Determination of encapsulation efficiency

The unbound CUR from CUR-CS-PEC-NPs pellet collected by centrifugation as described in section 2.2.2.2 was washed off twice in methanol. The amount of CUR in the methanol rinse and the supernatant were both analysed to determine the total unbound CUR using the HPLC method described in section 3.2.3. For the supernatant and rinse, 20 µl was injected directly onto the HPLC. The reported values are the means of three independent runs. The percentage of EE% was calculated as follows:

% EE% =Total CUR added−unbound CUR

Total CUR added X 100% Eq. 3.1 3.2.6 CUR release from CUR-CS-PEC-NPs

3.2.6.1 CUR release from CUR-CS-PEC-NPs in different simulated fluids

The variation in pH within the GIT and the effects of colonic enzymatic activity on the integrity of NPs were studied. For this purpose, CUR-CS-PEC-NPs were collected by centrifugation as described in section 2.2.2.2 and washed. A 20 mg/ml suspension of CUR-CS-PEC-NPs were suspended in phosphate buffer saline

114 (pH 6.4) containing 1% (w/v) tween 80 and 2.5% (w/v) pectinase enzyme as simulated colonic content. Similarly, CUR-CS-PEC-NPs were suspended in 0.1 N HCl (pH 1.2) and HEPES buffer (pH 6.8) mimicking the stomach and small intestine mediums, respectively (Dutta & Sahu, 2012; Saboktakin et al., 2011; Beaulieu et al., 2002; Luo et al., 2012; Chen & Subirade, 2005; Jain et al., 2006). Each of the three types of the fluids containing CUR-CS-PEC-NPs were seeded into eight sampling vials and subjected to rotary shaking (WiseCube®, Witeg Inc., Germany) at 180 rpm incubated at 37^oC. CUR release was studied over 6 hr at 20 min., 40 min., 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, and 6 hr by withdrawing one vial from each of the media and its content centrifuged at 4000 rpm for 10 min to pellet the particles. The amount of CUR released was determined in the supernatant using the HPLC analytical procedure described in section 3.2.3 in three independent runs. The percentage of CUR released was calculated from the calibration curve described in section 3.2.4 and calculated as follows:

% released CUR = Amount of released CUR

Amount of CUR initially added X 100% Eq. 3.2 To study the protective effects of PEC on the CUR-CS-PEC-NPs, PEC-free NPs were prepared as described in section 2.2.2.2 (i.e. without the addition of PEC). CUR-CS-PEC-NPs and PEC-free NPs were suspended in 0.1 N HCl (pH 1.2) for 1 hr, removed and air-dried followed by viewing under FESEM as described in section 2.2.4.

Since the CUR-CS-PEC-NPs are designed to transit the upper GIT followed by exposure in the alkaline conditions of the distal GIT, the effect that this variable pH might have on the physical integrity of the CUR-CS-PEC-NPs was studied by suspending the NPs in pH 1.2 for one hr, retrieving by centrifugation and then re-exposure in pH 6.8 for 2 hr. The zeta potential values and percentage retention of CUR were determined as described in sections 2.2.3 and 3.2.5, respectively.

115 3.2.6.2 CUR release from CUR-CS-PEC-NPs in simulated gastrointestinal tract

fluids

CUR-CS-PEC-NPs are designed to transit the upper GIT followed by the alkaline conditions of the distal GIT. Thus, the effects of such pH variation and enzymatic activity on CUR release from the CUR-CS-PEC-NPs were studied. Firstly, the collected CUR-CS-PEC-NPs were suspended in simulated gastric fluid (SGF) (0.1 N HCl, pH 1.2, containing pepsin at concentration of 0.1% (w/v)) for 1 hr. Then, CUR-CS-PEC-NPs were retrieved and suspended in simulated intestinal fluid (SIF) (HEPES buffer (pH 6.8) containing pancreatine and sodium deoxycholate at concentrations of 1% and 0.8% (w/v), respectively), for two hr. Finally, CUR-CS-PEC-NPs were retrieved and suspended in simulated colonic fluid (SCF) (PBS (pH 6.4) containing 1%

(w/v) tween 80 and 2.5% (w/v) pectinase enzyme) (Dutta & Sahu, 2012; Saboktakin et al., 2011; Beaulieu et al., 2002; Luo et al., 2012; Chen & Subirade, 2005; Jain et al., 2006). Figure 3.1 illustrates the aforementioned process. At designated time intervals (20 min., 40 min., 1 hr, 2 hr, 4 hr, and 6 hr) the amount of CUR released was determined in the supernatant using the HPLC method described in section 3.2.3 in three independent runs and reported as the mean of these runs. The physical integrity of CUR-CS-PEC-NPs were investigated by studying the morphological changes on them at 30 min, 2.5 hr, and 6 hr using the SEM imaging as described in section 2.2.4.

116 Figure 3.1 CUR release at simulated GIT fluids (adopted with modification

from www.jocuri-fotbal.info) 3.2.7 Stability Studies

3.2.7.1 Storage

The effects of storage on the physical integrity of CUR-CS-PEC-NPs was conducted at 4^oC for 14 days. Physical characterization of CUR-CS-PEC-NPs including particle size, zeta potential, and SEM imaging were conducted as described in sections 2.2.3 and 2.2.4, respectively. Studies were conducted on day 0, 7, and 14.

3.2.7.2 Photosensitivity

CUR is photosensitive, therefore, CS-PEC matrix must offer necessary protection to encapsulated CUR from the degradative effects of light. This was studied after exposing equivalent concentrations of CUR encapsulated in CUR-CS-PEC-NPs and free CUR within transparent vials to sunlight and UV light (263 nm) for 6 hr. At predetermined time intervals (2, 4, and 6 hr) CUR-CS-PEC-NPs were collected by centrifugation. CUR was retrieved by hydrolysing the CUR-CS-PEC-NPs in methanol with vortex mixing for 1 minute followed by filtration through 0.22 µm syringe filter.

117 The filtered solution was then run using the HPLC method described in section 3.2.3.

The reported values are the means of three independent runs. The percentage of retained CUR was calculated as follows:

% CUR retained = Amount of CUR determined from analysis

Amount of added CUR X100% Eq. 3.3 3.2.7.3 Thermal

To investigate the extent of protection offered by the CS-PEC matrix to CUR against hydrolysis induced by thermal exposure as is likely under physiological conditions, (37^oC), equivalent concentrations of CUR encapsulated in CUR-CS-PEC-NPs and free CUR were exposed to 37^oC for three days. At day 1, 2, and 3, samples were sacrificed and CUR-CS-PEC-NPs were collected by centrifugation at 4000 rpm for 10 min. and then hydrolysed by vortex shaking the samples in methanol. This was further centrifuged at 4000 rpm for 10 min and the supernatant filtered using a 0.22 µm syringe. CUR was quantified using the HPLC method described in section 3.2.4 and the % of retrieved CUR was calculated using Eq. 3.3.

3.3 Results and Discussion