Selenium nanoparticles as antibacterial agents for potential application in chronic wound healing

Abstract

Chronic wounds have become a global problem. The importance of microbial colonies in delaying chronic wound healing has been highlighted recently. Keeping the wound free of infection plays a vital role in fast and successful wound healing. The rapid development of antibiotic resistance and the inability of antibiotics to penetrate biofilms seriously limit the efficacy of antibiotics. Therefore, new effective antimicrobial treatments are urgently needed to realize rapid and successful chronic wound healing. One promising candidate to address this requirement is selenium nanoparticles (Se NPs) which have antimicrobial activity. In this thesis, we were working on developing new antibacterial Se NPs, and adopting 3D printing technology to fabricate latticed wound dressings with a controlled release of Se NPs.

First, the influence of size on the antibacterial activity and cytotoxicity of Se NPs was investigated. In Chapter 3, spherical Se NPs ranging from 43 to 205 nm in diameter were fabricated, and their mammalian cytotoxicity and antibacterial activity as a function of their size were systematically studied. The antibacterial activity of the Se NPs was shown to be strongly size dependent, with 81 nm Se NPs showing the maximal growth inhibition and killing effect of methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MSSA and MRSA). The Se NPs were shown to have multi-modal mechanisms of action that depended on their size, including depleting internal adenosine triphosphate (ATP), inducing reactive oxygen species (ROS) production, and disrupting membrane potential. All the Se NPs were non-toxic towards mammalian cells up to 25 microgram/mL. Furthermore, the minimum inhibitory concentration (MIC) for the 81 nm particles produced in this research against MSSA is 16 +/- 7 microgram/mL, significantly lower than previously reported MIC values for Se NPs. This data illustrates that Se NP size is a facile yet critical and previously underappreciated parameter that can be tailored for maximal antimicrobial efficacy. We have identified that using Se NPs with a size of 81 nm and concentration of 10 microgram/mL shows promise as a safe and efficient way to kill S. aureus without damaging mammalian cells.

Second, the effect of charge on the antibacterial activity of Se NPs was researched. It has been shown previously that Se NPs with a net negative surface charge have good antibacterial activity against Gram-positive bacteria but are less effective against Gram-negative bacteria. Gram-negative bacteria have been observed to be more sensitive to positively charged nanoparticles because the surface charge of Gram-negative bacteria is generally more negative than that of Gram-positive bacteria. Therefore, in Chapter 4, Se NPs were capped with a positively charged protein – recombinant spider silk protein eADF4 (kappa 16) – to give them a net positive charge. Compared to the negatively charged polyvinyl alcohol (PVA) capped Se NPs, the positively charged eADF4 (kappa 16) coated Se NPs demonstrated a much higher bactericidal efficacy against the Gram-negative bacteria E. coli in water. Particularly, the minimum bactericidal concentration (MBC) of 46 nm eADF4 (kappa 16) capped Se NPs (8 +/- 1 microgram/mL) was approximately 50 times lower than the 46 nm PVA capped Se NPs (405 +/- 80 microgram/mL). Scanning electron microscopy (SEM) images showed that the PVA capped Se NPs were repelled by the E. coli cells, while the eADF4 (kappa 16) capped Se NPs attached to or even coated the E. coli cells. In addition, antibacterial films were created by immobilising the eADF4 (kappa 16) capped Se NPs on positively charged spider silk and these were shown to retain good bactericidal efficacy and overcome the issue of low particle stability in culture broth. It was found that these Se NPs needed to be released from the film surface in order to exert their antibacterial effects and this can be achieved by regulating the surface charge of the film. Overall, eADF4 (kappa 16) coated Se NPs are promising new antibacterial agents against life-threatening bacteria.

Third, the optimal size of Se NPs combined with positive surface charge was fabricated to realize a high antibacterial efficacy. In Chapter 5, 81 nm Se NPs combined with epsilon-poly-L-lysine (Se NP-epsilon-PL) were fabricated and their antibacterial activity and cytotoxicity were investigated. Se NP-epsilon-PL exhibited effective antibacterial activities against all tested 8 different species of bacteria including both Gram-positive and Gram-negative bacteria, and some of them are drug-resistant bacteria types. Bacteria were found to be very difficult to develop resistance to Se NP-epsilon-PL comparing to the conventional antibiotic kanamycin. S. aureus and E. coli started to develop resistance to kanamycin from 44 and 52 generations, respectively. By contrast, S. aureus started to develop resistance to Se NP-epsilon-PL after 132 generations, and E. coli failed to develop resistance to Se NP-epsilon-PL during the whole tested 312 generations. On the other hand, Se NP-epsilon-PL showed low cytotoxicity to human dermal fibroblasts. Based on its efficient and wide-spectrum antibacterial activity, the difficulty to develop resistance in bacteria and its low cytotoxicity, Se NP-epsilon-PL might become a promising member of the new generation of antibacterial agents.

Finally, in Chapter 6, alginate dressings with pH-responsive release of Se NPs were fabricated using 3D printing technology. Although the antibacterial activity of Se NPs has been proved, relatively high concentrations of Se NPs are toxic to the mammalian cells. It has been reported that the pH of body fluid in bacteria infected wounds is higher than that in normal skin. To enable faster release of Se NPs at a relatively high pH to perform higher bactericidal efficacy and slower release of Se NPs at a relatively low pH to protect the normal cells, alginate wound dressings with pH-responsive release of Se NPs were 3D printed using a Bioplotter. Calcium phosphate nanoparticles (CaP NPs) were introduced into alginate dressings to make their degradation pH-responsive, resulting in pH-responsive release of Se NPs from these dressings. The dressings’ mechanical properties, degradation rates and releasing rates of Se NPs were investigated. The results showed that the addition of CaP NPs can increase both tensile strength and elongation of alginate dressings and make the degradation rate of alginate dressings faster at a relatively high pH than that at a relatively low pH. Similar to these degradation results, when Se NPs have been introduced, the release rate of Se NPs from the scaffolds at a relatively high pH also showed faster than that at a relatively low pH.

In conclusion, the potential of using Se NPs as antibacterial agents has been investigated, and 3D printed alginate dressings with pH-responsive release of Se NPs have been developed.