Pak. J. Pharm. Sci., Vol.32, No.6(Suppl), November 2019, pp.2849-2857 2849 Formulation and in-vitro characterization of Capsaicin loaded ethosomes Imtiaz Majeed 1,2 , Syed Atif Raza 2 , Naveed Akhtar 3 , Faheem Ahmed Siddiqui 1 and Beenish Iqbal 1 1 Faculty of Pharmacy, University of Central Punjab, Lahore, Pakistan 2 Punjab Univeristy College of Pharmacy, University of the Punjab, Lahore, Pakistan 3 Faculty of Pharmacy & Alternative Medicine, University of Bahawalpur, Bahawalpur, Pakistan Abstract: The ability of ethosomes to entrap capsaicin was evaluated using four methods of preparation that are; hot method, cold method, classic method and injection method. The ethosomes were prepared, optimized and characterized with the aim to identify a technique best suitable for their formulation. Vesicle shape, size and entrapment efficiency was determined by scanning electron microscopy, dynamic light scattering and ultracentrifugation techniques, respectively. Vesicle sizes varied from an average of 15nm - 400nm depending on the concentrations of phospholipid, ethanol and method of preparation. The formulations demonstrated entrapment efficiency of 29-81% with maximum entrapment obtained in formulations prepared with hot method having high concentration of ethanol. The homogeneity index was measured with Zetasizer that showed formulation prepared with hot method to be more uniform in size distribution having PDI 0.162 while injection method of preparation yielded a moderately broad polydispersity of vesicles (0.276). Physical stability assessment done by storing the selected formulation samples at 4 0 C and 25 0 C indicated the refrigerator temperature to be the best for retention of drug in ethosomal vesicles. All formulations kept in refrigerator adequately retained capsaicin during the two months of stability studies while those at ambient temperature noticeably showed leaked drug from vesicles. FTIR analysis showed capsaicin and phospholipid to be compatible with each other with no sign of interaction. DSC studies evidently showed lowering of transition temperature of phospholipid from 327.13 0 C to 111.63 0 C in ethosomal formulation due to the presence of ethanol. It was concluded that capsaicin ethosomes can be successfully prepared to employ four different methods and their characterization parameters indicate hot method to be effective for preparation of nano-sized uniform, homogeneous and stable capsaicin ethosomes. Keywords: Ethosomes, capsaicin, Zetasizer, FTIR. INTRODUCTION Ethosomes are a novel mode of drug delivery targeted to the skin which is an easily accessible route for drug delivery. Touitou was the first to develop this vesicular system and due to the presence of ethanol in their vesicular structure named them as ethosomes (Touitou, 1998). Ethosomes are formulated by ethanol, phospholipid and water and have evidently enhanced topical uptake of numerous drugs (Ainbinder and Touitou, 2005). Earlier lipid constructed preparations like ‘microemulsions’, ‘niosomes’ and ‘liposomes’ has considerably upgraded the skin permeation however their inability to distribute the drug to deep skin strata left room for developing further improved mode of drug delivery (Cevc, 2004). Ethosomes are generally comprised of phospholipids, ethanol and water. Their average size ranges from tens of nanometer to few microns, but the size is dependent on the concentration of phospholipid and ethanol so it can be varied and controlled. Ethosomes act as permeation enhancing carriers, that is, they are a lot better in distributing topical drugs to the skin in manner of amount and range of delivery, compared to liposomes or hydroalcoholic solutions (Ting et al., 2004). Their soft, flexible characteristics and unique properties allow their easier infiltration into deeper skin layers (Fang et al., 2008). The high ethanol content (up to 50%) was proposed to fluidize the ethosomal lipid component and stratum corneum lipid structures, thereby permitting soft malleable ethosomes to penetrate (Ainbinder and Touitou, 2005). Ethanol is proposed to interfere with the polar part of lipid molecules, reducing melting point of stratum corneum lipid and in turn boosting lipid fluidity and cell membrane absorptivity (Touitou et al., 2000). Capsaicin induces “heat sensation” which is ascribed to its binding with Transient receptor potential vanilloid (TRPV1) ion- channel receptors, also called Capsaicin receptors (Ramsey et al., 2006). Hogyes first observed this heating sensation in 1878 when extract of Capsicum was applied on human skin (Toh et al., 1955). Capsaicin has high affinity for TRPV1 and acts as an agonist at these receptors. TRPV1 is a nonselective cation channel favourably expressed on small-diameter sensory neurons (C-fibers and A-δ fibres) (Alawi and Keeble, 2010, Bley et al., 2012). Capsaicin primarily excites the sensory neurons followed by a persisting refractory state known as desensitization where the priorly excited neuron becomes insensitive to diverse stimuli. The persistent *Corresponding author: e-mail: imtiaz.majeed@ucp.edu.pk