Periodic Reporting for period 1 - BiofilmEradicate (Modification of liposomic nano-carriers: a novel strategy for improved drug-delivery and eradication of bacterial biofilms)
Berichtszeitraum: 2022-10-01 bis 2024-03-31
The problem we have been aiming to solve or, at the least, greatly alleviate, concerns the strong resistance to drug delivery, and hence to treatment and cure (eradication), of bacterial biofilm infections, which impose a large economic and health burden. This is done by exploiting a recent discovery concerning the properties of certain functionalized liposomes, i.e. vesicles whose membranes are lipid bilayers.
Pathogenic bacteria in the form of microbial biofilms, which form both at infections in vivo and on devices in contact with living tissue, are structured as communities which encase themselves in a dense hydrated matrix of polysaccharide and protein. This matrix strongly suppresses penetration of antibiotics to the bacteria, rendering them highly resistant to drug treatment. This resistance, and the limited success in overcoming it to date, constitutes the major objectives which this project addresses.
Because of their resistance to existing antibiotic-delivery systems, treatments available for wound infections are poorly efficient and very side-effects prone. There is therefore an urgent need for vehicles which enable efficient delivery of the drugs directly to the bacterial pathogens at the infected sites. In this context, lipid-bilayer vesicles, or liposomes, in particular have been proposed as suitable drug delivery nanovehicles, not least for bacterial infections, with their versatility arising from biocompatibility, ability to incorporate both water-loving and water-hating compounds (in contrast to micellar or polymer nanoparticle vehicles where such duality is considerably more challenging), and protection of their cargo prior to delivery. Such liposomic vehicles are most often functionalized by poly(ethylene glycol) moieties (PEG, the current gold-standard)) both for colloidal stability and, in the case of IV administration, to increase their blood circulation time through steric suppression of protein adsorption. Such PEGylation, however, is itself associated with significant problems, greatly reducing their efficiency for biofilm treatment.
Our solution to the above problem of suppressed drug delivery to bacterial biofilms is to implement a new functionalization strategy for liposomic drug carriers, by replacing the generally-used PEG moieties on liposomes with novel (IP-protected) biocompatible moieties, provides colloidal stability to the liposomal dispersions comparable to PEGylation, with extended shelf-life (more than one year). Crucially, however, it is expected to provide significant advantages relative to PEGylation for drug delivery to the bacterial cells in biofilms. This arises from the specific structure of the functionalizing groups on the liposomes, which . There is also independent evidence that novel functionalized lipid vesicles we use do not adsorb plasma proteins and so are likely not to readily trigger an immune response, resulting in long circulation time.
In this way the project creates novel drug-delivery vehicles based on drug-encapsulating functionalized liposomes, which can be used to treat drug-penetration-resistant bacterial biofilm infections far more efficiently than existing drug-delivery solutions, leading to their eradication. The implications of this are that a major health burden affecting millions, with large economic costs will be strongly alleviated.
Pathogenic bacteria in the form of microbial biofilms, which form both at infections in vivo and on devices in contact with living tissue, are structured as communities which encase themselves in a dense hydrated matrix of polysaccharide and protein. This matrix strongly suppresses penetration of antibiotics to the bacteria, rendering them highly resistant to drug treatment. This resistance, and the limited success in overcoming it to date, constitutes the major objectives which this project addresses.
Because of their resistance to existing antibiotic-delivery systems, treatments available for wound infections are poorly efficient and very side-effects prone. There is therefore an urgent need for vehicles which enable efficient delivery of the drugs directly to the bacterial pathogens at the infected sites. In this context, lipid-bilayer vesicles, or liposomes, in particular have been proposed as suitable drug delivery nanovehicles, not least for bacterial infections, with their versatility arising from biocompatibility, ability to incorporate both water-loving and water-hating compounds (in contrast to micellar or polymer nanoparticle vehicles where such duality is considerably more challenging), and protection of their cargo prior to delivery. Such liposomic vehicles are most often functionalized by poly(ethylene glycol) moieties (PEG, the current gold-standard)) both for colloidal stability and, in the case of IV administration, to increase their blood circulation time through steric suppression of protein adsorption. Such PEGylation, however, is itself associated with significant problems, greatly reducing their efficiency for biofilm treatment.
Our solution to the above problem of suppressed drug delivery to bacterial biofilms is to implement a new functionalization strategy for liposomic drug carriers, by replacing the generally-used PEG moieties on liposomes with novel (IP-protected) biocompatible moieties, provides colloidal stability to the liposomal dispersions comparable to PEGylation, with extended shelf-life (more than one year). Crucially, however, it is expected to provide significant advantages relative to PEGylation for drug delivery to the bacterial cells in biofilms. This arises from the specific structure of the functionalizing groups on the liposomes, which . There is also independent evidence that novel functionalized lipid vesicles we use do not adsorb plasma proteins and so are likely not to readily trigger an immune response, resulting in long circulation time.
In this way the project creates novel drug-delivery vehicles based on drug-encapsulating functionalized liposomes, which can be used to treat drug-penetration-resistant bacterial biofilm infections far more efficiently than existing drug-delivery solutions, leading to their eradication. The implications of this are that a major health burden affecting millions, with large economic costs will be strongly alleviated.
We assessed the best conditions for stronger interaction between the functionalized liposomic carriers and cell membranes, and confirmed that such affinity results in a more efficient delivery. We quantified the release of the liposomal luminal cargo in bacteria cells, using a range of parameters, varying the concentration and degree of polymerization of the functionalizing moieties in the vesicle membrane, and vesicle size. We examined a particular model system to compare payload release between PEGylated and our functionalized liposomes, as well as bare liposomes (no surface functionalization), and show that our functioalization significantly improves payload release compared to PEGylated liposomes. We also visualized this using superresolution microscopy to complement our fluorescence assay. These studies shed light on which main factors contribute to the mechanism of interaction and how to tune the functionalization of our liposomic carriers to achieve the highest uptake efficiency.
We examined how to optimize encapsulation of antimicrobial drugs into our new liposomal system through evaluation of its stability. Initially we loaded a clinically used antimicrobial agent (Sulfamethoxazole) within the vectors and verified its long shelf-life stability and retention time at long-term storage conditions. We also examined the release of the drug in-vitro under physiological conditions to determine the delivery kinetics. The measured parameters (encapsulation efficiency, release kinetics, shelf-life) were compared to results obtained with a similar antibiotic using PEGylated liposomic carriers. We also developed loading strategies for two agents to be loaded simultaneously inside a single carrier: this pointed to synergistic interaction that may help to overcome drug resistance. These different carrier configurations were then applied to examine eradication of biofilms of different bacterial strains.
We investigated the in vitro efficiency of our antimicrobial-loaded liposomes in delivering their content to bacteria and eradicating biofilms. As a model system, we employed Pseudomonas aeruginosa, as well as the clinical strain of cystic fibrosis - LESB58, to probe the breadth and potential of our novel functionalization in addressing several typologies of infections. We quantified the efficiency of eradication based on a high throughput screening assay, complemented with a fluorescence assay using confocal microscopy to quantify the fraction of dead bacteria within biofilms upon treatment.
In the final part of the project we examined its extension to a murine model to apply the in vitro ideas to a living organism. We used hairless mice and carried out an extensive toxicity study using differing doses of our novel functionalized carriers and comparison with PEGylated carriers. In particular our results demonstrated complete safety, and advanced us considerably towards treatment of biofilms on wound infections. Feedback from this was used to optimize dual-agent loading for additional bacterial eradication studies. In particular we evaluated the optimal carriers’ configurations achieving the highest biofilm treatment potential and with the best biosafety profile.
We examined how to optimize encapsulation of antimicrobial drugs into our new liposomal system through evaluation of its stability. Initially we loaded a clinically used antimicrobial agent (Sulfamethoxazole) within the vectors and verified its long shelf-life stability and retention time at long-term storage conditions. We also examined the release of the drug in-vitro under physiological conditions to determine the delivery kinetics. The measured parameters (encapsulation efficiency, release kinetics, shelf-life) were compared to results obtained with a similar antibiotic using PEGylated liposomic carriers. We also developed loading strategies for two agents to be loaded simultaneously inside a single carrier: this pointed to synergistic interaction that may help to overcome drug resistance. These different carrier configurations were then applied to examine eradication of biofilms of different bacterial strains.
We investigated the in vitro efficiency of our antimicrobial-loaded liposomes in delivering their content to bacteria and eradicating biofilms. As a model system, we employed Pseudomonas aeruginosa, as well as the clinical strain of cystic fibrosis - LESB58, to probe the breadth and potential of our novel functionalization in addressing several typologies of infections. We quantified the efficiency of eradication based on a high throughput screening assay, complemented with a fluorescence assay using confocal microscopy to quantify the fraction of dead bacteria within biofilms upon treatment.
In the final part of the project we examined its extension to a murine model to apply the in vitro ideas to a living organism. We used hairless mice and carried out an extensive toxicity study using differing doses of our novel functionalized carriers and comparison with PEGylated carriers. In particular our results demonstrated complete safety, and advanced us considerably towards treatment of biofilms on wound infections. Feedback from this was used to optimize dual-agent loading for additional bacterial eradication studies. In particular we evaluated the optimal carriers’ configurations achieving the highest biofilm treatment potential and with the best biosafety profile.
The potential of our results for better treatment of biofilms is clear, and some of the results contributed to a patent application on the used of our functionalized vesicles as novel drug carriers for biofilm treatment. For further success, our preliminary studies on a murine model need to be extended, while suitable licensing of our patent and further development and trials by commercial entities will ensure it further development and bringing to market