In vitro drug release study

The in vitro release of CHG from the NE formulations and control solution (loading dose for both equivalent to 20 mg/g CHG concentration) was investigated using dialysis membrane, and the amount of CHG released was plotted against time (Figure 5.10). The data shows an initial burst release of CHG from the NEs and control solution for up to 4 h, the first time point, followed by a slow release up to 24 h.

Figure 5.10 In vitro release profiles of CHG from NE formulations [C-EO-70(10), C-EO-75(10), C-OO- 70(10), C-OO-75(10)] and control solution (Mean ± SD, n = 6).

Release data shows lower CHG release from NE formulations compared to control solution, which might be due to the effect of formulation excipients. The amount of oil in the formulation restricts partitioning of the drug between the oil and water interface and

0 20 40 60 80 100 120 0 4 8 12 16 20 24 Concen tration of CH G release (μg/m l ) Time (h) Control C-EO-75(10) C-EO-70(10) C-OO-75(10) C-OO-70(10)

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dialysis membrane controls the rate of drug release (Sandhu et al., 2012). Literature study reported by Syed (2013) for in vitro evaluation of docetaxel OO-NEs (10 % w/w oil) formulated using similar production method. Results showed lower release of docetaxel from NE formulations compared to it control solution. Another study reported evaluation of in vitro release and epidermal permeation of dapsone NEs for topical delivery (Borges et al., 2013). Release of dapsone was found to be less from NEs compared to a control solution, confirming the role of formulation excipients and composition in controlled release of drug into receiver.

5.4.8 In vitro skin diffusion studies

In vitro skin permeation of all CHG-NE formulations and control solution was performed using Franz diffusion cells to study the localisation of drug within the skin layers and to determine if the formulation had any influence on this. Cumulative permeation was plotted against time (Figure 5.11), and permeability coefficient and steady state flux were calculated (Table 5.5). After 24 h less than 1 % w/w of the total applied dose of CHG reached the receiver compartment from NE formulations compared to 1.6 % w/w from the control solution. The reduced permeation through the skin may be as a result of altering the pathway into the skin. It has been reported that NEs may more effectively target hair follicles, thus differences will be seen between lipid formulations and the solution. The positive charge on the NEs in this study may also influence interactions with protein residues compared to non-charged equivalents (Yilmaz and Borchert, 2005).

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Figure 5.11 In vitro skin permeation of CHG from NE formulations [C-EO-70(10), C-EO-75(10), C-OO- 70(10), C-OO-75(10)] and control solution (Mean ± SD, n = 6).

In our study, permeation graphs show slow release of CHG from NEs, which unexpectedly plateaued after 12 h of diffusion experiment for CHG-OO NEs and control solution. Based on the literature study, CHG has been found to be stable at experimental conditions such as pH and temperature and also sink condition was maintained throughout experiment to establish concentration gradient across skin. The possible explanation could be binding of CHG with lipids and ceramides present in the SC of the epidermis, which enhances CHG penetration and retention after its application to the skin reducing its permeation into receiver compartment (Lorian, 2005). Similar permeation results were reported by Karpanen et al. (2008) in evaluation of CHG penetration into human skin, which had showed less CHG permeation into receiver compartment. Another study reported by Tsai et al., (2014) showed effect of NEs as carrier for hydrophilic ropinirole hydrochloride. Skin permeation study showed no detectable level of drug in receiver compartment after 12 h of diffusion experiment. Less permeation of CHG from NE formulations compared to control solution might be due to the presence of formulation excipients such as oil, surfactant and

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cosurfactant which enhances the penetration of CHG into skin by interacting and disrupting barrier properties of SC (Makraduli et al., 2013; Tsai et al., 2013).

Statistical analysis revealed a significant difference (p<0.05) in steady state flux values obtained for CHG-EO NEs and CHG-OO NEs. The flux of NEs and control solution was in order of control > C-EO-75(10) > C-EO-70(10) > C-OO-75(10) > C-OO-70(10).

Table 5.5 In vitro permeability parameters of CHG from NE formulations and control solution (Mean ± SD, n = 6).

Formulation Code Flux (Jss) µg/cm2/h Permeability coefficient (Kp) x 10-4 cm/h Control 1.23 2.81 C-EO-75(10) 0.91 1.29 C-EO-70(10) 0.87 1.52 C-OO-75(10) 0.84 0.26 C-OO-70(10) 0.76 0.22

5.4.8.1 Quantification of chlorhexidine digluconate in skin using adhesive tape stripping method

The adhesive tape stripping method is used for quantification of topically applied substances in the skin. It removes the superficial layers of SC and allows the determination of the amount of drug that has penetrated into the skin. The CHG levels found in the skin are presented in Figure 5.12.

CHG penetration into the skin was higher for NE formulations compared to the control, with the differences being statistically significant (p<0.05). In addition, CHG skin penetration was significantly higher (p<0.05) for CHG-EO NEs compared to CHG-OO NEs and the control solution. The amount of CHG recovered from the skin for the control

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solution was 3.01 ± 0.02 µg/mg, compared to 6.15 ± 0.12 µg/mg, 5.31 ± 0.08 µg/mg, 4.39 ± 0.04 µg/mg and 3.98 ± 0.06 µg/mg for C-EO-75(10), C-EO-70(10), C-OO-75(10) and C- OO-70(10) respectively. The C-EO-75(10) formulation had the CHG retention in skin, which is above the MIC range (2 g/ml) required to inhibit the growth of many Gram- positive and Gram-negative microorganisms reported in literature (Kärpänen, 2008; Popovich et al., 2012).

Figure 5.12 Penetration profiles showing the concentrations of CHG (µg/mg tissue) in porcine ear skin after 24 h exposure to the NE formulations [C-EO-70(10), C-EO-75(10), C-OO-70(10), C-OO-75(10)] and control solution (Mean ± SD, n = 6). HOMO refers to homogenised tissue after removal of the SC layers.

EO enhances the permeation of CHG into the skin, suggesting that a combination of CHG and EO may be a potential method to improve skin antisepsis in clinical practice. EO contains 1,8-cineole, which has been shown to bind in large quantities to the SC (Cornwell et al., 1996). It is thought to enhance lipophilic drug penetration by increasing the partition coefficient (partitioning of drug between vehicle and SC), as well as hydrophilic drug penetration by increasing the diffusion coefficient (Cal et al., 2001). Williams et al., (2006) also showed that 1,8-cineole partitioning in the skin lipids is heterogeneous, leading to both

0 1 2 3 4

Control C-EO-75(10) C-EO-70(10) C-OO-75(10) C-OO-70(10)

Am oun t of CH G ret ained in skin ( µg/ m g ) TAPE 1 TAPE (2-15) HOMO

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ordered and disordered areas in SC lipids. Furthermore, it has been shown in in vitro assays that cineole does not permeate through the skin but is retained in the skin (Cal et al., 2006). Biruss et al., (2007) reported increased skin penetration of steroid hormones using an EO (45 % v/v) microemulsion for topical delivery with EO shown to enhance percutaneous absorption by SC lipid extraction and loosening the hydrogen bond between the ceramides leading to fluidisation of lipid bilayers (Chena et al., 2014). Topical application of EO and other essential oil mixtures on necrotic ulcers on the neck areas of cancer patients, resulted in not only antibacterial but also an anti-inflammatory activity (Warnke et al., 2006). EO exhibited low toxicity and greater efficacy in reduction of morbidity associated with neoplastic ulcers. These studies indicate the potential of EO as a topical skin penetration enhancer and also suggest useful future work to determine the antibacterial effect of CHG- NE formulations in the prevention of bacterial skin infections. Another study reported amount of CHG recovered from top layers of human skin (100 µm thickness) with combination of EO with CHG was 0.157 µg/mg, which was higher than the concentration required to kill many common skin microorganisms (Kärpänen, T., 2008).

5.4.9 In vitro skin diffusion studies of chlorhexidine digluconate nanoemulsions using