Thermal stability study

Thermodynamic stability tests were performed to identify any unstable or metastable NEs compositions (Shakeel et al., 2014a, 2014b). All of the prepared formulations were

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subjected to stability studies that included centrifugation, heating-cooling and freeze-thaw cycles, as described in detail in chapter 4 (Section 4.4.4).

The C-EO-75(5) NE formulation underwent phase separation following centrifugation, while the other formulations were stable (Table 5.3). These were then subjected to thermal stress under two different conditions, 4°C and 25°C. Only the formulations that remained stable were then subjected to the freeze-thaw cycle. The formulations containing 10 % w/w Smix for both EO [C-EO-70(10), C-EO-75(10)] and OO [C-OO-70(10), C-OO-75(10)] were

found to be stable under all three testing conditions.

Based on the results obtained from studies of effect of different homogenisation speed, time and stability studies, the CHG-EO NEs [C-EO-70(10), C-EO-75(10)]and CHG-OO NEs [C- OO-70(10), C-OO-75(10)] each containing a surfactant concentration of 10 % w/w, were found to have the smallest droplet size, PDI and stable under all thermal stress studies, thus these formulations were selected for further physicochemical characterisation and in vitro diffusion studies.

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Table 5.3 Thermal stability assessments of CHG-loaded EO-NE and OO-NE formulations.

Formulation Code Centrifugation

Heating-cooling cycle Freeze-thaw cycle 4°C 25°C C-EO-70(5) √ X X N/A C-EO-70(10) √ √ √ √ C-EO-70(15) √ √ X X

C-EO-75(5) X N/A N/A N/A

C-EO-75(10) √ √ √ √

C-EO-75(15) √ √ X N/A

C-OO-70(5) √ X N/A N/A

C-OO-70(10) √ √ √ √

C-OO-70(15) √ √ X N/A

C-OO-75(5) √ √ √ X

C-OO-75(10) √ √ √ √

C-OO-75(15) √ √ X N/A

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5.4.4 Physicochemical characterisation of nanoemulsions

The formulations that passed the thermal stability testing were further characterised for ZP, % DEE, pH and viscosity and results are presented in Table 5.4. High zeta values have been suggested as an indicator of the physical stability of NEs, as they can ensure the creation of a high energy barrier against coalescence of the inner phase droplets (Zhao et al., 2010). All formulations had high positive ZPs, ranging from 39.53 ± 1.21 mV to 47.16 ± 1.72 mV. This indicates a reasonable electrostatic repulsion between the droplets (Baspinar et al., 2010). The positive charge of NE formulations was likely due to the presence of water soluble, positively charged CHG. The % DEE of all the NEs formulations were C- EO-70(10), C-EO-75(10), C-OO-70(10) and C-OO-75(10) were 79.83 ± 2.28, 82.14 ± 1.93, 73.49 ± 2.06 and 78.19 ± 1.35 respectively. The % DEE for both EO and OO formulations were found to be almost similar with C-EO-75(10) having the highest entrapment efficiency. Table 5.4 Physicochemical characterisation of NE formulation (Mean ± SD, n = 3).

Formulation ZP (mV) % DEE pH Viscosity (cP)

C-EO-70(10) 47.16 ± 1.72 79.83 ± 2.28 5.78 23.08 ± 1.14

C-EO-75(10) 44.82 ± 0.91 82.14 ± 1.93 5.65 25.15 ± 0.94

C-OO-70(10) 39.53 ± 1.21 73.49 ± 2.06 5.41 29.31 ± 1.29

C-OO-75(10) 41.91 ± 1.23 78.19 ± 1.35 5.36 33.46 ± 1.73

The pH of skin ranges between 5 and 6, with 5.5 considered to be average pH of the skin (Ohman and Vahlquist, 1998). Therefore, formulations intended for application to skin should have a pH close to this range. The pH values for all the NEs formulations prepared were found to be in the range of 5.36 to 5.78, suitable for topical application.

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Viscosity of EO and OO reported in literature is 30 cP and 40 cP at 40°C respectively (Diamante and Lan, 2014; Tarabet et al., 2012). Viscosities of the both CHG-EO NEs and CHG-OO NEs were less than the respective oils due to the presence of water in formulations. Similar results were reported by Shakeel et al., (2015), using W/O EO-NEs, which showed a direct relationship between oil concentration and viscosities of NEs formulations.

5.4.5 Morphological study

The CHG-NEs were examined using TEM to observe the droplet shape and verify the droplets size determined by NTA. The droplets of the CHG-NEs appeared dark, and the surrounding liquid appeared bright, as shown in Figure 5.7. As seen in the displayed image, the observed droplets were spherical in shape, and ranged in size from 200 nm – 300 nm. The TEM images for all the formulated NEs were similar in shape, and the droplet size range was in agreement with the NTA results (Section 5.4.2.2).

Figure 5.7 TEM image of CHG-loaded NEs [C-EO-70(10)].

Li et al. (2015) reported a similar droplet shape for a chlorhexidine acetate NE designed to improve chlorhexidine solubility and to enhance its antimicrobial activity against Spreptococcus mutans in vitro and in vivo. They reported minimum inhibitory concentration

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(MIC) of a chlorhexidine acetate control solution was 0.8 µg/ml, which was two times higher than chlorhexidine acetate NEs (0.4 µg/ml). Also the NE formulations exhibited a fast-acting bactericidal activity against Spreptococcus mutans, causing over 95 % death within 5 min, compared to chlorhexidine acetate solution (73 %). These data showed the potential role of NE formulation to prevent bacterial infection during the wound healing process.

5.4.6 Fourier transform infrared spectrometry

The FTIR absorption spectra were obtained to study the drug and excipient interaction in the NE formulations. The FTIR spectra of CHG, EO and OO, CHG free formulations (blank EO-NEs and OO-NEs) and CHG-loaded NEs are shown in Figures 5.8 and Figure 5.9 respectively.

The FTIR spectrum of CHG had a broad symmetrical absorption peak between 3700–2700 cm-1, representing OH stretching due to the presence of water. Sharper amide bands were seen at 1650 cm-1 (C = O stretch), 1538 cm-1 (secondary N-H bend and C-N stretch) corresponding presence of CHG characteristic peaks. As seen in Figures 5.8 and 5.9, EO and OO had multiple absorption bands, which are described in detail in the previous chapter (Section 4.4.8).

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Figure 5.8 FTIR spectra for CHG, EO, Blank EO-NEs and CHG-loaded EO-NEs [C-EO-70(10), C-EO- 75(10)].

Figure 5.9 FTIR spectra for CHG, OO, Blank OO-NEs and CHG-loaded OO-NEs [C-OO-70(10), C-OO- 75(10)].

The FTIR spectra for both blank and CHG-loaded NEs of EO and OO (Figure 5.8 and Figure 5.9) had a broad symmetrical absorption peak between 3700–2700 cm-1, representing the OH stretching mode of water superimposed over the CHG bands. The

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broad peak seen between 1500 cm-1 and 1700 cm-1 in the blank and the CHG-loaded NEs formulations is due to the C=O double bond stretching vibration, resulting in superimposed peaks for CHG and EO and OO in this region (Kim et al., 2008). Results obtained from FTIR spectra does not show any new peaks or modification of existing peaks between prepared NEs and individual components of NE system, which represents no chemical interaction between drug and excipients during formulation.