Advances in Pharmaceutical Research

Research Article [Artilce ID-1949694]

The Effect of Transferosomes on Skin Permeation of Ketorolac Tromethamine

Anand Jyoti1, Kumar Pramod2
1Pharmacy Department, S. Sinha College, Aurangabad, Bihar, India
2National University of Ireland, Galway, Ireland

Received: 19 Mar, 2019; Revised: 31 Mar, 2019; Accepted: 17 Apr, 2019; Published: 03 May, 2019

DOI - 10.36218/APR/1949694

Copyright 2019 This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Correnspondence should be addressed to Anand Jyoti:


To overcome lipophilicity problem, associated side effects and frequent dosing of Tromethamine, it is needed to develop some newer and safer. Ketorolac Tromethamine Transferosomes may help to increase the depth of skin permeation and the amount of drug delivered to systemic circulation (help in overcoming lipophilicity problem). At the same time if may provide the alternative of oral route (to minimize the side effects), or parenteral route (prevent the pain at injection site) and also can provide the long duration of action, to overcome the problem of frequent dosing. The transfersomal vesicles were prepared by conventional rotary evaporation sonication method using Soya phosphatidylcholine and edge activator (Span 80 and Sodium cholate). Optimizations of the transfersomal formulation were done by selecting various process variables such as effect of Soya phosphatidylcholine-edge activator ratio, effect of various edge activator and solvents. The skin permeation was tested using Franz diffusion cell and rat skin. Vesicles composition had a major effect on the morphological characterization, size and size distribution, percent entrapment efficiency, degree of deformability and in vitro release pattern of the vesicular system. Herein, it is observed that complex lipid molecules transfersomes can increase the transdermal flux and prolong the release of Ketorolac Tromethamine. Present investigation showed that Ketorolac tromethamine could be a potential drug carrier in the form of new ultra-flexible transferosomes.


Ketorolac tromethamine (KT) is a novel non- steroidal anti-inflammatory drug (NSAID) with potent analgesic and modest anti-inflammatory activity [1-4]. It inhibits prostaglandins (PG) synthesis and is believed to relieve pain by a peripheral mechanism. KT is currently administered intramuscularly and orally in multiple divided doses for short-term management of post-operative pain. Intra muscular injection is the preferred route of administration (30 mg four times a day).

It is administered for moderate to severe pain management, even though patient compliance is rather low for this route (create pain at injection site). The drug is administered orally as a conventional tablet (10 mg, four times a day) for management of mild to moderate pain. Its oral administration carries many risks when administered as a conventional dosage form. In postoperative pain it has equalled the efficacy of morphine, which does not interact with opioid receptors. The major side effect is gastrointestinal (GI) complications including peptic ulcers, and GI bleeding or perforations. The half-life of KT ranges from 4-6 hr. Therefore, frequent dosing is required to alleviate the pain due to its short half-life and it also reduced patient compliance. Moreover, most of adverse reactions to KT are dose related and it is strictly advised not to exceed this limit. To avoid an invasive drug delivery technique (i.e. intramuscular injection) and to decrease the gastrointestinal side effects produced by the oral tablets, there is an imperative need for an alternative non-invasive mode of a controlled drug delivery system for KT, which could improve therapeutic efficacy and reduce the severity of upper GI side effects through altering dosage forms of KT by modifying release of the drug.

Transdermal drug delivery system (TDDS) offers several advantages like bypassing the first pass effect, reducing frequency of administration, potentially decreasing the side effect, improved patient compliance, sustaining of drug delivery and interruption or termination of treatment if necessary. However, the of transdermal route is limited to the drugs having appropriate features such as appropriate low molecular weight and high lipophilicity. KT shows a small molecular weight but it appears to be not lipophilic enough (with reported log P of 1.04) to penetrate the skin. To overcome lipophilicity problem, side effects and frequent dosing, it is needed to develop the novel TK formulation. It is envisaged that Transferosomes may increase the depth of skin permeation and the amount of drug delivered to systemic circulation by depot action of transferosomes. Thus, the present work was envisaged to develop transfersomes of KT that will release the drug over a prolonged period barring frequent dosing, improving patient compliance and, provide more constant plasma level ultimately the approach will help to reduce the associated side effects and improved therapeutic index.



Ketorolac tromethamine was gift sample from Scott-Edil Pharmacia Ltd. (H.P., India), Soya lecithin and Sodium cholate were purchased from Himedia Laboratories (Mumbai, India) and Span 80 was purchased from CDH (Delhi, India). All other chemicals were used of analytical grade.


Preparatory of Transferosomes

Ketorolac tromethamine loaded Transferosomes Transferosomes were prepared by conventional rotary evaporation sonication method [5]. The method was comprised of two steps. First, a thin film of lipid was prepared, hydrated and then brought to the desired size by sonication; and secondly, sonicated vesicles were homogenized by extrusion through a sandwich of 200 and 450 nm polycarbonate membranes. Briefly, Phospholipid was mixed with edge activator [6], taken in a clean dry round bottom flask and the lipid mixture was dissolved in chloroform: methanol (3:1 v/v). The organic solvents were removed by rotary evaporator above the lipid transition temperature (430C) and traces of solvent were removed under vacuum overnight. The deposited lipid film was hydrated by hydro-alcoholic solution with drug as added to the desired concentration in the final preparation by rotation at 60 rpm for 1 hr at room temperature (25oC). The resulting vesicles were swollen for 2 hr at room temperature to get large multilamellar vesicles (LMLVs). The thick suspension thus obtained was vortexed to get homogenous small size transfereosomes. The resulting suspension was sonicated for 35 min at 4oC to achieve desired vesicle size [5,7, 8].

Formulation Optimization

There are three variables which can affect the preparation and properties of transferosomes, (i) saturated concentration of drug in formulation. (ii) lipid (Soya lecithin) and surfactant (Span 80, Sodium cholate) molar ratio, and (iii) alcohol concentration (Ethanol, Isopropyl alcohol). The optimization of transferosomes formulation was done based on morphological characterization, vesicle size, size distribution (polydispersity index), surface charge analysis, entrapment efficiency, elasticity (Deformability index) and in-vitro study.

Morphological characterization

Ketorolac tromethamine-loaded vesicles were spread on a slide and viewed under optical microscope with photograph auto-selecting system (Magnus analytical instrument) to observe the shape of vesicles. The photographs were prepared by auto-selecting system in 10×100 magnification. For determination of surface characteristic (three-dimensional structure), the formulation was coated uniformly with gold-palladium by using sputter coater (polaron SC 76430) for 5-7 minutes, after fixing the sample in individual stabs. All sample of transferosomes were then randomly examined for surface morphology by using scanning electron microscopy (LIO-430) at different magnification range. Transmission electron microscopy (TEM) was used to visualize the transfersomal vesicles (morphology of the two-dimensional vesicles justifying the vesicular characteristics). For this, the vesicles were dried on a copper grid and adsorbed with filter paper.

Before the film dried on the grid, it was negatively stained with 1% phosphotungastic acid (PTA). A drop of the staining solution was added on to the film and the excess of the solution was drained off with a filter paper. After drying, the sample was viewed under the microscope at 10–100 k magnification at an accelerating voltage of 100 kV [9].

Drug entrapment efficiency

Transferosomes entrapped Ketorolac tromethamine was estimated by centrifugation method. Ketorolac tromethamine loaded transferosomes preparations were kept overnight at 4oC and ultracentrifuge (Remi Equipments, Mumbai, India) at 14000 rpm at a temperature of 4°C for 30 minutes, where upon the pellets of transfersomes and the supernatant containing free drug were obtained. The transfersomes pellets were washed again with distilled water to remove any unentrapped drug by centrifugation. The combined supernatant was analyzed for the drug content after suitable dilution with phosphate buffer (pH 7.4) by measuring absorbance at 323.0 nm using Shimadzu U-V 1700 (U-V Spectrophotometer). The free Ketorolac tromethamine in the supernatant gives us the total amount of unentrapped drug. Encapsulation efficiency was calculated according to equation [10].

Percentage entrapment efficiency = [(A1 – A2) ÷ A1] ×1OO

Where; A1= Amount of Ketorolac tromethamine added initially, A2= Amount of Ketorolac tromethamine determined in the filtrate, (A1 – A2) = represents the amount of Ketorolac tromethamine entrapped in the formulation.

Vesicles elasticity measurement

Elasticity of vesicle membrane is an important and unique parameter of elastic liposomal formulations because it distinguishes elastic liposomes from other vesicular carriers e.g. liposomes that are unable to cross the stratum corneum intact. The deformability study was done for the elastic liposomal formulation against the pure water as standard using a home-built device. The elasticity of transfersomes vesicles were measured by extrusion method [5, 11]. The transfersomes formulation were extruded through polycarbonate membrane (pore size diameter 450 nm), using a stainless-steel filter holder having 50 mm diameter, by applying a pressure of 2.5 bar.

The quantity of vesicles suspension extruded in 5 minutes was measured. The degree of deformability was calculated by using the formula below;

E = J × (rv / rp)

Where, E = Elasticity of vesicles membrane, J = Amount of suspension extruded in 5 minutes, rv = Vesicles size (after passes), rp = pore diameter of the barrier.

Vesicles size, size distribution and zeta potential analysis

The vesicles size and size distribution (polydispersity index) of transferosomes vesicles were determined by Dynamic Light Scattering method (DLS) in a multimodal mode using a computerized inspection system (Malvern Zetamaster, Malvern, UK.). The zeta potentials of various transfersomal formulations were determined using a Zetasizer (Malvern instrument Ltd., Malvern, U.K.) [12].

In-vitro drug skin permeation study

The in-vitro skin permeation of Ketorolac tromethamine loaded formulation was studied by using a in house fabricated Franz diffusion cell and rat skin was used as natural membrane.

Preparation of the skin

Permeation and deposition studies were carried out through hairless abdominal skin of 6-8 weeks old male albino rat using fabricated Franz diffusion cell. The animals were sacrificed by overdose of chloroform inhalation. The hairs of dorsal side of animal were removed using electric clipper. The hair removed skin was separated from the animal and the hypodermis including blood vessels were surgically removed using surgical blade no. 23. The dermis part of the skin was wiped off with an isopropanol wet cotton swab gently to remove any adhering for material. The excised skin washed with distilled water and allowed to dry (exposed to ambient air conditions for 20 minutes). The skin was packed in aluminium foil and store in a polyethylene bag at 20oC.

In -vitro skin permeation studies

In-vitro drug permeation study was performed by using rat skin in phosphate buffer solution (pH 7.4). For this, Franz diffusion cell with a receiver compartment volume of 50 ml and effective diffusion area of 2.50 cm2 used.

Fresh abdominal skin of rat was prepared (described above) and used in the permeation experiments. To perform skin permeation study, treated skin was mounted horizontally on the receptor compartment with the stratum corneum side facing towards the donor compartment of Franz diffusion cell. The receptor compartment was filled with 50 ml of phosphate buffer (pH 7.4) saline maintained at 37 ± 0.5oC and stirred by a magnetic bar at 100 rpm. Ketorolac tromethamine loaded formulation (equivalent to 2.5 mg drug) was placed on the skin and the top of the diffusion cell was covered. At appropriate time (0.0, 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 12.0 and 24.0 hr) intervals, 1 ml aliquots of the receptor medium were withdrawn and immediately replaced by an equal volume of fresh phosphate buffers (pH 7.4) to maintain sink conditions. Correction factors for each aliquot were considered in calculation of release profile. The samples were analyzed spectrophotometrically at λmax 323.0 nm by appropriate dilution.

In- vitro skin deposition studies

At the end of the permeation experiments (after 24 hr), the skin surface was washed five times with ethanol: PBS pH 7.4 (1:1), then with water to remove excess drug from surface. The skin was cut into small pieces (1mm2 pieces). The tissue was further homogenized with ethanol: buffer solution pH 7.4 (1:1) and left for 6 hr at room temperature (25oC).

After shaking for 5 minutes and centrifuging for 5 minutes at 5000 rpm, the Ketorolac tromethamine content was analyzed by UV visible spectrophotometry method after appropriate dilutions with PBS (pH 7.4) at 323.0 nm [13].

Statistical analysis

Statistical analysis was carried out employing the one-way analysis of variance (ANOVA) by using the software PRISM (Graph Pad). A value of P < 0.05 was considered statistically significant.


Formulation development

The conventional rotary evaporation sonication method reported by Cevc et al. 2003 and Ahad et al 2012 [14, 15] was used to prepare the transfersomal formulation. Formulations were designed using different concentrations of lipid (Soya phosphatidylcholine), surfactant (Span 80 and Sodium cholate), where Sodium cholate was used due to its biocompatibility, ionic surfactant nature, HLB value as 18, to make it water soluble. Whereas Span-80 were selected because they are pharmaceutically acceptable, non-ionic surfactant, HLB value as 4.3 as water insoluble. Ethanol and Isopropyl alcohol solvents were investigated for hydration of lipid layer, where Ethanol is more water-soluble compare to Isopropyl alcohol while Isopropyl alcohol provides cosurfactant properties comparison to Ethanol due to higher branching nature.

Optimization of drug concentration in formulation

The maximum concentration of Ketorolac tromethamine that could be incorporated into the vesicle formulations was 5.0 mg/ml with percentage entrapment efficiency of 56.94 ± 0.63. After increasing the amount of drug, drug crystals were precipitated probably due to the saturation of the vesicular formulation (Tables 1, 2 & 3). It should be noted out that these saturation concentrations are the total concentrations of Ketorolac tromethamine that could be incorporated in the vesicular system.

Table 1: The effect of Soya lecithin (phosphatidylcholine) and surfactant (Span 80 and Sodium cholate) concentration on transferosomes formulation

Formulation Code

Composition PC: S (10%w/v)

Ethanol (% v/v)

Drug (mg) (0.5% w/v)









































Plain Drug




Table 2: Effect of alcohol concentration on transferosomes formulation

Formulation Code

PC: S (10% w/v)

Alcohol (% v/v)

Drug (mg) (0.5% w/v)



Without alcohol




7% Ethanol




14% Ethanol




7% 2-Propanol


Table 3: Incorporation of Ketorolac tromethamine into transferosomes formulation at saturated concentration

Formulation code

Drug (% w/v)

Microscopic Observation

(Drug Crystal)

% Entrapment efficiency



Not appeared

47.25 ± 0.92


Not appeared

51.16 ± 1.28


Not appeared

56.94 ± 0.63



42.15 ± 2.62

All value represented as mean ± SD (n=3)

Morphological characterization (Vesicle morphology)

The surface morphology of prepared formulation was examined by optical microscope. It is observed that the vesicles were formed in all formulation and at surfactant concentration from 5% to 15%, but at increased surfactant concentration above 15%, vesicles aggregates were appeared. These vesicles aggregates were also present in formulation having alcohol concentration above 7% (Fig. 1). The surface morphology and three-dimensional natures of transfersomes were studied by further analysis using SEM, confirmed the vesicular characteristics possessed by this novel carrier (Fig. 2). The morphology of two-dimensional vesicles was further evaluated by TEM justified vesicular characteristics. TEM studies show that Transfersomes appeared as multilamellar vesicles, with the lamellae of vesicles evenly spaced to the core (Fig. 3). The high amount of surfactant present in transferosomes reduce the surface tension and alcohol concentration above 7% also reduce interfacial tension, thus vesicles aggregates observed seen in formulation having surfactant concentration above 15% and/or alcohol concentration above 7%.

Fig. 1: Optical photomicrograph of optimized (KTTF-S803) Ketorolac tromethamine loaded transferosomes

Size and size distribution analysis

The vesicle size and size distribution were determined by light scattering method by Malvern Zetasizer of the optimized Ketorolac tromethamine loaded formulation. The results were expressed as the average vesicle size. There were insignificant differences in size of the transfersomal formulation containing different surfactants. These results correlate well because a similar method of preparation was involved. However, a reduction of vesicle size was observed when surfactant concentration increased above 15% w/w.

The vesicles size was larger in the presence of alcohol (KTTF-S803-E1) sample as compared to IPA(KTTF-S803-P1) and reduction in vesicles size was observed with increased alcohol concentration (> 7%) as in KTTF-S803-E2. This is due to the formation of a micellar structure instead of the vesicles, which are relatively smaller in size. After increasing the alcohol concentration (>7%) as in KTTF-S803-E2, polydispersity index increased (Fig. 4) as increased ethanol concentration decreased interfacial tension of product and more micelles was formed and prepared formulation results in heterogeneous formulation.

The vesicles size containing ethanol (KTTF-S803-E1) were larger as compared with vesicles contained IPA (KTTF-S803-P1) because ethanol have greater solubility with water so better hydration of vesicles and increase alcohol concentration (>7%) as in KTTF-S803-E2, vesicles size decreased this is due to ethanol causes a modification of the net charge of the system and confers it some degree of steric stabilization that may finally lead to decreased in the mean particles size. Size distribution confirmed the normal size distribution of the vesicles. The particle size distribution with a very low polydispersity index of less than 0.1 indicates a narrow size distribution of the transferosomes formulation and consequently a homogeneous distribution.

Although with increased surfactant concentration, polydispersity index increased suggesting formation of non-vesicular structures instead of vesicles. The polydispersity index of vesicles containing ethanol (KTTF-S803-E1) were lower than the vesicles contained IPA (KTTF-S803-P1), because IPA act as cosurfactant (due to more branching nature) at same concentration of ethanol. Overall, more micelles are formed, and prepared formulation results in more heterogeneous compared to ethanol product.

Fig. 2: SEM micrograph of optimized (KTTF-S803) Ketorolac tromethamine loaded transferosomes


Fig. 3: TEM micrograph of optimized (KTTF-S803) Ketorolac tromethamine loaded transferosomes

Fig. 4: Size distribution curve of optimized (KTTF-S803) Ketorolac tromethamine loaded transferosomes

Surface charge (zeta potential)

The zeta potential of all vesicle formulations was negative (Fig. 5) due to the net negative charge of the lipid composition in the formulations. PC (phophatidylcholine) is a zwitterionic compound with an isoelectric point between 6 and 7. Under experimental conditions (pH 7.4), where the pH was higher than its pKa making the formulation with net charge as negative value. In case of plain drug formulation in which zeta potential was also observed negative, might be due to combined effect of anionic nature of Ketorolac tromethamine and alcohol. However, the negative charge of formulations strongly improved skin permeation of drugs in transdermal delivery.

Fig. 5: Zeta potential curve of optimized (KTTF-S803) Ketorolac tromethamine loaded transferosomes

Entrapment efficiency

The maximum entrapment efficiency obtained was 61.92 ± 1.57% for transfersomes formulation KTTF-S801. The effect of surfactant concentration in the lipid components of vesicles on the entrapment efficiency of the drug clearly shows that entrapment efficiency decreased with an increase in concentration of surfactant. The effect may be due to the possible coexistence of mixed micelles and vesicles at higher concentrations of surfactant, with the consequence of lower drug entrapment in mixed micelles as described earlier [16, 17]. The reduction in entrapment efficiency also depended on the surfactant type. The pronounced effect exerted by sodium cholate could be attributed to the fact that the cholate has an ionic surfactant (water soluble) like Ketorolac tromethamine and thus displaces a part of drug from the bilayered vesicles. This displacement involves competition between species and thus, becomes noticeable at high concentrations of sodium cholate. The data indicated that entrapment efficiency also varies with ethanol concentration, and it is evident that increase in ethanol concentration decrease entrapment efficiency. The entrapment efficiency also depends on ethanol concentration, and increase ethanol concentration decrease entrapment efficiency. The possible region behind this is the fact that, with increase in ethanol concentration in the presence of surfactant lead to increase fluidity of membrane and vesicles become leakier so decrease in entrapment efficiency. The formulation without alcohol (KTTF-S80-WTE) show low entrapment efficiency as 47.52 ± 0.68, might be due to improper hydration of lipid layer during transfersomes preparation.

Measurement of elasticity

The results indicate that elasticity of vesicles depends on both surfactant concentration and type. With increase in the surfactant concentration from 5% to 15% w/w, the elasticity value increases from 25.68 ± 0.57 to 49.93 ± 2.25 for formulation KTTF-S801 and KTTF-S803 respectively. However, with further increase in surfactant concentration, the elasticity value of vesicle membranes decreases significantly (from 49.93 ± 2.25 to 9.35 ± 1.40 for formulation KTTF-S803 (15%) and KTTF-S805 (25%) respectively. An increase in alcohol concentration decreased membrane elasticity from 49.93 ± 2.25 to 13.26 ± 1.25 for formulation KTTF-S803-E1 and KTTF-S803-E2 respectively. This decrease in membrane elasticity with increase in alcohol concentration might be due to the formation of mixed micelles at higher concentration of ethanol.

The extremely high flexibility of the membrane permits transfersomes to squeeze themselves even through pores much smaller than their own diameters. This is due to the high flexibility of the transfersomes membrane and is achieved by judiciously combining at least two lipophilic/amphiphilic components (phospholipid plus surfactant), with sufficiently different packing characteristics, into a single bilayer. The HLB values of Span-80 and sodium cholate are 4.3 and 18 respectively.

Based on these HLB values, at similar molar ratios, the extent of surfactant interaction with lipid bilayers should be in the following order: Span 80 > sodium cholate. Overall, span 80 should produce better effect in comparison with the other surfactants by providing greater flexibility to the vesicle membrane. However, once the concentration increases above 15% of surfactant (span 80 and sodium cholate) flexibility decreased due to micelle structure formed instead of vesicles, which are less flexible compare to vesicles. The resulting high-aggregate deformability permits transfersomes to penetrate the skin spontaneously and minimizes the risk of complete vesicle rupture in the skin.

In-vitro drug release and drug deposition studies

In-vitro drug permeation studies give us valuable information about the product behaviour in- vivo. The drug permeability suggests about the amount of drug available for absorption.

Better transdermal flux and lower lag phase with our formulation was perhaps due to combination of one or more of following mechanisms: (i) increase in thermodynamic activity, (ii) increased skin vehicle partitioning of drug, (iii) altering the barrier properties by interacting with skin component and (iv) elasticity of vesicle membrane. The results of skin permeation study through rat skin from transfersomal formulation, and plain drug at the same drug concentration showed that transdermal flux (Jss) of vesicle formulation is significantly higher. For plain drug transdermal flux is 1.13 ± 0.02 (µg/cm2/hr) and while 10.71 ± 0.09 (µg/cm2/hr) for KTTF-S803 and 6.33 ± 0.06 for (µg/cm2/hr) for KTTF-SC3 (Fig. 6).