INTRODUCTION:

Human skin has great elasticity due to a natural protein called collagen present in the dermal layer of the skin. Collagen is produced inside the body naturally but as a person ages the production of collagen decreases and hence elasticity of the skin is lost thus wrinkles are formed. At young age, epidermis of the skin stretches and holds large amount of moisture due to the presence of fibers called elastin and the protein collagen, also a layer of fat in the subcutaneous level of skin gives plump look ^{1, 2}. All this is lost as person ages. So dermis begins to have difficulty in moving adequate amount of moisture up to the epidermis. This leads to the sagging and hence wrinkling of the epidermal layer of the skin ³. Also exposure of the skin to UV irradiations and absorption of photons by endogenous photosensitizer molecules results in formation of different reactive oxygen species, which affects appearance of the skin. It also leads to different degenerative effects and skin damages such as photoaging, sunburn, photocarcinogenesis, etc ⁴.

Amongst the approaches used to avoid wrinkling of the skin and protect it from these degenerative effects, use of synthetic peptides and antioxidants has been adopted as an important strategy in cosmeceutical industry. They are incorporated in cosmeceutical formulations to prevent the muscle contraction and to scavenge free radicals in skin produced by UV light and environmental pollutants and also provide moisture barrier. There are various types of facial rejuvenation treatments available and they carry their own set of risks, benefits, costs, and techniques. Some of them are facelift, contour threads, dermabrasion. Some minimally invasive techniques are microdermabrasion, facial peel, hyaluronic acid injections, botox injections, collagen injections, fat injections, gortex implants ^{5, 6}. But these treatments need repeated injection of toxin in chronic disorders which can lead to mild pain during injection, minimal local edema, and erythema. Thus, there is a need to design and validate non-toxic molecules that mimic the action of BoNTs .The various peptides recommended in literature are argireline, SNAP-7, SNAP-8 (elongation of Argireline), leuphasyl and BoNT-L-Peptide.

BoNT-L-Peptide mimics the amino acid sequence of the C-terminal domain of SNAP- 25, blocking its activity. It produces its therapeutic effect by acting selectively on peripheral cholinergic motor nerve endings to inhibit the release of the neurotransmitter acetylcholine, which is mainly responsible for contraction of the muscle, at the neuromuscular junction. Thus muscles do not contract and wrinkles can be prevented. Recently antioxidants such as vitamins A, C and E are widely used in cosmeceutical products due to their obvious advantages for the skin. Antioxidants are promising in photoprotection with negligible side effects at physiological concentrations ⁷. It is expected that desirable amount of active should reach at the site for effectiveness. However, delivery of drugs through topical preparations viz. creams, gels, lotions, emulsion, etc. limits the effectiveness of actives due to barrier properties of the skin which hinder the drug deposition and relative poor stability of vitamins due to direct exposure of actives to UV light.

Thus selection of proper carrier is extremely important by considering the views in mind that they should increase drug penetration and flux and should protect drug from photodegradation ^{8, 9}. In last two decades, number of innovative microparticulate carrier systems viz. microemulsion, nanoemulsion, nanoparticles, liposomes, ethosomes, etc. have been reported for improving delivery of drug to the skin ^{10, 11}. Recently, liposome based formulations for topical delivery have been shown to be extremely promising for enhancement of drug penetration, improved pharmacological effects, decreased side effects, controlled drug release and drug photoprotection ¹². Also liposomes have an additional advantage of carrying both water soluble and lipid soluble ingredients. Improved delivery of drugs using liposomes is based on similarity of vesicle bilayer structure to that of natural membranes, which can alter cell membrane fluidity and fuse with cells. However the exact mechanistic basis of the same still remains unclear ^{13, 14}. The phenomenon of improved drug delivery is based on factors viz. lipid concentration, composition, lamellarity, vesicle size, surface charge, type of formulation. Vitamin E plays a role against aging, particularly of the skin since lipid per oxidation in tissue may be one of the causes of skin aging. It is also expected to ameliorate excessive pigmentation in the skin, a possible cause of decreased elasticity and poor water retention. Vitamin E is an excellent lipid radical scavenger, thus it is especially useful in terminating lipid radical chain reactions ¹⁵.Vitamin E is considered to be essential for the stabilization of biological membranes, particularly those containing large amounts of polyunsaturated fatty acids ¹⁶. Drug deposition and vesicle size were the key parameters involved in formulation of topical liposomes. The number of formulation and processing variables are involved during liposome preparation may affect these parameters and hence the performance of the formulation Thus it becomes extremely difficult to study the effect of interactions between various variables and preparation of liposome by a conventional method. Factorial design and response surface methodology is an important statistical tool to study the effect of several factors influencing responses by varying them simultaneously by carrying out limited number of experiments ¹⁷.

Thus the aim of the present investigation was to prepare liposomal formulation, using factorial design approach and to explore its application for topical delivery of synthetic peptide and Vitamin E Acetate. Relative quantities of phospholipid and cholesterol are an important parameter in light of the stability and cost consideration. Their ratio also greatly affects vesicle size and drug deposition in the skin. In the present study, liposomes were prepared by 3² factorial design using a modified ethanol injection method. Liposomes were characterized by vesicle size, zeta potential and drug release. Gels containing liposomal dispersion (batch with desired drug release of vitamin E Acetate and synthetic peptide) were prepared using Carbopol and were characterized for viscosity, pH, spreadability and drug release. Stability of liposome dispersion and lipogel was studied at 25ºC/60% RH, 30ºC/65% RH & 40 ºC/75% RH for 3.

MATERIALS AND METHODS:

Materials:

Phospholipon 85G® (P85G) (unsaturated phosphotidylcholine (PC) 85% and lysophopsho tidylcholine 3%), was a kind gift from Phospholipid GmbH, Nattermann, Germany. Cholesterol (CH) and stearic acid (SA) were purchased from Loba chemi, India. Synthetic peptide (BoNT-L-Peptide, INCI name- palmitoyl hexapeptide, 50 ppm solution) of pharmaceutical purity was obtained as generous gift sample from Infinitec Activos S.L., Barcelona, Spain through Pioma Chemicals, Mumbai, Vitamin E acetate (vit E) was obtained as generous gift sample from Inland pharma, Mumbai. Carbopol® (U21) was gift sample from Arihant trading, India. All other chemicals used were of analytical grade.

Preparation of liposomes:

Liposomes were prepared by modified ethanol injection method ¹⁸. In brief, required amount of phospholipid 85G, cholesterol and stearic acid were dissolved in ethanol (10%) containing Vitamin E acetate (0.12%). Ethanolic solution was added dropwise with the help of syringe into 10 ml of double distilled water (DDW) under stirring for 15 min using Teflon coated magnetic bead. To study the effect of variables on liposome characteristics, different batches were prepared using 3² factorial design. Phospholipids and cholesterol are the main components of the lipid bilayer which are responsible for their rigidity, lamellarity and stability. Thus amount of Phospholipid 85G and Cholesterol were selected as two independent variables and they were studied at three different levels. Stearic acid gives rigidity to the bilayer, optimum concentration was used in the formulation. All the batches were prepared with 0.12% vitamin E acetate.

Effect of variables:

To study the effect of variables on liposome performance and characteristics, different batches were prepared using 3² factorial design. Amount of phospholipid 85G and Cholesterol were selected as two independent variables. Effect of these variables on particle size is given in Fig 1. Amount of Stearic acid and vitamin E acetate were kept constant. Values of all variables and batch codes are as shown in Table I.

Table I: Experimental design with coded levels of variables and actual values:

Batch No.	Phospholipid 85G (mg)	Cholesterol (mg)
LPE1	60	12
LPE2	60	9
LPE3	60	6
LPE4	50	12
LPE5	50	9
LPE6	50	6
LPE7	40	12
LPE8	40	9
LPE9	40	6

Preparation of Lipogel and conventional gel:

Lipogel: Lipogel was prepared by adding liposomal dispersion (LPE7) into a suitable gel base like carbopol U21. Various batches of gels were prepared using Carbopol U 21 at different concentrations (0.1%, 0.3%, 0.5% and 1% w/w). Definite amount of Carbopol U 21 was sprinkled into the vortex created by stirring double distilled water containing preservatives and the solution was stirred for 15—20 minutes. Liposomal dispersion (LPE7) was added to hydrated carbopol solution with stirring. Gelling was induced by neutralization using triethanolamine.

Conventional gel (0.5%): They were prepared by sprinkling Carbopol U 21 into the vortex created by stirring double distilled water containing preservatives and the solution was stirred for 15—20 minutes. To this peptide (4%) and vitamin E acetate (0.12%) was added under stirring. Gelling was induced by neutralization using triethanolamine.

Evaluation of Liposomal dispersion and Lipogel:

Physicochemical Properties:

Liposomal dispersion: The Liposomal dispersions were characterized for color, odour, pH.

Lipogel: Lipogels were evaluated for color, appearance and pH.

Drug Content:

Liposomal dispersion: Peptide and vitamin E acetate are soluble in ethanol so they were extracted with ethanol from the liposomal dispersion by shaking the mixture of liposomal dispersion with ethanol for 2 minutes and sonicating further for 30 minutes. The sample was then spotted on TLC plate and analyzed by HPTLC method using Camag TLC scanner 3 operated by Cats Planar Chromatography version 1.1.3.0. The stationary phase used was silica gel 60 F₂₅₄and toluene: ethanol (9:1) as mobile phase at λ_max 221 nm for BoNT-L-Peptide using chloroform: cyclohexane (5:5) as mobile phase at λ_max 200nm for vitamin E acetate ¹⁹.

Lipogel: Appropriate quantity of gel was taken in a volumetric flask and the actives (Peptide and vitamin E acetate) were extracted in the similar way as described above for liposomal dispersion using ethanol as a solvent.

Drug content uniformity: Drug content uniformity was determined by analyzing drug concentration in gel taken from three to four different points using HPTLC.

Viscosity of gel:

Viscosity of conventional gel containing peptide (4%) and vitamin E acetate (0.12%) and lipogel (containing liposomal dispersion – LPE7) was measured using Brookfield viscometer model LVT, LV spindle set was used for finding rheological properties. Spindle no.4 was selected for finding the viscosity. Spindle was lowered into beaker containing gel and rotated at speeds 0.3 – 12 rpm. At each speed the corresponding dial reading on the viscometer was recorded. Direct multiplication of the dial reading with the factors gave the viscosity in centipoises ²⁰.

Spreadability of Lipogel:

Spreadability is one of the important characteristics of any topical preparation as far as patient compliance is concerned. It was determined according to the method described by Riffin et. al. About 1gm of gel was placed between the two glass slides onto which weights were allowed to rest. The top slide was then subjected to pull of 100gm. The time in seconds required for the top slide to travel 100m distance gave an idea of the relative spreadability of gel ^[20].

Particle size analysis of liposomal dispersion:

The particle size analysis was performed using laser diffraction theory by Beckman particle size analyzer (N5).

The particle size and shape of liposome was confirmed by TEM. Transmission electron microscopy was performed using JEOL 1010 (JEOL Ltd, Tokyo, Japan). A small aliquot of liposomal dispersion was placed on the grid, dried for 3 to 5 minutes, and drained on the filter paper. The grid was further air dried then it was loaded in the transmission electron microscope, and areas were scanned. The picture was taken under the electron microscope and is shown in Fig. 1.

Figure 1: Effect of concentration of phospholipids (mg) and cholesterol (mg) on particle size (nm):

Zeta potential (ζ) determination of liposomes:

Charge vesicles surface were determined using Zetasizer 300HSA. Charge and mobility of liposomes were determined.

In -Vitro drug Release from liposomal dispersion and lipogel:

In- vitro release studies were performed using Franz diffusion cells. Phosphate buffer pH 6.8 was used as receptor fluid. Dialysis membrane filters (0.22-μm pore diameter) /guinea pig skin (shaved, 0.5mm thickness) were soaked in phosphate buffer pH 6.8. One milliliter of liposomal dispersion or 1 g of the lipogel was placed in the donor compartment. Samples were collected at 1, 2, 3, 4, 5, 6, 7 & 24 hrs intervals and analyzed by HPTLC.

Primary skin irritation studies of lipogel:

Primary skin irritation studies of the selected formulation were performed using albino rabbits in accordance with the guidelines of the Consumer Product Safety Commission ^{21, 22}. Formalin was taken as positive control and plain gel was used as negative control in the study. The study was approved by the Institutional Ethics Committee (IAEC C. U. Shah College of Pharmacy, Mumbai, India).

Stability of liposomal dispersion and lipogel:

Stability of selected liposome dispersion (LP-7) and 0.5% (w/w) gel formulation were carried out at 25ºC/60% RH, 30ºC/65% RH & 40 ºC/75% RH for 3 months. Effects of temperature and RH on the vesicle size for liposomal dispersion and drug content, spreadability, pH, were studied for lipogel during stability period.

RESULTS AND DISCUSSIONS:

Preparation of liposomal dispersion:

Liposomal dispersion of synthetic peptide and vitamin E acetate were successfully prepared by modified ethanol injection method. The concentrations of phosphilipid 85G, cholesterol and stearic acid were optimized by factorial design to obtain stable liposomal dispersion devoid of aggregation, fusion and sedimentation (visual observation). Stearic acid (0.03%) was found to be optimum to maintain required bilayer rigidity, with increased concentration grittiness was observed in the formulation, hence optimum concentration of the stearic acid was used and kept constant in all the batches. Amount phosphilipid 85G and cholesterol were found to be critical in preparation and stabilization of liposomes and hence selected as variables in the 3² factorial design (Table I).

Responses of different batches obtained using factorial design are shown in Table II.

Effect of variables on size distribution:

A positive correlation was observed for both variables phopspholipid 85G and cholesterol on vesicle size of liposomes. Thus with increase in concentration of lipids vesicle size was found to be increased. Fig. 1.

Preparation of lipogel:

Lipogels were off white, opaque. Carbopol gels prepared with 0.1%, 0.3% w/w carbopol were not of good consistency, they were fluidy / watery in nature. Whereas gels with 1% w/w carbopol was stiff. 0.5% w/w carbopol gel was found to be of good consistency and acceptable feel. Hence 0.5% carbopol gel was used to make lipogel.

Physicochemical Properties:

Liposomal dispersion: The liposomal dispersion was off-white in color, odourless, and fluid in nature. It was stable and did not show sedimentation, pH was found to be in the range of 4.7-5.2.

Lipogel: The lipogels were off-white in color, opaque, odorless, with smooth appearance devoid of any aggregation. The pH of the gels was in the range of 6.4-6.6 (Table III).

Drug content and content uniformity:

Liposomal dispersion: Drug content for liposomal dispersion ranges from 97.13 % - 101.56 %. (Table II)

Lipogel: Drug content for lipogels ranges from 97.88 % - 101.53 % and content uniformity obtained was 99.26 % + 0.96% (Table III).

Viscosity of gel:

Viscosity of 0.5% lipogel and conventional gel (0.5%) were found to be 8550cps – 9000cps and 35000cps-38000cps respectively (Table III).

Spreadability of gel:

Increase in the concentration of carbopol from 0.1%-1% w/w, the spreadability was found to increase. The spreadability of 0.1%, 0.3%, 0.5% and 1% was found to be 4, 5, 7 and

10 sec/gm respectively (Table III).

Particle size analysis of liposome:

Ethanol injection method was found to produce unilamellar and homogeneous population of liposomes as indicated by the low polydispersity index (Table II). The particle size analysis of the nanoparticulate dispersion by Beckman particle size analyzer (N5) showed particle size range of 203.1nm - 347.8nm. Transmission electron micrograph (TEM) of liposomal dispersion of vitamin E confirms the spherical shape of vesicle. Fig. 2.

Figure 2: Negative-staining transmission electron microscopy images of liposomes. Magnification 1,000,000×

Table II: Responses obtained for studied parameters from experimental batches (n=3)

Batch No.	Appearance	pH	Odour	Particle size (nm) ± S.D.	Polydispersibility index (PI)	Drug content		Zeta potential (mV) ± S.D.
Batch No.	Appearance	pH	Odour	Particle size (nm) ± S.D.	Polydispersibility index (PI)	Synthetic peptide	Vitamin E acetate	Zeta potential (mV) ± S.D.
LPE1	Milky white	4.8	Odourless	347.8 ± 15.89	0.265	98.25	98.76	−53.87 ± 0.51
LPE2	Milky white	4.9	Odourless	338.1 ± 15.56	0.302	99.68	99.59	−52.93 ± 0.85
LPE3	Milky white	5.1	Odourless	326.2 ± 11.26	0.257	97.96	98.28	−48.70 ± 0.62
LPE4	Milky white	5.0	Odourless	305.1 ± 13.68	0.280	99.03	97.13	−50.27 ± 0.42
LPE5	Milky white	5.2	Odourless	288.1 ± 14.55	0.222	101.56	100.03	−51.07 ± 1.57
LPE6	Milky white	4.8	Odourless	276.1 ± 13.23	0.210	98.03	98.89	−35.33 ± 2.71
LPE7	Milky white	4.7	Odourless	252.5 ± 11.58	0.330	100.46	101.20	−29.30 ± 1.87
LPE8	Milky white	4.8	Odourless	213.4 ± 14.59	0.274	97.96	97.89	−33.57 ± 1.16
LPE9	Milky white	5.2	Odourless	203.1 ± 15.56	0.335	99.65	98.35	−45.33 ± 1.00

Table III: Evaluation of plain gel and lipogel of synthetic peptide and Vitamin E acetate

Parameters	Observation
Parameters	Conventional gel (0.5%)	Lipogel (0.5%)
Appearance	Transparent, homogeneous gel	Opaque, homogeneous gel
Colour	Colourless	Off white
pH	6.7-7.0	6.4-6.6
Spreadability	7 sec/1gm	5 sec/1gm
Viscocity (cps)	35000cps-38000cps.	8550cps – 9000cps.
Assay (%)	Peptide – 98.56 – 100.23% Vit E - 98.42 – 101.26%	Peptide – 98.97 – 101.86 % Vit E - 97.6 – 101.03%
Drug content uniformity (%)	98.95 + 0.56	99.26 + 0.96
Microscopic evaluation	No aggregation or lumps were seen	No aggregation or lumps were seen

Primary Skin Irritation Studies of lipogel (As per open patch test):

The scores for erythema and edema were totaled for intact and abraded skin for all rabbits at 24 and 72 hours. The primary irritation index (PII) was calculated based on the sum of the scored reactions divided by 24 (2 scoring intervals multiplied by 2 test parameters multiplied by 6 rabbits). The developed formulation showed no erythema or edema on the intact and abraded rabbit skin Fig. 6. The Primary irritation index of the formulation was calculated to be 0.00. Thus the formulation can be classified as a nonirritant to the rabbit skin.

Figure 5: In-vitro release from lipogel through guinea pig skin (LPE7).

Stability study:

Liposomal dispersion: Stability of liposomal dispersion was carried out for 3 months at various temperatures as per ICH guidelines. At 25ºC/60% RH, 30ºC/65% RH insignificant changes was observed on drug content and vesicle size. Slight increase in the vesicle size (insignificant) was observed which might be attributed to very slight fusion of the liposomes. This insignificant increase in vesicle size might be due to presence of surface negative charge on liposomes, which either avoids or delays formation of liposome aggregates due to electrostatic repulsion (the results for vesicle size and drug content is given in Table IV).

Lipogel: No change was observed on pH, Drug content, spreadability and viscosity at 25ºC/60% RH, 30ºC/65% RH.

Both the liposomal dispersion and lipogel batches were not stable at accelerated temperature this may be due to hydrolysis of phospholipids in aqueous form at accelerated temperature.

CONCLUSIONS:

A stable liposomal dispersion containing BoNT-L-Peptide (hydrophilic) and vitamin E acetate (lipophilic) was developed with narrow particle size distribution ranging from 203.1 nm – 347.8 nm and PI ranging from 0.210 – 0.335. A 3²factorial design demonstrated that increase in lipid concentration from 0.46% to 0.72% resulted in increase in vesicle size. The drug content obtained was 97.13% - 101.56% for peptide and vitamin E acetate. Lipid content of 0.52% with phospholipids: cholesterol ratio of 1:0.3 (LPE7) gave optimum release of both the drugs. The release of 64.38% (synthetic peptide) and 88.471% (Vitamin E Acetate) was obtained at the end of 24 hours. Lipogel (0.5%) was found to be of good consistency and acceptable feel with viscosity of 8550 – 9000 cps and spreadability of 5sec/gm.

Figure 6: Photographs of rabbit skin treated with developed Lipogel (0.5%): (A) intact and (B) abraded after 24 hours; (C) intact and (D) abraded after 72 hours.

The release of actives was slower from the gel formulation, 49.36% (peptide) and 66.42% (vitamin E acetate) at the end of 24 hours. Liposomal dispersion and lipogel were found to be non irritant on rabbit skin and also stable at refrigeration temperature and room temperature. The developed lipogel can be beneficial to improve drug penetration to the dermal layer of the skin due to nano size. Thus a stable and safe nano cosmeceutical lipogel of peptide and vitamin E acetate was prepared and evaluated.

ACKNOWLEDGEMENTS:

Authors are grateful to Dr. Torsten Kromp from Phospholipid GmbH, Nattermannalee, Germany for providing gift sample of phospholipids. Authors are thankful to Inland pharma Mumbai, India and Arihant trading, India for providing gift samples of Vitamin E acetate and Carbopol® U 21, respectively. Authors are also thankful to Mr. Vijay Doshi from Pioma chemicals for providing gift sample of BoNT-L-Peptide. Authors are also thankful to Mr. Thete and Gauri charegaonkar from Anchrom labs for readily allowing touse their instrument for carrying out method development and HPTLC analysis.

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Received on 24.12.2009 Accepted on 20.02.2010

Res. J. Topical and Cosmetic Sci. 1(1): Jan. – June 2010 page 18-24