(W1130-01-06) Development and Characterization of Ciprofloxacin Nanoemulsion-Loaded Bigels of Different Oleogel to Hydrogel Ratios: A Comparative Study

Purpose: To develop and evaluate ciprofloxacin nanoemulsion-loaded bigels prepared by incorporating ciprofloxacin hydrochloride nanoemulsion into two types of bigels, an oleogel-in-hydrogel and a hydrogel-in-oleogel at different ratios, to enhance drug penetration through the skin for topical treatment of bacterial infections.

Methods: Initially, an oil-in-water (O/W) ciprofloxacin hydrochloride nanoemulsion (CIP.HCl NE) was prepared by spontaneous emulsification technique. CIP.HCl NE was characterized for droplet size, ζ-potential, and polydispersity index (PDI) using a Zetasizer, droplet morphology using transmission electron microscopy (TEM), drug content using an HPLC analytical method, thermodynamic stability by subjecting CIP.HCl NE to centrifugation, six heating/cooling cycles, and three freezing/thawing cycles, and chemical stability under various storage conditions (refrigerator, room temperature, 30°C/65% relative humidity (RH), and 4°C/75% RH) for 3 months. Then, the two types of bigels (oleogel-in-hydrogel (OL-in-WH, Type I) and hydrogel-in-oleogel (WH-in-OL, Type II) were prepared by dispersing CIP.HCl NE into OL or WH phase. The OL was prepared using the oleogelator ethyl cellulose (EC) and WH was prepared using the hydrogelator Carbopol® 971P (CP). To prepare bigels, OL and WH were then be mixed at 1:1, 1:2, and 1:4 ratios. The bigels were investigated in terms of physical appearance , microstructural characterization using TEM and light-field microscopy, drug content using an HPLC analytical method, rheological properties using a controlled-stress rheometer, in vitro release using Franz diffusion cells, antimicrbial activity by well diffusion method and ex vivo skin infection model, rheological stability after 3 months of storage at room temperature, and a short-term physical and chemical stability under various storage conditions for 3 months.

Results: CIP.HCl NE was composed of oil phase (6.67% oleic acid), surfactant/cosurfactant (60% Labrasol®/propylene glycol at 1:1 ratio), and aqueous phase (33.3% acetic acid (5%), selected based on the maximum solubility of CIP.HCl and the clarity of CIP.HCl NE. The droplet size, PDI, and ζ-potential of CIP.HCl NE were 17.27±2.74 nm, 0.21±0.01, and 0.70±0.01 mV, respectively. TEM images displayed spherical droplets of CIP.HCl NE that were uniformly dispersed, showing no aggregation. The CIP.HCl content was 100.6±2.9%. CIP.HCl NE was physically stable showing no cracking or phase separation after passing centrifugation and heating/cooling (45/4°C) cycles. However, during the freezing/thawing cycles, CIP.HCl NE crystallized at -21ºC, however, crystallization decreased upon thawing at room temperature. The stored samples of CIP.HCl NE were chemically stable with CIP.HCl content ranging between 96.5 and 105.6%. The primary physical evaluation showed that Type I bigels (OL-in-WH) were physically stable, showing no phase separation when tested visually. Whereas Type II bigels (WH-in-OL) showed phase separation, hence they were excluded from the study. TEM images showed a highly structured bigel network formed from dense EC fibers of OL that were enclosed within the CP honeycomb-like structure of WH with nanoemulsion droplets dispersed within the bigel network. The images of light-field microscope showed blue-dark spheres of OL phase dispersed within the continuous WH phase, confirming Type I bigels. Additionally, the smaller blue-dark spherical droplets of CIP.HCl NE were dispersed within the bigel network. Bigels exhibited pseudoplastic flow and viscoelastic properties with elastic modulus (G’) dominating viscous modulus (G") that varied based on the OL:WH ratios. The cumulative amount of CIP.HCl release from bigels was between 568.1 and 603.2 µg/cm². The rate of CIP.HCl release from bigels varied based on the OL:WH ratio, attaining a complete release after 4-5 h. The CIP.HCl NE and bigels exhibited an effective antimicrobial activity against Gram-positive and Gram-negative bacteria. The zone of inhibition of bigels was dependent on the amount of CIP.HCl diffused from the bigel network, where bigels with high OL proportion showed relatively lower zones of inhibition compared to those of low OL proportion. Moreover, the ex vivo skin infection model provided a precise quantitative assessment of the antibacterial effectiveness of bigels. The rheological stability studies demonstrated that the viscosity and viscoelastic properties of bigels decreased after 3 months of storage at room temperature and the degree of reduction varied based on the composition of bigels. The physical stability studies demonstrated that bigels with 1:2 OL:WH ratio displayed phase separation, whereas those with1:4 OL:WH ratio remained stable for three months under various storage conditions. Chemically, bigel with 1:4 OL:WH ratio and CIP.HCl NE dispersed into WH phase showed the highest CIP.HCl content over time, ranging between 89.8 and 94.1%.

Conclusion: The comparative evaluation of bigels revealed that the physicochemical, microstructural, rheological, and antimicrobial properties, in vitro release, and stability of bigels varied based on the ratio of OL to WH, thus could be tailored by altering the OL:WH ratio. Therefore, the two drug delivery systems (O/W nanoemulsion and bigel) could potentially be used for the topical delivery of antimicrobial drugs to treat skin infections, where nanoemulsion retained the antimicrobial activity of the drug and bigel released it in a controlled-release manner.

Acknowledgements:This study was funded by Al-Zaytoonah University of Jordan, grants # ‏13/08/2021-2022 and 09/18/2018-2019. Additionally, authors would like to express gratitude to Mrs Duaa Qattan from Faculty of Medicine-University of Jordan for her assistance in TEM.