Formulation of pH-Responsive Quatsomes from Quaternary Bicephalic Surfactants and Cholesterol for Enhanced Delivery of Vancomycin against Methicillin Resistant Staphylococcus aureus

Globally, human beings continue to be at high risk of infectious diseases caused by methicillin-resistant Staphylococcus aureus (MRSA); and current treatments are being depleted due to antimicrobial resistance. Therefore, the synthesis and formulation of novel materials is essential for combating antimicrobial resistance. The study aimed to synthesize a quaternary bicephalic surfactant (StBAclm) and thereof to formulate pH-responsive vancomycin (VCM)-loaded quatsomes to enhance the activity of the antibiotic against MRSA. The surfactant structure was confirmed using 1H, 13C nuclear magnetic resonance (NMR), Fourier-transform infrared spectroscopy (FT-IR), and high-resolution mass spectrometry (HRMS). The quatsomes were prepared using a sonication/dispersion method and were characterized using various in vitro, in vivo, and in silico techniques. The in vitro cell biocompatibility studies of the surfactant and pH-responsive vancomycin-loaded quatsomes (VCM-StBAclm-Qt1) revealed that they are biosafe. The prepared quatsomes had a mean hydrodynamic diameter (MHD), polydispersity index (PDI), and drug encapsulation efficiency (DEE) of 122.9 ± 3.78 nm, 0.169 ± 0.02 mV, and 52.22 ± 8.4%, respectively, with surface charge switching from negative to positive at pH 7.4 and pH 6.0, respectively. High-resolution transmission electron microscopy (HR-TEM) characterization of the quatsomes showed spherical vesicles with MHD similar to the one obtained from the zeta-sizer. The in vitro drug release of VCM from the quatsomes was faster at pH 6.0 compared to pH 7.4. The minimum inhibitory concentration (MIC) of the drug loaded quatsomes against MRSA was 32-fold and 8-fold lower at pH 6.0 and pH 7.4, respectively, compared to bare VCM, demonstrating the pH-responsiveness of the quatsomes and the enhanced activity of VCM at acidic pH. The drug-loaded quatsomes demonstrated higher electrical conductivity and a decrease in protein and deoxyribonucleic acid (DNA) concentrations as compared to the bare drug. This confirmed greater MRSA membrane damage, compared to treatment with bare VCM. The flow cytometry study showed that the drug-loaded quatsomes had a similar bactericidal killing effect on MRSA despite a lower (8-fold) VCM concentration when compared to the bare VCM. Fluorescence microscopy revealed the ability of the drug-loaded quatsomes to eradicate MRSA biofilms. The in vivo studies in a skin infection mice model showed that groups treated with VCM-loaded quatsomes had a 13-fold decrease in MRSA CFUs when compared to the bare VCM treated groups. This study confirmed the potential of pH-responsive VCM-StBAclm quatsomes as an effective delivery system for targeted delivery and for enhancing the activity of antibiotics.


S1. Synthesis of Compound 3 (di-tert-butyl 3,3'-(octadecylazanediyl)dipropionate
Compound 3 was synthesized following a reported procedure [1]. In brief, tert-butyl acrylate (3.5 g; 27.3 mmol) dissolved in methanol (30 mL) and the solution was added to stearyl amine in methanol (3.4 g; 12.6 mmol) (20 mL). The mixture was stirred and refluxed at 80 °C for 24 h and the progress of the reaction was monitored via thin layer chromatography. The crude product was washed with chloroform several times to remove excess tert-butyl acrylate (tBA) and the solvent and tBA were removed in vacuo. The crude product was purified via column chromatography (silica, mesh size 60-100) ( Figure S1-S4).

S4. Thermal profile using Differential scanning calorimetry (DSC)
The thermographs for quatsomes and their constituents were shown in Figure S13 were determined by DSC (Shimadzu DSC-60, Tokyo Japan). VCM, StBAclm, CHol, physical mixture (VCM, StBAclm and CHol) and the lyophilised VCM-StBAclm-Qt1 quatsomes thermoprofiles displayed their distinctive endothermic melting points at 126.97 °C, 170.05 °C, 155.68 °C, and (129.49 °C, 151.18 °C and 199.08 °C) respectively. In the physical mixture, showed the respective peaks of all the ingredients used to formulate the quatsome. In the lyophilised formulation, the thermal peaks of the VCM disappeared, which could indicate the encapsulation of VCM. Furthermore, such disappearance of the VCM peaks might be due to the conversion of the crystalline form of VCM molecules into an amorphous form.

S5. Drug entrapment efficiency (DEE %) and drug loading capacity (DLC%)
The VCM-StBAclm-Qt quatsomes were further characterized to determine DEE% and DLC%. The DEE% was determined by an ultrafiltration method, as previously reported [4,5]. Briefly, exactly, 2 mL of the drug-loaded quatsomes was loaded into the Amicon® Ultra-4 centrifugal filter tubes (Millipore Corp., USA) having 10 kDa pore size and centrifuged at 3000 rpm for exactly 30 min at 25 °C. The amount of the unentrapped drug in the filtrate was determined spectrophotometrically using Shimadzu UV 1601, Japan at 280 nm. The regression equation of the calibration curve (y = 0.0038x − 0.0031), with a linear regression coefficient (R²) of 0.9998, was used to determine the unknown drug concentration values. The experiment was done in triplicate, and the DEE% was calculated using the equation below. The DLC% was determined by freeze-drying the quatsomes, and the weight of VCM was calculated using the Equation (1) and Equation (2)

S6. Fractional inhibitory concentration (FIC)
The combined antimicrobial effect of blank quatsome and VCM in the VCM StBAclm-Qt quatsomes against MRSA were assessed using the FIC method [6]. The method is based on the Loewe additivity zero-interaction theory. The theory states that a drug cannot interact with itself and if the effect of combinations are synergistic less of the drug is needed for activity. On the other hand, when antagonism occurs, more of the drug would be needed to produce the same effect as the drug alone. The FIC index was calculated using the Equation (3)

S7. Downstream physiological phenomena: electrical conductivity quantification
1% MRSA was inoculated in 10 mL MHB with bacteria (control), bare VCM and VCM-StBAclm-Qt quatsomes (100 µg/mL) and cultured for 16-hours. 3 mL of the cultured samples were centrifuged at 4000 rpm for 15-min and the supernatant was collected in order to determine its electrical conductivity [7]. The electrical conductivity was determined using an electrical conductivity meter (OHAUS USA) at pH 7.4 and calculated using the equation below (n = 3). Where Q refers to conductivity change rate, Qs and Qc are the conductivity of the test groups and the control group respectively (Equation (4)).