Taking on microbial resistance
Anna Demming reports on efforts to harness nanoparticles as alternative bacteria-killing agents
Alexander Fleming’s 1928 isolation of penicillin was a step change in the fight against infectious disease. In just a few short years after the discovery, medical science progressed from folkloric preparations of medicinal moulds to a barrage of purified drugs, many of which are still in use today. Yet as bacterial strains evolve resistance to antibiotics, fatal infectious diseases are now showing signs of re-emerging, with microbial infection predicted to become a bigger killer than cancer by 2050.
One of the more promising strategies for fending off this microbial Armageddon involves the increased use of nanotechnology, either instead of traditional antibiotics or in conjunction with them. Today’s widespread use of antibiotics can be traced to 19th-century studies of fungal and bacterial colonies, which led to the idea that the antagonistic substances released by competing micro-organisms could be harnessed to defend humans too. However, silver also has antimicrobial effects that have long been exploited, from its use in cutlery and tableware to its inclusion in wound dressings. Despite this long history, we are only now coming to understand the mechanism behind the antimicrobial activities of silver and other types of nanoparticles.
Enhancing the alternatives
In 2004 an influential report by Miguel Jose Yacaman and colleagues in the journal Nanotechnology concluded that the bactericidal effects of silver nanoparticles depended on the sizes of the particles. Working at the University of Texas at Austin in the US and Centro de Investigaciones y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN) in Mexico, the researchers investigated the antimicrobial activity of silver nanoparticles between 1 and 100 nm in size and found that only those under 10 nm presented a direct preferential interaction with the bacteria.
This result was groundbreaking at the time, but later studies suggested a more nuanced picture. In particular, a 2012 report by Pedro J J Alvarez and colleagues at Rice University in the US indicated that silver’s bactericidal activity stems from the presence of silver ions as opposed to the nanoparticles themselves. Their work suggests that apparent size and morphological dependencies are, in fact, due to the higher surface areas of small particles (relative to their volume) giving rise to higher ion concentrations. Building on this work, other researchers found that the antibiotic properties of silver extend not only to the silver nanoparticles themselves, but even to bacteria that have died from silver exposure. This “zombie effect”, as the ingested silver in dead bacteria proves fatal to living bacteria that come into contact with it, was first documented in Scientific Reports in 2015.
Silver is far from the only nanoparticle in the arsenal against harmful micro-organisms. Plants, too, have defence mechanisms to combat microbial infections. A plant’s primary metabolism caters for all the organism’s basic needs, including growth and seed production. But alongside this, secondary metabolic processes are also at play, interacting with other organisms and the outside world by, for example, encouraging pollinators or deterring pathogens. The small molecules produced in these secondary metabolic processes, and used by the plant to deter pathogens, might seem the perfect alternative to the antibiotic substances produced by competing micro-organisms. Discouragingly, however, at dosage levels that avoid adverse side effects, the antibiotic activity of naturally occurring plant secondary metabolites (PSMs) is insufficient to combat disease.
Researchers have tried several approaches to enhancing the microbicidal activity of PSMs. One notable strategy is that adopted by Kateryna Bazaka and colleagues in Australia, Singapore and the US, who reported in 2017 that plasma treatments are an effective way of enhancing PSM antibiotic activity. This work was significant because whereas other successful strategies tend to be both complex and specific to a particular PSM, the plasma treatment is generic – meaning that it could potentially be applied to various other substances, providing a greatly extended range of alternatives to traditional antibiotics. In fact, the plasma-enhanced effects are so generic that even water and PSMs with no or negligible antimicrobial activity can be made to demonstrate a degree of hostility to micro-organisms. “When this plasma-activated water was used in conjunction with other drugs, the treatment outcome was enhanced, even though in those experiments the drugs were not treated by plasma directly,” Bazaka explains.
While further work is required to determine why plasma treatments are so effective, the researchers have hypothesized that the enhanced biological activity stems from chemically active oxygenated derivatives. In the meantime, the plasma device used in the study has already been certified in Europe for use by medical and veterinary practitioners in the treatment of wounds, bringing the approach closer to implementation for fighting disease.
Besides offering alternatives to antibiotic drugs, nanostructures have also demonstrated an ability to clamp down on the main microbial cell-defence mechanism against antibiotics: the so-called “efflux pump” that bacteria use to flush out drugs (see figure). Nanomaterials tend to lyse, or break, cell membranes indiscriminately, and combining this with antibiotics’ more targeted methods of destruction makes it possible to get past the efflux pump defence. “We realized this was a way to resurrect antibiotics that bacteria have become resistant to,” says Vincent Rotello, a materials scientist at the University of Massachusetts at Amherst in the US who has led research into the symbiotic effect of delivering gold nanoparticles with antibiotics.
Rotello’s team functionalized gold nanoparticles with hydrophobic ligands that lock in with the hydrophobic cell membrane, thereby maximizing the nanoparticles’ effect. The researchers’ nanoparticles also repress other transporter proteins crucial for the survival of the bacteria, further aiding the effectiveness of the antibiotics. Ultimately, they demonstrated that, when combined with the functionalized gold nanoparticles, the minimum concentration of the drugs levofloxacin and ciprofloxacin needed to inhibit E. coli fell by a factor of 16. As Rotello points out, this equates to the difference between “eating [drugs] like cereal and eating a pill”.
While treating infectious diseases and fighting cancer generally involve significant differences in strategy, both strands of medicine currently suffer from evolved drug resistance that stems from the same efflux pump action. Rotello’s group was able to shut this resistance down with functionalized nanoparticles, but groups working in physical oncology have adopted a gene-based approach. In 2017 Leu-wei Lo and colleagues in Taiwan used microporous silicon nanoparticles to co-deliver the anticancer drug doxorubicin with a DNA-active enzyme, or DNAzyme, that targets the transcription factor for the multi-drug-resistant proteins that cells produce to flush drugs out. Crucially, the microporous silicon nanoparticle allows the genetic and chemotherapeutic drugs to be released sequentially, so the DNAzyme has time to cleave the transcription factor and inhibit expression of the efflux proteins before the doxorubicin is released. Since the transcription factors targeted are key to cell growth, metastasis of the cancer was also inhibited.
Limited time only
Antibiotics have been so effective against micro-organisms that some infectious diseases have been effectively eradicated, and invasive surgery can be performed relatively safely. It is possible that, either through the alternatives or the synergies they offer, nanomaterials may contribute to our continued protection from micro-organisms in the years to come. The challenge for researchers in this field, however, is that time is not necessarily on the humans’ side. The fate of Ernest Duchesne, who submitted one of the first scholarly works on the antimicrobial activities of moulds, provides a cautionary tale. Duchesne’s seminal work was published in 1897 as part of his doctoral thesis, but a few years later he contracted tuberculosis. He died in 1912 at the tragically young age of 37. It would be more tragic still if the rise of drug-resistant strains of bacteria left us as vulnerable to bacterial infections as we were 120 years ago.