total amount of valsartan found in the studied formulations at the end of the preparation procedure, was determined by HPLC method21 after disruption of vesicles with Triton X-100.22 The analyses were carried out with a liquid chromatograph (Model-1120 Compact LC; Agilent Technologies, Santa Clara, California), equipped with an ultraviolet (UV) detector. Separation was achieved using Shiseido C-18 column (250 × 4.6 mm, internal diameter 5 μm). Binary elution was carried out at a flow rate of 1.3 mL/min with the mobile phase containing 45% acetonitrile and 55% phosphate buffer solution (PBS), pH 3.0. Mobile phase was prepared daily, filtered by passing through a 0.45-μm membrane filter, and degassed. All chromatographic separations were performed at room temperature. Detection was carried out at 265 nm with a UV detector. The amount of entrapment drug expressed as a percentage was calculated from the following equation:
EEk =
Entrapped drug × 100 Total drug
Ex vivo skin permeation studies The ex vivo skin permeation of valsartan from nanotransfersomes was studied23-26 using a locally fabricated Franz diffusion cell with an effective permeation area and receptor cell volume of 1.0 cm2 and 15 mL, respectively. The temperature of the receiver vehicle (ethanol-PBS pH 7.4, 40:60 ratio) was maintained at 37 ± 1°C and was constantly stirred by magnetic stirrer at 100 rpm. An amount of nanotransfersomes equivalent to 20 mg of valsartan (transdermal dose of valsartan) was placed in the donor compartment. Samples of 500 μL were withdrawn from the receptor compartment via the sampling port at different time intervals (0, 1, 2, 3, 4, 6, 8, 10, 12, and 24 hours) and analyzed for drug content by HPLC method as stated above. The receptor phase was immediately replenished with an equal volume of fresh diffusion buffer. Similar experiments were performed with conventional liposomal formulations and ethanolic phosphate buffer solution of pure drug sample.13,17 To determine the extent of enhancement, enhancement ratio (ER) was calculated as follows:
ER =
Steady state flux of formulation Steady state flux of control
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Table 3 Groups of experimental rats, treatment given to different groups, and time intervals for measurement of BP Groups
Treatments
Group A Normal control Group B Hypertensive control Group C Oral suspension Group D Placebo-TTS Group E VLGF-TTS Group F VTGF-OPT-TTS
from the tail was recorded at predetermined time intervals up to 48 hours (Table 3).
No. of rats Measurement of systolic BP at in a group different time intervals (hr) 6 6
0,1, 3 , 6 , 9 , 12, 24 , 36, and 48 0,1, 3 , 6 , 9 , 12, 24 , 36, and 48
6 6 6 6
0,1, 3 , 6 , 9 , 12, 24 , 36, and 48 0,1, 3 , 6 , 9 , 12, 24 , 36, and 48 0,1, 3 , 6 , 9 , 12, 24 , 36, and 48 0,1, 3 , 6 , 9 , 12, 24 , 36, and 48
Confocal laser scanning microscopy (CLSM) study CLSM was used to scan the fluorescence signal of VLFs and VTF-OPT at different skin depths. For CLSM study, the ex vivo skin permeation study was carried out as described above. After 8 hours the skin was removed and washed with distilled water. The treated area was cut out and tested for probe penetration.27,28 The excised nude rat skin was positioned on the microscopic slide with the SC side face to the coverglass. CLSM was carried out with rhe Laser Confocal Microscope with Fluorescence Correlation Spectroscope-Olympus FluoView FV1000 (Olympus, Melville, New York) with an argon laser beam with excitation at 488 nm and emission at 590 nm. Each skin sample was sliced in sections of 6–10 μm thickness through the z-axis by CLSM. The VTF-OPT intensity and the permeated depth were detected by CLSM with Fluoview software. In vivo antihypertensive studies The preclinical assessment of antihypertensive activity of the developed transdermal therapeutic system (TTS) was performed on experimentally hypertensive rats. Hypertension was induced by injecting methyl prednisolone acetate (MPA, Depo-Medrol; Pfizer, Mumbai, India) (20 mg/kg/week) subcutaneously for 2 weeks. The studies were carried out using small Animal Tail Noninvasive Blood Pressure system (NIBP 200A; Biopac System, Inc., Goleta, California) based on cuff tail technique. The experimental (MPA-induced) hypertensive rats with minimum mean systolic blood pressure (BP) of 150 mm Hg were selected for further study. According the initial BP of rats, the animals were divided into six groups (group A to F) of six animals each. Treatments given to each group are shown in Table 3. Group A was taken as normal control. Hypertension was induced in the remaining groups (groups B to F) by subcutaneous injection of MPA for 2 weeks as per the method29 of Aqil et al. Group B served as hypertensive control and received no further treatment. After MPA treatment group C received an oral suspension of valsartan (3.6 mg/kg); the dose for the rats was calculated based on the body weight of the rats as per the surface area ratio method.30 Groups D, E, and F were subjected to placeboTTS, VLGF-TTS, and VTGF-OPT-TTS, respectively. Each TTS was applied to the previously shaven abdominal area of rat skin. The rat was then placed in the restrainer and the BP
Results Optimization of nanotransfersomes preparation Effect of drug concentration EE % is the percentage fraction of the total drug incorporated into the transfersomes. The maximum and minimum EE % values obtained were 93.56% ± 5.45% for VTF7 and 62.73% ± 5.35% for VTF5, respectively (Table 2). It was found that upon increasing valsartan concentration from 40 mg to 60 mg in the nanotransfersomes prepared, the EE % significantly increased from 65.75% ± 6.12% (VTF24, with least concentration PL90G and SDC) to 70.59% ± 5.07% (VTF10) (P b 0.001). However, a further increase in drug concentration to 80 mg led to a significant decrease in EE % to 62.73% ± 5.35% (VTF5) (P b 0.001); this may be due to the leakage of excess drug from the vesicular structure. Effects of independent variables on EE % are presented by three-dimensional graph in Figure 1. According to Lopes et al,31 the entrapment of drug occurs in both the bilayers and the aqueous compartment of the vesicles. When the lipid compartment and aqueous phase became saturated with the drug, the vesicles provided limited entrapment capacity.32 Highest flux (626.57 ± 37.19 μg/cm2/hr) was obtained for transfersomal formulation VTF28 having 60 mg of valsartan (Table 2). Effect of PL90G/SDC ratio Initially, the EE % increased significantly with increasing EA concentration from 5 to 15 mg (w/w). Further increase in EA concentration from 15 up to 25 mg (w/w) showed a decrease in EE % (Table 2). The ratio (85 mg PL90G/15 mg EA) showed optimum EE %. Upon incorporation of EA in low concentration, growth in vesicle size occurred,33 whereas further increase in the content of EA may have led to pore formation in the bilayers. It was observed that with increased EA concentration in the lipid components of the vesicles, the EE % of the valsartan decreased (Table 2). Ex vivo skin permeation studies Ex vivo skin permeation studies from nanotransfersomes containing SDC (with different ratios of PL90G/EA/drug) (Table 2) were performed. Steady-state fluxes from nanotransfersomes at 24 hours first increased with increasing EA concentration (from 5 to 15 mg) via rat skin, and then decreased (Table 2). The ex vivo permeation profile of transfersomal (VTF) and liposomal formulation shows that Transfersomes formulation (VTF28) presented maximum flux value (i.e., 626.57 ± 37.19 μg/cm2/hr over rigid liposome formulation (18.47 ± 0.95 μg/cm2/hr) with ER of 33.92 through rat skin. Effects of independent variables on flux are presented by three-dimensional graph in Figure 2. In our study we observed that the transdermal flux first increased with increasing EA concentration and then decreased (Table 2).
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Figure 1. Response surface plot showing effect of independent variables on percent entrapment efficiency.
Vesicle size analysis The mean vesicle sizes of various VTFs are presented in Table 2. The smallest mean vesicle size was observed for valsartanloaded Transfersomes formulation VTF18 (72 ± 4 nm), whereas the maximum vesicle size was obtained as 176 ± 9 nm for VTF7. This was a smaller mean vesicle size than that of traditional liposomes (209 ± 15 nm) prepared by the same method. These variations in vesicles size were highly significant (P b 0.001). Effects of independent variables on vesicles size are presented by three-dimensional graph in Figure 3. We conclude that small amounts of an EA in liposomal membranes increases the flexibility of vesicles, thereby enabling them to pass more easily through the pores of the polycarbonate filter during the vesicle preparation. Figure 4 quantitatively compares the resultant experimental values of the responses with those of the predicted values. Optimization The optimum formulation of valsartan-loaded nanotransfersomes systems was selected based on the criteria of attaining the maximum value of transdermal flux and EE %, minimizing the
vesicles size by applying the point prediction method of the Design Expert software.34,35 Upon “trading off” various response variables and comprehensive evaluation of feasibility search and exhaustive grid search, the formulation composition with phospholipid (85 mg), SDC (15 mg), valsartan (60 mg), and sonication time (25 minutes) was found to fulfill requisites of an optimum formulation (i.e., VTF-OPT). The optimized formulation has the EE % of 85.77% ± 2.97% with vesicles size and transdermal flux across rat skin of 130 ± 10 nm and 627.47 ± 30.45 μg/cm2/hr, respectively. Electron micrographs of VTF-OPT are shown in Figure 5, A. They show the outline and core of the wellidentified spherical vesicles, displaying sealed vesicular structure. Size distribution of optimized VTF-OPT nanotransfersomes loaded with valsartan is presented in Figure 5, B. The liposomes formulation showed EE % of 80% ± 3.25%, having a mean vesicles size of 209 ± 15 nm. The valsartan flux of 27.11 ± 2.90 μg/cm2/hr was achieved from the ethanolic PBS of valsartan through rat skin. The VTF-OPT and ethanolic PBS of valsartan showed ERs of 33.97 ± 1.25 and 1.46 ± 0.024 over the liposomes formulation, which produced the least transdermal
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Figure 2. Response surface plot showing effect of independent variables on transdermal flux.
flux of 18.47 ± 0.95 μg/cm2/hr through rat skin. On the basis of factorial design approach, the nanotransfersomal formulation (VTF-OPT) was selected for further in vivo studies. The VTFOPT was converted into gel. Briefly, Carbopol 940 (S. D. Fine Chemicals Ltd., Mumbai, India) (1% w/w) was added into water and kept overnight for complete humectation of polymer chains. Optimized Transfersomes dispersion (VTF-OPT) equivalent to 40 mg of valsartan (transdermal dose of valsartan for 48 hours) was added to hydrated Carbopol solution with stirring. Other ingredients, such as 15% w/v polyethylene glycol-400 (PEG400) and triethanolamine (0.5% w/v), were added to obtain homogeneous dispersion of gel, and this optimized valsartan transfersomal gel formulation (VTGF-OPT) was incorporated into reservoir-type TTS for in vivo antihypertensive studies.
RR) clearly defined the transdermal potential of nanotransfersomal carrier. CLSM study results revealed that the VTF-OPT formulation was fairly evenly distributed throughout the SC, viable epidermis, and dermis with high fluorescence intensity (Figure 6). CLSM studies conducted to measure the extent of penetration and transdermal potency of the nanotransfersomes system (VTF-OPT) depicted an increase in both the depth (up to 170 μm) of penetration and fluorescence intensity (Max FI = 160 arbitrary units, AU) on application of RR-loaded VTF-OPT as compared to rigid liposomes that were confined to a few microns (50 μm) depth only, and Max FI was found to be 40 AU with depth of penetration up to 80 μm (Figure 6). Ethanolic PBS was also effective in permeating probe up to 160 μm, although very low FI was observed as compared to the nanotransfersomal system with a Max FI of 80 AU (Figure 6).
CLSM study The extent of vesicular penetration measured by CLSM after application of three systems (i.e., ethanolic PBS of drug, conventional liposomes, and VTF-OPT-each containing 0.03%
In vivo antihypertensive study Hypertension was successfully induced in the normotensive rats by MPA administration for a period of 2 weeks, and they
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Figure 3. Response surface plot showing effect of independent variables on vesicles size.
remained hypertensive for 72 hours after stopping the MPA injection, as shown by high significant difference (t-test, P b 0.001) found in the pre- and post-treatment values (Table 4). This was authenticated by Dunnet test, which showed significant difference (P b 0.001) in BP values of normal control (group A) and hypertensive control (group B). The oral administration of valsartan suspension significantly (P b 0.05) controlled the hypertension within 2 hours, with the maximum antihypertensive effect (108.75 ± 9.75 mm Hg) observed at 3 hours, but after 3 hours the BP started rising gradually. The BP rose above the normal BP value (126.35 ± 8.75 mm Hg) after 9 hours and progressively reached 151.72 ± 14.83 mm Hg at 48 hours (Table 4). Oral suspension of valsartan acted rapidly and showed utmost changes in rat BP as shown in Table 4, but its effect dipped rapidly and BP increased to above normal value. Although liposome gel formulation TTS (VLGF-TTS) failed to reduce the BP value to normal, the maximum decrease (130.63 ± 11.05 mm Hg) observed for BP was at 12 hours. In contrast, the administration of valsartan through transdermal route (VTGFOPT-TTS) resulted in a gradual decrease of BP, with the
maximum effect (108.85 ± 7.98 mm Hg) observed at 6 hours on treatment of the experimentally hypertensive rats (P N 0.05). Table 4 reveals that the systolic BP of hypertensive rats was controlled up to 48 hours; however, at the 48-hour time point BP was comparable to the normotensive rats (P N 0.05). VTGFOPT-TTS decreased the BP insignificantly at the first hour (P N 0.05), but the BP gradually declined to normal value (122.42 ± 5.07 mm Hg) after 3 hours and the effect continued for 48 hours (125.76 ± 9.45 mm Hg). Discussion Effect of drug concentration Nanotransfersomes could entrap valsartan only to an optimum extent, after which any further increase in drug concentration led to leakage of valsartan from vesicle bilayers.31 Transdermal flux first increased with increasing valsartan concentration from 40 mg to 60 mg; with further increase in the drug concentration to 80 mg the flux decreased as a result of
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Figure 4. Linear correlation plots (A, C, E) between actual and predicted values and the corresponding residual plots (B, D, F) for various responses.
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mixed micelles and vesicles at higher concentrations of EA, with the consequence of lower drug entrapment in mixed micelles.13,39 Ex vivo skin permeation studies
Figure 5. (A) Transmission electron micrography following negative staining (20,000×). (Inset) Detailed picture, where unilamellarity and size of vesicles can be seen (180,000×). (B) Size distribution of optimized VTFOPT nanotransfersomes loaded with valsartan.
The reason for this better performance of nanotransfersome formulations in comparison with the traditional liposomes is the flexibility of the nanotransfersomes that allow them to pass through the skin more easily. The extremely high flexibility of the membrane permits nanotransfersomes to squeeze themselves even through pores much smaller than their own diameters.40 We observed that too low or too high concentration of EA (SDC) is not beneficial in vesicular delivery through skin. These findings are in agreement with published data.13,41-44 A possible explanation for lower drug delivery at a high surfactant concentration may be that the surfactant at high concentration decreased the EE % and disrupted the lipid membrane so that it became more leaky to the entrapped drug. This will, in turn, reduce the delivery, especially if we consider the possible carrier function of these nanotransfersomes. These mixed micelles are reported to be less effective in transdermal drug delivery as compared with a transfersomal system, because micelles are much less sensitive to a water activity gradient than transfersomes. This hypothesis is supported by the report45 of Cevc et al, who compared the penetration ability of transfersomes, liposomes, and mixed micelles by CLSM and observed that mixed micelles were restricted to the topmost part of the SC and that nanotransfersomes penetrate to a deeper skin layer. Vesicles size analysis
leakage of valsartan from vesicle bilayers. Results of ex vivo skin permeation suggested that a too low or a too high concentration of drug (valsartan) is not beneficial in vesicular delivery through skin and also indicated that the possible penetration-enhancing effect of drug is not mainly responsible for improved valsartan skin delivery from deformable vesicles. Other variables such as amount of phospholipids and surfactant present are also affecting the EE %. Effect of PL90G/SDC ratio Initially, the EE % increased significantly with increasing EA concentration; further increase in EA concentration showed a decrease in EE %, possibly due to the coexistence of mixed micelles and vesicles at higher concentrations of EA, with the consequence of lower drug entrapment in mixed micelles.17 The formation of micellar structure at higher concentrations of EA is an established fact. The studies36,37 by Lasch et al and Lopez et al prove this fact. At the same time the EA also causes fluidization of the bilayer that is responsible for increase in elasticity of vesicle membrane. At higher EA concentration, conversion of lipid vesicles into mixed micelles begins. These mixed micelles have a diameter b10 nm and are reported to be less deformable in nature and also have less skin permeation ability across the skin in comparison to transfersomes.13,36,37,38 Studying the effect of EA concentration in the lipid components of vesicles on the EE % of the lipophilic model drug, valsartan, clearly shows that EE % decreased with an increase in concentration of EA. This is due to the possible coexistence of
Inclusion of SDC (EA), an anionic surfactant used for formulation of nanotransfersomes which interact with lipid bilayers,46 instead of cholesterol (traditional liposomes), could explain this reduction in vesicle size. The size distribution of vesicles was determined by dynamic light scattering. We observed an initial decrease in the average size of the vesicles with increasing amounts of SDC (from 5 to 15 mg). However, a further increase in the SDC concentration from 15 up to 25 mg led to a reduction in the average size of vesicles. This is due to the formation of a micellar structure instead of the vesicles, which are relatively smaller in size.13 Fitting of data to the model Fitting of the data for observed responses to various models; it was observed that the best-fitted model for all the four dependent variables was the quadratic model (Table 5). Higher values of the standard error (SE) for coefficients indicate the quadratic (nonlinear) nature of the relationship. A positive value in regression equation for a response represents an effect that favors the optimization (synergistic effect), whereas a negative value indicates an inverse relationship (antagonistic effect) between the factor and the response.47 From Table 5 it is evident that the two independent variables (i.e., the concentrations of the phospholipid and the drug) have positive effects on the response Y1 (EE %), whereas the response Y2 (vesicle size) has an inverse relationship with SDC and sonication time. Response Y3 (flux) was affected by EA and drug concentrations, whereas it can be retarded if their concentrations are further increased beyond the
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Figure 6. CLS micrographs 8 hours after application of RR-loaded elastic liposomes optical cross-sections perpendicular to the rat skin surface. (A) Ethanolic PBS. (B) Valsartan liposomes formulation. (C) VTF-OPT formulation.
optimum limits (Table 2). The EA concentration had a negative effect on the response Y3 (flux). There existed a direct relationship between the phospholipid concentration and EA (SDC) on the vesicles size, EE % of the vesicles, and transdermal flux of vesicles loaded with valsartan. The lowest EE % was found (62.73% ± 5.35% for the formulation VTF5, and maximum EE % was found (93.56% ± 5.45%) for VTF7. It is observed from the experimental design that EE % has a direct positive relationship with concentration of phospholipid as revealed by the equation Y1 = +88.62 +9.73X1 +0.078X2 −2.65X3 −0.19X4 −3.79X1 X2 −3.72X1 X3 −1.59 X 1 X 4 −0.47 X 2 X 3 −1.20 X 2 X 4 −0.33 X 3 X 4 −5.0X1 2−6.57X2 2 −7.97X3 2 −1.58X4 2. As the concentration of the phospholipid increases, the vesicle EE %, but it also depends upon other variables such as amount of EA and drug content. The vesicle size is a very important criterion for the nanovesicular formulation; the size of the nanovesicles was found to vary between 72 ± 04 nm and 176 ± 09 nm. It was observed that the vesicle size has a direct positive relationship with the phospholipid concentration but a negative relationship with the amount of EA. We observed an initial decrease in the average size of the vesicles with increasing amounts of SDC (from 5 to 15 mg). However, an additional increase in the SDC concentration from 15 up to 25 mg led to a further decrease in the average size of vesicles. This is due to the formation of a micellar structure instead of the vesicles, which are relatively smaller in size. This relationship is presented by the following equation Y2 = +135.00 +36.42X1 −23.17X2+2.25X3 −15.83X4 +1.25X1 X2 −2.00 X1 X3 +5.50 X1 X4 +1.50X2 X3 −0.75 X2 X4 −0.25 X3 X4 −3.75 X1 2 +2.13 X2 2 −4.25 X3 2−3.88X4 2. Transdermal flux of valsartan loaded in transfersomal formulation is another important criterion for optimization of VTF; it was observed from the equation below that the
transdermal flux of VTF increased on increasing the lipid content in the formulation whereas the transdermal flux is decreased on increasing the EA: Y3 = +620.07+78.47X1 −25.94X2 +6.11X3 +23.82 X4 +18.56X1 X2 +10.37 X1 X3 +0.85 X1 X4 +16.62 X2 X3 +3.61 X2 X4 +22.10 X3 X4 −208.72X12 −91.67X2 2 −31.49X3 2−41.65X4 2 A possible explanation for lower flux at a high EA concentration may be that the EA at high concentration disrupted the lipid membrane so that it became more leaky to the entrapped drug. This will in turn reduce the flux. The valsartan concentration had a positive relationship with transdermal flux up to 60 mg of valsartan, but beyond this concentration it showed a negative relationship with transdermal flux. Further increasing the drug concentration up to 80 mg resulted in a decrease in the transdermal flux, possibly due to leakage of valsartan from vesicle bilayers at higher concentration. CLSM study The extent of vesicular penetration measured by CLSM after application of three systems (VTF-OPT, ethanolic PBS, and conventional liposomes each containing 0.03% RR (Figure 6) distinctly described the transdermal potential of nanotransfersomal carrier. CLSM results revealed that VTF-OPT were fairly evenly distributed throughout the SC, viable epidermis, and dermis with high fluorescence intensity. VTF-OPT penetrated deeply into rat skin, followed by ethanolic PBS solution of valsartan and conventional liposomes. VTF-OPT were delivered to a maximum possible depth of dermatomed skin. The prominently efficient delivery of RR by transfersomal carriers suggests their enhanced penetration and consequent fusion with the membrane lipids in the depths of the skin, supporting the hypothesis of many researchers.28,39,48,49
6.05 0.85 2.56 22.13 121.57 ± 5.35 161.84 ± 10.75 145.26 ± 12.44 160.44 ± 14.12 146.52 ± 10.57 121.65 ± 8.64 115.52 ± 7.25 163.09 ± 12.86 139.29 ± 10.14 161.58 ± 15.65 134.63 ± 10.55 120.42 ± 10.75 109.36 ± 4.75 165.78 ± 8.34 126.35 ± 8.75 162.75 ± 11.74 130.63 ± 11.05 116.34 ± 9.36
122.38 ± 4.86 161.50 ± 12.65 151.72 ± 14.83 160.12 ± 15.89 157.36 ± 13.14 125.76 ± 9.45
Quadratic model
R2
Adjusted R2
Predicted R2
SD
% CV
Response (Y1) 0.9866 0.9732 0.9227 1.39 1.74 Response (Y2) 0.9944 0.9888 0.9679 3.22 2.46 0.9878 0.9756 0.9298 18.71 4.02 Response (Y3) Regression equation of the fitted quadratic model⁎ Y1 = +88.62 + 9.73X1 + 0.078X2 − 2.65X3 − 0.19 X4− 3.79X1 X2 − 3.72X1 X3 − 1.59X1 X4 − 0.47X2 X3 − 1.20 X2 X4 − 0.33 X3 X4 − 5.02 X1 2 − 6.57 X2 2 − 7.97 X3 2− 1.58X4 2 Y2 = +135.00 + 36.42X1 − 23.17X2 + 2.25X3 − 15.83X4 +1.25X1 X2 − 2.00X1 X3 + 5.50 X1 X4 + 1.50X2 X3 − 0.75 X2 X4 − 0.25 X3 X4 − 3.75 X12 +2.13 X2 2 − 4.25 X3 2− 3.88X4 2 Y3 = +620.07 + 78.47X1 − 25.94X2 + 6.11X3 + 23.82X4 + 18.56X1 X2 + 10.37 X1 X3 + 0.85X1 X4 + 16.62X2 X3 + 3.61 X2 X4 + 22.10 X3 X4 − 208.72 X21 − 91.67 X2 2 − 31.49 X3 2 − 41.65 X4 2 CV, coefficient of variation. ⁎ Only the terms with statistical significance are included.
In vivo antihypertensive study
123.87 ± 6.74 165.74 ± 7.14 110.42 ± 9.78 162.08 ± 13.77 156.16 ± 10.44 108.85 ± 7.98 8.35 5.25 9.75 13.29 ± 10.06 5.07 ⁎ Control rats without hypertension induced and no further treatment. † Percentage reduction in BP at 48-hr time point. ‡ Hypertension was induced with MPA, and no treatment was given.
110.56 ± 179.26 ± 108.75 ± 178.62 ± 172.45 ± 122.42 ± 119.20 ± 8.53 182.65 ± 8.42 155.42 ± 10.58 182.45 ± 15.22 179.78 ± 10.24 162.25 ± 8.25 120.45 ± 10.32 183.37 ± 7.53 180.24 ± 5.65 179.48 ± 14.54 181.92 ± 7.85 179.64 ± 8.96 Normal control⁎ Hypertensive control‡ Oral suspension Placebo-TTS VLGF-TTS VTGF-OPT-TTS A B C D E F
247
Table 5 Summary of results of regression analysis for responses Y1, Y2, and Y3 for fitting to quadratic model
122.08 ± 5.12 169.42 ± 5.63 119.37 ± 9.68 166.59 ± 12.02 153.35 ± 10.32 110.74 ± 8.45
36 hr 24 hr 12 hr 9 hr
Mean systolic blood pressure (mmHg)
6 hr 3 hr 1 hr Initial Treatments Groups
Table 4 Influence of various optimized transdermal systems of valsartan on mean BP in MPA-induced hypertensive rats
48 h
Reduction in BP (%)†
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The developed VTGF-OPT-TTS was found to decrease the BP significantly (P b 0.001) in proximity of the normal value. The effect was maintained for 48 hours. On comparing the effects of all the TTS systems, the percentage reductions in mean systolic BP of rats by VTGF-OPT-TTS and placebo-TTS were 22.13% and 0.85%, respectively. However, percentage reduction in mean systolic BP of rats by oral valsartan solution and VLGFTTS was found to be 6.05% and 2.56%, respectively. Results of in vivo antihypertensive activity clearly indicates that the VTGF-OPT-TTS released the drug gradually over a period of time, which resulted in prolonged control of hypertension up to 48 hours. Formulation VTGF-OPT-TTS was successful in reverting the rat BP to normal values, whereas VLGF-TTS failed to reduce the BP to normal value. The above results suggest that the developed nanotransfersomal system of valsartan holds promise for the management of hypertension that must be validated by clinical trials. The results of the present study showed that deformable lipid vesicles, nanotransfersomes, improve the transdermal delivery of the lipophilic drug, valsartan. The formulation-optimizing study using statistical experimental design shows that optimum concentrations of phospholipid, surfactant, and valsartan are required to provide the maximum value of transdermal flux and EE %, minimizing the vesicles size. The optimized nanotransfersomal gel formulation showed better antihypertensive activity in a rat model in comparison with placebo-TTS and liposomes formulations. The developed valsartan nanotransfersomal gel formulation TTS was found to decrease the BP significantly (P b 0.001) in experimental hypertensive rats, which was maintained for 48 hours. The results of the present study demonstrated that introduction of nanotransfersomes as a vesicular drug carrier overcomes the limitation of low penetration ability of liposomes across the skin. Hence, it could be concluded that nanotransfersomes are a potentially suitable carrier for transdermal delivery of valsartan. Further studies are needed to establish their therapeutic utility in human beings.
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Research Article
nanomedjournal.com
Formulation and optimization of nanotransfersomes using experimental design technique for accentuated transdermal delivery of valsartan Abdul Ahad, MPharm, PhD, Mohammed Aqil, MPharm, PhD⁎, Kanchan Kohli, MPharm, PhD, Yasmin Sultana, MPharm, PhD, Mohammed Mujeeb, MPharm, PhD, Asgar Ali, MPharm, PhD Faculty of Pharmacy, Hamdard University, New Delhi, India Received 8 November 2010; accepted 4 June 2011
Abstract The purpose of this work was to develop and statistically optimize nanotransfersomes for enhanced transdermal of valsartan vis-àvis traditional liposomes. Nanotransfersomes bearing valsartan were prepared by conventional rotary evaporation method and characterized for various parameters including entrapment efficiency, vesicles shape, size, size distribution, and skin permeation. In vivo antihypertensive activity conducted on Wistar rats was also taken as a measure of performance of nanotransfersomes and liposomes. Nanotransfersomes proved significantly superior in terms of amount of drug permeated in the skin, with an enhancement ratio of 33.97 ± 1.25 when compared to rigid liposomes. This was further confirmed through a confocal laser scanning microscopy study. Nanotransfersomes showed better antihypertensive activity in comparison to liposomes by virtue of better permeation through Wistar rat skin. Finally, it could be concluded that the nanotransfersomes accentuates the transdermal flux of valsartan and could be used as a carrier for effective transdermal delivery of valsartan. From the Clinical Editor: In this paper, the authors discuss the development and optimization of nanotransfersomes for enhanced transdermal of valsartan and demonstrate accentuated transdermal compared to standard preparations. © 2012 Elsevier Inc. All rights reserved. Key words: Box-Behnken design; Elastic liposomes; Hypertension; Transfersomes; Valsartan
The greatest obstacle for transdermal delivery is the barrier property of the stratum corneum (SC).1,2 Many approaches have been used to breach the skin barrier; for example, the use of lipid vesicles to modulate the SC is gaining interest. The first articles to report on the effectiveness of vesicles for skin delivery were published in the early 1980s. Most groups concluded that liposomes did not act as transport systems.3 Conventional liposomes have been generally reported to remain confined to the upper layer of the SC and to accumulate in the skin appendages, with minimal penetration to deeper tissues, because of their large size and lack of flexibility.4,5 Further intensive research over the past two decades led to the introduction and development of a new class of lipid vesicles, the ultradeformable (elastic or ultraflexible) liposomes that have been termed Transfersomes (IDEA AG, Munich, Germany). Several studies have reported that TransferAbdul Ahad thanks the Council for Scientific and Industrial Research (CSIR), India (File No. 09/591 (0084)/2009-EMR-I), for providing financial assistance in the form of a senior research fellowship. ⁎Corresponding author: Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University, New Delhi 110 062, India. E-mail address: [email protected] (M. Aqil).
somes were able to improve in vitro skin delivery of various drugs6 and to penetrate intact skin in vivo, transferring therapeutic amounts of drugs with efficiency comparable with subcutaneous administration.7-12 The key factors that confer ultradeformability to the liposomes have been considered to be edge activators (EAs), the special surfactants incorporated into Transfersomes (e.g., sodium cholate or sodium deoxycholate, SDC). Because Transfersomes are composed of surfactant, they have better rheology and hydration properties, which are responsible for their superior skin penetration ability. Less deformable vesicles, including traditional liposomes, are confined to the skin surface where they dehydrate completely and fuse, so they have less penetration power than Transfersomes. Transfersomes are optimized in this respect, thus attaining maximum flexibility, and hence they can take full advantage of the transepidermal osmotic gradient (water concentration gradient).13 The aim of the present study was to optimize the nanovesicles formulations (nanotransfersomes) for enhanced skin delivery of a model drug valsartan, a lipophilic antihypertensive drug having low oral bioavailability of about 25%. It has low molecular weight (435.5) and melting point (116–117°C) with a log partition coefficient of 4.5 and a mean biological half-life of 7.5 hours; there are no reports of skin irritation attributed to valsartan.
1549-9634/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2011.06.004 Please cite this article as: A. Ahad, M. Aqil, K. Kohli, Y. Sultana, M. Mujeeb, A. Ali, Formulation and optimization of nanotransfersomes using experimental design technique for accentuated transdermal delivery of vals.... Nanomedicine: NBM 2012;8:237-249, doi:10.1016/j.nano.2011.06.004
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Valsartan has been previously identified as a promising candidate for transdermal drug delivery.14-16
Table 1 Variables in Box-Behnken design for preparation of valsartan nanotransfersomes Factor
Methods Materials Valsartan was received as a gratis sample from Ranbaxy Research Laboratories Ltd. (Gurgaon, India). Phospholipon 90G (PL90G) was received as a gift sample from Phospholipid GmbH (Nattermannallee, Germany). Water for high-performance liquid chromatography (HPLC) was purchased from Thomas Baker Chemicals Ltd. (Mumbai, India). SDC and cholesterol were purchased from Spectrochem Pvt Ltd. (Mumbai, India). Rhodamine Red-X 1,2 dihexadecanoyl-sn-glycero-3-phosphoethanolamine trimethylammonium salt (RR) was purchased from Molecular Probes (Eugene, Oregon). Absolute ethanol was purchased from Merck (Darmstadt, Germany). All other chemicals used were of reagent grade and were used as received. Double-distilled water was used for all experiments. Animals Albino Wistar rats (6-8 weeks old, 100-125 g) were supplied by Central Animal House of Hamdard University and kept under standard laboratory conditions in 12 hours light/dark cycle at 25 ± 2°C. Animals were nourished with a pellet diet (Lipton India Ltd., Bangalore, India) and water ad libitum. The animals were received after the study was duly approved by the University Animal Ethics Committee, and Committee for the Purpose of Control and Supervision on Experiments on Animals (CPSCEA), government of India. Wistar rats were euthanized with prolonged ether anesthesia and the abdominal skin of each rat was excised. Hairs on the skin of animals (thickness ∼0.6 mm) were removed with electrical clippers, subcutaneous tissues were surgically removed, and the dermis side was wiped with isopropyl alcohol to remove residual adhering fat. The skin was washed with phosphate buffered saline, wrapped in aluminum foil, and stored in a deep freezer at −20°C until further use (used within 2 weeks of preparation). On the day of the experiment, skin was brought to room temperature (22°C) and skin samples were mounted over the diffusion cells in such a way that the SC side faced the donor compartment whereas the dermis faced the receiver compartment. Preparation of nanotransfersomes and liposomes using the factorial designs Valsartan transfersomal formulations (VTFs) were prepared by conventional thin-layer evaporation technique13,17 using factorial design, A four-factor three-level Box-Behnken design was employed to study the effect of independent variables on dependent variables as shown in Table 1. Twenty-nine formulations were prepared according to the experimental design shown in Table 2. The entrapment efficiency (EE %), vesicle size, and transdermal flux obtained from the skin permeation study of nanotransfersomes bearing valsartan are presented in Table 2. The valsartan liposomes formulation (VLF; ratio of PL90G to cholesterol, 85:15) that were used as a control in the present study were prepared by the rotary evaporation sonication method as
Independent variables X1 = Phospholipon 90G (mg) X2 = Sodium deoxycholate (mg) X3 = Valsartan (mg) X4 = Sonication time (minutes) Dependent variables Y1 = EE % Y2 = Vesicles size (nm) Y3 = Flux (μg/cm2/hr)
Level used, actual (coded) Low (−1)
Medium (0)
High (+1)
75 5 40 15
85 15 60 25
95 25 80 35
described above. Similarly, RR (0.03% w/v)-loaded transfersomes and liposomes were prepared for confocal laser microscopy. Experimental design A four-factor, three-level Box-Behnken design was used to explore the quadratic response surfaces and for constructing a second-order polynomial models using Design Expert (Version 7.1.6; Stat-Ease Inc., Minneapolis, Minnesota). A design matrix comprising 29 experimental runs was constructed, for which the nonlinear computer-generated quadratic model is defined as: Y = b0 + b1 X1 + b2 X2 + b3 X3 + b12 X1 X2 + b13 X1 X3 + b23 X2 X3 + b11 X12 + b22 X22 + b33 X32 where Y is the measured response associated with each factor level combination; b0 is constant; b1, b2, b3 are linear coefficients, b12, b13, b23 are interaction coefficients between the three factors, b11, b22, b33 are quadratic coefficients computed from the observed experimental values of Y from experimental runs; and X1, X2, and X3 are the coded levels of independent variables. The terms X1X2 and X1 2 (i = 1, 2, or 3) represent the interaction and quadratic terms, respectively.18 The independent variables selected were the amount of the phospholipids 90G (X1), SDC (X2), valsartan (X3), and sonication time (X4). The dependent variables were EE % (Y1), vesicle size (Y2), and flux (Y3), with constraints applied on the formulation of nanotransfersomes. The concentration range of independent variables under study is shown in Table 1 along with their low, medium, and high levels, which were selected based on the results from preliminary experimentation. The concentration range of phospholipids 90G (X1), SDC (X2), valsartan (X3), and sonication time (X4) used to prepare the 29 formulations and the respective observed responses are given in Table 2. Vesicles shape, size, and size distribution Nanotransfersomes vesicles were visualized by using a Morgagni 268D (Fei Electron Optics, Eindhoven, the Netherlands) transmission electron microscope; digital micrograph and soft imaging viewer software were used to perform the image capture and analysis.19 The vesicles size and size distribution were determined by dynamic light scattering method, using a
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Table 2 Observed response in Box-Behnken design for valsartan transfersomal formulation (VTF) Formulation code
VTF1 VTF2 VTF3 VTF4⁎ VTF5 VTF6⁎ VTF7 VTF8 VTF9⁎ VTF10 VTF11 VTF12 VTF13 VTF14 VTF15 VTF16 VTF17 VTF18 VTF19 VTF20 VTF21 VTF22 VTF23 VTF24 VTF25 VTF26 VTF27 VTF28⁎ VTF29⁎
Independent variables
Dependent variables
X1 (mg)
X2 (mg)
X3 (mg)
X4 (minutes)
Y1 (%) (mean ± SD)
Y2 (nm) (mean ± SD)
Y3 (μg/cm2/hr) (mean ± SD)
0 −1 −1 0 −1 0 +1 0 0 −1 +1 0 +1 0 0 0 0 −1 0 +1 0 +1 +1 −1 0 0 0 0 0
−1 0 −1 0 0 0 0 +1 0 0 0 +1 0 −1 −1 0 0 0 0 +1 0 −1 0 0 −1 +1 +1 0 0
−1 0 0 0 +1 0 0 0 0 0 0 0 +1 0 +1 +1 −1 0 +1 0 −1 0 −1 −1 0 −1 +1 0 0
0 0 0 0 0 0 −1 +1 0 −1 +1 −1 0 −1 0 0 +1 +1 +1 0 −1 0 0 0 +1 0 0 0 0
76.24 ± 4.23 69.76 ± 3.20 63.94 ± 3.65 88.62 ± 6.36 62.73 ± 5.35 87.56 ± 6.42 93.56 ± 5.45 79.46 ± 4.78 89.74 ± 6.47 70.59 ± 5.07 91.84 ± 8.12 82.82 ± 4.54 77.44 ± 3.74 78.45 ± 5.30 73.23 ± 4.21 77.53 ± 3.52 80.36 ± 5.04 75.23 ± 3.65 75.23 ± 4.32 81.62 ± 5.20 81.33 ± 5.77 90.98 ± 7.53 92.34 ± 7.09 65.75 ± 6.12 79.88 ± 6.22 77.45 ± 4.27 72.55 ± 4.52 86.74 ± 5.11 87.21 ± 4.75
158 ± 8 75 ± 4 120 ± 8 135 ± 10 90 ± 4 132 ± 7 176 ± 9 90 ± 5 138 ± 6 115 ± 5 155 ± 9 125 ± 7 160 ± 10 170 ± 8 162 ± 10 145 ± 7 110 ± 5 72 ± 4 115 ± 6 150 ± 8 139 ± 7 190 ± 12 163 ± 9 85 ± 5 138 ± 7 105 ± 6 115 ± 6 131 ± 7 133 ± 7
525.63 ± 25.47 210.71 ± 15.41 298.89 ± 25.85 620.06 ± 42.63 286.21 ± 22.42 625.32 ± 45.89 419.53 ± 32.28 470.26 ± 40.78 618.44 ± 49.05 255.74 ± 22.44 471.25 ± 32.74 425.26 ± 29.84 448.26 ± 37.54 490.32 ± 32.08 500.84 ± 25.75 531.02 ± 28.63 552.86 ± 24.70 304.04 ± 20.81 600.35 ± 42.78 411.83 ± 29.63 541.94 ± 32.74 425.76 ± 27.89 432.63 ± 32.74 312.05 ± 19.74 520.86 ± 30.44 445.64 ± 29.32 487.34 ± 29.85 626.57 ± 37.19 617.32 ± 32.25
X1, Phospholipon 90G (mg); X2, sodium deoxycholate (mg); X3, valsartan (mg); X4, sonication time (minutes); Y1, EE % ; Y2, = vesicle size (nm) ; Y3, flux (μg/cm2/hr). ⁎ Indicates the center point of the design.
computerized inspection system (Zetasizer, HAS 3000; Malvern Instruments, Malvern, United Kingdom).20 Entrapment efficiency EE %, expressed as a percentage of the total amount of valsartan found in the studied formulations at the end of the preparation procedure, was determined by HPLC method21 after disruption of vesicles with Triton X-100.22 The analyses were carried out with a liquid chromatograph (Model-1120 Compact LC; Agilent Technologies, Santa Clara, California), equipped with an ultraviolet (UV) detector. Separation was achieved using Shiseido C-18 column (250 × 4.6 mm, internal diameter 5 μm). Binary elution was carried out at a flow rate of 1.3 mL/min with the mobile phase containing 45% acetonitrile and 55% phosphate buffer solution (PBS), pH 3.0. Mobile phase was prepared daily, filtered by passing through a 0.45-μm membrane filter, and degassed. All chromatographic separations were performed at room temperature. Detection was carried out at 265 nm with a UV detector. The amount of entrapment drug expressed as a percentage was calculated from the following equation:
EEk =
Entrapped drug × 100 Total drug
Ex vivo skin permeation studies The ex vivo skin permeation of valsartan from nanotransfersomes was studied23-26 using a locally fabricated Franz diffusion cell with an effective permeation area and receptor cell volume of 1.0 cm2 and 15 mL, respectively. The temperature of the receiver vehicle (ethanol-PBS pH 7.4, 40:60 ratio) was maintained at 37 ± 1°C and was constantly stirred by magnetic stirrer at 100 rpm. An amount of nanotransfersomes equivalent to 20 mg of valsartan (transdermal dose of valsartan) was placed in the donor compartment. Samples of 500 μL were withdrawn from the receptor compartment via the sampling port at different time intervals (0, 1, 2, 3, 4, 6, 8, 10, 12, and 24 hours) and analyzed for drug content by HPLC method as stated above. The receptor phase was immediately replenished with an equal volume of fresh diffusion buffer. Similar experiments were performed with conventional liposomal formulations and ethanolic phosphate buffer solution of pure drug sample.13,17 To determine the extent of enhancement, enhancement ratio (ER) was calculated as follows:
ER =
Steady state flux of formulation Steady state flux of control
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Table 3 Groups of experimental rats, treatment given to different groups, and time intervals for measurement of BP Groups
Treatments
Group A Normal control Group B Hypertensive control Group C Oral suspension Group D Placebo-TTS Group E VLGF-TTS Group F VTGF-OPT-TTS
from the tail was recorded at predetermined time intervals up to 48 hours (Table 3).
No. of rats Measurement of systolic BP at in a group different time intervals (hr) 6 6
0,1, 3 , 6 , 9 , 12, 24 , 36, and 48 0,1, 3 , 6 , 9 , 12, 24 , 36, and 48
6 6 6 6
0,1, 3 , 6 , 9 , 12, 24 , 36, and 48 0,1, 3 , 6 , 9 , 12, 24 , 36, and 48 0,1, 3 , 6 , 9 , 12, 24 , 36, and 48 0,1, 3 , 6 , 9 , 12, 24 , 36, and 48
Confocal laser scanning microscopy (CLSM) study CLSM was used to scan the fluorescence signal of VLFs and VTF-OPT at different skin depths. For CLSM study, the ex vivo skin permeation study was carried out as described above. After 8 hours the skin was removed and washed with distilled water. The treated area was cut out and tested for probe penetration.27,28 The excised nude rat skin was positioned on the microscopic slide with the SC side face to the coverglass. CLSM was carried out with rhe Laser Confocal Microscope with Fluorescence Correlation Spectroscope-Olympus FluoView FV1000 (Olympus, Melville, New York) with an argon laser beam with excitation at 488 nm and emission at 590 nm. Each skin sample was sliced in sections of 6–10 μm thickness through the z-axis by CLSM. The VTF-OPT intensity and the permeated depth were detected by CLSM with Fluoview software. In vivo antihypertensive studies The preclinical assessment of antihypertensive activity of the developed transdermal therapeutic system (TTS) was performed on experimentally hypertensive rats. Hypertension was induced by injecting methyl prednisolone acetate (MPA, Depo-Medrol; Pfizer, Mumbai, India) (20 mg/kg/week) subcutaneously for 2 weeks. The studies were carried out using small Animal Tail Noninvasive Blood Pressure system (NIBP 200A; Biopac System, Inc., Goleta, California) based on cuff tail technique. The experimental (MPA-induced) hypertensive rats with minimum mean systolic blood pressure (BP) of 150 mm Hg were selected for further study. According the initial BP of rats, the animals were divided into six groups (group A to F) of six animals each. Treatments given to each group are shown in Table 3. Group A was taken as normal control. Hypertension was induced in the remaining groups (groups B to F) by subcutaneous injection of MPA for 2 weeks as per the method29 of Aqil et al. Group B served as hypertensive control and received no further treatment. After MPA treatment group C received an oral suspension of valsartan (3.6 mg/kg); the dose for the rats was calculated based on the body weight of the rats as per the surface area ratio method.30 Groups D, E, and F were subjected to placeboTTS, VLGF-TTS, and VTGF-OPT-TTS, respectively. Each TTS was applied to the previously shaven abdominal area of rat skin. The rat was then placed in the restrainer and the BP
Results Optimization of nanotransfersomes preparation Effect of drug concentration EE % is the percentage fraction of the total drug incorporated into the transfersomes. The maximum and minimum EE % values obtained were 93.56% ± 5.45% for VTF7 and 62.73% ± 5.35% for VTF5, respectively (Table 2). It was found that upon increasing valsartan concentration from 40 mg to 60 mg in the nanotransfersomes prepared, the EE % significantly increased from 65.75% ± 6.12% (VTF24, with least concentration PL90G and SDC) to 70.59% ± 5.07% (VTF10) (P b 0.001). However, a further increase in drug concentration to 80 mg led to a significant decrease in EE % to 62.73% ± 5.35% (VTF5) (P b 0.001); this may be due to the leakage of excess drug from the vesicular structure. Effects of independent variables on EE % are presented by three-dimensional graph in Figure 1. According to Lopes et al,31 the entrapment of drug occurs in both the bilayers and the aqueous compartment of the vesicles. When the lipid compartment and aqueous phase became saturated with the drug, the vesicles provided limited entrapment capacity.32 Highest flux (626.57 ± 37.19 μg/cm2/hr) was obtained for transfersomal formulation VTF28 having 60 mg of valsartan (Table 2). Effect of PL90G/SDC ratio Initially, the EE % increased significantly with increasing EA concentration from 5 to 15 mg (w/w). Further increase in EA concentration from 15 up to 25 mg (w/w) showed a decrease in EE % (Table 2). The ratio (85 mg PL90G/15 mg EA) showed optimum EE %. Upon incorporation of EA in low concentration, growth in vesicle size occurred,33 whereas further increase in the content of EA may have led to pore formation in the bilayers. It was observed that with increased EA concentration in the lipid components of the vesicles, the EE % of the valsartan decreased (Table 2). Ex vivo skin permeation studies Ex vivo skin permeation studies from nanotransfersomes containing SDC (with different ratios of PL90G/EA/drug) (Table 2) were performed. Steady-state fluxes from nanotransfersomes at 24 hours first increased with increasing EA concentration (from 5 to 15 mg) via rat skin, and then decreased (Table 2). The ex vivo permeation profile of transfersomal (VTF) and liposomal formulation shows that Transfersomes formulation (VTF28) presented maximum flux value (i.e., 626.57 ± 37.19 μg/cm2/hr over rigid liposome formulation (18.47 ± 0.95 μg/cm2/hr) with ER of 33.92 through rat skin. Effects of independent variables on flux are presented by three-dimensional graph in Figure 2. In our study we observed that the transdermal flux first increased with increasing EA concentration and then decreased (Table 2).
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Figure 1. Response surface plot showing effect of independent variables on percent entrapment efficiency.
Vesicle size analysis The mean vesicle sizes of various VTFs are presented in Table 2. The smallest mean vesicle size was observed for valsartanloaded Transfersomes formulation VTF18 (72 ± 4 nm), whereas the maximum vesicle size was obtained as 176 ± 9 nm for VTF7. This was a smaller mean vesicle size than that of traditional liposomes (209 ± 15 nm) prepared by the same method. These variations in vesicles size were highly significant (P b 0.001). Effects of independent variables on vesicles size are presented by three-dimensional graph in Figure 3. We conclude that small amounts of an EA in liposomal membranes increases the flexibility of vesicles, thereby enabling them to pass more easily through the pores of the polycarbonate filter during the vesicle preparation. Figure 4 quantitatively compares the resultant experimental values of the responses with those of the predicted values. Optimization The optimum formulation of valsartan-loaded nanotransfersomes systems was selected based on the criteria of attaining the maximum value of transdermal flux and EE %, minimizing the
vesicles size by applying the point prediction method of the Design Expert software.34,35 Upon “trading off” various response variables and comprehensive evaluation of feasibility search and exhaustive grid search, the formulation composition with phospholipid (85 mg), SDC (15 mg), valsartan (60 mg), and sonication time (25 minutes) was found to fulfill requisites of an optimum formulation (i.e., VTF-OPT). The optimized formulation has the EE % of 85.77% ± 2.97% with vesicles size and transdermal flux across rat skin of 130 ± 10 nm and 627.47 ± 30.45 μg/cm2/hr, respectively. Electron micrographs of VTF-OPT are shown in Figure 5, A. They show the outline and core of the wellidentified spherical vesicles, displaying sealed vesicular structure. Size distribution of optimized VTF-OPT nanotransfersomes loaded with valsartan is presented in Figure 5, B. The liposomes formulation showed EE % of 80% ± 3.25%, having a mean vesicles size of 209 ± 15 nm. The valsartan flux of 27.11 ± 2.90 μg/cm2/hr was achieved from the ethanolic PBS of valsartan through rat skin. The VTF-OPT and ethanolic PBS of valsartan showed ERs of 33.97 ± 1.25 and 1.46 ± 0.024 over the liposomes formulation, which produced the least transdermal
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Figure 2. Response surface plot showing effect of independent variables on transdermal flux.
flux of 18.47 ± 0.95 μg/cm2/hr through rat skin. On the basis of factorial design approach, the nanotransfersomal formulation (VTF-OPT) was selected for further in vivo studies. The VTFOPT was converted into gel. Briefly, Carbopol 940 (S. D. Fine Chemicals Ltd., Mumbai, India) (1% w/w) was added into water and kept overnight for complete humectation of polymer chains. Optimized Transfersomes dispersion (VTF-OPT) equivalent to 40 mg of valsartan (transdermal dose of valsartan for 48 hours) was added to hydrated Carbopol solution with stirring. Other ingredients, such as 15% w/v polyethylene glycol-400 (PEG400) and triethanolamine (0.5% w/v), were added to obtain homogeneous dispersion of gel, and this optimized valsartan transfersomal gel formulation (VTGF-OPT) was incorporated into reservoir-type TTS for in vivo antihypertensive studies.
RR) clearly defined the transdermal potential of nanotransfersomal carrier. CLSM study results revealed that the VTF-OPT formulation was fairly evenly distributed throughout the SC, viable epidermis, and dermis with high fluorescence intensity (Figure 6). CLSM studies conducted to measure the extent of penetration and transdermal potency of the nanotransfersomes system (VTF-OPT) depicted an increase in both the depth (up to 170 μm) of penetration and fluorescence intensity (Max FI = 160 arbitrary units, AU) on application of RR-loaded VTF-OPT as compared to rigid liposomes that were confined to a few microns (50 μm) depth only, and Max FI was found to be 40 AU with depth of penetration up to 80 μm (Figure 6). Ethanolic PBS was also effective in permeating probe up to 160 μm, although very low FI was observed as compared to the nanotransfersomal system with a Max FI of 80 AU (Figure 6).
CLSM study The extent of vesicular penetration measured by CLSM after application of three systems (i.e., ethanolic PBS of drug, conventional liposomes, and VTF-OPT-each containing 0.03%
In vivo antihypertensive study Hypertension was successfully induced in the normotensive rats by MPA administration for a period of 2 weeks, and they
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Figure 3. Response surface plot showing effect of independent variables on vesicles size.
remained hypertensive for 72 hours after stopping the MPA injection, as shown by high significant difference (t-test, P b 0.001) found in the pre- and post-treatment values (Table 4). This was authenticated by Dunnet test, which showed significant difference (P b 0.001) in BP values of normal control (group A) and hypertensive control (group B). The oral administration of valsartan suspension significantly (P b 0.05) controlled the hypertension within 2 hours, with the maximum antihypertensive effect (108.75 ± 9.75 mm Hg) observed at 3 hours, but after 3 hours the BP started rising gradually. The BP rose above the normal BP value (126.35 ± 8.75 mm Hg) after 9 hours and progressively reached 151.72 ± 14.83 mm Hg at 48 hours (Table 4). Oral suspension of valsartan acted rapidly and showed utmost changes in rat BP as shown in Table 4, but its effect dipped rapidly and BP increased to above normal value. Although liposome gel formulation TTS (VLGF-TTS) failed to reduce the BP value to normal, the maximum decrease (130.63 ± 11.05 mm Hg) observed for BP was at 12 hours. In contrast, the administration of valsartan through transdermal route (VTGFOPT-TTS) resulted in a gradual decrease of BP, with the
maximum effect (108.85 ± 7.98 mm Hg) observed at 6 hours on treatment of the experimentally hypertensive rats (P N 0.05). Table 4 reveals that the systolic BP of hypertensive rats was controlled up to 48 hours; however, at the 48-hour time point BP was comparable to the normotensive rats (P N 0.05). VTGFOPT-TTS decreased the BP insignificantly at the first hour (P N 0.05), but the BP gradually declined to normal value (122.42 ± 5.07 mm Hg) after 3 hours and the effect continued for 48 hours (125.76 ± 9.45 mm Hg). Discussion Effect of drug concentration Nanotransfersomes could entrap valsartan only to an optimum extent, after which any further increase in drug concentration led to leakage of valsartan from vesicle bilayers.31 Transdermal flux first increased with increasing valsartan concentration from 40 mg to 60 mg; with further increase in the drug concentration to 80 mg the flux decreased as a result of
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Figure 4. Linear correlation plots (A, C, E) between actual and predicted values and the corresponding residual plots (B, D, F) for various responses.
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mixed micelles and vesicles at higher concentrations of EA, with the consequence of lower drug entrapment in mixed micelles.13,39 Ex vivo skin permeation studies
Figure 5. (A) Transmission electron micrography following negative staining (20,000×). (Inset) Detailed picture, where unilamellarity and size of vesicles can be seen (180,000×). (B) Size distribution of optimized VTFOPT nanotransfersomes loaded with valsartan.
The reason for this better performance of nanotransfersome formulations in comparison with the traditional liposomes is the flexibility of the nanotransfersomes that allow them to pass through the skin more easily. The extremely high flexibility of the membrane permits nanotransfersomes to squeeze themselves even through pores much smaller than their own diameters.40 We observed that too low or too high concentration of EA (SDC) is not beneficial in vesicular delivery through skin. These findings are in agreement with published data.13,41-44 A possible explanation for lower drug delivery at a high surfactant concentration may be that the surfactant at high concentration decreased the EE % and disrupted the lipid membrane so that it became more leaky to the entrapped drug. This will, in turn, reduce the delivery, especially if we consider the possible carrier function of these nanotransfersomes. These mixed micelles are reported to be less effective in transdermal drug delivery as compared with a transfersomal system, because micelles are much less sensitive to a water activity gradient than transfersomes. This hypothesis is supported by the report45 of Cevc et al, who compared the penetration ability of transfersomes, liposomes, and mixed micelles by CLSM and observed that mixed micelles were restricted to the topmost part of the SC and that nanotransfersomes penetrate to a deeper skin layer. Vesicles size analysis
leakage of valsartan from vesicle bilayers. Results of ex vivo skin permeation suggested that a too low or a too high concentration of drug (valsartan) is not beneficial in vesicular delivery through skin and also indicated that the possible penetration-enhancing effect of drug is not mainly responsible for improved valsartan skin delivery from deformable vesicles. Other variables such as amount of phospholipids and surfactant present are also affecting the EE %. Effect of PL90G/SDC ratio Initially, the EE % increased significantly with increasing EA concentration; further increase in EA concentration showed a decrease in EE %, possibly due to the coexistence of mixed micelles and vesicles at higher concentrations of EA, with the consequence of lower drug entrapment in mixed micelles.17 The formation of micellar structure at higher concentrations of EA is an established fact. The studies36,37 by Lasch et al and Lopez et al prove this fact. At the same time the EA also causes fluidization of the bilayer that is responsible for increase in elasticity of vesicle membrane. At higher EA concentration, conversion of lipid vesicles into mixed micelles begins. These mixed micelles have a diameter b10 nm and are reported to be less deformable in nature and also have less skin permeation ability across the skin in comparison to transfersomes.13,36,37,38 Studying the effect of EA concentration in the lipid components of vesicles on the EE % of the lipophilic model drug, valsartan, clearly shows that EE % decreased with an increase in concentration of EA. This is due to the possible coexistence of
Inclusion of SDC (EA), an anionic surfactant used for formulation of nanotransfersomes which interact with lipid bilayers,46 instead of cholesterol (traditional liposomes), could explain this reduction in vesicle size. The size distribution of vesicles was determined by dynamic light scattering. We observed an initial decrease in the average size of the vesicles with increasing amounts of SDC (from 5 to 15 mg). However, a further increase in the SDC concentration from 15 up to 25 mg led to a reduction in the average size of vesicles. This is due to the formation of a micellar structure instead of the vesicles, which are relatively smaller in size.13 Fitting of data to the model Fitting of the data for observed responses to various models; it was observed that the best-fitted model for all the four dependent variables was the quadratic model (Table 5). Higher values of the standard error (SE) for coefficients indicate the quadratic (nonlinear) nature of the relationship. A positive value in regression equation for a response represents an effect that favors the optimization (synergistic effect), whereas a negative value indicates an inverse relationship (antagonistic effect) between the factor and the response.47 From Table 5 it is evident that the two independent variables (i.e., the concentrations of the phospholipid and the drug) have positive effects on the response Y1 (EE %), whereas the response Y2 (vesicle size) has an inverse relationship with SDC and sonication time. Response Y3 (flux) was affected by EA and drug concentrations, whereas it can be retarded if their concentrations are further increased beyond the
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Figure 6. CLS micrographs 8 hours after application of RR-loaded elastic liposomes optical cross-sections perpendicular to the rat skin surface. (A) Ethanolic PBS. (B) Valsartan liposomes formulation. (C) VTF-OPT formulation.
optimum limits (Table 2). The EA concentration had a negative effect on the response Y3 (flux). There existed a direct relationship between the phospholipid concentration and EA (SDC) on the vesicles size, EE % of the vesicles, and transdermal flux of vesicles loaded with valsartan. The lowest EE % was found (62.73% ± 5.35% for the formulation VTF5, and maximum EE % was found (93.56% ± 5.45%) for VTF7. It is observed from the experimental design that EE % has a direct positive relationship with concentration of phospholipid as revealed by the equation Y1 = +88.62 +9.73X1 +0.078X2 −2.65X3 −0.19X4 −3.79X1 X2 −3.72X1 X3 −1.59 X 1 X 4 −0.47 X 2 X 3 −1.20 X 2 X 4 −0.33 X 3 X 4 −5.0X1 2−6.57X2 2 −7.97X3 2 −1.58X4 2. As the concentration of the phospholipid increases, the vesicle EE %, but it also depends upon other variables such as amount of EA and drug content. The vesicle size is a very important criterion for the nanovesicular formulation; the size of the nanovesicles was found to vary between 72 ± 04 nm and 176 ± 09 nm. It was observed that the vesicle size has a direct positive relationship with the phospholipid concentration but a negative relationship with the amount of EA. We observed an initial decrease in the average size of the vesicles with increasing amounts of SDC (from 5 to 15 mg). However, an additional increase in the SDC concentration from 15 up to 25 mg led to a further decrease in the average size of vesicles. This is due to the formation of a micellar structure instead of the vesicles, which are relatively smaller in size. This relationship is presented by the following equation Y2 = +135.00 +36.42X1 −23.17X2+2.25X3 −15.83X4 +1.25X1 X2 −2.00 X1 X3 +5.50 X1 X4 +1.50X2 X3 −0.75 X2 X4 −0.25 X3 X4 −3.75 X1 2 +2.13 X2 2 −4.25 X3 2−3.88X4 2. Transdermal flux of valsartan loaded in transfersomal formulation is another important criterion for optimization of VTF; it was observed from the equation below that the
transdermal flux of VTF increased on increasing the lipid content in the formulation whereas the transdermal flux is decreased on increasing the EA: Y3 = +620.07+78.47X1 −25.94X2 +6.11X3 +23.82 X4 +18.56X1 X2 +10.37 X1 X3 +0.85 X1 X4 +16.62 X2 X3 +3.61 X2 X4 +22.10 X3 X4 −208.72X12 −91.67X2 2 −31.49X3 2−41.65X4 2 A possible explanation for lower flux at a high EA concentration may be that the EA at high concentration disrupted the lipid membrane so that it became more leaky to the entrapped drug. This will in turn reduce the flux. The valsartan concentration had a positive relationship with transdermal flux up to 60 mg of valsartan, but beyond this concentration it showed a negative relationship with transdermal flux. Further increasing the drug concentration up to 80 mg resulted in a decrease in the transdermal flux, possibly due to leakage of valsartan from vesicle bilayers at higher concentration. CLSM study The extent of vesicular penetration measured by CLSM after application of three systems (VTF-OPT, ethanolic PBS, and conventional liposomes each containing 0.03% RR (Figure 6) distinctly described the transdermal potential of nanotransfersomal carrier. CLSM results revealed that VTF-OPT were fairly evenly distributed throughout the SC, viable epidermis, and dermis with high fluorescence intensity. VTF-OPT penetrated deeply into rat skin, followed by ethanolic PBS solution of valsartan and conventional liposomes. VTF-OPT were delivered to a maximum possible depth of dermatomed skin. The prominently efficient delivery of RR by transfersomal carriers suggests their enhanced penetration and consequent fusion with the membrane lipids in the depths of the skin, supporting the hypothesis of many researchers.28,39,48,49
6.05 0.85 2.56 22.13 121.57 ± 5.35 161.84 ± 10.75 145.26 ± 12.44 160.44 ± 14.12 146.52 ± 10.57 121.65 ± 8.64 115.52 ± 7.25 163.09 ± 12.86 139.29 ± 10.14 161.58 ± 15.65 134.63 ± 10.55 120.42 ± 10.75 109.36 ± 4.75 165.78 ± 8.34 126.35 ± 8.75 162.75 ± 11.74 130.63 ± 11.05 116.34 ± 9.36
122.38 ± 4.86 161.50 ± 12.65 151.72 ± 14.83 160.12 ± 15.89 157.36 ± 13.14 125.76 ± 9.45
Quadratic model
R2
Adjusted R2
Predicted R2
SD
% CV
Response (Y1) 0.9866 0.9732 0.9227 1.39 1.74 Response (Y2) 0.9944 0.9888 0.9679 3.22 2.46 0.9878 0.9756 0.9298 18.71 4.02 Response (Y3) Regression equation of the fitted quadratic model⁎ Y1 = +88.62 + 9.73X1 + 0.078X2 − 2.65X3 − 0.19 X4− 3.79X1 X2 − 3.72X1 X3 − 1.59X1 X4 − 0.47X2 X3 − 1.20 X2 X4 − 0.33 X3 X4 − 5.02 X1 2 − 6.57 X2 2 − 7.97 X3 2− 1.58X4 2 Y2 = +135.00 + 36.42X1 − 23.17X2 + 2.25X3 − 15.83X4 +1.25X1 X2 − 2.00X1 X3 + 5.50 X1 X4 + 1.50X2 X3 − 0.75 X2 X4 − 0.25 X3 X4 − 3.75 X12 +2.13 X2 2 − 4.25 X3 2− 3.88X4 2 Y3 = +620.07 + 78.47X1 − 25.94X2 + 6.11X3 + 23.82X4 + 18.56X1 X2 + 10.37 X1 X3 + 0.85X1 X4 + 16.62X2 X3 + 3.61 X2 X4 + 22.10 X3 X4 − 208.72 X21 − 91.67 X2 2 − 31.49 X3 2 − 41.65 X4 2 CV, coefficient of variation. ⁎ Only the terms with statistical significance are included.
In vivo antihypertensive study
123.87 ± 6.74 165.74 ± 7.14 110.42 ± 9.78 162.08 ± 13.77 156.16 ± 10.44 108.85 ± 7.98 8.35 5.25 9.75 13.29 ± 10.06 5.07 ⁎ Control rats without hypertension induced and no further treatment. † Percentage reduction in BP at 48-hr time point. ‡ Hypertension was induced with MPA, and no treatment was given.
110.56 ± 179.26 ± 108.75 ± 178.62 ± 172.45 ± 122.42 ± 119.20 ± 8.53 182.65 ± 8.42 155.42 ± 10.58 182.45 ± 15.22 179.78 ± 10.24 162.25 ± 8.25 120.45 ± 10.32 183.37 ± 7.53 180.24 ± 5.65 179.48 ± 14.54 181.92 ± 7.85 179.64 ± 8.96 Normal control⁎ Hypertensive control‡ Oral suspension Placebo-TTS VLGF-TTS VTGF-OPT-TTS A B C D E F
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Table 5 Summary of results of regression analysis for responses Y1, Y2, and Y3 for fitting to quadratic model
122.08 ± 5.12 169.42 ± 5.63 119.37 ± 9.68 166.59 ± 12.02 153.35 ± 10.32 110.74 ± 8.45
36 hr 24 hr 12 hr 9 hr
Mean systolic blood pressure (mmHg)
6 hr 3 hr 1 hr Initial Treatments Groups
Table 4 Influence of various optimized transdermal systems of valsartan on mean BP in MPA-induced hypertensive rats
48 h
Reduction in BP (%)†
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The developed VTGF-OPT-TTS was found to decrease the BP significantly (P b 0.001) in proximity of the normal value. The effect was maintained for 48 hours. On comparing the effects of all the TTS systems, the percentage reductions in mean systolic BP of rats by VTGF-OPT-TTS and placebo-TTS were 22.13% and 0.85%, respectively. However, percentage reduction in mean systolic BP of rats by oral valsartan solution and VLGFTTS was found to be 6.05% and 2.56%, respectively. Results of in vivo antihypertensive activity clearly indicates that the VTGF-OPT-TTS released the drug gradually over a period of time, which resulted in prolonged control of hypertension up to 48 hours. Formulation VTGF-OPT-TTS was successful in reverting the rat BP to normal values, whereas VLGF-TTS failed to reduce the BP to normal value. The above results suggest that the developed nanotransfersomal system of valsartan holds promise for the management of hypertension that must be validated by clinical trials. The results of the present study showed that deformable lipid vesicles, nanotransfersomes, improve the transdermal delivery of the lipophilic drug, valsartan. The formulation-optimizing study using statistical experimental design shows that optimum concentrations of phospholipid, surfactant, and valsartan are required to provide the maximum value of transdermal flux and EE %, minimizing the vesicles size. The optimized nanotransfersomal gel formulation showed better antihypertensive activity in a rat model in comparison with placebo-TTS and liposomes formulations. The developed valsartan nanotransfersomal gel formulation TTS was found to decrease the BP significantly (P b 0.001) in experimental hypertensive rats, which was maintained for 48 hours. The results of the present study demonstrated that introduction of nanotransfersomes as a vesicular drug carrier overcomes the limitation of low penetration ability of liposomes across the skin. Hence, it could be concluded that nanotransfersomes are a potentially suitable carrier for transdermal delivery of valsartan. Further studies are needed to establish their therapeutic utility in human beings.
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