Date of Completion

2024

Document Type

Thesis

Degree Name

Bachelor of Science in Pharmacy

Keywords

Antimicrobial photodynamic combination therapy, aPDT, aloe-emodin solid lipid nanoparticles, sodium thiocyanate, Methicillin-resistant Staphylococcus aureus, reactive oxygen species

Abstract

Antibacterial activity of aloe-emodin (AE) was enhanced by adding NaSCN and exposing this combination to 488-nm light. AE/NaSCN in antimicrobial photodynamic combination therapy showed potential in treating drug-susceptible staphylococcal infections. However, its use in clinical settings is limited by suboptimal aqueous solubility of AE. This study aimed to synthesize AE solid lipid nanoparticles (AE-SLNs) with enhanced aqueous solubility and photodynamic bacterial inhibition against methicillin-resistant Staphylococcus aureus (MRSA) when used in combination with NaSCN. AE-SLNs were prepared by combining AE, glyceryl monostearate (GMS, 10 mg/mL), and Tween 80 (2:5 w/w surfactant-lipid ratio) in a pre-emulsion, which was homogenized by bath ultrasonication (BU) or high-speed stirring (HSS). AE-SLNs were collected and oven-dried at 40-degree Celsius for 48 hours. Drug-entrapment efficiency (%DEE) was determined colorimetrically to assess the effectiveness of AE-encapsulation in lipid matrix. Particle size, distribution, and dispersion stability of AE-SLNs were evaluated using dynamic light scattering, SEM, and zeta-potential determination. Photodynamic antibacterial activity of AE-SLNs (16.9–270.4 µg/mL) in combination with NaSCN (5.1 µg/mL) was determined by exposing cultures containing clinically-isolated MRSA (1.5E+8 cfu/mL) to 488-nm LED light (20 mW/cm^2) for 10 min at 1-cm distance (12.2 J/cm^2). Dark-treated control groups were similarly prepared, but were not exposed to LED light. Aliquots from light-exposed and dark-treated wells were transferred to mannitol-salt agar in 24-well plates and incubated overnight at 37-degree Celsius. Log reduction and time-kill kinetics were determined by colony-counting of treated and untreated groups using ImageJ software. ROS generation was indirectly quantified by using colorimetric cyanide and sulfite assays. %DEE of AE-SLNs was determined 86.5% in BU and 70.6% in HSS (p>0.05). AE-SLNs had an average particle size ranging from 605–660 nm, and showed remarkable solubility compared to AE. Zeta potential (–29.16±4.63 mV) revealed AE-SLNs forming stable dispersions. Mean log reduction of light-exposed AE-SLNs was increased by 22x when combined with NaSCN. Light-exposed AE-SLNs/NaSCN showed 91.38% inhibition against MRSA at 270.24 µg/mL, and 61x reduction in MRSA colonies compared with dark-treated AE-SLNs/NaSCN. There is a positive correlation between the concentration of AE-SLNs with constant amount of NaSCN and sulfite and cyanide concentrations, indicating increase in ROS generated after irradiation. AE-SLNs significantly enhanced the photodynamic antibacterial activity against MRSA when combined with NaSCN and exposed to 488-nm light, offering a promising approach to overcoming AE’s solubility limitations in clinical applications.

First Advisor

SIGFREDO B. MATA, RPh

Download

Share

COinS