Antibacterial Nanomedicine

Bio-inspired Antibacterial Nanomedicines

The following studies describe the application of nanoparticles of biological origin in their natural or in a modified form as antibacterials. The first example involves an additional application of electrospun nanofiber mats; however, in this case, they were spun from the polysaccharide chitosan. Polymers with intrinsic bacteriostatic and/or bactericidal activity and, in particular, polysaccharides are considered as promising for wound-healing and -dressing applications. The natural polysaccharide chitosan was reported to possess advantageous biological properties, such as hemostatic activity, nontoxicity, biodegradability, intrinsic antibacterial properties and the ability to affect macrophage function, which contributes to faster wound healing.^[56,57] Ignatova et al.^[58] studied the antibacterial properties of nanoparticles that were fabricated from quaternized chitosan (QCh) and poly(vinyl alcohol). QCh derivatives illustrate a higher activity against bacteria, broader spectrum of activity and higher killing rate as compared with those of chitosan.^[59,60] Nanofibers have been electrospun successfully from water-soluble nonionogenic polymers, such as polyoxyethylene^[61] and poly(vinyl alcohol) (PVA).^[62] PVA is interesting for wound treatment because it is highly hydrophilic, has an inherent fiber- and film-forming ability and can be cross-linked easily. The cross-linking of electrospun PVA mat in the solid state has been reported.^[9,63] After immobilization of model drugs (8-hydroxyquinoline derivatives) in chitosan- or N-carboxyethylchitosan-containing nanofibers, they acquire antimicrobial and antimycotic activity.^[64,65] They intended to study the preparation of QCh-containing nanofibers by electrospinning of mixed QCh/PVA aqueous solutions, as well as the antibacterial properties of photo-cross-linked electrospun QCh/PVA mats.

Ignatova et al. reported that the measured average diameters of the QCh/PVA fibers were 600 and 145 nm for total polymer concentration of 13 and 8 wt%, respectively. The free form of QCh was reported to show high antibacterial activity against S. aureus.^[66] In the current study, the antibacterial activity of photo-cross-linked electrospun QCh/PVA mats was evaluated against S. aureus and E. coli. Toxicity was tested by the viable cell-counting method. S. aureus bacteria were killed within 60 min of contact with cross-linked QCh/PVA electrospun mat containing 2845 µg/ml QCh, in contrast to electrospun photo-cross-linked PVA mat, used as a control, which did not modulate bacterial growth. The electrospun QCh/PVA mats were also exposed to the Gram-negative bacteria E. coli. A reduction of bacterial growth by 98% was observed after 120 min exposure to the photo-cross-linked QCh/PVA nanofibers.

To conclude, the electrospun QCh/ PVA fibers had diameters of 60–200 nm with narrow diameter distribution. Moreover, the higher the content of QCh, the smaller was the diameter of the nanofibers. The electrospun QCh/PVA nanofibrous mats were stabilized successfully against dissolution in the aqueous environment using photo-mediated cross-linking. Finally, photo-cross-linked electrospun nanofibrous QCh/PVA mats had good bactericidal activity against the Gram-negative bacteria E. coli and also against the Gram-positive bacteria S. aureus.

In another bio-inspired study by Salmaso et al.,^[67] the fabrication of nisin-loaded poly-L-lactide nanoparticles and their antimicrobial activity was reported. Nisin belongs to the lantinobiotics, which are a family of antimicrobial proteins of bacterial origin containing unusual amino acids, such as lanthionine.^[68] These bacteriocins display a broad inhibitory spectrum against a variety of Gram-positive bacteria. Recently, the US FDA recognized nisin, a bacteriocin produced by Lactococccus lactis, as a food additive.^[69] However, the use of nisin in food preservation is strongly limited by its structural instability, deprivation by interaction with food and cell matrixes and development of tolerant and resistant Listeria.^[70] Therefore, usage of excessive amounts of nisin is required to guarantee effective pathogen growth inhibition. Nisin-loaded polymeric micro- and nanoparticles seem to be promising formulations to achieve long-lasting antimicrobial activity. Poly-L-lactide nanoparticle systems have been investigated actively as protein-drug-delivery systems because they can enhance the biological performance of bioactive molecules.^[71–73] With respect to other slow-release systems, polymeric micro-/nano-colloids are stable physically and can be formulated easily with a variety of materials, obtaining a controlled rate of drug release. Polymeric-drug-delivery systems are widely evaluated in the field of oncology^[74] and here we see the first example of an antibacterial application of such systems.

Salmaso et al. dealt with the preparation of stable long-lasting antimicrobial nisin-loaded polymeric micro-/nanoparticles, which can be dispersed in food, pharmaceutical products or other materials with different physical consistence. Nisin–poly-L-lactide (PLA) nanoparticles were prepared from the peptide nisin and PLA by using the supercritical CO₂ in mixed solvent method.^[75] This material was chosen owing to its biodegradable and nontoxic characteristic, which is common in protein formulation.^[76] Furthermore, PLA possesses suitable properties for compressed CO₂ antisolvent precipitation that was used for fabrication. The observed particle size was in the range of 250 to 400 nm.

Next, the antimicrobial properties were tested against Lactobacillus. A total of 1 mg of free nisin or nisin-equivalent nanoparticles (20 mg of 5% nisin A-loaded nanoparticles or 5 mg of 20% nisin A-loaded nanoparticles) was dissolved or suspended in 5 ml of sterile MRS medium. A total of 1% Lactobacillus delbrueckeii spp. bulgaricus culture was added to the medium and incubated overnight, after which dilutions were made to obtain a colony-forming unit count. The two formulations, 5 and 20% nisin-loaded nanoparticles, displayed similar behavior and prolonged activity, as measure by inhibition of bacterial growth for up to 40 days. In comparison, free nisin samples displayed antibacterial activity for 7 days, whereas the unloaded PLA nanoparticles had no antibacterial activity.

To conclude, the nisin-loaded nanoparticles are a typical example of the great potential of this technique in protein formulation. Nisin-loaded polymeric nanoparticles fabricated by the GAS precipitation technique demonstrated long-lasting antimicrobial activity. Indeed, this formulation provides for slow protein release and protein stabilization, which yields an efficient antimicrobial system that should prove to be useful in food and pharmaceutical preservation.

These last examples are studies by the author's group that are focused at applying filamentous bacteriophages (phages) as a targeted drug-carrying platform.^[77,78] The goal of these studies is the fabrication of a universal, modular solution for the eradication of pathogenic bacteria and other cells that are bearers of disease. Phage nanoparticles can actually be described as nanoneedles, with diameters of approximately 8 nm, which can be used to deliver a large payload of a cytotoxic drug to the target cells(Figure 1C). To apply this platform against pathogenic bacteria, a drug was linked to genetically modified phages by means of chemical conjugation through an esterase-cleavable linker subject to controlled release by serum esterases. Thus, as long as the drug is in a conjugated state, it is essentially a prodrug devoid of cytotoxic activity, which is activated post cleavage from the phage in proximity to the target site. The guidance of the drug-carrying phages to target cells was mediated by genetic expression of a targeting moiety on the phage coat. The main achievement of our studies was the substitution of the drug selectivity itself to a target selectivity borne by the targeting moiety. This approach may enable the use of neglected drugs that, owing to toxicity or low selectivity, have thus far been excluded from clinical use. The feasibly of this approach was demonstrated by using the bacteriostatic, nonpotent antibiotic chloramphenicol (which is mostly excluded from systemic therapeutic application owing to it hemolytic properties ^[79]) as a model drug. In our proof-of-concept study,^[78] targeting was accomplished by using two different targeting moieties: target-specific peptides, selected from a peptide phage-display library, which were displayed on the major coat protein of the phage; or antibodies linked to the phages via an IgG Fc-binding ZZ domain that is fused to the g3p minor coat protein of the phage. As a model target, we used the bacterial pathogen S. aureus. The preliminary system^[78] suffered from limited ability to inhibit bacterial growth owing to a limited arming capacity of less than 3000 drug molecules/phage and limited solubility of the entire platform, limitations that were mainly due to the hydrophobicity of the drug. To overcome these limitations, we developed a ‘second generation' of the platform by developing a unique drug-conjugation chemistry, based on the application of hydrophilic aminoglycoside antibiotics as branched, solubility-enhancing linkers.^[77] The replacement of the arming chemistry and a modification of the antibody–phage conjugation method, improved our system into a viable and versatile tool for the targeting of a broad range of pathogenic bacteria, such as S. aureus, Streptococcus pyogenes and E. coli, each targeted by microbe-specific antibodies. Experimentally, the new drug-conjugation approach led to an arming rate of over 40,000 chloramphenicol molecules/phage. These results had provided impressive improvement in drug potency of approximately 20,000 in comparison to the free drug, as measured by bacterial growth inhibition in liquid culture. This large drug-carrying capacity was made possible by using the exterior of the phage coat that offers many docking sits on the 3000 coat-protein monomers. Other phage- or virus-based drug-delivery model systems pack the drug within the particle and hence have a limited drug-carrying capacity.^[80]

In conclusion, drug-carrying phage represents a versatile therapeutic nanoparticle that, owing to the tailoring of its coat, by the simplicity of which it can be equipped with a targeting moiety, and its massive drug-carrying capacity, may become an important general targeting drug-delivery platform. By comparison to particulate drug-carrying devices, such as liposomes or virus-like particles, the arrangement of drug that is conjugated in high density on the exterior of the targeted particle is unique. A dense coating of the phage with aminoglycosides and drugs might produce advantages that are regarded as challenges in the application of phages as therapeutics; the primary, of course, is the immunogenicity on in vivo administration. Indeed, our unpublished results have shown that drug-carrying phages are hardly recognized by commercial antiphage antibodies and generate significantly lower antiphage antibody titers when used to vaccinate mice (in comparison to ‘naked' phages) [Vaks et al., Unpublished Data]. Further, we found that drug-carrying phages are nontoxic to BALB/c mice up to a high dose of 10¹² phage particles injected intravenously or intraperitoneally. The therapeutic capacities of targeted drug-carrying phages in mouse models are being studied currently.

Nanomedicine. 2008;3(3):329-341. © 2008  Future Medicine Ltd.

No writing assistance was utilized in the production of this manuscript.

Cite this: Antibacterial Nanomedicine - Medscape - May 01, 2008.