Design and Evaluation of Nanoparticle Systems for Bacterial Targeting

The rise of antimicrobial resistance poses a major threat to modern medicine, presenting an urgent need for new treatment strategies beyond traditional antibiotics. Nanoparticles (NPs) have emerged as promising tools for antimicrobial applications due to their potential for high drug-loading capacity, tuneable surface chemistry, and target specificity. However, their interactions with the diverse and complex structures of bacterial surfaces remain poorly understood, limiting their progress toward clinical translation. Thus, this thesis investigates the design of two NP systems: 1) cationic fusogenic liposomes (cFLs) and 2) metal-organic frameworks (MOFs) for bacterial targeting, with a focus on evaluating their molecular interactions with bacterial envelopes using super-resolution microscopy. cFLs, lipid-based NPs engineered to fuse with cellular membranes, have been previously studied for their ability to deliver antimicrobial agents across the permeability barrier of the bacterial outer membrane (OM). In contrast, MOFs are solid NPs characterised by their high drug-loading capacity and reactive surfaces, making them promising candidates for antimicrobial delivery applications. We observed heterogeneous interactions of NPs with bacteria. In Chapter 4, we first confirmed the previously reported fusion of cFLs with the OM of Gram-negative bacteria. Surprisingly, however, we revealed previously unknown interactions with Gram-positive bacteria, which exhibited lipid uptake rather than envelope fusion. The different observed interaction phenomena can be rationalised due to the distinct outer envelope structures presented by these two groups of bacteria, with the lipid-based OM presented by Gram-negative cells and a thick and rigid sugar-peptide construct (the peptidoglycan) exposed by Gram-positive cells. Furthermore, we found that cFL fusion with the OM of Gram-negative cells compromised the cells’ membrane integrity leading to reduced bacterial viability even in the absence of antibiotics. This result indicates that the integration of artificial lipids impaired the OM’s biophysical properties which limits bacterial survival. However, as presented in Chapter 5, the cationic charge of cFLs, essential for bacterial targeting (with bacteria presenting negative surface charges), also caused bacterial aggregation through electrostatic interactions, highlighting the challenge of using non-specific targeting mechanisms. While reducing the cationic content lowered aggregation, it also decreased fusogenicity confirming that the cationic charge is necessary to drive fusion with the OM. The introduction of a DNA-based targeting moiety, designed to promote specific bacterial targeting, facilitated liposome attachment to the cell surface but did not induce membrane fusion, likely due to steric hindrance or insufficient membrane contact. In addition to the efforts on tuning the cFL composition, the lipid-NP interactions were studied with bacteria of varying lipopolysaccharide (LPS)1 complexity and growth stages, as highlighted in Chapter 6. Super-resolution microscopy revealed that the cFLs used for this study demonstrated fusion with all cells, independent of OM complexity. However, exponentially grown Gram-negative bacteria showed lipid uptake at high cFL concentrations. Whereas, stationary-phase bacteria primarily exhibited membrane fusion with the exception of Gram-negative cells lacking the steric oligosaccharide barrier (part of the OM), which also indicated lipid uptake in the stationary phase. These results suggest a higher susceptibility of bacteria in an exponential growth phase and with reduced LPS complexity. In Chapter 7, the focus shifts to MOFs. Unlike cFLs, MOFs can only attach to bacterial surfaces rather than fuse with membranes. The model MOF used in this study, PCN-222, is strongly positively charged, complicating post-synthesis functionalization with a DNA-based targeting assembly that we designed to specifically target bacteria. To address the nonspecific interactions, the surface of PCN-222 was functionalized with peptide nucleic acid (PNA), a DNA mimic with a neutral backbone used to reduce charge-driven binding. PNA-functionalized MOFs demonstrated improved attachment specificity and allowed for further functionalization with the DNA-linked targeting moiety carrying wheat germ agglutinin (WGA), a protein that binds to N-acetyl-D-glucosamine of the LPS. However, drug-loaded PNA-coated MOFs did not significantly affect bacterial growth potentially due to the slow release profile of the MOF system being used. In summary, the work presented here highlights the complexity of designing NPs for bacterial targeting and the need for a deeper understanding of NP-bacteria interactions at the molecular level. With the use of super-resolution microscopy, the dynamic and static interactions of NPs with bacteria have been studied, revealing new interaction phenomena (e.g. lipid uptake by Gram-negative cells) and complications (e.g. bacterial clumping through non-specific interactions) that would have remained undiscovered through imaging with lower resolution systems and bulk measurement techniques. Therefore, this thesis provides valuable insights into the design, performance, and existing challenges of cFLs and PCN-222 MOFs for bacterial targeting. Further research is needed to improve our current understanding of NP interactions with bacterial targets to design more effective treatment strategies. 1 Component of the OM with an oligosaccharide chain attached to a lipid base.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2025

Author dc:creator

• Scheeder, Anna

Advisor dc:contributor.advisor

• Kaminski, Clemens

• Nanoparticle
• Bacteria
• Super-resolution Microscopy

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/fff6c1c5-845f-4ec4-9c37-cba5d09ada33/download
• http://purl.org/NET/rdflicense/allrightsreserved

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.119631

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/386404