The following points summarises the research tasks and results from the BacDrug project:
1. Lipid-based vesicles required for the encapsulation of antimicrobial agents were prepared and characterised. To harvest naturally derived vesicles several different bacterial growth conditions were tested to maximise bacterial membrane vesicle (BMV) production. Purified BMVs were studied with TEM, Cryo-TEM and DLS to verify vesicle formation. Synthetic vesicles (liposomes) were prepared using thin-film hydration technology and multiple lipid formulations were tested to identify the ideal composition to implement a controlled cargo release mechanism.
2. Optimisation of drug loading into liposomes and BMVs was performed in several stages throughout the funding period to overcome challenges encountered with BMVs active and passive loading. For the passive loading of BMVs a mutant library including several bacterial strains carrying different constructs to express antibacterial enzymes was generated and confirmed by sequencing. In the case of passively loading BMVs several techniques were used including extrusion, freeze-thaw, and ultrasound. To overcome low loading efficiency of BMVs multilayered particle were engineered to localise BMVs or liposomes around a core particle. Liposomes or BMVs were either loaded with dyes to characterise assembly or with different antimicrobial agents.
3. The layer-by-layer technique was used to assemble multilayered particles composed of alternating layers of lipid-based vesicles and polymers around a core particle. For the lipid-based layers either synthetic liposomes or naturally derived BMVs were used. For characterisation studies liposomes were labelled with dyes and the successful assembly was confirmed using fluorescence microscopy and flow cytometry. Particles with one, two, three and four layers of liposomes were achieved.
4. Controlled drug release was introduced by developing liposomes that mimic human cells which are lysed by bacterial toxins. Selective cargo release from all liposome layers was demonstrated only in the present of toxin producing MRSA.
5. For a proof-of-concept study capsosomes delivering either one antibiotic (vancomycin) or two (vancomycin and antimicrobial peptide) were prepared and the antibacterial activity was tested towards MRSA. Incubation of bacteria with either single or dual loaded capsosomes demonstrated significant reduction of bacterial numbers in vitro with an improved effect seen for drug delivery systems with dual antimicrobial agents. Additionally, antibacterial activity was also proven in an in vivo fly model resulting in an increased survival of infected flies compared to empty capsosomes.
The key findings from the BacDrug project were disseminated by presenting at conferences including GW4 AMR Symposium as well as CMBI centre wide seminars. The work of the multilayered drug delivery system engineered in this fellowship has led to a manuscript accepted in Advanced Healthcare Materials. In all cases the Marie Skłodowaska-Curie Action Fellowship and the European Commission were acknowledged as funding body. Despite the difficulties caused by the pandemic I participated during the funding period in multiple outreach activities. I participated in sessions such as ‘Meet a Scientist’ (online) and organised events like ‘Bugs and More’ (in-person).
