In the present scenario of fast pace drug discovery system, various drug delivery systems are being developed to further enhance the therapeutic efficacy of new drugs as well as to improve upon the side effects of the active ingredient by means of formulation development. Further, it has become important for the drug delivery system to afford controlled drug delivery at the specific site [
Liposome has been an important lipid based formulation indicated for topical and transdermal applications. Structurally, liposome contains an outer mono- or bilayer of molecules surrounding hollow core which serves as storage for the therapeutic agent. Liposomes can accommodate physicochemically different drugs in liposome membrane (hydrophobic) and internal core (hydrophilic) [
In the literature, the term liposome refers to vesicles containing phospholipid as main lipid component, in particular, phosphatidylcholine; however, the stability and permeation profile for liposome are two important areas needing consideration [
Owing to the above-mentioned drawbacks, there is a need to explore new lipid molecules for formulating into liposome with improved stability and permeation profile. In the present study, we have developed a nanovesicular formulation containing guggul lipid as main lipid component to improve the stability profile of the formulation. Further, the developed formulation was evaluated for its transdermal application since drug administration through skin offers advantages of avoidance of GIT and first pass metabolism and also can improve upon the drugs’ side effect, mainly gastrointestinal irritation [
Guggul lipid is guggulsterone (4,17(20)-pregnadiene-3,16-dione), which is present in a concentration of 4.0–6.0% in ethyl acetate extract obtained from
Aceclofenac is an analgesic, antipyretic, and anti-inflammatory drug and is indicated in rheumatoid arthritis, osteoarthritis, and ankylosing spondylitis. It acts on COX-2 isozyme to reduce the production of inflammation mediators [
It is practically water insoluble, having a molecular weight of 354.19, pKa value 4.7, and log
In the present study, we have developed a nanovesicle formulation using guggul lipid as main lipid component for Aceclofenac. The formulations were evaluated for physicochemical parameters, transdermal permeation, stability, and anti-inflammatory activity. The selected formulation was compared with an established transdermal aceclofenac formulation (ACE-PROXYVON GEL, aceclofenac 1.5% w/w) in permeation studies. A gel formulation containing plain aceclofenac was also prepared and compared with designed formulation. This study will be useful in devising improved formulations for transdermal application.
Aceclofenac was the gift sample from Akums Drugs & Pharmaceuticals Ltd., Haridwar, India. Guggul lipid was purchased from Sami Labs Limited, Bangalore, Karnataka, India. Cholesterol (Chol) was purchased from Himedia, Mumbai, India. Phosphatidylcholine (PC) and dicetyl phosphate (DCP) were purchased from Sigma-Aldrich (New Delhi, India). All other materials were of analytical grade. Commercial formulation was ACE-PROXYVON GEL (aceclofenac 1.5% w/w) manufactured by Wockhardt Merind Limited (Wockhardt. Ltd. Enterprise), Mumbai, India.
Lipid film hydration method was used to formulate the nanovesicles as per Table
Composition of the nanovesicle formulations (lipid drug ratio = 3 : 1).
S. No. | Formulation code | Composition | Molar ratio |
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1. | PC-1 | PC/Chol/DCP | 7/3/1 |
2. | PC-2 | PC/Chol/DCP | 5/3/1 |
3. | PC-3 | PC/Chol/DCP | 3/3/1 |
4. | GL-1 | GL/Chol/DCP | 7/3/1 |
5. | GL-2 | GL/Chol/DCP | 5/3/1 |
6. | GL-2 | GL/Chol/DCP | 3/3/1 |
PC: Phosphatidylcholine nanovesicle; GL: Guggul Lipid nanovesicle; Chol: cholesterol; DCP: Dicetyl phosphate.
This dispersion was passed through sephadex G-20 minicolumn to remove unentrapped drug [
Negative staining followed by TEM was employed to estimate the shape and size of formulation. An aliquot of test sample was located over the copper grid followed by phosphotungstic acid (1%). Then test sample was dried at room temperature and analyzed by using TEM (Philips CM-10, acceleration voltage: 100 kV; magnification: up to 450,000x; Cryoattachment).
Zetasizer (Nano-ZS, Malvern Instruments, UK) fitted with a 4 mW He-Ne laser was used to analyze polydispersity indices and zeta potential. The test sample was lyophilized and then suspended in phosphate buffer (5.5). It was then placed into microcentrifuge tube to determine the number of photons (kilo count per second) for analyzing the result.
Ultracentrifugation method was employed for determining the entrapment efficiency. An aliquot of formulation was centrifuged at 12000 rpm using ultracentrifuge (Remi C-24 BL with angular R-241 rotor, Remi House, Mumbai, India) and content of drug was estimated separately in the sediment and the supernatant [
HPLC method using reverse phase adsorption chromatography was used for determination of drug concentration. The instrument consisted of a Shimadzu LC-10AT VP pump, a SIL-10AF autoinjector, an SPD-10A UV-VIS detector, and an SCL-10A VP system controller (Shimadzu, Japan). The column was Shim-pack VP-ODS, having 4.6 mm I.D. and 150 mm bed length with adsorbent particle size 5
Calibration curve was plotted by taking concentration in range of 1–50 ng /mL with respect to peak area. A linear correlation between peak area and concentration was obtained within 2–40 ng/mL concentration range. Calibration curve equation was
Cellulose acetate synthetic membrane was used for determining the drug release. The membrane had molecular cutoff of 12000 Da. At first, the membrane was kept in physiological saline solution at
The formulations were evaluated for stability by initially storing the vesicles at 4°C ± 2°C and
Further, the nanovesicles were formulated into gel for ease in application. Carbopol 934 was dispersed in water and dispersion (1%) was prepared. The dispersion was mechanically stirred and then neutralized with triethanolamine solution (0.5%). The neutralized dispersion was kept overnight to remove any air bubble. Nanovesicles were then added to the dispersion [
Brookfield DV III ultra V6.0 RV cone and plate rheometer (Brookfield Engineering Laboratories, Inc., Middleboro, MA, USA) was used to determine the viscosity using spindle number CPE40 at
Following the experiment, the skin was stripped ten times using scotch crystal tape. After stripping, tapes were transferred into a glass vial of suitable size according to the following plan: vial 1 = strip 1, vial 2 = strips 2-3, vial 3 = strips 4–6, and vial 4 = strips 7–10. Then the rest of epidermis was removed using surgical scalpel. Residual skin sample was homogenized in methanol and analyzed for drug content [
The experiment protocol was reviewed and approved by Institutional Animal Ethics Committee, Department of Pharmacology, ITS Paramedical College, Muradnagar (vide letter number 1044/c/07/CPCSEA-2011-MPh-07), dated 08/11/2011. Three groups of six male albino rats were used in the study. The animals were 7–9 weeks old and housed in polypropylene cages under standard laboratory conditions (temperature:
For transdermal administration, the animals were sedated with ketamine hydrochloride (75 mg/kg) and xylazine (5 mg/kg). The abdominal area was cleaned with distilled water after trimming the abdominal hairs. The treatment was applied according to the following plan with a gentle rub and held in place by open containers glued to the skin by a silicon rubber having a nominal area of 3.14 cm2. At appropriate time interval, 0.2 mL blood sample was taken in vacutainer tubes and processed to separate the plasma by means of centrifugation at 8000 rpm for 15 min. Plasma was stored at −21°C before performing the drug content analysis with HPLC Assay [ Group I: CF (commercial formulation; 1 g, 1.5%; ~15 mg Aceclofenac), Group II: GLG-1 (500 mg, 1% ~5 mg Aceclofenac), Group III: PCG-1 (500 mg, 1% ~5 mg Aceclofenac).
Carrageenan induced edema model was employed for determining anti-inflammatory activity. The study protocol was designed and approved by the Institutional Animal Ethical Committee. Selected formulations (GLG-1 and PCG-1) were compared with a standard anti-inflammatory drug (Aceclofenac suspension) and a CF in four groups containing six animals in each group. Animals were fasted for 24 h before the experiment with free access to water.
Treatments were administered as per the following plan: Group I: Aceclofenac suspension, p.o., 20 mg/Kg, Group II: CF (1 g), Group III: GLG-1 (500 mg), Group IV: PCG-1 (500 mg).
Transdermal administration was kept in place by open containers glued to the abdominal skin by a silicon rubber. The untreated paw was taken as negative control. After one hour, 1% carrageenan suspension in saline was prepared and 0.1 mL was injected into right hind paw. After every hour, the paw volume was measured to yield the values for initial and at 1, 2, 3, 4, 5, and 6 h using digital plethysmograph. Percentage of inflammation was calculated by using formula given in data analysis [
Two groups of 6 males in each group were used. All the subjects were properly educated about the study procedure and consent forms were signed. Irritation potential was evaluated by visual observations in comparison to 5% Sodium Lauryl Sulfate (SLS) solution as positive control and untreated skin as negative control and scores were given as follows no reaction: 0, weak spotty or diffuse erythema: 1, weak but well perceptible erythema covering the total exposure area: 2, moderate erythema: 3, severe erythema with edema: 4, very severe erythema with epidermal defects: 5. Group I: positive control (SLS treated), Group II: GLG-1.
For the study, upper arm area was thoroughly examined for any irregularities and 1 g formulation was administered by gentle rubbing and held onto place with a bandage. After 24 hours, the bandage was detached and application site was cleaned with cotton. Then again application of treatment was done, for seven consecutive days. After seven days, the scores were given based on the observations [
For determination of permeation parameters, cumulative amount of drug permeated was plotted against time. The linear portion of the curve was extrapolated and projected
Permeability coefficient through the membrane (
Pharmacokinetic parameters, that is,
Percentage of higher edema inhibition provided by the treatment was calculated to determine the anti-inflammatory potential as per the following formula [
All the experiments involving live subjects (animal or human) were done on a group of 6 whereas other studies, for example, physicochemical characterization and stability studies, were performed in triplicate. Data is expressed as mean ± S.D. Statistical analyses were performed using the Graph pad Prism Version 4 software. Statistical comparisons were made using analysis of variance (ANOVA) or the paired
The developed formulations were characterized for physicochemical parameters, for example, size, PDI, zeta potential, entrapment efficiency, and
Figure
Physicochemical evaluation of developed nanovesicle formulations.
Formulation code | Size* (nm) | PDI* |
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Entrapment efficiency* (%) | Viscosity† (Cps) |
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PC-1 |
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PC-2 |
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PC-3 |
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GL-1 |
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GL-2 |
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GL-2 |
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All data expressed as mean ± S.D.;
Photomicrograph of PC-1 and GL-1 (×10000) in TEM: (a) PC-1 and (b) GL-1.
Figure
Based on physicochemical characterization, PC-1 and GL-1 were considered for stability evaluation at accelerated conditions for duration of 180 days at the temperature of 4°C and 25°C. The maximum damage was done by 25°C in 180 days in both types of formulations.
PC-1 was most affected by accelerated conditions. It showed 2.8 and 4.7 times value for vesicle size at 4 and 25°C after 180 days whereas PDI became 1.89 and 2.43 times at similar temperature. At 25°C, PC-1 showed considerable aggregation of vesicles after 30 days only as vesicle size and PDI are increased by 44% and 58% and entrapment efficiency decreased by 26.89%. Zeta potential increased by 21.42% after storing at 25°C for 180 days.
GL-1 showed maximum instability after 180 days as vesicle size increased by 56.19%; however, increase in vesicle size was 14% if stored at 4°C for 180 days and 3% if stored at 25°C for 30 days. Further, entrapment efficiency was decreased by 10.77% after storing at 25°C for 180 days (Table
Physicochemical characterization of selected nanovesicle formulations (PC-1 and GL-1) after stability studies.
Parameters | Formulation code | 0th Day | 30th Day | 90th Day | 180th Day | |||
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4°C | 25°C | 4°C | 25°C | 4°C | 25°C | |||
Size (nm) | PC-1 | 147 ± 2.5 | 163 ± 1.3 | 212 ± 4.2 | 285 ± 2.4 | 346 ± 2.8 | 412 ± 3.7 | 698 ± 4.7 |
GL-1 | 121 ± 1.1 | 122 ± 1.5 | 125 ± 1.7 | 129 ± 2.8 | 143 ± 2.5 | 138 ± 1.6 | 189 ± 1.9 | |
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PDI | PC-1 | 0.297 ± 0.001 | 0.351 ± 0.003 | 0.472 ± 0.007 | 0.449 ± 0.002 | 0.548 ± 0.003 | 0.562 ± 0.009 | 0.723 ± 0.007 |
GL-1 | 0.153 ± 0.004 | 0.167 ± 0.002 | 0.171 ± 0.001 | 0.179 ± 0.001 | 0.185 ± 0.007 | 0.182 ± 0.005 | 0.198 ± 0.001 | |
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PC-1 | −42 ± 1.2 | −43 ± 1.8 | −44 ± 1.3 | −46 ± 3.1 | −48 ± 3.5 | −48 ± 2.9 | −51 ± 2.6 |
GL-1 | −25 ± 1.1 | −26 ± 1.7 | −27 ± 2.9 | −26 ± 2.9 | −28 ± 1.6 | −27 ± 1.9 | −29 ± 2.4 | |
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Entrapment efficiency (%) | PC-1 | 47.6 ± 1.8 | 46.8 ± 1.3 | 34.8 ± 2.1 | 38.2 ± 1.5 | 28.1 ± 2.6 | 32.6 ± 2.1 | 17.2 ± 2.2 |
GL-1 | 78.9 ± 1.1 | 78.4 ± 1.3 | 77.3 ± 1.5 | 77.6 ± 1.5 | 73.2 ± 1.4 | 74.1 ± 1.8 | 70.4 ± 1.7 | |
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PC-1 | 73.91 ± 1.4 |
79.2 ± 1.9 |
91.4 ± 2.2 |
85.5 ± 2.8 |
94.3 ± 2.3 |
93.7 ± 1.4 |
95.3 ± 1.9 |
GL-1 | 70.06 ± 1.3 |
72.4 ± 1.9 |
75.6 ± 1.5 |
78.8 ± 2.2 |
81.3 ± 1.9 |
89.5 ± 1.6 |
95.2 ± 1.4 |
All data expressed as mean ± S.D.;
Full thickness human skin was used to study the permeation profile of the developed formulations and for computing the permeation parameters. Figure
The total content of drug accumulated in various skin layers was determined by analyzing drug content in different strata of skin after separation of these layers by stripping. Figure
Drug deposition profile of developed nanovesicle gels in different layers of skin with respect to CPG and CF;
Based on
Formulation code | Flux ( |
Lag time (hrs) | Permeability coefficient |
Distribution coefficient |
Enhancement ratio |
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CPG | 0.049 | 4.55 | 4.9 × 10−6 | 0.769 | 1 |
CF | 0.2218 | 3.2 | 2.218 × 10−5 | 1.093 | 4.52 |
PCG-1 | 0.806 | 1.675 | 8.06 × 10−5 | 2.08 | 16.44 |
PCG-2 | 0.6997 | 1.875 | 6.997 × 10−5 | 1.866 | 14.27 |
PCG-3 | 0.4941 | 1.95 | 4.941 × 10−5 | 1.794 | 10.08 |
GLG-1 | 1.4295 | 1.15 | 1.4295 × 10−4 | 3.043 | 29.17 |
GLG-2 | 1.1928 | 1.35 | 1.1928 × 10−4 | 2.59 | 24.34 |
GLG-3 | 0.9047 | 1.55 | 9.047 × 10−5 | 2.258 | 18.46 |
Pharmacokinetic parameters of selected nanovesicle gel formulations (PCG-1 and GLG-1) with respect to CF.
Formulation code |
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AUC ( |
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CF* |
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4 | 11.6 |
PCG-1 |
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4 | 83.9 |
GLG-1† |
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8 | 141.2 |
†500 mg formulation equivalent to 5 mg of Aceclofenac.
All data expressed as mean ± S.D.;
Plasma drug concentration profile of selected nanovesicle gels with respect to CF.
Carrageenan induced paw edema method was used for determining edema inhibition provided by selected formulation in comparison to CF and a standard treatment, oral Aceclofenac. In the initial hour, edema inhibition was the same for all the treatment; however, in second phase of edema production, GLG-1 provided 90.81% edema inhibition closely followed by PCG-1 at 85.62%. Standard treatment produced 74.84% edema inhibition in 6 hours whereas CF afforded 52.89% edema inhibition. It was special to note that PCG-1 afforded almost the same edema inhibition which might be due to topical nature of inflammation present in this kind of
% edema inhibition provided by selected formulations (GLG-1, PCG-1, CF, and Aceclofenac).
No group showed any severe irritation except the group treated with SLS.
The formulations PC-1 and GL-1 showed optimum physicochemical parameters. Smaller vesicle size produces larger surface area and appropriately charged zeta potential keeps them away so that the vesicle formulations remain stable.
The entrapment efficiency increased with increase in lipid content. Cholesterol is present in the same concentration in all the vesicle formulation. It is also reported to have an additive effect on increasing the drug entrapment.
Zeta potential is an important parameter owing to the role of surface charge in stabilization of the vesicle formulation; however, it may be affected by vesicle size, surface area, spatial localization of various components, and their state of ionization at the pH of application site. DCP is the only compound carrying electric charge and the difference in vesicle size is also nonsignificant.
The drug release profile shows that the highest drug releases were found in PC-3 and GL-3, having lower concentration of lipid. PC-1 and GL-1 showed the lowest drug release in their category having higher lipid content. PC and GL vesicles contain gradually decreasing fraction of PC, and guggul lipid, respectively. The content of cholesterol and DCP remained the same. PC is a phospholipid and forms bilayer in aqueous medium. Cholesterol seals the gap in PC membrane thereby used as an integral part of liposome composition to increase the stability. It increases drug entrapment and decreases drug release. The PC-1 and GL-1 were selected based on their physicochemical parameters and stability profile.
The selected formulations showed the difference in stability evaluation as PC-1 showed severe instability at higher temperature range in short duration and even at 4°C in 6 months. Temperature and time period both have shown detrimental effect on PC-1. Phosphatidylcholine is prone to hydrolysis and oxidation during storage producing lysolecithin. The presence of lysolecithin in lipid bilayers greatly enhances the permeability of liposomes. This is evident in reduction of time for drug release in PC-1 after storage at higher temperature. The similar event occurred at 4°C albeit after a longer time period. GL-1 showed commendable stability at 4°C; however, even at 25°C the stability is acceptable. Guggul lipid is composed of guggulsterones which are isomeric compounds of steroids category. The chemical structure is similar to cholesterol minus the side chain of cholesterol. The structure of guggulsterone is planar. The authors hypothesize the molecule by molecule stacking of guggulsterone and cholesterol considering steric hindrance due to cholesterol side chain. Further, due to this kind of membrane structure, more content of drug is retained even after 24 hours.
The drug content in receptor fluid was almost double in GLG formulation in comparison to the PCG formulations of the same ratio. An interesting finding is that for PCG formulations content of drug was higher in upper layers of skin. This is in accordance with the previous findings about rupture of PC liposome in upper layers of skin and drug deposition in superficial layers. In Guggul lipid vesicles, drug content in receptor fluid was in equilibrium with drug content in dermis which means that guggul lipid vesicles enhance the drug permeation inside the skin.
The study revealed that guggul lipid vesicles showed the optimum physical parameters and permeation profile; however, more significantly it shows good stability profile over PC liposomes. The most promising formulation was found to be GLG-1 with a composition of 7 : 3 : 1 (guggul lipid : cholesterol : DCP). We suggest that guggul lipid vesicles would be beneficial for transdermal drug delivery.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors want to acknowledge Akums Drugs & Pharmaceuticals Ltd., Haridwar, India, for genuine supply of Aceclofenac.