Nanomedicine: Nanotechnology, Biology, and Medicine8 (2012) 237–249
Research Article
Formulation and optimization of nanotransfersomes using experimentaldesign 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
nanomedjournal.com
Abstract
The purpose of this work was to develop and statistically optimize nanotransfersomes for enhanced transdermal of valsartan vis-à-vis traditional liposomes. Nanotransfersomes bearing valsartan were prepared by conventional rotary evaporation method andcharacterized for various parameters including entrapment efficiency, vesicles shape, size, size distribution, and skin permeation. Invivo antihypertensive activity conducted on Wistar rats was also taken as a measure of performance of nanotransfersomes andliposomes. Nanotransfersomes proved significantly superior in terms of amount of drug permeated in the skin, with an enhancementratio of 33.97 ± 1.25 when compared to rigid liposomes. This was further confirmed through a confocal laser scanning microscopystudy. Nanotransfersomes showed better antihypertensive activity in comparison to liposomes by virtue of better permeation throughWistar rat skin. Finally, it could be concluded that the nanotransfersomes accentuates the transdermal flux of valsartan and could beused as a carrier for effective transdermal delivery of valsartan.
The greatest obstacle for transdermal delivery is the barrierproperty of the stratum corneum (SC).1,2 Many approaches havebeen used to breach the skin barrier; for example, the use of lipidvesicles to modulate the SC is gaining interest. The first articles toreport on the effectiveness of vesicles for skin delivery werepublished in the early 1980s. Most groups concluded thatliposomes did not act as transport systems.3 Conventionalliposomes have been generally reported to remain confined tothe upper layer of the SC and to accumulate in the skin appendages,with minimal penetration to deeper tissues, because of their largesize and lack of flexibility.4,5 Further intensive research over thepast two decades led to the introduction and development of a newclass of lipid vesicles, the ultradeformable (elastic or ultraflexible)liposomes that have been termed Transfersomes (IDEA AG,Munich, Germany). Several studies have reported that Transfer-
Abdul Ahad thanks the Council for Scientific and Industrial Research(CSIR), India (File No. 09/591 (0084)/2009-EMR-I), for providing financialassistance in the form of a senior research fellowship.
⁎Corresponding author: Department of Pharmaceutics, Faculty ofPharmacy, Hamdard University, New Delhi 110 062, India.
Please cite this article as: A. Ahad, M. Aqil, K. Kohli, Y. Sultana, M. Muexperimental design technique for accentuated transdermal delivery of vals....
somes were able to improve in vitro skin delivery of various drugs6
and to penetrate intact skin in vivo, transferring therapeuticamounts of drugs with efficiency comparable with subcutaneousadministration.7-12 The key factors that confer ultradeformability tothe liposomes have been considered to be edge activators (EAs),the special surfactants incorporated into Transfersomes (e.g.,sodium cholate or sodium deoxycholate, SDC). Because Transfer-somes are composed of surfactant, they have better rheology andhydration properties, which are responsible for their superior skinpenetration ability. Less deformable vesicles, including traditionalliposomes, are confined to the skin surface where they dehydratecompletely and fuse, so they have less penetration power thanTransfersomes. Transfersomes are optimized in this respect, thusattaining maximum flexibility, and hence they can take fulladvantage of the transepidermal osmotic gradient (water concen-tration gradient).13 The aim of the present studywas to optimize thenanovesicles formulations (nanotransfersomes) for enhanced skindelivery of a model drug valsartan, a lipophilic antihypertensivedrug having low oral bioavailability of about 25%. It has lowmolecular weight (435.5) and melting point (116–117°C) with alog partition coefficient of 4.5 and amean biological half-life of 7.5hours; there are no reports of skin irritation attributed to valsartan.
jeeb, A. Ali, Formulation and optimization of nanotransfersomes usingNanomedicine: NBM 2012;8:237-249, doi:10.1016/j.nano.2011.06.004
Valsartan was received as a gratis sample from RanbaxyResearch Laboratories Ltd. (Gurgaon, India). Phospholipon 90G(PL90G) was received as a gift sample from Phospholipid GmbH(Nattermannallee, Germany). Water for high-performance liquidchromatography (HPLC) was purchased from Thomas BakerChemicals Ltd. (Mumbai, India). SDC and cholesterol werepurchased from Spectrochem Pvt Ltd. (Mumbai, India). Rhoda-mine Red-X 1,2 dihexadecanoyl-sn-glycero-3-phosphoethanola-mine trimethylammonium salt (RR) was purchased fromMolecular Probes (Eugene, Oregon). Absolute ethanol waspurchased from Merck (Darmstadt, Germany). All otherchemicals 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 suppliedby Central Animal House of Hamdard University and kept understandard 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 werereceived after the study was duly approved by the UniversityAnimal Ethics Committee, and Committee for the Purpose ofControl and Supervision on Experiments on Animals (CPSCEA),government of India. Wistar rats were euthanized with prolongedether anesthesia and the abdominal skin of each rat was excised.Hairs on the skin of animals (thickness ∼0.6 mm) were removedwith electrical clippers, subcutaneous tissues were surgicallyremoved, and the dermis side waswipedwith isopropyl alcohol toremove residual adhering fat. The skin was washed withphosphate buffered saline, wrapped in aluminum foil, and storedin a deep freezer at −20°C until further use (used within 2 weeksof preparation). On the day of the experiment, skin was brought toroom temperature (22°C) and skin samples were mounted overthe diffusion cells in such a way that the SC side faced the donorcompartment whereas the dermis faced the receiver compartment.
Preparation of nanotransfersomes and liposomes using thefactorial designs
Valsartan transfersomal formulations (VTFs) were prepared byconventional thin-layer evaporation technique13,17 using factorialdesign, A four-factor three-level Box-Behnken design wasemployed to study the effect of independent variables ondependent variables as shown in Table 1. Twenty-nine formula-tions were prepared according to the experimental design shown inTable 2. The entrapment efficiency (EE %), vesicle size, andtransdermal flux obtained from the skin permeation study ofnanotransfersomes bearing valsartan are presented in Table 2.The valsartan liposomes formulation (VLF; ratio of PL90G tocholesterol, 85:15) that were used as a control in the present studywere prepared by the rotary evaporation sonication method as
described above. Similarly, RR (0.03%w/v)-loaded transfersomesand liposomes were prepared for confocal laser microscopy.
Experimental design
A four-factor, three-level Box-Behnken design was used toexplore the quadratic response surfaces and for constructing asecond-order polynomial models using Design Expert (Version7.1.6; Stat-Ease Inc., Minneapolis, Minnesota). A design matrixcomprising 29 experimental runs was constructed, for which thenonlinear computer-generated quadratic model is defined as:
Y = b0 + b1X1 + b2X2 + b3X3 + b12X1X2 + b13X1X3
+ b23X2X3 + b11X 21 + b22X 2
2 + b33X 23
where Y is the measured response associated with each factorlevel combination; b0 is constant; b1, b2, b3 are linear coeffi-cients, b12, b13, b23 are interaction coefficients between the threefactors, b11, b22, b33 are quadratic coefficients computed fromthe 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 interactionand quadratic terms, respectively.18 The independent variablesselected were the amount of the phospholipids 90G (X1), SDC(X2), valsartan (X3), and sonication time (X4). The dependentvariables were EE % (Y1), vesicle size (Y2), and flux (Y3), withconstraints applied on the formulation of nanotransfersomes.The concentration range of independent variables under studyis shown in Table 1 along with their low, medium, and highlevels, which were selected based on the results from preliminaryexperimentation. The concentration range of phospholipids90G (X1), SDC (X2), valsartan (X3), and sonication time (X4)used to prepare the 29 formulations and the respective observedresponses are given in Table 2.
Vesicles shape, size, and size distribution
Nanotransfersomes vesicles were visualized by using aMorgagni 268D (Fei Electron Optics, Eindhoven, the Nether-lands) transmission electron microscope; digital micrograph andsoft imaging viewer software were used to perform the imagecapture and analysis.19 The vesicles size and size distributionwere determined by dynamic light scattering method, using a
Table 2Observed response in Box-Behnken design for valsartan transfersomal formulation (VTF)
239A. Ahad et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 237–249
computerized inspection system (Zetasizer, HAS 3000; MalvernInstruments, Malvern, United Kingdom).20
Entrapment efficiency
EE %, expressed as a percentage of the total amount ofvalsartan found in the studied formulations at the end of thepreparation procedure, was determined by HPLC method21 afterdisruption of vesicles with Triton X-100.22 The analyses werecarried out with a liquid chromatograph (Model-1120 CompactLC; Agilent Technologies, Santa Clara, California), equippedwith an ultraviolet (UV) detector. Separation was achieved usingShiseido 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 withthe mobile phase containing 45% acetonitrile and 55% phosphatebuffer solution (PBS), pH 3.0. Mobile phase was prepared daily,filtered by passing through a 0.45-μm membrane filter, anddegassed. All chromatographic separations were performed atroom temperature. Detection was carried out at 265 nmwith a UVdetector. The amount of entrapment drug expressed as apercentage was calculated from the following equation:
EEk =Entrapped drugTotal drug
× 100
Ex vivo skin permeation studies
The ex vivo skin permeation of valsartan from nanotransfer-somes was studied23-26 using a locally fabricated Franzdiffusion cell with an effective permeation area and receptorcell volume of 1.0 cm2 and 15 mL, respectively. Thetemperature of the receiver vehicle (ethanol-PBS pH 7.4,40:60 ratio) was maintained at 37 ± 1°C and was constantlystirred by magnetic stirrer at 100 rpm. An amount ofnanotransfersomes equivalent to 20 mg of valsartan (transder-mal dose of valsartan) was placed in the donor compartment.Samples of 500 μL were withdrawn from the receptorcompartment via the sampling port at different time intervals(0, 1, 2, 3, 4, 6, 8, 10, 12, and 24 hours) and analyzed for drugcontent by HPLC method as stated above. The receptor phasewas immediately replenished with an equal volume of freshdiffusion buffer. Similar experiments were performed withconventional liposomal formulations and ethanolic phosphatebuffer solution of pure drug sample.13,17 To determine theextent of enhancement, enhancement ratio (ER) was calculatedas follows:
ER =Steady state flux of formulationSteady state flux of control
Table 3Groups of experimental rats, treatment given to different groups, and timeintervals for measurement of BP
Groups Treatments No. of ratsin a group
Measurement of systolic BP atdifferent time intervals (hr)
Group A Normal control 6 0,1, 3 , 6 , 9 , 12, 24 , 36, and 48Group B Hypertensive
control6 0,1, 3 , 6 , 9 , 12, 24 , 36, and 48
Group C Oral suspension 6 0,1, 3 , 6 , 9 , 12, 24 , 36, and 48Group D Placebo-TTS 6 0,1, 3 , 6 , 9 , 12, 24 , 36, and 48Group E VLGF-TTS 6 0,1, 3 , 6 , 9 , 12, 24 , 36, and 48Group F VTGF-OPT-TTS 6 0,1, 3 , 6 , 9 , 12, 24 , 36, and 48
240 A. Ahad et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 237–249
Confocal laser scanning microscopy (CLSM) study
CLSM was used to scan the fluorescence signal of VLFsand VTF-OPT at different skin depths. For CLSM study, theex vivo skin permeation study was carried out as describedabove. After 8 hours the skin was removed and washed withdistilled water. The treated area was cut out and tested forprobe penetration.27,28 The excised nude rat skin waspositioned on the microscopic slide with the SC side face tothe coverglass. CLSM was carried out with rhe Laser ConfocalMicroscope with Fluorescence Correlation Spectroscope-Olym-pus FluoView FV1000 (Olympus, Melville, New York) withan argon laser beam with excitation at 488 nm and emission at590 nm. Each skin sample was sliced in sections of 6–10 μmthickness through the z-axis by CLSM. The VTF-OPTintensity and the permeated depth were detected by CLSMwith Fluoview software.
In vivo antihypertensive studies
The preclinical assessment of antihypertensive activity ofthe developed transdermal therapeutic system (TTS) wasperformed on experimentally hypertensive rats. Hypertensionwas induced by injecting methyl prednisolone acetate (MPA,Depo-Medrol; Pfizer, Mumbai, India) (20 mg/kg/week)subcutaneously for 2 weeks. The studies were carried outusing small Animal Tail Noninvasive Blood Pressure system(NIBP 200A; Biopac System, Inc., Goleta, California) based oncuff tail technique. The experimental (MPA-induced) hyper-tensive rats with minimum mean systolic blood pressure (BP)of 150 mm Hg were selected for further study. According theinitial BP of rats, the animals were divided into six groups(group A to F) of six animals each. Treatments given to eachgroup are shown in Table 3. Group A was taken as normalcontrol. Hypertension was induced in the remaining groups(groups B to F) by subcutaneous injection of MPA for 2 weeksas per the method29 of Aqil et al. Group B served ashypertensive control and received no further treatment. AfterMPA treatment group C received an oral suspension ofvalsartan (3.6 mg/kg); the dose for the rats was calculatedbased on the body weight of the rats as per the surface arearatio method.30 Groups D, E, and F were subjected to placebo-TTS, VLGF-TTS, and VTGF-OPT-TTS, respectively. EachTTS was applied to the previously shaven abdominal area ofrat skin. The rat was then placed in the restrainer and the BP
from the tail was recorded at predetermined time intervals up to48 hours (Table 3).
Results
Optimization of nanotransfersomes preparation
Effect of drug concentrationEE % is the percentage fraction of the total drug incorporated
into the transfersomes. Themaximum andminimumEE% valuesobtained were 93.56% ± 5.45% for VTF7 and 62.73% ± 5.35%for VTF5, respectively (Table 2). It was found that uponincreasing valsartan concentration from 40 mg to 60 mg in thenanotransfersomes prepared, the EE % significantly increasedfrom 65.75% ± 6.12% (VTF24, with least concentration PL90Gand SDC) to 70.59% ± 5.07% (VTF10) (P b 0.001). However, afurther increase in drug concentration to 80mg led to a significantdecrease in EE % to 62.73% ± 5.35% (VTF5) (P b 0.001); thismay be due to the leakage of excess drug from the vesicularstructure. Effects of independent variables on EE % arepresented by three-dimensional graph in Figure 1. According toLopes et al,31 the entrapment of drug occurs in both thebilayers and the aqueous compartment of the vesicles. Whenthe lipid compartment and aqueous phase became saturatedwith the drug, the vesicles provided limited entrapmentcapacity.32 Highest flux (626.57 ± 37.19 μg/cm2/hr) wasobtained for transfersomal formulation VTF28 having 60 mg ofvalsartan (Table 2).
Effect of PL90G/SDC ratioInitially, the EE % increased significantly with increasing
EA concentration from 5 to 15 mg (w/w). Further increase inEA concentration from 15 up to 25 mg (w/w) showed adecrease in EE % (Table 2). The ratio (85 mg PL90G/15 mgEA) showed optimum EE %. Upon incorporation of EA in lowconcentration, growth in vesicle size occurred,33 whereasfurther increase in the content of EA may have led to poreformation in the bilayers. It was observed that with increasedEA concentration in the lipid components of the vesicles, theEE % of the valsartan decreased (Table 2).
Ex vivo skin permeation studiesEx vivo skin permeation studies from nanotransfersomes
containing SDC (with different ratios of PL90G/EA/drug)(Table 2) were performed. Steady-state fluxes from nanotrans-fersomes at 24 hours first increased with increasing EAconcentration (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 formula-tion (VTF28) presented maximum flux value (i.e., 626.57 ±37.19 μg/cm2/hr over rigid liposome formulation (18.47 ± 0.95μg/cm2/hr) with ER of 33.92 through rat skin. Effects ofindependent variables on flux are presented by three-dimensionalgraph in Figure 2. In our study we observed that the transdermalflux first increased with increasing EA concentration and thendecreased (Table 2).
Figure 1. Response surface plot showing effect of independent variables on percent entrapment efficiency.
241A. Ahad et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 237–249
Vesicle size analysisThe mean vesicle sizes of various VTFs are presented in
Table 2. The smallest mean vesicle sizewas observed for valsartan-loaded Transfersomes formulation VTF18 (72 ± 4 nm), whereasthe maximum vesicle size was obtained as 176 ± 9 nm for VTF7.This was a smaller mean vesicle size than that of traditionalliposomes (209 ± 15 nm) prepared by the same method. Thesevariations in vesicles size were highly significant (P b 0.001).Effects of independent variables on vesicles size are presented bythree-dimensional graph in Figure 3. We conclude that smallamounts of an EA in liposomal membranes increases the flexibilityof vesicles, thereby enabling them to pass more easily through thepores of the polycarbonate filter during the vesicle preparation.Figure 4 quantitatively compares the resultant experimental valuesof the responses with those of the predicted values.
OptimizationThe optimum formulation of valsartan-loaded nanotransfer-
somes systems was selected based on the criteria of attaining themaximum value of transdermal flux and EE %, minimizing the
vesicles size by applying the point predictionmethod of theDesignExpert software.34,35 Upon “trading off” various response vari-ables and comprehensive evaluation of feasibility search andexhaustive grid search, the formulation composition withphospholipid (85 mg), SDC (15 mg), valsartan (60 mg), andsonication time (25 minutes) was found to fulfill requisites of anoptimum formulation (i.e., VTF-OPT). The optimized formulationhas the EE % of 85.77% ± 2.97% with vesicles size andtransdermal flux across rat skin of 130 ± 10 nm and 627.47 ± 30.45μg/cm2/hr, respectively. Electron micrographs of VTF-OPT areshown in Figure 5, A. They show the outline and core of the well-identified spherical vesicles, displaying sealed vesicular structure.Size distribution of optimized VTF-OPT nanotransfersomesloaded 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 of27.11 ± 2.90 μg/cm2/hr was achieved from the ethanolic PBS ofvalsartan through rat skin. The VTF-OPT and ethanolic PBS ofvalsartan showed ERs of 33.97 ± 1.25 and 1.46 ± 0.024 over theliposomes formulation, which produced the least transdermal
Figure 2. Response surface plot showing effect of independent variables on transdermal flux.
242 A. Ahad et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 237–249
flux of 18.47 ± 0.95 μg/cm2/hr through rat skin. On the basis offactorial design approach, the nanotransfersomal formulation(VTF-OPT) was selected for further in vivo studies. The VTF-OPT was converted into gel. Briefly, Carbopol 940 (S. D. FineChemicals Ltd., Mumbai, India) (1% w/w) was added into waterand kept overnight for complete humectation of polymer chains.Optimized Transfersomes dispersion (VTF-OPT) equivalent to40 mg of valsartan (transdermal dose of valsartan for 48 hours)was added to hydrated Carbopol solution with stirring. Otheringredients, such as 15% w/v polyethylene glycol-400 (PEG-400) and triethanolamine (0.5% w/v), were added to obtainhomogeneous dispersion of gel, and this optimized valsartantransfersomal gel formulation (VTGF-OPT) was incorporatedinto reservoir-type TTS for in vivo antihypertensive studies.
CLSM study
The extent of vesicular penetration measured by CLSM afterapplication of three systems (i.e., ethanolic PBS of drug,conventional liposomes, and VTF-OPT-each containing 0.03%
RR) clearly defined the transdermal potential of nanotransferso-mal carrier. CLSM study results revealed that the VTF-OPTformulation was fairly evenly distributed throughout the SC,viable epidermis, and dermis with high fluorescence intensity(Figure 6). CLSM studies conducted to measure the extent ofpenetration and transdermal potency of the nanotransfersomessystem (VTF-OPT) depicted an increase in both the depth (up to170 μm) of penetration and fluorescence intensity (Max FI = 160arbitrary units, AU) on application of RR-loaded VTF-OPT ascompared to rigid liposomes that were confined to a few microns(50 μm) depth only, and Max FI was found to be 40 AU withdepth of penetration up to 80 μm (Figure 6). Ethanolic PBS wasalso effective in permeating probe up to 160 μm, although verylow FI was observed as compared to the nanotransfersomalsystem with a Max FI of 80 AU (Figure 6).
In vivo antihypertensive study
Hypertension was successfully induced in the normotensiverats by MPA administration for a period of 2 weeks, and they
Figure 3. Response surface plot showing effect of independent variables on vesicles size.
243A. Ahad et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 237–249
remained hypertensive for 72 hours after stopping the MPAinjection, as shown by high significant difference (t-test, P b0.001) found in the pre- and post-treatment values (Table 4).This was authenticated by Dunnet test, which showed significantdifference (P b 0.001) in BP values of normal control (group A)and hypertensive control (group B). The oral administration ofvalsartan suspension significantly (P b 0.05) controlled thehypertension within 2 hours, with the maximum antihyperten-sive effect (108.75 ± 9.75 mm Hg) observed at 3 hours, butafter 3 hours the BP started rising gradually. The BP rose abovethe normal BP value (126.35 ± 8.75 mm Hg) after 9 hoursand progressively reached 151.72 ± 14.83 mm Hg at 48 hours(Table 4). Oral suspension of valsartan acted rapidly and showedutmost changes in rat BP as shown in Table 4, but its effectdipped rapidly and BP increased to above normal value.Although liposome gel formulation TTS (VLGF-TTS) failed toreduce the BP value to normal, the maximum decrease (130.63 ±11.05 mm Hg) observed for BP was at 12 hours. In contrast, theadministration of valsartan through transdermal route (VTGF-OPT-TTS) resulted in a gradual decrease of BP, with the
maximum effect (108.85 ± 7.98 mm Hg) observed at 6 hourson treatment of the experimentally hypertensive rats (P N 0.05).Table 4 reveals that the systolic BP of hypertensive rats wascontrolled up to 48 hours; however, at the 48-hour time point BPwas comparable to the normotensive rats (P N 0.05). VTGF-OPT-TTS decreased the BP insignificantly at the first hour (P N0.05), 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 anoptimum extent, after which any further increase in drugconcentration led to leakage of valsartan from vesicle bilayers.31
Transdermal flux first increased with increasing valsartanconcentration from 40 mg to 60 mg; with further increase in thedrug concentration to 80 mg the flux decreased as a result of
Figure 5. (A) Transmission electron micrography following negativestaining (20,000×). (Inset) Detailed picture, where unilamellarity and sizeof vesicles can be seen (180,000×). (B) Size distribution of optimized VTF-OPT nanotransfersomes loaded with valsartan.
245A. Ahad et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 237–249
leakage of valsartan from vesicle bilayers. Results of ex vivo skinpermeation suggested that a too low or a too high concentrationof drug (valsartan) is not beneficial in vesicular delivery throughskin and also indicated that the possible penetration-enhancingeffect of drug is not mainly responsible for improved valsartanskin delivery from deformable vesicles. Other variables such asamount of phospholipids and surfactant present are also affectingthe EE %.
Effect of PL90G/SDC ratio
Initially, the EE % increased significantly with increasingEA concentration; further increase in EA concentration showeda decrease in EE %, possibly due to the coexistence of mixedmicelles and vesicles at higher concentrations of EA, with theconsequence of lower drug entrapment in mixed micelles.17 Theformation of micellar structure at higher concentrations of EAis an established fact. The studies36,37 by Lasch et al and Lopezet al prove this fact. At the same time the EA also causesfluidization of the bilayer that is responsible for increase inelasticity of vesicle membrane. At higher EA concentration,conversion of lipid vesicles into mixed micelles begins. Thesemixedmicelles have a diameter b10 nm and are reported to be lessdeformable in nature and also have less skin permeation abilityacross the skin in comparison to transfersomes.13,36,37,38
Studying the effect of EA concentration in the lipid componentsof vesicles on the EE % of the lipophilic model drug, valsartan,clearly shows that EE % decreased with an increase inconcentration of EA. This is due to the possible coexistence of
mixed micelles and vesicles at higher concentrations of EA, withthe consequence of lower drug entrapment inmixedmicelles.13,39
Ex vivo skin permeation studies
The reason for this better performance of nanotransfersomeformulations in comparison with the traditional liposomes is theflexibility of the nanotransfersomes that allow them to passthrough the skin more easily. The extremely high flexibility ofthe membrane permits nanotransfersomes to squeeze themselveseven 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. Thesefindings are in agreement with published data.13,41-44 A possibleexplanation for lower drug delivery at a high surfactantconcentration may be that the surfactant at high concentrationdecreased the EE % and disrupted the lipid membrane so thatit became more leaky to the entrapped drug. This will, in turn,reduce the delivery, especially if we consider the possible carrierfunction of these nanotransfersomes. These mixed micelles arereported to be less effective in transdermal drug delivery ascompared with a transfersomal system, because micelles aremuch less sensitive to a water activity gradient than transfer-somes. This hypothesis is supported by the report45 of Cevc et al,who compared the penetration ability of transfersomes, lipo-somes, and mixed micelles by CLSM and observed that mixedmicelles were restricted to the topmost part of the SC and thatnanotransfersomes penetrate to a deeper skin layer.
Vesicles size analysis
Inclusion of SDC (EA), an anionic surfactant used forformulation of nanotransfersomes which interact with lipidbilayers,46 instead of cholesterol (traditional liposomes), couldexplain this reduction in vesicle size. The size distribution ofvesicles was determined by dynamic light scattering. Weobserved an initial decrease in the average size of the vesicleswith increasing amounts of SDC (from 5 to 15 mg). However, afurther increase in the SDC concentration from 15 up to 25 mgled to a reduction in the average size of vesicles. This is due tothe 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 fourdependent variables was the quadratic model (Table 5). Highervalues of the standard error (SE) for coefficients indicate thequadratic (nonlinear) nature of the relationship. A positive valuein regression equation for a response represents an effect thatfavors the optimization (synergistic effect), whereas a negativevalue indicates an inverse relationship (antagonistic effect)between the factor and the response.47 From Table 5 it is evidentthat the two independent variables (i.e., the concentrations of thephospholipid and the drug) have positive effects on the responseY1 (EE %), whereas the response Y2 (vesicle size) has an inverserelationship with SDC and sonication time. Response Y3 (flux)was affected by EA and drug concentrations, whereas it can beretarded if their concentrations are further increased beyond the
Figure 6. CLS micrographs 8 hours after application of RR-loaded elastic liposomes optical cross-sections perpendicular to the rat skin surface. (A) EthanolicPBS. (B) Valsartan liposomes formulation. (C) VTF-OPT formulation.
246 A. Ahad et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 237–249
optimum limits (Table 2). The EA concentration had a negativeeffect on the response Y3 (flux).
There existed a direct relationship between the phospholipidconcentration and EA (SDC) on the vesicles size, EE % of thevesicles, and transdermal flux of vesicles loaded with valsartan.The lowest EE % was found (62.73% ± 5.35% for theformulation VTF5, and maximum EE % was found (93.56% ±5.45%) for VTF7. It is observed from the experimental designthat EE % has a direct positive relationship with concentrationof phospholipid as revealed by the equation Y1 = +88.62+9.73X1 +0.078X2 −2.65X3 −0.19X4 −3.79X1 X2 −3.72X1 X3
−1.59X1 X4−0.47X2 X3 −1.20X2 X4 −0.33X3 X4
−5.0X12−6.57X2
2 −7.97X32 −1.58X4
2. As the concentrationof the phospholipid increases, the vesicle EE %, but it alsodepends upon other variables such as amount of EA and drugcontent. The vesicle size is a very important criterion for thenanovesicular formulation; the size of the nanovesicles wasfound to vary between 72 ± 04 nm and 176 ± 09 nm. It wasobserved that the vesicle size has a direct positive relationshipwith the phospholipid concentration but a negative relationshipwith the amount of EA. We observed an initial decrease in theaverage size of the vesicles with increasing amounts of SDC(from 5 to 15 mg). However, an additional increase in the SDCconcentration from 15 up to 25 mg led to a further decrease inthe average size of vesicles. This is due to the formation of amicellar structure instead of the vesicles, which are relativelysmaller in size. This relationship is presented by the followingequation Y2 = +135.00 +36.42X1 −23.17X2+2.25X3 −15.83X4
A possible explanation for lower flux at a high EA concentrationmay be that the EA at high concentration disrupted the lipidmembrane so that it became more leaky to the entrapped drug.This will in turn reduce the flux. The valsartan concentration hada positive relationship with transdermal flux up to 60 mg ofvalsartan, but beyond this concentration it showed a negativerelationship with transdermal flux. Further increasing the drugconcentration up to 80 mg resulted in a decrease in thetransdermal flux, possibly due to leakage of valsartan fromvesicle bilayers at higher concentration.
CLSM study
The extent of vesicular penetration measured by CLSM afterapplication of three systems (VTF-OPT, ethanolic PBS, andconventional liposomes each containing 0.03% RR (Figure 6)distinctly described the transdermal potential of nanotransferso-mal carrier. CLSM results revealed that VTF-OPT were fairlyevenly distributed throughout the SC, viable epidermis, anddermis with high fluorescence intensity. VTF-OPT penetrateddeeply into rat skin, followed by ethanolic PBS solution ofvalsartan and conventional liposomes. VTF-OPT were deliveredto a maximum possible depth of dermatomed skin. Theprominently efficient delivery of RR by transfersomal carrierssuggests their enhanced penetration and consequent fusion withthe membrane lipids in the depths of the skin, supporting thehypothesis of many researchers.28,39,48,49
CV, coefficient of variation.⁎ Only the terms with statistical significance are included.
Table
4Influenceof
variou
sop
timized
transdermal
system
sof
valsartanon
meanBPin
MPA
-ind
uced
hypertensive
rats
Groups
Treatments
Meansystolic
bloo
dpressure
(mmHg)
Reductio
nin
BP
(%)†
Initial
1hr
3hr
6hr
9hr
12hr
24hr
36hr
48h
ANormal
control⁎
120.45
±10
.32
119.20
±8.53
110.56
±8.35
123.87
±6.74
122.08
±5.12
109.36
±4.75
115.52
±7.25
121.57
±5.35
122.38
±4.86
-B
Hypertensivecontrol‡
183.37
±7.53
182.65
±8.42
179.26
±5.25
165.74
±7.14
169.42
±5.63
165.78
±8.34
163.09
±12
.86
161.84
±10
.75
161.50
±12
.65
-C
Oralsuspension
180.24
±5.65
155.42
±10
.58
108.75
±9.75
110.42
±9.78
119.37
±9.68
126.35
±8.75
139.29
±10
.14
145.26
±12
.44
151.72
±14
.83
6.05
DPlacebo
-TTS
179.48
±14
.54
182.45
±15
.22
178.62
±13
.29
162.08
±13
.77
166.59
±12
.02
162.75
±11
.74
161.58
±15
.65
160.44
±14
.12
160.12
±15
.89
0.85
EVLGF-TTS
181.92
±7.85
179.78
±10
.24
172.45
±±10
.06
156.16
±10
.44
153.35
±10
.32
130.63
±11
.05
134.63
±10
.55
146.52
±10
.57
157.36
±13
.14
2.56
FVTGF-O
PT-TTS
179.64
±8.96
162.25
±8.25
122.42
±5.07
108.85
±7.98
110.74
±8.45
116.34
±9.36
120.42
±10
.75
121.65
±8.64
125.76
±9.45
22.13
⁎Control
ratswith
outhypertension
inducedandno
furthertreatm
ent.
†Percentagereductionin
BPat
48-hrtim
epo
int.
‡Hyp
ertensionwas
indu
cedwith
MPA
,andno
treatm
entwas
given.
247A. Ahad et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 237–249
In vivo antihypertensive study
The developed VTGF-OPT-TTS was found to decrease theBP significantly (P b 0.001) in proximity of the normal value.The effect was maintained for 48 hours. On comparing theeffects of all the TTS systems, the percentage reductions in meansystolic BP of rats by VTGF-OPT-TTS and placebo-TTS were22.13% and 0.85%, respectively. However, percentage reductionin mean systolic BP of rats by oral valsartan solution and VLGF-TTS was found to be 6.05% and 2.56%, respectively.
Results of in vivo antihypertensive activity clearly indicatesthat the VTGF-OPT-TTS released the drug gradually over aperiod of time, which resulted in prolonged control ofhypertension up to 48 hours. Formulation VTGF-OPT-TTSwas successful in reverting the rat BP to normal values, whereasVLGF-TTS failed to reduce the BP to normal value. The aboveresults suggest that the developed nanotransfersomal system ofvalsartan holds promise for the management of hypertensionthat must be validated by clinical trials.
The results of the present study showed that deformable lipidvesicles, nanotransfersomes, improve the transdermal delivery ofthe lipophilic drug, valsartan. The formulation-optimizing studyusing statistical experimental design shows that optimumconcentrations of phospholipid, surfactant, and valsartan arerequired to provide the maximum value of transdermal flux andEE %, minimizing the vesicles size. The optimized nanotrans-fersomal gel formulation showed better antihypertensive activityin a rat model in comparison with placebo-TTS and liposomesformulations. The developed valsartan nanotransfersomal gelformulation TTS was found to decrease the BP significantly (P b0.001) in experimental hypertensive rats, which was maintainedfor 48 hours. The results of the present study demonstrated thatintroduction of nanotransfersomes as a vesicular drug carrierovercomes the limitation of low penetration ability of liposomesacross the skin. Hence, it could be concluded that nanotransfer-somes are a potentially suitable carrier for transdermal deliveryof valsartan. Further studies are needed to establish theirtherapeutic utility in human beings.
248 A. Ahad et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 237–249
References
1. Aqil M, Ahad A, Sultana Y, Ali A. Status of terpenes as skin penetrationenhancers. Drug Discov Today 2007;12:1601-7.
2. Ahad A, Aqil M, Kohli K, Chaudhary H, Sultana Y, Mujeeb M, et al.Chemical penetration enhancers: a patent review. Expert Opin TherPatents 2009;19:969-88.
3. Honeywell-Nguyen PL, Bouwstra JA. Vesicles as a tool for transdermaland dermal delivery. Drug Discov Today 2005;2:67-74.
4. Manosroi A, Jantrawut P, Manosroi J. Anti-inflammatory activity ofgel containing novel elastic niosomes entrapped with Diclofenacdiethylammonium. Int J Pharm 2008;360:156-63.
5. Verma DD, Verma S, Blume G, Fahr A. Liposomes increase skinpenetration of entrapped and non-entrapped hydrophilic substances intohuman skin: a skin penetration and confocal laser scanning microscopystudy. Eur J Pharm Biopharm 2003;55:271-7.
6. Elsayed MMA, Abdallah OY, Naggar VF, Khalafallah NM. Lipidvesicles for skin delivery of drugs: reviewing three decades of research.Int J Pharm 2007;332:1-16.
7. Cevc G, Blume G. New, highly efficient formulation of diclofenacfor the topical, transdermal administration in ultradeformable drugcarriers, Transfersomes. Biochim Biophys Acta 2001;1514:191-205.
8. Cevc G, Blume G. Biological activity and characteristics of triamcin-olone-acetonide formulated with the self-regulating drug carriers,Transfersomes. Biochim Biophys Acta 2003;1614:156-64.
9. Cevc G, Blume G. Hydrocortisone and dexamethasone in very defor-mable drug carriers have increased biological potency, prolonged effect,and reduced therapeutic dosage. Biochim Biophys Acta 2004;1663:61-73.
10. Lee EH, Kim A, Oh Y, Kim C. Effect of edge activators on the formationand transfection efficiency of ultradeformable liposomes. Biomaterials2005;26:205-10.
11. Cevc G, Blume G. Lipid vesicles penetrate into intact skin owing to thetransdermal osmotic gradients and hydration force. Biochim BiophysActa 1992;1104:226-32.
12. Cevc G, Schatzlein A, Richardsen H. Ultra deformable lipid vesicles canpenetrate the skin and other semi-permeable barriers unfragmented.Evidence from double label CLSM experiments and direct sizemeasurements. Biochim Biophys Acta 2002;1564:21-30.
13. Jain S, Jain P, Umamaheshwari RB, Jain NK. Transfersomes—a novelvesicular carrier for enhanced transdermal delivery: development,characterization, and performance evaluation. Drug Dev Ind Pharm2003;29:1013-26.
14. Ahad A, Aqil M, Kohli K, Sultana Y, Mujeeb M. Role of novel terpenesin transcutaneous permeation of valsartan: effectiveness and mechanismof action. Drug Dev Ind Pharm 2011;37:583-96.
15. Rizwan MD, Aqil M, Ahad A, Sultana Y, Ali M. Transdermal deliveryof valsartan: I. Effect of various terpenes. Drug Dev Ind Pharm2008;34:618-26.
16. Nishida N, Taniyama K, Sawabe T, Manome Y. Development andevaluation of a monolithic drug-in-adhesive patch for valsartan. Int JPharm 2010;402:103-9.
17. Mishra D, Garg M, Dubey V, Jain S, Jain NK. Elastic liposomesmediated transdermal delivery of an anti-hypertensive agent: propranololhydrochloride. J Pharm Sci 2007;96:145-55.
18. Khajeh M. Application of Box-Behnken design in the optimization of amagnetic nanoparticle procedure for zinc determination in analyticalsamples by inductively coupled plasma optical emission spectrometry.J Hazard Mater 2009;172:385-9.
19. Guo J, Ping Q, Sun G, Jiao C. Lecithin vesicular carriers for transdermaldelivery of cyclosporine. Int J Pharm 2000;194:201-7.
20. El Maghraby GMM, Williams AC, Barry BW. Skin delivery fromultradeformable liposomes: refinement of surfactant concentration.J Pharm Pharmacol 1999;51:1123-34.
21. Tatar S, Sasglik S. Comparison of UV- and second derivativespectrophotometric andHPLCmethods for the determination of valsartanin pharmaceutical formulation. J Pharm Biomed Anal 2002;30:371-5.
22. Mura S, Manconi M, Sinico C, Valenti D, Fadda AM. Penetrationenhancer-containing vesicles (PEVs) as carriers for cutaneous delivery ofminoxidil. Int J Pharm 2009;380:72-9.
23. Ahad A, Aqil M, Kohli K, Sultana Y, Mujeeb M. Interactions betweennovel terpenes and main components of rat and human skin: mechanisticview for transdermal delivery of propranolol hydrochloride. Curr DrugDeliv 2011;8:213-24.
24. Narishetty STK, PanchagnulaR. Transdermal delivery of zidovudine: effectof terpenes and their mechanism of action. J Control Rel 2004;95:367-79.
25. Panchagnula R, Salve PS, Thomas NS, Jain AK, Ramarao P.Transdermal delivery of naloxone: effect of water, propylene glycol,ethanol and their binary combinations on permeation through rat skin.Int J Pharm 2001;219:95-105.
26. Shakeel F, Baboota S, Ahuja A, Ali J, Shafiq S. Skin permeationmechanism and bioavailability enhancement of celecoxib from trans-dermally applied nanoemulsion. J Nanobiotechnology 2008;6:8.
27. Dayan N, Touitou E. Carriers for skin delivery of trihexyphenidyl HCl:ethosomes vs. liposomes. Biomaterials 2000;21:1879-85.
28. Touitou E, Dayan N, Bergelson L, Godin B, Eliaz M. Ethosomes–novelvesicular carriers for enhanced delivery: characterization and skinpenetration properties. J Control Rel 2000;65:403-18.
29. Aqil M, Ali A, Sultana Y, Parvez N. Matrix type transdermal drugdelivery systems of metoprolol tartrate: skin toxicity and in vivocharacterization. Ethiop Pharm J 2004;22:27-35.
30. Ghosh MN. Fundamentals of experimental pharmacology. 3rd ed.Kolkata: Hilton and Company; 2005.
31. Lopes LB, Scarpa MV, Silva GVJ, Rodrigues DC, Santilli CV, OliveiraAG. Studies on the encapsulation of Diclofenac in small unilamellarliposomes of soya phosphatidylcholine. Colloids Surf B: Biointerfaces2004;39:151-8.
32. Ning MY, Guo YZ, Pan HZ, Yu HM, Gu ZW. Preparation andevaluation of proliposomes containing clotrimazole. Chem Pharm Bull(Tokyo) 2005;53:620-4.
33. Van den Bergh BA, Wertz PW, Junginger HE, Bouwstra JA. Elasticityof vesicles assessed by electron spin resonance, electron microscopy andextrusion measurements. Int J Pharm 2001;217:13-24.
34. Sultana S, Bhavna, Iqbal Z, Panda BP, Talegaonkar S, Bhatnagar A, et al.Lacidipine encapsulated gastroretentive microspheres prepared by chem-ical denaturation for Pylorospasm. J Microencapsul 2009;26:385-93.
35. Gannu R, Palem CR, Yamsani SK, Yamsani VV, Yamsani MR.Enhanced bioavailability of buspirone from reservoir-based transdermaltherapeutic system, optimization of formulation employing Box–Behnken statistical design. AAPS PharmSciTech 2010;11:976-85.
36. Lasch J, Hoffman J, Amelyaneenka WG, Klibanov AA, Torchilin VP,Binder H, et al. Interaction of Triton X-100 and Octyl glycoside withliposomal membranes at sublytic and lytic concentration: spectroscopicstudies. Biochim Biophys Acta 1990;1022:171-80.
37. Lopez C, Maza A, Coderch L, Lpez Iglesias L, Wehli E, Parra JL. Directformation of mixed micelles in the solubilization of phospholipidsliposome by Triton X-100. FEBS Lett 1998;426:314-8.
38. Cevc G. Lipid vesicles and other colloids as drug carriers on the skin.Adv Drug Del Rev 2004;56:675-711.
39. Jain SK, Gupta Y, Jain A, Rai K. Enhanced transdermal delivery ofacyclovir sodium via elastic liposomes. Drug Deliv 2008;15:141-7.
40. Cevc G, Gebauer D, Schatzlein A, Blume G. Ultraflexible vesiclestransfersomes have an extremely therapeutic amount of insulin across theintact mammalian skin. Biochim Biophys Acta 1998;1368:201-15.
41. Cevc G, Schatzlein A, Blume G. Transdermal drug carrier basicproperties, optimization and transfer efficiency in the case ofepicutaneously applied peptides. J Control Rel 1995;36:3-16.
42. Cevc G, Blume G, Schatzlein A. Transfersomes mediated transepidermaldelivery improves the regiospecificity and biological activity ofcorticosteriods in vivo. J Control Rel 1997;45:211-26.
43. El Maghraby GMM, Williams AC, Barry BW. Oestradiol skin deliveryfrom ultradeformable liposomes, refinement of surfactant concentration.Int J Pharm 2000;196:63-74.
249A. Ahad et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 237–249
44. Hiruta Y, Hattori Y, Kawano K, Obata Y, Maitani Y. Novelultradeformable vesicles entrapped with bleomycin and enhanced topenetrate rat skin. J Control Rel 2006;113:146-54.
45. Cevc G, Blume G, Schatzlein A, Gebauer D, Paul A. The skin: a pathwayfor the systemic treatment with patches and lipid based agent carrier. AdvDrug Deliv Rev 1996;18:349-78.
46. El Maghraby GMM, Williams AC, Barry BW. Skin delivery ofoestradiol from lipid vesicles: importance of liposome structure. Int JPharm 2000;204:159-69.
47. Chopra S, Patil GV, Motwani SK. Release modulating hydrophilicmatrix systems of losartan potassium: optimisation of formulation usingstatistical experimental design. Eur J Pharm Biopharm 2007;66:73-82.
48. Godin B, Touitou E. Mechanism of bacitracin permeation enhance-ment through the skin and cellular membranes from an ethosomal carrier.J Control Rel 2004;94:365-79.
49. Dubey V, Mishra D, Jain NK. Melatonin loaded ethanolic liposomes:physicochemical characterization and enhanced transdermal delivery.Eur J Pharm Biopharm 2007;67:398-405.
