Nanoantibiotics

Are polymer nanoparticles a new weapon in the fight against bacteria?

Arkady Kuramshin, "Elements"

Polymer nanoparticles that mimic the structure of the virus envelope can selectively kill various types of bacteria (including strains that have developed resistance to antibiotics) without affecting human cells. Varying the size and shape of nanoparticles changes their specific activity. The results of the study can become the basis for a new strategy for the development of antimicrobial drugs that will fight infections without contributing to the development of resistance in bacteria.

The resistance of organisms to the action of antibiotics – antibiotic resistance – is currently one of the most serious health problems. The report of the World Health Organization, published in April 2014, says the following: "Resistance is no longer just a forecast for the future, because it is already manifesting itself right now in every region of the world and can negatively affect everyone, regardless of age, in every country. Antibiotic resistance is a phenomenon when bacteria change so much that antibiotics no longer have any effect on the body of people who need them to fight infection, and this is now one of the most serious threats to human health." Of course, progress in the development of new antibiotics also does not stand still, it is regularly reported about the creation of drugs to which resistance in bacteria is developed very slowly, but the strategies used to develop antimicrobial drugs, alas, do not shine with diversity. These include blocking the mechanisms that control the construction of the bacterial cell membrane, disruption of the bacterial ribosome responsible for protein synthesis processes, or disruption of bacterial DNA replication. Unfortunately, no matter how powerful a new antibiotic is, sooner or later natural selection leads to the appearance of bacterial strains resistant to its effects. The defense mechanisms that are produced in bacteria may include a biochemical modification of an antibiotic that reduces its activity, a change in the molecular target affected by the antibiotic by the bacterium, or the active removal of the drug from the bacterial cell.

Solving the problem of developing new antimicrobial drugs, researchers are trying to invent new types of strategies – to obtain systems that can literally tear apart bacterial cells, without giving them time to develop resistance. One of such promising approaches is the use of antimicrobial peptides (antimicrobial peptides). These are short chains consisting of amino acid residues produced by various organisms to protect against biological pathogens (bacteria, parasites or fungi). The action of antimicrobial peptides is based on their incorporation into the cell membrane of a pathogenic organism, violation of the structural integrity of the membrane and, as a consequence, destruction of the cell.

Antimicrobial nanoparticles were previously obtained from antimicrobial peptides of natural origin (see, for example, Tew et al., 2010. De novo design of antimicrobial polymers, foldamers, and small molecules: From discovery to practical applications), as well as from polymers modeling these peptides (E. F. Palermo, K. Kuroda, 2010. Structural determinants of antimicrobial activity in polymers which mimic host defense peptides). However, nanoparticles from both proteins and synthetic polymers imitating their structure have a common disadvantage: their hydrophobic fragments also destroy mammalian cell membranes. This limits the possibility of using such systems as antibiotics.

Researchers from the group of Hongjun Liang (Hongjun Liang) from Texas Tech University suggested that proteins of bacteriophages – viruses selectively attacking bacterial cells that do not have hydrophobic fragments can demonstrate greater selectivity in the fight against bacteria. More precisely, not the proteins of bacteriophages themselves, but their synthetic model.

The researchers obtained three nanoparticles that mimic the structure of bacteriophages: a sphere with a diameter of 8 nanometers and two rods with a diameter of 7 nanometers and a length of 18, the other 70 nanometers. For the synthesis of polymer nanostructures, block copolymerization was used at the first stage: to β-cyclodextrin (this structure served as the basis for spherical nanoparticles) or poly[2-(bromisobutyryl)ethyl methacrylate] (this polymer was the basis for rod-shaped nanoparticles) was grafted with poly(4-vinyl-N-methylpyridine) filaments. The obtained block copolymers containing pyridine fragments in the framing groups were modified at the second stage of obtaining nanoparticles by treating with iodomethane. A quaternization reaction occurred (in this reaction, an uncharged trivalent nitrogen atom (less often phosphorus) reacts with something (most often with an acid or alkyl halide), turning into tetravalent nitrogen carrying a positive charge, which is balanced by a negative charge of the acid residue or halide ion from the alkyl halide composition; see Quaternary ammonium cation), which allowed convert methylpyridine fragments into salt methylpyridine iodide groups.

The choice of shapes and sizes of nanoparticles was dictated by the desire to model the structural motifs of some elements of bacteriophages; for example, the tail tube of the bacteriophage T4 is characterized by a length of 94 nm and a diameter of 9.6 nm. Nanoparticles consist of a polymer core framed by "villi" of a hydrophilic polymer carrying a positive charge – poly(4-vinyl-N-methylpyridinium iodide) (Fig. 1).

Fig. 1. Spherical (a) and rod-shaped (b) polymer molecular brushes synthesized during the study mimic the two main motifs of the bacteriophage structure. Their chemical structure is shown in diagrams c and d. The blue chains in images a and b and the blue fragments of structural formulas in schemes c and d correspond to poly(4-vinyl-N-methylpyridinium iodide); the red elements and fragments of structural formulas correspond to β-cyclodextrin (for a and c) and poly[2-(bromisobutyryl)ethyl methacrylate] (for b and d). Figure from the discussed article in ACS Infectious Diseases

The hydrophilicity (i.e., the ability to form strong intermolecular interactions with water) of polymer fragments surrounding the core is achieved due to the presence of an ionic chemical bond between the pyridinium cation (pyridinium – ionic derivative of pyridine) and the iodide anion as part of the structural units of the outer shell of the obtained polymer particles. Water is a polar solvent that forms strong associates with charged polymer fragments with the help of non–chemical bonds.

All three types of nanoparticles were tested for activity against gram-negative strains of Escherichia coli (Fig. 2), gram-positive strains of Staphylococcus aureus, a strain of Pseudomonas aeruginosa resistant to a number of antibiotics, as well as cytotoxicity against human red blood cells. Experiments have shown that spherical nanoparticles work most effectively. To kill 99.9% of gram-negative E. coli, no more than 32 micrograms/ml of this type of nanoparticles are needed, and their concentration, fatal for gram-positive S. aureus, is 4 mgk/ml. Finally, a concentration of only 2 micrograms/ml is sufficient to destroy 99.9% of cells resistant to a wide range of P. aeruginosa antibiotics.

Fig. 2. Images of E. coli bacteria obtained using a scanning electron microscope. c is a control sample, d–f are samples incubated with nanoparticles: a long rod (d), a short rod (e) and a sphere (f). Violation of the integrity of the bacterial shell manifests itself already 200 seconds after the start of incubation of E. coli with spherical nanoparticles and with nanoparticles representing a short rod. Figure from the discussed article in ACS Infectious Diseases

Rod-shaped particles showed less antimicrobial activity. For a nanorad with a length of 70 nm, the minimum bactericidal concentration in relation to two different gram-positive strains of S. aureus was at least 512 micrograms/ml. This is a very high concentration, which actually excludes the use of nanoparticles of this type as bactericides in the fight against gram-positive microbes: in such a concentration, nanoparticles are too harmful to the body (primarily for its filtering organs, such as the liver and kidneys) and expensive for mass use. But nanorods are dangerous for gram-negative bacteria: the minimum bactericidal concentration for gram-negative P. aeruginosa is 2 and 4 micrograms/ml for nanorods with a length of 18 and 70 nm, respectively.

Selectivity towards gram-negative bacteria is due to the fact that gram-positive bacteria have a dense peptidoglycan shell, the pores of which with a diameter of 5 to 50 nm are poorly permeable or impervious to rod-shaped nanoparticles; at the same time, the same nanorods are quite capable of damaging the thin outer lipid shell of gram-negative bacteria. It is assumed that the results obtained may be useful for controlling the activity and selectivity of a bactericidal nanoparticle by changing its size or/and shape.

The experiments also showed that all three nanoparticles are safe for human erythrocytes, they do not cause either destruction of their membrane or their agglutination (sticking). The different attitude to cells of different types is explained by the difference in the structure of the membranes of these cells. The shell of human erythrocytes, consisting mainly of nonpolar and hydrophobic phospholipids, should not interact with polar nanoparticles, therefore, they will not be able to integrate into the shell and violate its integrity or contact the shell of two or more erythrocytes at once and cause them to stick together. The cell membranes of bacterial cells include sphingolipids, in which polar fragments are present in a noticeable amount – phosphocholine (see Phosphocholine) or phosphoethanolamine (see Phosphorylethanolamine), with which hydrophilic polymers bind, forming the shell of nanospheres or nanorods.

It is still premature to talk about the clinical use of bactericidal nanoparticles, but the researchers hope that the results of their work will allow changing approaches to the fight against pathogenic microorganisms at the global level. Their work can become the basis for the creation of a new class of antibiotics – "nanoantibiotics", the directed design of the structure of which will make it possible to control the activity, selectivity and biological compatibility of nanoparticles exhibiting bactericidal properties.

Source: Yunjiang Jiang et al., Hydrophilic Phage-Mimicking Membrane Active Antimicrobials Reveal Nanostructure-Dependent Activity and Selectivity Selectivity // ACS Infectious Diseases. 2017.

Portal "Eternal youth" http://vechnayamolodost.ru 04.09.2017