Formulation and topical delivery of niosomes and proniosomes containing carnosine

Wiechers (2008:1-18) explains that the efficacy of a biologically active cosmetic product depends on both the intrinsic activity of the active, as well as the delivery of the active. This study aimed to deliver the cosmeceutical active, carnosine, topically. Carnosine is a naturally occurring compound with both anti-oxidation and anti-glycation properties (Kyriazis, 2010:45-49). The biological functions of carnosine vary amongst different tissues, as well as within one kind of tissue (Boldyrev et al., 2013:1820). Both oxidation and glycation are associated with ageing in skin, and with carnosine's aforementioned properties, it could possibly provide the skin with anti-ageing benefits (Hipkiss, 1998:864). The outermost layer of the skin is the most important physical barrier to overcome during topical delivery. This layer, the stratum corneum, is also considered as the rate-limiting barrier (El Maghraby et al., 2008:203-206; Wickett & Visscher, 2006:98-106). The stratum corneum consists of intercellular spaces filled with a lipophilic matrix and corneocytes aligned in a scaffold-like framework, which contribute to the difficulty of delivering hydrophilic compounds topically (Goebel et al., 2012:281-286; Venus et al., 2011:471-474). Unfortunately, carnosine is a hydrophilic compound and has unfavourable properties when considering the relevant physico-chemical properties for skin diffusion. The important physicochemical properties of the active to consider for skin permeation include the octanol-water partition coefficient (log P), molecular size and aqueous solubility (Benson & Watkinson, 2012). The ideal log P of an active for topical delivery is 1 to 3, whereas the general rule is normally for molecular size and aqueous solubility to be less than 500 Da and more than 1 mg/ml, respectively (Khalid et al., 2016:129; Naik et al., 2000:319). According to the Material Safety Data Sheet (MSDS, 2013), carnosine has a molecular weight of 226.23 Da. The aqueous solubility was determined as 122.804 ± 0.716 mg/ml in phosphate buffer solution (PBS) at pH 7.4 and 25 °C, whilst the octanol-buffer distribution coefficient (log D) was determined as - 2.891 ± 0.013 in PBS (pH 7.4). Due to carnosine's unfavourable log D, poor skin diffusion can be predicted and external interventions might be necessary to enhance skin permeation. Vesicle systems have been used since the 80s to improve skin permeation (Nasir et al., 2012:484). Vesicles are colloidal particles consisting of a hydrophilic headgroup and a hydrophobic tail (Honeywell-Nguyen & Bouwstra, 2005:67-74). Non-ionic surfactant based vesicles, better known as niosomes, can encapsulate both amphiphilic and lipophilic actives (Nasir et al., 2012:479-487; Uchegbu & Vyas, 1998:33-70). The use of niosomes to encapsulate drug molecules provides a number of advantages for topical delivery. These advantages include, but are not limited to, increased penetration of the stratum corneum, prolonged residence time of active ingredients in the skin and reduced systemic absorption (Mali et al., 2013:587). In cosmetic delivery, niosomes also increase the stability and improve the bioavailability of the active (Nasir et al., 2012:484). Proniosomes are a dry form of niosomes, which are more stable than niosomes, but easily hydrated with an aqueous phase upon use (Kumar & Rajeshwarrao, 2011:214; Marianecci et al., 2013:71). Niosomes can only be considered as a pre-formulation due to problems such as instability, visual appearance and mostly the very low viscosity. According to Barry (2007:595), topical preparations must be acceptable for patients. Patient preference generally includes products, which are easily transferred from the container, spread freely to leave no residue and are not difficult to remove from the skin. In order to benefit from the advantages of niosomes and increase patient compliance, a proper semi-solid dosage form is necessary. Stahl (2015:209-218) classifies semi-solid dosage forms into gels, creams, ointments and pastes. These dosage forms have sufficient viscosity to stay on the skin for a prolonged time, resulting in an increased chance for diffusion of the active ingredient through the formulation into the skin (Stahl, 2015:209-218). In this study, niosomes and proniosomes were used as pre-formulations. After a trial and error approach, the ideal carnosine concentration to be encapsulated was determined as 3%. Tests were performed on the two final pre-formulations to characterise them and ensure the quality of the dispersions. The characteristics tested included vesicle size, polydispersity index (PdI), zeta-potential, pH, viscosity and entrapment efficiency (EE%). Except for the low viscosities, the pre-formulations had overall good characteristics. The niosomes were chosen to formulate the semi-solids, since they had better overall characteristics and were easier and quicker to prepare prepared to the proniosomes. Two semi-solid formulations, a gel and a cream containing carnosine encapsulated in niosomes, were then formulated. Membrane release experiments, followed by transdermal diffusion studies and tape stripping experiments were performed on all four of the preparations. The membrane release experiments proved that carnosine was released from all four of the preparations. The niosomes had the best median flux (1 139.10 μg/cm2.h) of the four preparations, followed by the proniosomes, the gel and finally the cream. A slight negative effect of the formulations on the release from the pre-formulations was noticed. None of the samples had carnosine in the receptor phase after transdermal diffusion studies; whilst all four of the experiments successfully delivered carnosine to the stratum corneum-epidermis (SCE) and epidermis-dermis (ED). The gel delivered the highest median concentration carnosine (2.458 μg/ml) to the SCE, followed by niosomes, proniosomes and finally the cream. The niosomes delivered the highest median concentration to the ED (2.465 μg/ml), followed by the gel, the cream and finally the proniosomes. The niosomes and the gel were the best pre-formulation and semi-solid formulation considering topical delivery of carnosine. The median values were preferred, as they represented the skewed data more accurately than the average values (Dawson & Trapp, 2001:30; Gerber et al., 2008:190). The two semi-solid formulations underwent accelerated stability tests for three months. The stability tests were performed following the International Conference on Harmonisation (ICH) Guidelines. The formulation changes were assessed during accelerated (40 ± 2 °C/75 ± 5% RH (relative humidity)), intermediate (30 ± 2 °C/60 ± 5% RH) and long-term (25 ± 2 °C/60 ± 5% RH) storage conditions (ICH, 2003:3). The stability tests included concentration assays on the active and preservatives, pH, conductivity, viscosity and zeta-potential measurements, mass loss determination, as well as a microscopic and macroscopic examination. Neither of the products was considered stable after three months and was not suitable for manufacture.