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Posts Tagged ‘gram-positive’


Author: Tilda Barliya PhD.

I recently read this beautiful paper by Fredrik Nederberg from the IBM Almaden Research Center  and A*STAR Institute of Bioengineeringtitled “Biodegradable nanostructures with selective lysis of microbial membranes” (http://www.nature.com/nchem/journal/v3/n5/full/nchem.1012.html)

This paper gained a lot of attention as it merged as an innovation in nanotechnology and antibacterial therapeutics and therefore I have decided to introduce it here to the audience.

“Bacteria are increasingly resistant to conventional antibiotics and, as a result, macromolecular peptide-based antimicrobial agents are now receiving a significant level of attention. Most conventional antibiotics (such as ciprofloxacin, doxycycline and ceftazidime) do not physically damage the cell wall, but penetrate into the target microorganism and act on specific targets (for example, causing the breakage of double-stranded DNA due to inhibition of DNA gyrase, blockage of cell division and triggering of intrinsic autolysins). Bacterial morphology is preserved and, as a consequence, the bacteria can easily develop resistance. In contrast, many cationic peptides (for example, magainins, alamethicin, protegrins and defensins) do not have a specific target in the microbes, and instead interact with the microbial membranes through an electrostatic interaction, causing damage to the membranes by forming pores in them3. It is the physical nature of this action that prevents the microbes from developing resistance to the peptides. Indeed, it has been proven that cationic antimicrobial peptides can overcome bacterial resistance”.

“Most antimicrobial peptides have cationic and amphiphilic features, and their antimicrobial activities largely depend on the formation of facially amphiphilic a-helical or b-sheet-like tubular structures when interacting with negatively charged cell walls, followed by diffusion through the cell walls and insertion into the lipophilic domain of the cell membrane after recruiting additional
peptide monomers. The disintegration of the cell membrane eventually leads to cell death. Over the last two decades, efforts have been made to design peptides with a variety of structures, but there has been limited success in clinical settings, and only a few cationic synthetic peptides have entered phase III clinical trials. This is largely due to the cytotoxicity (for example, haemolysis) resulting from their cationic nature, their short half-lives in vivo (they are labile to proteases) and their high manufacturing costs”

A number of cationic polymers thatmimic the facially amphiphilic structure and antimicrobial functionalities of peptides have been proposed as a better approach, because they can be prepared more easily and their synthesis can be more readily scaled up compared with peptides.

The authors Yang, Hedrick and their co-workers have developed a polymer-based peptide alternative which avoids all of these problems. The polymer incorporates three key components: a non-polar hydrophobic head and tail, which drives the polymer to self-assemble into a nanoparticle; a positively charged block that selectively interacts with the bacterial cell membrane; and a carbonate backbone that slowly breaks down inside the cell, ensuring good biocompatibility. “The starting materials of our synthesis are inexpensive, and the synthesis of the antimicrobial nanoparticles is simple and can be scaled up easily for future clinical application.

“Polycarbonates are attractive biomaterials because of their biocompatibility, biodegradability, low inherent toxicity and tunable mechanical properties”

 

In general, Preclinical results confirm that the nanoparticles can efficiently kill fungi and multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE), even at low concentrations. The nanoparticles also showed insignificant activity against red blood cells, and no significant toxicity was observed during the in vivo studies in mice, even at concentrations well above their effective dose.

In more specific, the authors evaluated the minimal inhibitory concentrations (MICs) of the polymers against Gram-positive bacteria such as Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus and methicillin-resistant S. aureus (MRSA), and the fungus Cryptococcus neoformans. MIC is an important parameter commonly used to evaluate the activity of new antimicrobial agents, and is generally defined as the minimum concentration of an antimicrobial agent at which no visible growth of microbes is observed

Some of the MIC were evaluated against conventional antimicrobial agents that are used in clinical settings to treat infections caused by these microbes, such as vancomycin for S. aureus, MRSA and E. faecalis, and amphotericin B for C. neoformans.  When compared with these conventional antimicrobial agents, the polymers demonstrated comparable antimicrobial activities against all the microbes except for E. faecalis. This is important, because vancomycin-resistant E. faecalis, and S. aureus, as well as amphotericin B-resistant C. neoformans have been reported, and the resistant strains of these microbes are not susceptible to conventional antimicrobial agents. This suggests that there is an urgent need to develop safe and efficient macromolecular antimicrobial agents.

The hypothesis was that the cationic micelles can interact easily with the negatively charged cell wall by means of an electrostatic interaction, and the steric hindrance imposed by the mass of micelles in the cell wall and the hydrogen-binding/electrostatic interaction between the cationic micelle and the cell wall may inhibit cell wall synthesis and/or damage the cell wall, resulting in cell lysis. In addition, the micelles may easily permeate the cytoplasmic membrane of the organisms due to the relatively large volume of the micelle, destabilizing the membrane as a result of electroporation and/or the sinking raft model, leading to cell death.

Haemolysis is a major harmful side effect of many cationic antimicrobial peptides and polymers. The haemolysis of mouse red blood cells was evaluated after incubation with polymers 1 and 3 at various concentrations. Although the polymers disrupt microbial walls/membranes efficiently, they do not damage red blood cell membranes.

Toxicity tests in vivo showed that the micelles do not cause significant acute damage to liver and kidney functions, nor do they interfere with the electrolyte balance in the blood. Importantly, these parameters remain unchanged, even at 14 days post-injection.

In addition, no mouse treated with the polymer died, and no colour change was observed in the serum samples and urine of the mice treated with the polymer when compared with the control group. These findings demonstrate that the polymer did not induce significant toxicity to the mice during the period of testing. Nonetheless, preclinical studies should be conducted in the future to further evaluate potential long-term toxicity of the antimicrobial polymers before clinical applications.

In summary, the authors  have designed and synthesized novel biodegradable, cationic and amphiphilic polycarbonates that can easily self-assemble into cationic micellar nanoparticles by direct dissolution in water. The cationic nanoparticles formed from the polymers, with optimal compositions, can efficiently kill Gram positive bacteria, MRSA and fungi, even at low concentrations. Importantly, they have no significant haemolytic activity over a wide range of concentrations, and cause no obvious acute toxicity to the major organs and the electrolyte balance in the blood of mice at a concentration well above the MICs. These antimicrobial polycarbonate nanoparticles could be promising as antimicrobial drugs for the decolonization of MRSA and for the treatment of various infectious diseases, including MRSA associated infections.

The data presented by the authors is very promising and open a new door to antimicrobial therapy. Several questions and new avenues comes in minds:

  • Can these polymers be proven for there efficiency in specific disease animal models?
  • Can these NPs or similar approach can be applied to gram-negative bacteria?
  • Can these polycarbonate  affect massive biofilms?!

Looking forward to reading more news and results from this research group.

 

 

 

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