The diffusive and abusive use of antibiotics have accelerated the evolution of multidrug-resistant microbes, while the development of new antibiotics is lagging behind as many big pharmaceutical companies shut down their antibiotic research department due to insufficient profit margin. Infectious disease has become a major threat to human society again. Membrane-active antibacterial nanomaterials, or nanoantibiotics, are attractive alternatives when conventional antibiotics fail, because their different antibacterial mechanism could potentially evade the pathways of antibiotic resistance.1, 2 Various membrane-active nanoantibiotics have been reported in recent years,3, 4 the roles of nanostructures, however, remain elusive.
In our work, we developed nanoparticle-pinched polymer brushes (NPPBs) to dissect the antibiotic role of nanostructures.5 NPPBs consists of chemically inert silica nanospheres of systematically varied diameters (7-270 nm) covalently grafted with hydrophilic polymer brushes that are non-toxic and non-bactericidal. Assembly of the hydrophilic polymers into nanostructured NPPBs doesn’t alter their compatibility with mammalian cells, but it incurs a transformation of their antimicrobial potential against bacteria that depends critically on the nanoparticle sizes. This study illuminates nanoengineering as a viable approach to develop nanoantibiotics that kill bacteria upon contact yet remain nontoxic when engulfed by mammalian cells.
To isolate the roles of nanostructure, model nanoparticles need to be built with bioinert materials. Silica nanoparticle is known for its biocompatibility. On the other hand, we have previously demonstrated that hydrophilic polymer poly(4-vinyl-Nmethylpyridine iodide) (P4MVP) is neither toxic to human cells nor active to bacteria when its degree of polymerization (DP) is small (e.g., ~30).6 We therefore designed model NPPBs consisting of inert silica nanospheres of systematically varied diameters (7-270nm) grafted with P4MVP with a similar DP around 30. NPPBs were first demonstrated to have low cytotoxicity regardless of sizes (Figure 1c). Interestingly, they exhibited antibacterial activity in a size-dependent manner: while the large NPPBs with size ＞50 nm show low activity to both Gram-negative and Gram-positive bacteria (Figure 1e), the small NPPBs with size ≤ 50 nm become highly active to bacteria (Figure 1d).
To understand this size-dependent antibacterial activity, we utilized a variety of biophysical methods to characterize the interaction between NPPBs and bacteria or mammalian cells. For example, when NPPBs encounter lipid vesicles with membrane composition mimicking either the bacterial or mammalian membrane, we found that NPPBs are selectively destructive to bacteria-mimicking lipid vesicles and the small ones (d ≤ 50 nm) are more destructive than the large ones (d > 50 nm). In another example, we directly visualized the surface and cross-sectional morphology of Gram-negative E. coli after treatment with NPPBs. Clearly, the surface morphology of E. coli treated with small NPPBs (d = 25 nm) was completely destroyed and many nanoparticles penetrated inside of bacteria (Figure 1f and g). In contrast, when E. coli cells were treated with large NPPBs (d = 270 nm), their surface and cross-sectional morphologies were nearly intact (Figure 1h and i). A similar case was also found for Gram-positive S. aureus. One possible reason for this size-dependent activity is because of the nanoporous peptidoglycan cell wall covering the inner membrane, which precludes the large nanoparticles from gaining access to bacterial inner membrane.6 To further unveil the size-dependent antibacterial activity against Gram-negative bacteria, we characterized the self-assembled structures of NPPBs with bacteria-mimicking lipid vesicles. The results showed that while large NPPBs (d > 50 nm) induce the membrane to form a bicontinous cubic phase (Figure 1l), the small NPPBs (d ≤ 50 nm) induce the membrane to form a porous hexagonal phase (Figure 1j and k). The different membrane remodeling capability gives rise to the size-dependent antibacterial activity.
Figure 1. Hydrophilic nanoparticles that kill bacteria while sparing mammalian cells reveal the antibiotic role of nanostructures. Model NPPBs with core sizes ranging from 7 nm (a) to 270 nm (b) were synthesized and they have low cytotoxicity to HEK cells (c). However, they exhibited size-dependent activity to E. coli with the small ones (d ≤ 50 nm) being highly active (d) and the large ones (d > 50 nm) remain inactive (e). The biophysical studies revealed that the small NPPBs (f and g) are more membrane destructive and penetrative than the large ones (h and j). Further mechanistic studies revealed the insight of the interaction between bacteria-mimicking membrane with NPPBs: while the large ones induce the membrane to form a bicontinuous cubic phase (l), the small ones induce the membrane to form a porous inverted hexagonal phase (j and k) (i.e., formation of membrane pores). Refer to the original article for more details.
The major discovery of this study includes: 1) the difference in lipid composition between bacterial and mammalian membrane determines the selectivity of NPPBs toward bacterial cells; 2) the different sizes of NPPBs give rise to their different membrane remodeling capability against bacterial cells, and the existence of nanoporous peptidoglycan cell wall further contributes to the size-dependent antibacterial activity; 3) when nanostructures come into play, hydrophilic non-toxic and antimicrobially-inactive polymers can be transformed into potent bacteria killers without compromising their biocompatibility with mammalian cells.
This work has uncovered many other interesting findings beyond what have been briefly mentioned here. If interested, please refer to the original article for more details: Jiang, Y. Zheng, W., Tran, K. Kamilar, E., Bariwal, J., Ma, H., Liang, H. Hydrophilic nanoparticles that kill bacteria while sparing mammalian cells reveal the antibiotic role of nanostructures. Nat Commun 13, 197 (2022). https://doi.org/10.1038/s41467-021-27193-9
- Pelgrift, R.Y. & Friedman, A.J. Nanotechnology as a therapeutic tool to combat microbial resistance. Adv Drug Deliver Rev 65, 1803-1815 (2013).
- Huh, A.J. & Kwon, Y.J. "Nanoantibiotics": A new paradigm for treating infectious diseases using nanomaterials in the antibiotics resistant era. J Control Release 156, 128-145 (2011).
- Lam, S.J., Wong, E.H.H., Boyer, C. & Qiao, G.G. Antimicrobial polymeric nanoparticles. Prog Polym Sci 76, 40-64 (2018).
- Beyth, N., Houri-Haddad, Y., Domb, A., Khan, W. & Hazan, R. Alternative Antimicrobial Approach: Nano-Antimicrobial Materials. Evid-Based Compl Alt 2015 (2015).
- Jiang, Y. Zheng, W., Tran, K. Kamilar, E., Bariwal, J., Ma, H., Liang, H. Hydrophilic nanoparticles that kill bacteria while sparing mammalian cells reveal the antibiotic role of nanostructures. Nat Commun 13, 197 (2022).
- Jiang, Y., Zheng, W., Kuang, L., Ma, H. & Liang, H. Hydrophilic Phage-Mimicking Membrane Active Antimicrobials Reveal Nanostructure-Dependent Activity and Selectivity. Acs Infect Dis 3, 676-687 (2017).