Calcium Phosphate Nanoparticles as Intrinsic Inorganic Antimicrobials: The Antibacterial Effect

Abstract

Cheap and simple to make, calcium phosphate (CP), thanks to its unusual functional pleiotropy, belongs to the new wave of abundant and naturally accessible nanomaterials applicable as a means to various technological ends. It is used in a number of industries, including the biomedical, but its intrinsic antibacterial activity in the nanoparticle form has not been sufficiently explored to date. In this study, we report on this intrinsic antibacterial effect exhibited by two distinct CP phases: an amorphous CP (ACP) and hydroxyapatite (HAp). The effect is prominent against a number of regular bacterial species, including Staphylococcus aureus, Staphylococcus epidermis, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa, but also their multidrug-resistant (MDR) analogues. Although ACP and HAp displayed similar levels of activity against Gram-negative organisms, ACP proved to be more effective against the Gram-positive ones, with respect to which HAp was mostly inert, yet this trend became reversed for the MDR strains. In addition to the intrinsic antimicrobial effect of CP nanoparticles, we have also observed a synergistic effect between the nanoparticles and certain antibiotics. Both forms of CP were engaged in a synergistic relationship with a variety of concomitantly delivered antibiotics, including ampicillin, kanamycin, oxacillin, vancomycin, minocycline, erythromycin, linezolid, and clindamycin, and enabled even antibiotics completely ineffective against particular bacterial strains to significantly suppress their growth. This relationship was complex; depending on a particular CP phase, bacterial strain and antibiotic, the antibacterial activity (i) intensified proportionally to the nanoparticle concentration, (ii) plateaued immediately after the introduction of nanoparticles in minute amounts, or (iii) exhibited concentration-dependent minima due to stress-induced biofilm formation. These findings present grounds for the further optimization of CP properties and maximization of this intriguing effect, which could in the long run make this material comparable in activity to the inorganics of choice for this application, including silver, copper, or zinc oxide, while retaining its superb safety profile and positive eukaryotic versus prokaryotic cell selectivity.

Calcium phosphate nanoparticles as intrinsic inorganic antimicrobials: In search of the key particle property

“…Their more detailed physicochemical characterization, such as particle size, morphology, crystallinity, phase composition, and thermal stability, including the basic information about their antibacterial efficacy and selectivity, was given in the first part of this study. 1 This section follows an innovative, but logical form by being divided to nine subsections, each dealing with the investigation of a single effect potentially causative of the observed antibacterial activity and ending up with an estimate of how realistic and/or critical its role in the process is. This approach is justified by the fact that will become obvious in the course of the discussion: rather than a single particle property being the determinant of the antibacterial effect, a number of them acting in synergy are responsible for causing this effect.…”

“…Raw CP powders of different monophasic compositions were prepared as described in detail in the first part of this study. 1 Briefly, HAp [Ca 5 (PO 4 ) 3 OH] was synthesized by adding an aqueous solution containing 0.06 M NH 4 H 2 PO 4 and 1.75 vol. % NH 4 OH dropwise to the same volume of an aqueous solution containing 0.1 M Ca(NO 3 ) 2 and 3.5 vol.…”

“…Antibacterial assays performed by dispersing nanoparticles in Luria broths (LB) and by depositing them on LB agar plates were described in the first part of the study. 1 Briefly, 1 ml of 1:50 diluted overnight cultures of bacteria mixed with CP nanopowders were incubated overnight at 37°C and 200 rpm, with or without the presence of concomitantly added antibiotics. After the overnight incubation, 200 μl of the bacterial suspension was centrifuged at 10 000 rpm and resuspended in 200 μl of 110 mM solution of NaCl in 250 mM HCl (150 μl 0.85 wt.…”

“…Traditional antibiotics are notorious for their promotion of microbial resistance, low targeting potential, and an array of systemic side effects, which are all issues amendable with the use of appropriate materials science platforms. In the first part of this study, 1 we reported on an intriguing antibacterial effect caused by calcium orthophosphate (CP) nanoparticles, including hydroxyapatite (HAp) and amorphous CP (ACP). The nanoparticles were not only effective against both lab strains and clinical isolates when delivered alone, but were also able to synergize with a number of different antibiotics and significantly increase their activity against a variety of pathogenic and opportunistic bacteria, including some of the strains that are insensitive to antibiotics per se.…”

“…The nanoparticles also exhibited a selective response whose origin was unclear: namely, while HAp was more effective than ACP against Gram-negative species and drug-resistant strains, it was not effective against Gram-positive strains, against which ACP was very effective. 1 HAp is the most sparsely soluble nondoped CP phase and is also the mineral component of all hard tissues in the body except for the minor calcite portions of the middle ear. In contrast, ACP has been detected as the solid intermediate in the crystallization of both biogenic and synthetic HAp, forming as the result of the Ostwald law of stages, which predicts the successive formation of phases in the order of their stability.…”