Abstract
A persistent challenge in drug development lies in the disparity between in vitro therapeutic efficacy and in vivo targeted delivery, accentuating the importance of advanced drug delivery systems. Phospholipids, which are fundamental to the formation of cellular membranes and numerous biological processes essential for life, have garnered prominence in biomedical applications through their repurposing for therapeutic delivery enhancement and pathological symptom mitigation. Liposomal nanocarriers, self-assembled phospholipid vesicles, have emerged as a central example in this field. This thesis systematically explores strategies to optimise liposomal drug nanocarrier performance across two objectives: (1) engineering stimuli-responsive release mechanisms, and (2) optimising antimicrobial delivery to combat antibiotic resistance.
Liposomal encapsulation of the dopaminergic agonist apomorphine, used in the treatment of Parkinson’s disease, has been previously observed to induce intraliposomal drug precipitation, presenting as amorphous side-pockets associated with the liposome’s phospholipid bilayer. To better understand the preparation conditions favouring formation of these morphologies over conventional spherical vesicles, a systematic investigation of formulation variables was conducted. Optimisation studies revealed that cholesterol and phospholipid ratios modulated both drug loading efficiency and liposome morphology. Alterations to the transmembrane salt gradient, necessary for the active drug loading process, identified 500 mM ammonium sulphate as optimal for side-pocket formation, though other ammonium salts exhibited comparable effects on drug precipitation.
Prior studies have demonstrated that apomorphine-containing liposomes with drug side-pockets exhibit enhanced ultrasound-triggered drug release and increased optical density at wavelengths >600 nm compared to spherical counterparts. Building on these findings, this optical property was leveraged to assess the influence of side-pocket architecture on drug release sensitivity under 808 nm near-infrared (NIR) irradiation. The results confirmed the potential of these structures for photothermal activation. To further enhance release of the liposome payload in response to NIR laser irradiation, an aza-BODIPY (aza-borondipyrromethene) derivative optimised for photothermal conversion was synthesised and incorporated into liposomes through two strategies: direct integration into the lipid bilayer or conjugation to bovine serum albumin prior to encapsulation. Combining the aza-BODIPY dye with apomorphine side-pockets enabled efficient NIR-triggered payload release, achieving >90% release upon prolonged irradiation with minimal passive leakage (<5% over 24 hours).
The second objective of this work focused on antimicrobial delivery applications. Drug encapsulation within liposomes has been previously employed to address the challenge of poor drug solubility. This principle was applied to the effectively aqueous insoluble oxazolidinone antibiotic tedizolid. An optimised liposome formulation achieved intra-liposomal drug concentrations of 0.9 mg/mL, representing a 10-fold increase over the aqueous solubility limit of the non-derivatised tedizolid, while simultaneously reducing residual solubilisation excipients by 50% compared to conventional formulations.
While liposomal stability and circulation half-life in vivo are well characterised, their response to hydrolytic enzymes (host- or pathogen-derived) remains poorly understood. To investigate this, liposomes containing the fluorescent dye 5(6)-carboxyfluorescein (CF) were incubated with enzymes to determine their release kinetics. Pancreatic sPLA2 exhibited a five-fold greater activity against liposomes comprising natural enantiomer diacyl phospholipid compared to unnatural diacyl and diether counterparts. To measure the response to pathogen derived enzymes, extracellular bacterial lipases isolated from Staphylococcus aureus were incubated with liposomes. Exposure to these enzymes demonstrated significantly enhanced payload release from liposomes, despite minimal activity against free long-chain fatty acid substrates in solution.
To further develop this concept of controlled release in the presence of hydrolytic enzymes excreted by pathogens, liposomes incorporating the ionisable lipid ALC-0315, used in the Pfizer COMIRNATY® mRNA COVID-19 vaccine to improve transfection through enhanced endosomal escape, were formulated. The efficacy of liposomal antibiotic was compared to free antibiotic in inhibiting the growth of both methicillin susceptible and methicillin resistant strains of S. aureus (SA and MRSA, respectively). Incorporation of the narrow spectrum antibiotic penicillin G within conventional or ALC-0315 containing liposomes had no negative effect on the minimum inhibitory concentration (MIC) compared to free penicillin G against susceptible strains. Interestingly, encapsulation within liposomes also appeared to decrease the antibiotic concentration required to inhibit the growth of MRSA.
This work aimed to advance the rational design of liposomal systems, offering insights into stimuli-responsive formulations as well as antimicrobial encapsulation and delivery optimisation. The findings underscore the potential of engineered liposomes to overcome critical barriers in controlled drug delivery and antimicrobial therapy.