Abstract

Background: The active loading of drugs into liposomes was proven to be a very successful technique in drug loading to enhance the percentage of drug loaded. However, numerous knowledge gaps have been identified in active drug loading in niosomes. The purpose of this thesis is to investigate the effect of different cosurfactants (Solutol HS-15, Cremophor ELP, and Cremophor RH 40) on the niosomal membrane stability and their effect on active drug loading and niosomes' physicochemical properties.
Methods: A new technique to study pH gradient in nanovesicles was developed utilising the pH indicator bromocresol green (BCG). BCG was used as a probe to investigate the presence and stability of the pH gradient across the niosomal membrane, as it undergoes colour changes at different pH values. It helps studying the effects of different parameters on active drug loading via a pH gradient, including buffer types, surfactants, and co-surfactants. The optimised formulations were used to actively load doxorubicin using an ammonium sulfate pH gradient. Preliminary studies included the formulation of niosomes using the thin-film hydration technique. However, the microfluidic technique was also used to formulate optimised niosomes for active pH-gradient doxorubicin loading, as this has not been studied before.
Results: The results demonstrated that the compatibility between the main surfactant and the co-surfactant is critical for maintaining pH gradient stability. For instance, Span 65 showed greater compatibility with Cremophor RH 40, maintaining a pH gradient for more than 1 hour, compared to Solutol HS-15, which maintained a pH gradient for less than 15 minutes. In contrast, Span 60 exhibited the opposite behaviour, maintaining a longer pH gradient with Solutol HS 15 than with Cremophor RH 40. Additional factors influencing the entrapment efficiency of actively loaded drugs and the size of niosomes included the buffer system used, the vesicle size and morphology, the temperature during drug loading, and the cholesterol content of the formulation.
Among the formulations tested, Span 65 niosomes exhibited smaller vesicle size and greater stability than Span 60 niosomes. Doxorubicin was successfully loaded into Span 65–Cremophor RH 40 niosomes with high entrapment efficiency using the pH gradient method, reaching 78.79 % EE, and achieved a size of 237.8 nm after sonication when 5% of Cremophor RH 40 was included as the co-surfactant and 76.99% EE and 191.9 nm after sonication when 10% of Cremophor RH 40 was included as the co-surfactant
with size distribution PDI for both formulations was 0.27 and 0.28 respectively. Given the promising performance of this formulation, a microfluidic technique was employed to prepare it, yielding niosomes with smaller sizes and a more controlled size distribution. The microfluidic approach produced Span 65 niosomes with controlled size and narrow size distribution when 0.3 mg/mL of doxorubicin was loaded (~ 108 nm, PDI = 0.12) and 0.5mg/mL of doxorubicin was loaded (~ 140 nm, PDI = 0.11) while maintaining high entrapment efficiency (~ 70% in both drug loading concentrations) through active drug loading, resulting in a stable niosomal formulation (no significant size change over 3 months period with both drug loading concentrations.