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Bacterial membrane vesicles a novel delivery system for the treatment of multi-drug resistant Gram-negative bacterial infections.

Periodic Reporting for period 1 - BacDrug (Bacterial membrane vesicles a novel delivery system for the treatment of multi-drug resistant Gram-negative bacterial infections.)

Reporting period: 2020-03-01 to 2022-02-28

One of the milestones in medicine was the discovery of antibiotics which prevent/treat bacterial infections and by this saved millions of lives. Conventional systemic antibiotic therapy is often limited by instability of the drug, cytotoxicity and negative effects on the patient’s microbiota. Additionally the global spread of antimicrobial resistant (AMR) bacteria, such as Pseudomonas aeruginosa and Staphylococcus aureus, combined with a dearth of R&D of novel antibiotics are a significant public health challenge and alternative treatment strategies are urgently needed. The overall goal of this Marie Skłodowaska-Curie Fellowship was to develop a drug delivery platform to overcome the limitation of conventional antibiotic therapy and tackle AMR. In this project multilayered particles (based on capsosomes) were engineered with a high carry capacity for lipid-based vesicles. This vesicles can be loaded with single drugs or offer the benefit of delivering drug combinations. In this project lipid-based vesicles mimicking human cells were used to implement a bacterial toxin triggered release mechanism. As a proof-of-concept single (vancomycin) or dual loaded (vancomycin and antimicrobial peptide) capsosomes were developed and their activity towards the multi-drug resistant methicillin-resistant Staphylococcus aureus (MRSA) was tested. The capsosomes engineered in this project released their cargo only in the presence of toxin producing MRSA and exhibited excellent antibacterial activity in vitro and in vivo. Additionally, the delivery of dual drugs resulted in an enhanced killing effect even at lower antibiotic concentrations. Overall, the results provided in this project have made significant progress towards a drug delivery system for multi-drug resistant bacteria with the potential to tackle one of the biggest health care crises of our day.

The success of this ambitious project was highly dependent on the placement within the world-renowned Stevens Group and the close collaboration with the Edwards Group at Imperial College London. The interdisciplinary expertise of these groups was the ideal host for this project and critical for the highly promising outcomes of this work. As a result of this fellowship an ongoing close collaboration between the Stevens Group and the Edwards Group was established which is the steppingstone to further develop this drug delivery system for clinical applications and tailor it towards other critical pathogens.
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).
The multidisciplinary nature of this fellowship has advanced the state-of -the-art across several fields including antibiotic delivery systems, drug development and bioengineered particles. In this project I developed a multilayered drug delivery platform with a bacterial toxin triggered cargo release mechanism. Due to its individual compartments this system offers a straightforward way to encapsulate and deliver single and/or multiple antibiotics. The multilayered nature of this system and the possibility to use natural and synthetic vesicles makes this approach inherently tuneable as a drug delivery system. The flexibility in cargo loading, release mechanism and delivery routes enabling researcher to use this system not only as an antibacterial drug delivery system but also offers the possibility for other applications. The implementation of a bacterial toxin triggered release mechanism is the foundation of a control release mechanism, in ongoing work with the Stevens Group other avenues are explored including fusogenic particles and enzyme triggered release. The control antibiotic release at the side of infection would have tremendous benefits for the treatment of bacterial infection by reducing the negative side effects of drugs and protecting the patient’s microbiota. This work could have several positive impacts on society and economy, the developed drug delivery system has potential to improve the treatment of MRSA infections as well as its flexible design offers the possibility to tailor it towards the most dangerous pathogens.
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