(A-7) Nitric Oxide releasing N-acetylcysteine Decorated Hyperbranched Polyethyleneimine brush interface for Anti-infective Biomedical Applications.

Introduction:: Healthcare associated infections (HAI) are a major public health concern that has been exacerbated during the current pandemic. In the United States alone, HAIs cause around 90,000 deaths and cost 28 - 45 billion USD annually. The treatment of these infections is managed by administration of antibiotics, which has contributed to the development of antibiotic resistance in bacteria. Biomedical device infections are characterized and worsen by the formation of biofilms, which consist of a consortium of bacterial cells embedded within a protective extracellular polymeric matrix. The formation of biofilms presents a challenge in the eradication of bacteria from medical device surfaces as it enables evasion from antibiotics and the immune system. To address this issue, research efforts have been directed towards developing surfaces that possess inherent resistance against infection and biofilm formation. The objective of this project is to create an anti-biofilm surface that can effectively prevent biofilm formation upon infection.

We designed a poly-vinyl chloride (PVC) surface with anti-bacterial molecule polyethyleneimine (PEI) and N-acetyl cysteine (NAC) and combined it with Nitric oxide (NO) technology. PEI is a polycationic molecule that possesses remarkable anti-biofilm properties, while NAC is a mucolytic agent that also exhibits anti-bacterial capabilities. NO, a small molecule gasotransmitter that is naturally produced in endothelial cells, is a broad-spectrum antibacterial agent with a short half-life and multi-mechanistic bacterial killing abilities, which helps prevent the development of bacterial resistance.


Materials and Methods:: In this study, we produced PVC films that were mixed with a NO donor molecule known as S-nitroso-N-acetylpenicillamine (SNAP) and a plasticizer called Triethylhexyl trimellitate (TOTM). We then covalently linked PEI with PVC and subsequently conjugated NAC using EDC-NHS coupling. To evaluate the physical and mechanical characteristics of the biomaterial, we utilized FTIR, water contact angle, and tensile testing techniques.

NO release was assessed using a chemiluminescence based NO analyzer and diffusion of NO donor over a period of 2 weeks. We further evaluated the cytocompatibility of the biomaterial with human fibroblast and endothelial cells. The effectiveness of the films in preventing bacterial growth was evaluated by subjecting them to two medically significant strains, namely S. aureus and E. coli, for a period of 24 hours. The number of bacteria that adhered to the film and those that remained in a planktonic state were recorded. Moreover, the films were examined for their ability to prevent the formation of biofilms, and the number of bacteria that adhered to the film was quantified after 48 hours.


Results, Conclusions, and Discussions:: Results:

Our results demonstrated successful attachment of PEI and NAC on PVC surface. The novel biomaterial had mechanical strength comparable to commercial medical grade PVC tubing. The films released physiologically relevant levels of NO (4 to 0.5 ´ 10^-10 mol cm^-2 min^-1) for at least 7 days followed by a lower release for another week indicating their applicability for long term usage. Moreover, the films significantly decreased both adhered ( >95% reduction) and planktonic ( >95% reduction) bacterial load compared to control for both the strains tested. Furthermore, the films exhibited remarkable efficacy against biofilm formation for a minimum of 48 hours, resulting in an approximately 80% reduction in E. coli and a nearly 95% reduction in S. aureus. Additionally, the films demonstrated cytocompatibility, with human fibroblast and endothelial cells exhibiting over 70% relative viability after a 24-hour exposure.

Conclusion:

In summary, we have successfully created a unique biomaterial that exhibits inherent resistance to biofilm formation and possesses the capability to eradicate any bacteria that may attach to its surface. This biomaterial utilizes a multi-faceted approach to bacterial elimination, suggesting its potential to combat antibiotic-resistant bacteria and prevent biofilm formation. The biomaterial fabricated is also compatible with relevant cell types, an essential criteria for clinical usage. Upon conducting in vivo testing in animal models, this material can be presented as an excellent candidate for clinical application.


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