(i) Paint Composites, containing Lytic Enzyme-Nanotube Bioconjugate, for Biological Decontamination
Figure1. Lysostaphin-based paint composites that kill MRSA on contact.
Pathogen–mediated infectious diseases constitute an emerging societal problem. Various approaches are being explored to design coatings that prevent pathogens from proliferating on common surfaces such as hospital equipment, surgical suits etc. In our recent article in ACS Nano, we have demonstrated the effective use of carbon nanotube–enzyme conjugates in preparing antimicrobial coatings that are highly effective against antibiotic–resistant Staphylococcus aureus. Specifically, nanotube conjugates of lysostaphin (a cell lytic enzyme effective against Staphylococci) have been incorporated into nanocomposite films to impart antimicrobial properties (Figure 1). We probed into effect of PEG linker in enhancing the activity of enzyme when attached to nanosupport. We reasoned that by adding a small, hydrophilic and inert linker between the enzyme and the nanotube, the enzyme will have better mobility and hence accessibility to its target, i.e. bacterial cell. Indeed, we have found that presence of linker leads to ca. 2-fold increase in rate of bacterial killing compared to directly attached enzyme. What makes such enzyme-based formulations interesting and unique is their ability to target specific pathogens and not affect irrelevant microflora. Along those lines, we have demonstrated species-specific activity of lysostaphin against S. aureus and Staphylococcus epidermidis, and ineffectiveness against Escherichia coli. Additionally, we have assessed effectiveness of these coatings against four different methicillin–resistant Staphylococcus aureus strains that are responsible for hospital- and community-acquired infections (Figure 2). The use of nanotubes as enzyme support was well justified based on the experimental results that showed little or no release of bactericidal conjugates. The feasibility of using these composites for practical purposes was demonstrated by showing their reusable and stable nature when tested under different storage conditions for up to 6 months. In addition to enzymes showing bactericidal activity, we also prepared films containing proteases that can prohibit microbial adhesion and proliferation on common surfaces.
Figure 2. Antistaphylococcal activity lysostaphin-CNT-paint films against four different strains of methicillin-resistant Staphylococcus aureus (MRSA). The plot shows % viability in case of lysostaphin-containing films normalized to that obtained for control paint samples.
Currently, we are exploring general applicability of the above approach against different bacterial and hard-to-kill spore targets. Protein engineering, domain-swapping and controlled protein orientation is being employed in generating a general reactive engineered enzyme-based neutralization (GREEN) surface that is effective against biological agents.
(ii) Perhydrolase-based polymer/paint composites
Peracetic acid (PAA) is a potent oxidant that exhibits excellent and rapid disinfection activity against a broad spectrum of pathogens and is more effective than hydrogen peroxide. Commercial PAA is generally produced by reacting acetic acid with H2O2 using sulfuric acid as the catalyst. However, this reaction is slow (requiring up to several days to yield high amount of PAA), and moreover, residual levels of acetic acid, H2O2, and corrosive sulfuric acid in the product are high.
As an alternative to chemical synthesis, several biocatalytic routes have been devised, one of which involves use of perhydrolases. In our work, in collaboration with Prof. Ravi Kane, we used a perhydrolase, namely AcT (from Mycobacterium smegmatis,), a homo-octamer of 184 kDa with 72 x 72 x 60 Ao dimensions to catalyze the perhydrolysis of propylene glycol diacetate (PGD) to generate peracetic acid (PAA) (Scheme 1).2,3
Scheme 1. AcT-catalyzed perhydrolysis of propylene glycol diacetate (PGD) to generate peracetic acid (PAA).
We have exploited the interaction of AcT with multi-walled carbon nanotubes (MWNTs) to produce bioactive composites that allow efficient incorporation of the enzyme into polymeric coatings and paint (Figure 1). From two conjugate formulations tested, the one with PEG linker showed enhanced activity making it a better choice for incorporation in paint films. AcT–carbon nanotube conjugates were incorporated into polymeric and latex-based paints to yield active and stable composites. The paint films were capable of killing >98% of the spores within 15 min, initially challenged at 106 CFU/ml (2x109/m2), and essentially 100% killing was observed after 30 min (Figure 2). Despite a 10-fold drop in H2O2 concentration, >98% sporicidal activity against B. cereus spores was achieved within 30 min (Figure 3).
Figure 1. Direct covalent attachment of AcT onto MWNTs (a) and by using a PEG linker (b).
Figure 2. Sporicidal activity of AcT-PEG12-MWNT conjugate based paint films (%, w/w of AcT in paints) against Bacillus cereus spores. Experimental conditions: B. cereus spores, 106 CFU/ml; [PGD], 100 mM; [H2O2], 100 mM.
Figure 3. Sporicidal activity of AcT-PEG12-MWNT conjugate based paint films (%, w/w of AcT in paints) against B. cereus spores. Experimental conditions: B. cereus spores, 106 CFU/ml; [PGD], 100 mM; [H2O2], 10 mM.
The incorporation of AcT-MWNT conjugates into polymers and paints is the first step in the preparation of bioactive composites with enhanced strength and extended lifetime. These composites may be applied as decontaminating coatings on surfaces in hospitals, kitchens, and bathrooms, where effective killing of a variety of infectious organisms is critical.
(iii) Laccase-based polymer/paint composites
Iodine is well known as an efficient microbicide, and is commonly used in households and hospitals. It has a wide range of activity against principal pathogens, including enteric bacteria and viruses. Iodine may also be effective against spores. We are using laccase to continuously generate iodine through the simultaneous catalytic reduction of oxygen to water, and the oxidation of iodide to iodine (Scheme 2). We have exploited the interaction between laccase and MWNTs to produce bioactive composites that allow efficient incorporation of the enzyme into polymeric coatings and paint. The I2 generated is effective against E. coli, S. aureus, and Bacillus spores, and our results demonstrate that latex paints containing laccase-MWNT conjugates can kill more than 90% of spores initially charged at 104 CFU/mL in 3h (Figure 4).
Scheme 2. The generation of an antimicrobial agent, I2, using laccase as a catalyst and methyl syringate (MS) as a mediator.
Figure 4. Laccase-mediated killing of (a) E. coli and S. aureus, and (b) B. cereus spores through the generation of iodine from paint films containing laccase-MWNT conjugates.
(iv) Organophosphorus hysdrolase (OPH)
Enzyme-based composites that rapidly and effectively detoxify simulants of V- and G-type chemical warfare nerve agents have been developed in our group (Scheme 3)4. Our approach is based on the efﬁcient immobilization of organophosphorus hydrolase (OPH) onto carbon nanotubes to form active and stable conjugates that can be easily entrapped within commercially available paints, which do not leach out of the paint. These catalytic-based composites have been found to decontaminate more than 99% of 10 g/m2 of paraoxon, a simulant of the V-type nerve agent, in 30 min (Figure 5a), and more than 95% decontamination of diisopropylﬂuorophosphate (DPF), a simulant of G-type nerve agent, in 45 min (Figure 5b). These formulations are expected to be environmentally friendly and offer an easy to use, on demand, decontamination alternative to chemical approaches for sustainable material self-decontamination.
Scheme 3. Mechanism of (a) paraoxon and (b) DFP decontamination by OPH.
Figure 5. OPH-mediated decontamination of (a) paraoxon and (b) DFP using paint films containing OPH-MWCNT conjugates.
- R.C. Pangule, S.J. Brooks, C.Z. Dinu, G. Zhu, S.S. Bale, S. Salmon, D.W. Metzger, R.S. Kane, and J.S. Dordick (2010), “Antistaphylococcal Nanocomposite Films Based on Enzyme−Nanotube Conjugates”, ACS Nano 4, 3993-4000. (Selected News Highlights; Thomson Reuters, ‘Bacteria-killing Paint’, August 2010; Wall Street Journal, ‘Swatting Superbugs in Hospitals, Homes’, September 2010; RPI, ‘Coatings that Safely Kill MRSA on Contact’, August 2010).
- C.Z. Dinu, G. Zhu, S.S. Bale, G. Anand, P.J. Reeder, K. Sanford, G. Whited, R.S. Kane, and J.S. Dordick (2009), “Enzyme-Based Nanoscale Composites for Use as Active Decontamination Surfaces“, Adv. Funct. Mater. 20, 392-398.
- C.Z. Dinu, I.V. Borkar, S.S. Bale, G. Zhu, K. Sanford, G. Whited, R.S. Kane, and J.S. Dordick, “Enzyme-Nanotube Based Composites Used for Chemical and Biological Decontamination”, Green Polymer Chemistry: Biocatalysis and Biomaterials, ACS Symp. Ser. 2010, pp. 103-107.
- I.V. Borkar, C.Z. Dinu, G. Zhu, R.S. Kane, and J.S. Dordick (2010), “Bionanoconjugate-Based Composites for Decontamination of Nerve Agents”, Biotechnol. Prog. 26, 1622-1628.