Calcium Phosphate Nanoparticles as Intrinsic Inorganic Antimicrobials

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Cite This: ACS Appl. Mater. Interfaces 2018, 10, 34013−34028

Calcium Phosphate Nanoparticles as Intrinsic Inorganic Antimicrobials: The Antibacterial Effect Victoria M. Wu,† Sean Tang,† and Vuk Uskokovic*́ ,†,‡ †

Advanced Materials and Nanobiotechnology Laboratory, Department of Biomedical and Pharmaceutical Sciences, Center for Targeted Drug Delivery, Chapman University, Irvine, California 92618-1908, United States ‡ Advanced Materials and Nanobiotechnology Laboratory, Department of Bioengineering, University of Illinois, Chicago, Illinois 60607-7052, United States

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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. KEYWORDS: amorphous, antibacterial, calcium phosphate, hydroxyapatite, multidrug-resistant, nanoparticle, synergy

1. INTRODUCTION Disparity between the high rate at which pathogenic microorganisms gain resistance to traditional, small-molecule antibiotic therapies and the low rate at which new antibiotics of this type are being discovered,1 approved by the regulatory agencies, and introduced into the clinic has instigated a search for resistance-free alternatives.2 Inorganic nanoparticles present one such option because bacteria are much less likely to acquire resistance to them than to small-molecule antibiotics.3 Therefore, one of the most important objectives at the frontier of materials science is to replace traditional antimicrobial (and other) pharmacotherapies with advanced materials exhibiting precisely optimized physicochemical properties. Necessity for this type of materials is justified by their minimal propensity for causing bacterial resistance compared to small-molecule antibiotics. With the introduction of nanomaterials, medically © 2018 American Chemical Society

utilizable properties in materials have multiplied, given the extraordinary sensitivity of nanomaterials to the subtlest changes in their constituent nanoparticle properties,4 such as size, shape, topography, surface charge, faceting, termination, and so on. Nanoscience is indeed expected to revolutionize every single human craft and discipline, including medicine. Nanoparticles provide for broader spectra of potential properties optimizable using rational approaches than their bulk analogues and are additionally more active and more tunable owing to their high surface-to-volume ratios and quantum mechanical resonance effects, respectively. When it comes to antimicrobial nanomaterials, all of the applications on Received: July 27, 2018 Accepted: September 18, 2018 Published: September 18, 2018 34013

DOI: 10.1021/acsami.8b12784 ACS Appl. Mater. Interfaces 2018, 10, 34013−34028

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ACS Applied Materials & Interfaces

cell, this protean character of CP can be of advantage when it comes to its consideration for the role of an antibacterial agent. Although a constant demand for the tradeoff of medically relevant properties, such as the mechanical stability and bioresorbability or sustained release kinetics and flowability, is the downside of this qualitative versatility, it also presents grounds for smart, personally tailorable biomaterial-based therapies that are bound to become more clinically prominent in the coming eras. However, the mild antibacterial activity of CP nanoparticles, especially pronounced against Gram-negative strains, has not been a prime field of interest until recently. Our recent studies demonstrated that the microstructure of CP nanoparticles could be tuned to elicit a finite antibacterial response even in the absence of the delivered antibiotics.21 The action of antibiotics with little specificity toward specific bacterial strains can also be augmented when they are being delivered by CP nanoparticles. Simultaneously, CP nanoparticles elicit a positive effect on fibroblastic, osteoblastic, and osteoclastic cells in vitro, both at the phenotypic and genotypic levels.22,23 This selectivity presents the basis for the optimization of these nanoparticles for a more intense antibacterial effect, such that it would make them therapeutically competitive with at least silver nanoparticles, if not traditional antibiotics. The excellent safety profile, bioactivity, and biodegradability of CPs also provide a major advantage over nanosilver and traditional antibiotics, frequently tied to the problematic side effects.24,25 Here, we provide the report of a systematic study of the intrinsic antibacterial effect of different CPs against different Gram-positive and Gram-negative species, including the clinically relevant, multidrug-resistant (MDR) subspecies. We have also explored the synergistic effect of CP nanoparticles and antibiotics using different delivery regimens, including concomitant administration and nanoparticle preloading.

the market have utilized silver. However, nanoparticulate silver as the most marketed nanomaterial and the most medically utilized inorganic antimicrobial has fallen out of favor with the regulators of medical devices in the US and the EU because of its frequently detected genotoxic5 and cytotoxic6 effects on primary cells,7 prompting the search for novel inorganic materials with antibacterial properties and a more favorable primary versus pathogenic cell selectivity. Most of these materials have been photocatalytic metal oxides and chalcogenides, such as zinc oxide, titanium oxide, magnesium oxide, vanadium oxide, cadmium sulfide, and others, but their toxicity at high concentrations, often required to elicit a sufficient antibacterial effect, has also been an issue,8 adding up to the fact that the bactericidal activity of many of them is conditioned by the exposure to UV light.9 Calcium phosphate (CP) is not only one of the most pervasive but also the most protean, pleiotropic, and enigmatic of materials from the physicochemical and biological standpoints. It is also an inexpensive material naturally favorited by the new wave in biomaterials science, interested not in complexities for the sake of complexity, but in simplicity and elegance that bear a high translational potential.10 It holds a promise of being at the forefront of the new wave of cheap, abundant, and naturally accessible nanomaterials dispersible in water, including the likes of photoluminescent carbon dots,11 α-Zn3P2 photovoltaics,12 CaVO3 transparent conductors,13 and cellulose nanocrystals14 or paper-based diagnostics.15 CP is also an unusually versatile material, both from the compositional and the applicative standpoint.16 Today, it is used in a number of industries, from food to agricultural to environmental to chemical to energy to cosmetic to biomedical. For example, CP, aka E341, is a common leavening agent and dietary supplement in food industry, whereas in agriculture, it is used as the main ingredient of superphosphate fertilizers. In the environmental industry, it is used in soil, ash, and wastewater filters to capture organics and heavy metals and in chemical industry as a sorbent in chromatographic columns for separation of a range of compound types, thanks to its superb adsorption profile. In the energy sector, CP has been used as a catalyst for fuel production, especially in one of its numerous ion-doped forms.17 CP is a common addition to cosmetic products, where it controls abrasion, bulking, and opacity, whereas in the biomedical field, CPs are utilized as self-setting bone cements, dental remineralization and antisensitivity agents, nonviral gene delivery carriers, reinforcement components of tissue engineering constructs, bulking agents for the treatment of urinary incontinence, and so on. The breadth of this expansion of CP across a variety of disciplines as well as the ability of the material to be engineered to possess a broad array of oftentimes diametrically opposite properties are illustrated by the recent studies effectively utilizing hydroxyapatite (HAp), the most pervasive CP in biology and geology, albeit in a composite form, as a separator in lithium-ion batteries, thanks to its superior electrolyte wettability, thermal stability, mechanical robustness, and excellent safety profile,18 but also as the major component of a flexible, electrically conductive paper with a superhydrophobic surface, electrothermal behavior, and flame retardancy.19 This pleiotropy-typifying CPs may also explain their promising role as a component of a range of theranostic hybrids in the research stage.20 Because narrow-spectrum antibiotics are more prone to induce resistance in bacteria than molecules that act on a broad range of targets in the bacterial

2. MATERIALS AND METHODS 2.1. Synthesis of CP Nanopowders. Three stoichiometrically different CP nanopowders were synthesized and compared in this study: HAp (Ca5(PO4)3OH), dicalcium phosphate (DCP, CaHPO4), and amorphous calcium phosphate (ACP). The synthesis of different CP powders involved precipitation from aqueous solutions. Specifically, to make HAp, 400 mL of 0.06 M aqueous solution of NH4H2PO4 (Fisher Scientific) containing 25 mL of 28% NH4OH was added dropwise to the same volume of 0.1 M aqueous solution of Ca(NO3)2 (Fisher Scientific) containing 50 mL of 28% NH4OH (Sigma-Aldrich), vigorously stirred with a magnetic bar (400 rpm) and kept on a plate heated to 60−80 °C. After the addition of NH4H2PO4 was complete, the suspension was brought to boiling, then immediately removed from the heater, and air cooled to room temperature. Stirring was suspended and the precipitate together with its parent solution, the final pH of which was 10.6, was left to age under atmospheric conditions for 24 h. After the given time, the precipitate was washed once with deionized H2O, centrifuged (5 min at 3500 rpm), and let dry overnight in air. To synthesize DCP, the same procedure as that used to precipitate HAp was run, but using 50 mL of 0.33 M Ca(NO3)2 and 50 mL of 0.25 M NH4H2PO4 containing 0.1 mL of concentrated, 28% NH4OH to make pH 6.8, without bringing the suspension to boiling afterward unless noted otherwise. The final pH of the supernatant was 5.2. ACP was made by abruptly adding a solution containing 100 mL of 0.5 M Ca(NO3)2 and 7 mL of 28% NH4OH into a solution comprising 100 mL of 0.2 M NH4H2PO4 and 4 mL of 28% NH4OH. The fine precipitate formed upon mixing was aged for 15 s, before it was collected, centrifuged, washed with 0.14 w/v % NH4OH, centrifuged again, washed with ethanol, dried overnight in air, and stored at 4 °C to 34014


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Figure 1. SEM and HR-TEM images of HAp (a,d), ACP (b,e), and DCP (c) nanoparticles. Dashed lines in (d,e) represent the nanoparticle boundary. prevent spontaneous transformation to HAp. The pH of the supernatant following the precipitation reaction was 9.3. 2.2. Physicochemical Characterization. The morphology of the CP nanopowders was analyzed using a Hitachi S-4300SE/N scanning electron microscope at the Lawrence Berkeley National Lab, with the electron beam energy of 15 kV. High-resolution transmission electron microscopy (HR-TEM) analysis was carried out at the National Center for Electron Microscopy, on an FEI monochromated F20 UT Tecnai HR-TEM under the electron acceleration voltage of 200 kV. The phase composition of CP particles was confirmed on a Bruker D2 Phaser X-ray diffractometer using polychromatic Cu as the irradiation source. Kβ line was stripped off with an inbuilt filter, whereas Kα2 line, the source of peak asymmetry artifacts at high diffraction angles, was stripped off automatically, together with the instrumental line broadening. Diffractograms were recorded in the 10°−90° 2θ range, whereas the step size was 0.002° and the irradiation time per step was 1 s. Raw X-ray diffraction (XRD) patterns were smoothened either using the FFT filter or the Lowess method using the proportion for a span of 0.003. Thermal properties were determined in a simultaneous thermogravimetric/differential thermal analysis (TGA/DTA) on a Setsys (SETARAM Instrumentation, Caluire, France) instrument in an Al2O3 pan, the temperature range between 25 and 1000 °C, and under the air flow of 20 mL· min−1. Prior to the measurements, the materials were stabilized at 25 °C for 5 min, then heated to 1000 °C at the heating rate of 10 °C· min−1. 2.3. Antibacterial Assays. Antibacterial assays were performed against the following laboratory strains of bacteria: Gram-positive Staphylococcus aureus ATCC27661 (Carolina Biologicals), Staphylococcus epidermis ATCC12228 (gift of J. Yamaki), and Enterococcus faecalis ATCC29212 (gift of J. Yamaki) and Gram-negative Pseudomonas aeruginosa ATCC27853 (Carolina Biologicals) and Escherichia coli ATCC14948 (Carolina Biologicals), as well as MDR clinical isolates: Gram-positive methicillin-resistant S. aureus (MRSA, gift of J. Yamaki), Gram-negative MDR P. aeruginosa (gift of J. Yamaki), ESBL(+) E. coli (gift of J. Yamaki), and MDR Klebsiella pneumoniae ATCC1705 (gift of J. Yamaki). For broth assays, bacteria were grown overnight in 5 mL of cultures in either Luria broth (LB) or Vegatone broth at 37 °C and 200 rpm. To determine if CP nanopowders inhibited the bacterial growth, they were weighed out into bacterial culture tubes and 1 mL of 1:50 diluted overnight culture of bacteria was added. Bacteria were grown in the presence of CP nanoparticles overnight at 37 °C and 200 rpm. For assays involving nanoparticles and antibiotics, the two were added concomitantly to diluted bacterial broths, unless noted otherwise. After the overnight incubation, 200 μL of the bacterial suspension was centrifuged and resuspended in 200 μL of 110 mM solution of NaCl in 250 mM HCl (150 μL 0.85 wt % mixed with 50 μL of 1 M HCl) to dissolve the CP

nanoparticles and avoid their interference with the optical density (OD) readings. OD was measured at 600 nm (BMG LABTECH FLUOstar Omega) and converted to the volume concentration of colony-forming units by multiplying with 8 × 108. In the agar diffusion antibacterial assay, 5 mg of nanoparticles were placed onto a bacterium-infused LB agar plate with the spot radius of 1 cm. The agar plates were then allowed to incubate for 24 h at 37 °C. The radius of the zone of inhibition was then used to gauge the antibacterial activity of the cements. All of the samples for both antibacterial assays were analyzed in triplicates. IC50 values were obtained from exponential decay fits of the OD versus particle concentration plots in the 0−100 mg/mL range.

3. RESULTS 3.1. Physicochemical Characterization of CPs. The goal of this study was to assess the newly discovered antibacterial activity of CP nanoparticles under different experimental conditions before making an attempt to gain an insight into their mechanism of action using an approach analogous to digging the tunnel from the materials science side and from the cell biology side, hoping that the two will eventually meet in the middle. Two forms of CP were analyzed in parallel in this study: HAp, the only hydroxylated CP phase and the one comprising the mineral component of mammalian boney tissues, and ACP, a phase that is highly active, metastable, and quick to transition to HAp, acting as its precursor in the process of biomineralization. However, the understanding of the mechanism of action necessitated the inclusion of DCP, a hydrogenated CP phase forming in acidic solutions and strictly found in pathogenic-calcified tissues, as an additional CP phase. To ensure that any difference in the antibacterial activity or the mechanism of action is not because of a variation in the particle size, shape, or surface charge, all CP phases, albeit chemically and crystallographically distinct, were prepared with the goal to produce as similar size, shape, and charge as possible. Figure 1 displays SEM images of the three different CP nanomaterials, showing an indistinguishable level of similarity between their particle sizes and morphologies. CP nanoparticles for all three compositions appeared spheroidal under SEM and averaged at ∼100 nm in diameter (Figure 1a−c). Crystallinity and specific surface area (Sa) are often inversely proportional,26 and Sa and antibacterial activity can be directly27,28 or inversely29 proportional, but the highly similar particle sizes and morphologies ensured that Sa values 34015


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at 10 °C·min−1, and this peak was present only in ACP but not in HAp (Figure 3a). This crystallization exotherm is due to the release of excess enthalpy, the result of distorted bonds and higher bond energy in the amorphous structure. Its presence demonstrates the inherently far-from-equilibrium structure of ACP, potentially more active as such compared to the equilibrium structure of HAp. The water content was also higher in ACP, as demonstrated by the four times larger surface area of the DTA endotherm centered at ∼100 °C and the correspondingly higher mass loss in this temperature range. Namely, as it could be seen from Figure 3b, the majority of the weight loss during annealing of both CP powders occurs in the 60−300 °C window, which corresponds to the water loss endotherm. Because of the higher water content in ACP than in HAp, the total weight loss after heating up to 1000 °C is higher in ACP than in HAp: 22.25 wt % versus 6.57 wt % in HAp. The shift of the endotherm peak from 88.4 °C for HAp to 112.1 °C for ACP also suggests a tighter bonding and a higher concentration of lattice water in ACP, as opposed to mostly adsorbed, surface water in HAp. All of this indicates a higher hydration degree in ACP. Compared to anhydrous HAp, it is estimated at ∼15−20 wt % for ACP per its general stoichiometric formula, CaxHy(PO4)z·nH2O, where n = 3− 4.5.30 This implies that a hypothetically equal biological response elicited by these two powders would still imply a greater activity of ACP because of the significantly higher content of water in it. The TGA diagrams also showed a more significant weight loss in ACP than in HAp in the 450−635 °C region (1.7 vs 0.3 wt %, Figure 3b). This minor loss compared to the magnitude of water loss is caused by the formation of volatile P2O3 in response to an increase in the Ca/P molar ratio from ∼1.5 to 1.67, accompanying the transition from ACP to HAp. 3.2. Antibacterial Effect of Pure CPs: Regular Strains. When dispersed in broths, the two tested CP powders, ACP and HAp, exhibited comparable levels of antibacterial activity against Gram-negative bacteria, specifically E. coli and P. aeruginosa (Figure 4). In contrast, the activity of ACP was significantly higher than that of HAp when measured against all three Gram-positive bacterial species tested: S. aureus, S. epidermis, and E. faecalis (Figure 5). On the basis of IC50 values (Table 1), the activity of HAp was consistently higher than that of ACP against Gram-negative species and the activity of ACP was consistently higher than that of HAp against Grampositive species, suggesting that these two forms of CP exhibit two distinct mechanisms of action. Even though ACP

were almost identical, in the 80−90 m2/g range for all CP powders, thus excluding Sa as a potential cause of difference in the activity between different CP phases. TEM images in Figure 1d,e demonstrated that ACP was largely amorphous, although it contained occasional crystalline pockets, whereas HAp was not completely crystalline. Poorly crystalline HAp, such as that shown in Figure 1e, was composed of pockets of crystalline regions interspersed in an amorphous matrix. The ratio between the crystalline and the amorphous phases in HAp was markedly higher than in ACP. XRD patterns presented in Figure 2 confirm the monophasic composition

Figure 2. XRD patterns of ACP, HAp and DCP. Diffraction peaks labeled with *, # and o in the diffractogram of DCP originate from brushite (DCP dihydrate), monetite (DCP anhydrous) and HAp, respectively.

of HAp and a mixed phase composition of DCP, comprising its anhydrous form, monetite, its dihydrate form, brushite, and a minor amount of HAp. ACP, meanwhile, displayed a broad, diffuse pattern typical of amorphous structures. DTA analysis performed on HAp and ACP powders showed the presence of an exothermic peak at ∼650 °C upon heating

Figure 3. DTA (a) and TGA (b) diagrams obtained by heating ACP and HAp powders separately up to 1000 °C at the rate of 10 °C·min−1. 34016


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Figure 4. Reduction in the concentration of colony-forming units in broths of two different Gram-negative bacteria, including E. coli (a) and P. aeruginosa (b) treated with ACP or HAp nanoparticles in the 0−100 mg/mL concentration range. Data points represent averages (n = 3), and error bars represent the standard deviation. Data points statistically significantly lower (p < 0.05) compared to the negative control, that is, population challenged with 0 mg/mL ACP/HAp, are marked with an asterisk.

Figure 5. Reduction in the concentration of colony-forming units in broths of two different Gram-positive bacteria, including S. aureus (a), S. epidermis (b), and E. faecalis (c) treated with ACP or HAp nanoparticles in the 0−100 mg/mL concentration range. Data points represent averages (n = 3), and error bars represent the standard deviation. Data points statistically significantly lower (p < 0.05) compared to the negative control, that is, population challenged with 0 mg/mL ACP/HAp, are marked with an asterisk.

Compared to our previous studies on CP cements, which showed the highest antibacterial activity against E. coli and moderate activities against S. aureus and P. aeruginosa,31 CP powders analyzed here exhibited a greater difference in activity between Gram-positive versus Gram-negative organisms depending on the phase composition and also a more pronounced activity against Gram-positive species, albeit exhibited only by ACP. 3.3. Antibacterial Effect of Pure CPs: Clinical Isolate Strains. Clinical isolates, a.k.a. MDR strains, are typified by usually a series of interconnected cellular mechanisms by which they inhibit the toxic effect of antibiotic agents. Compared to the antibiotic-susceptible strains, the clinical strains were most affected by HAp rather than by ACP. Most noticeably, the effect of HAp on MRSA is dramatic compared to its lack of effect against standard S. aureus. Thus, although antibiotic-sensitive S. aureus showed little response to HAp up to 100 mg/mL, the viability of MRSA was reduced starting at 20 mg/mL of HAp (Figure 6a). An even more drastic effect was detected against MDR P. aeruginosa, whose viability HAp reduced by 50% already at 10 mg/mL (Figure 6b). In contrast, the first effect of ACP against MRSA was observed at 40 mg/ mL (Figure 6a), as opposed to 10 mg/mL against the regular S. aureus strain (Figure 5a). Similarly, although ACP was effective

Table 1. IC50 Values for ACP and HAp against Regular Bacterial Strains, as Determined from ConcentrationDependent Viability Curves bacterial group Gram-negative

Gram-positive

bacterial strain

IC50 (ACP) (mg/mL)

IC50 (HAp) (mg/mL)

E. coli P. aeruginosa

65 90

29 59

S. aureus S. epidermis E. faecalis

33 15 52

>100 >100 93

comprises 22.2 wt % water compared to only 6.6 wt % water in HAp (Figure 3b), the activity of ACP at the lowest concentration tested in these assays was significantly higher than that of HAp against both bacterial species. Specifically, at the concentration of 10 mg/mL, ACP statistically significantly lowered the viability of all five bacterial species except E. faecalis, as opposed to HAp, which lowered the viability of E. coli and S. epidermis only at this dose. The most obvious disparity between the two CP phases was observed against S. aureus; namely, the activity of ACP was pronounced, whereas the activity of HAp was minor and statistically insignificant even at the highest dose of 100 mg/mL (Figure 5a). 34017


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Figure 6. Reduction in the concentration of colony-forming units in broths of one Gram-positive (MRSA, a) and one Gram-negative (MDR P. aeruginosa, b) MDR bacterial strain treated with ACP or HAp nanoparticles in the 0−100 mg/mL concentration range. Data points represent averages (n = 3), and error bars represent the standard deviation. Data points statistically significantly lower (p < 0.05) compared to the negative control, that is, population challenged with 0 mg/mL ACP/HAp, are marked with an asterisk.

mg/mL for Gram-negative MDR P. aeruginosa (Table 2), whereas they equaled over 100 and 59 mg/mL for their laboratory analogues, respectively (Table 1). Intriguingly, the activity of ACP dropped against the clinical strains when compared to their regular counterparts (Figure 7b), whereas the activity of HAp was higher against the clinical, MDR bacterial strains than against their regular counterparts (Figure 7b). Therefore, the selective effect of ACP/HAp against Grampositive bacteria, respectively, becomes inverted for their clinical, MDR analogues. As seen from Figure 8, at an even lower concentration of 5 mg/mL, where the effect of ACP against P. aeruginosa was significant and the effect of HAp was not, the opposite is observed against the MDR version of the same bacterium, namely the significant effect of HAp, but not that of ACP too. 3.4. Synergy between Antibiotics and CPs: Regular Strains. After we realized that CP nanoparticles alone exert an intrinsic antimicrobial effect, we were curious to determine whether the use of nanoparticles in concert with antibiotics could have an effect on bacteria resistant to the given antibiotics. P. aeruginosa was chosen as the model organism for several reasons: (a) P. aeruginosa is a common opportunistic human pathogen and is ubiquitously found in

against regular P. aeruginosa already at 10 mg/mL (Figure 4b), it took doses higher or equal to 70 mg/mL to significantly affect the growth of the comparative clinical strain (Figure 6b). For both Gram-positive MRSA and Gram-negative MDR P. aeruginosa, the IC50 values of ACP were greater than 100 mg/ mL (Table 2) and equaled 33 and 90 mg/mL for their Table 2. IC50 Values for ACP and HAp against MDR Bacterial Strains, as Determined from ConcentrationDependent Viability Curves bacterial group Gram-negative Gram-positive

bacterial strain MDR P. aeruginosa MRSA

IC50 (ACP) (mg/mL)

IC50 (HAp) (mg/mL)

>100

10

>100

98

laboratory analogues, respectively (Table 1). Although these values imply only a slightly lower activity of ACP against MDR P. aeruginosa compared to the regular, laboratory P. aeruginosa, they also demonstrate more than threefold reduction in the activity of ACP against MRSA when compared to its regular analogue (Tables 1 and 2). In contrast, the IC50 values of HAp were under 100 mg/mL for Gram-positive MRSA and only 10

Figure 7. Comparative reduction in the concentration of colony-forming units in broths of regular and MDR versions of one Gram-positive (MRSA) and one Gram-negative (MDR P. aeruginosa) bacterial strain treated with ACP (a) or HAp (b) nanoparticles in the 0−100 mg/mL concentration range. Data points represent averages (n = 3), and error bars represent the standard deviation. Data points statistically significantly lower (p < 0.05) compared to the negative control, that is, population challenged with 0 mg/mL ACP/HAp, are marked with an asterisk. 34018


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cin, minocycline, and erythromycin for ACP and ampicillin, minocycline, and erythromycin for HAp. Vancomycin, for example, which is not effective in treating P. aeruginosa-related infections, in conjunction with ACP nanoparticles at the concentration as low as 0.5 mg/mL effectively reduces the CFU/mL in vitro broth assays by 75% (Figure 9). Agar assays in which vancomycin has been loaded onto ACP particles also show a strong effect, as evidenced by the increased zone of inhibition around the ACP-vancomycin particles and none around vancomycin alone (Figure 10). Interestingly, the synergetic effect was either similar to both ACP and HAp or drastically different and the most striking example was vancomycin. Namely, although it engaged in intense synergy with ACP, it was completely immune to it when codelivered with HAp. The synergetic effect also had a complex relationship with nanoparticle concentration. For some antibiotics, the activity at a constant antibiotic dose increased proportionally to the nanoparticle concentration, as was the case for oxacillin in combination with both ACP and HAp or clindamycin combined with ACP. Some antibiotics produced a drastic drop in bacterial viability at lowest nanoparticle concentrations and remained at a steady state from there on, as was the case with the combinations of ACP with ampicillin, vancomycin, and kanamycin. Finally, there were combinations for which a relatively fine concentration of nanoparticles proved to be most optimal, as everything above it increased the bacterial viability. Such was the case with the combinations of HAp and ACP with minocycline and erythromycin (Figure 9). One way of explaining this effect, which was absent in broths treated with nanoparticles only, is by assuming that the combination of nanoparticles and antibiotics, some of which are bactericidal, imposed a greater stress on the bacteria than the treatment with nanoparticles only and that the bacteria responded to this stress by forming more biofilm, turning nanoparticles into a substrate for biofilm formation and effectively hindering the synergy between the nanoparticles and antibiotics. One such stress-dependent biofilm formation is a common occurrence in P. aeruginosa35 and was frequently observed in our experiments. Experiments testing the ability of CP nanoparticles to synergistically augment the activity of antibiotics against planktonic P. aeruginosa corroborated the higher intrinsic antibacterial activity of ACP compared to HAp. Thus, the ability of ACP to boost the activity of ampicillin and kanamycin was comparable to that of HAp at higher dosages (>1 mg/mL) but also significantly higher at the lowest dosage (1 mg/mL). The biggest disparity between ACP and HAp was observed in relation to vancomycin; namely, ACP increased its activity at all five concentrations higher than 0.25 mg/mL, whereas HAp did not promote any antibiotic activity, but with increasing the nanoparticle concentration, both the planktonic bacterial population and the biofilm amount were also significantly higher than in the particle-untreated control. The synergetic activities of ACP and HAp were comparable during the co-delivery with minocycline and erythromycin in a sense that only the lower concentrations of nanoparticles produced an effect, whereas higher particle concentrations resulted in the loss of antibiotic activity. ACP activity was

Figure 8. Broth concentrations of colony-forming units of regular (left) and MDR (right) strains of P. aeruginosa treated with the low, 5 mg/mL concentration of ACP or HAp nanoparticles and no antibiotics. Data points represent averages (n = 3), and error bars represent the standard deviation. Data points statistically significantly lower (p < 0.05) compared to the untreated negative control are marked with an asterisk. Data points statistically insignificantly different (p > 0.05) compared to the untreated control broth are marked with “n.s.”.

the natural environment, urban settings, and clinical centers, with hospital-acquired P. aeruginosa infections being one of the leading causes of nosocomial mortalities worldwide;32 (b) the bacterium displays an intrinsic resistance against many antibiotics, with MDR isolates fast becoming a global health threat; (c) it forms biofilm notoriously difficult to eradicate;33 (d) antibiotics in broth tests were rather ineffective or partially effective against it, as compared to S. aureus (Figure S1) and E. coli (Figure 12a), whose populations responded more sensitively; (e) the effect of HAp and ACP on P. aeruginosa viability is comparable, meaning that any synergy seen cannot be assigned to a difference between HAp and ACP effectiveness. To examine the possible synergy between CP nanoparticles and antibiotics, we selected several antibiotics with different known mechanisms of action that P. aeruginosa showed, either complete resistance or slight sensitivity (Figure S1). Among a dozen tested antibiotics, rifampicin was left out of synergy experiments because it itself had a strong effect against P. aeruginosa, reducing its population 20fold. Ciprofloxacin was equally effective, but, similar to tetracycline, its solubility gets drastically reduced upon chelation of Ca2+ ions34 released by ACP/HAp and only negative synergy resulted from such combinations. Although minocycline belongs to the tetracycline family, it does not chelate Ca2+ and was used in the synergy study. As shown in Figure 9, a strong synergetic effect was observed for combinations of CP and the great majority of antibiotics tested, including ampicillin, kanamycin, oxacillin, vancomycin, minocycline, erythromycin, linezolid, and clindamycin. The combination with cephalexin was least effective, as it significantly reduced the bacterial population only at one out of five tested particle concentrations in the 0.25−20 mg/mL range for ACP and none for HAp. Many combinations acted synergistically and significantly reduced the colony-forming unit concentration compared to the antibiotic-only treatment already at the lowest of all tested particle concentrations of 250−500 μg/mL, including ampicillin, kanamycin, vancomy34019


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Figure 9. Broth concentrations of colony-forming units of P. aeruginosa treated synergistically with different concentrations of ACP (a) or HAp (b) nanoparticles and fixed doses of antibiotics adjusted to their activity: 100 μg/mL ampicillin, 50 μg/mL kanamycin, 5 μg/mL oxacillin, 100 μg/mL vancomycin, 5 μg/mL minocycline, 20 μg/mL erythromycin, 5 μg/mL linezolid, 5 μg/mL cephalexin, and 5 μg/mL clindamycin. Dashed line indicates the viability of control cultures treated with neither the antibiotic nor the CP particles. Data points represent averages (n = 3), and error bars represent the standard deviation. Data points statistically significantly lower (p < 0.05) compared to the only antibiotic-treated, 0 mg/mL ACP/HAp sample group are marked with an asterisk.

further higher than that of HAp during the codelivery of linezolid and cephalexin, even though it was generally significant for the former and very weak, detected only at the moderate concentration of 1 mg/mL for the latter. In turn, the activity of HAp was completely absent for cephalexin and present only at the highest tested concentration (20 mg/mL) for linezolid. ACP was also able to increase the activity of clindamycin, the frequent broad-range antibiotic of choice for treating bone infection. Further tests were conducted against P. aeruginosa at a fixed, low concentration of CP nanoparticles (5 mg/mL) and clinically relevant, blood serum concentrations of different antibiotics following systemic administration. For this assay, we selected ampicillin, erythromycin, and vancomycin because the original working stocks for these three antibiotics were high and not clinically relevant, in contrast to other antibiotics, for which the working stock was 5 μg/mL. Ampicillin and erythromycin were also selected because of their high safety profiles, availability as generic therapeutics, and low cost. Vancomycin was selected because under normal circum-

stances, a Gram-negative bacterium is not affected by vancomycin, and therefore it is unlikely that the resistance to the drug would occur. In general, the antibiotic per se did not significantly reduce the bacterial count, but in combination with CP nanoparticles it did (Figure 11). Vancomycin, for example, produced one such effect at each of the four tested antibiotic concentrations in the 0.5−10 μg/mL range in combination with ACP. It also produced an effect at three out of four concentrations in combination with HAp, although the synergistic effect decreased as the antibiotic concentration increased, similar to what was observed in experiments varying not antibiotic, but nanoparticle concentrations. A similarly strong effect was observed for the combination of HAp and erythromycin, but was present only in one out of four tested concentrations of erythromycin and ACP. These results demonstrate that the optimization of the concentrations of both the antibiotics and the CP nanoparticles, alongside their phase and crystallinity, is essential for producing the effective synergy between the two and the augmented therapeutic effect. 34020


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Finally, it is important to point out that although the positive synergy was observed for almost all antibiotics tested, albeit not necessarily at all concentrations, the negative synergy, where the activity of the combination of CP and an antibiotic would be significantly lower than that of the antibiotic alone, was almost completely absent. The antibacterial efficacy of some antibiotics, such as tetracycline, did become diminished when delivered using CP, the reason being the inactivation of this antibiotic through binding to Ca2+ ions diffusing through the double charge layer of the nanoparticles, explaining why its consumption together with milk is not recommended. However, although one would expect that CP may bring in an additional surface susceptible for biofilm formation or anchorage and protection of bacteria within pores and crevices of nanoparticle aggregates, where they would be less susceptible to antibiotic action, such an effect was not observed for any of the CP/antibiotic combinations except in a few cases, such as the combinations of linezolid and cephalexin with HAp at high antibiotic dosages (Figure 9) and erythromycin with ACP and vancomycin with HAp at low dosages (Figure 11). 3.5. Synergy between Antibiotics and CPs: Clinical Isolate Strains. Although the synergistic interaction between CP nanoparticles and antibiotics was promising, it was applied to the laboratory strains of bacteria rather than MDR, clinical isolates. To determine if this synergy exists in the clinical isolates, we performed two assays: (1) comparing the effect of antibiotic alone against the effect of an equivalent dose of the antibiotic-loaded CP nanoparticles and (2) comparing the effect of antibiotic alone against the effect of the concomitantly delivered equivalent dose of the antibiotic and the nanoparticles. The first set of assays showed that although no CP particles produced an effect at 5 mg/mL when delivered alone and the antibiotics ampicillin, erythromycin, and vancomycin similarly had no effect when delivered alone, their combination with CP concentrations as low as 1 mg/mL produced a statistically significant effect against MDR P. aeruginosa and MRSA, but not ESBL(+) E. coli and MDR K. pneumoniae, which were immune to the treatment (Figure 14). The effect was slightly more pronounced for HAp than for ACP, as in agreement with their intrinsic activities against the MDR strains. However, although HAp alone was more effective against MDR P. aeruginosa than ACP, ACP did engage in a synergistic interaction with ampicillin, a β-lactam antibiotic to which MDR P. aeruginosa is completely resistant. It should be noted that drug-loaded CP nanoparticles did release a larger amount of the drug than the standard dose, but the drug-only controls equaled the amount of the drug released by the CP nanoparticles during the overnight incubation. Experiments assessing the benefits of concomitant delivery also showed a clear synergistic effect against both Grampositive MRSA and Gram-negative MDR P. aeruginosa (Figure 15). Although nanoparticle concentrations as low as 1 mg/mL for both ACP and HAp produced a significant synergetic effect against MRSA when codelivered with ampicillin or erythromycin, the effect was opposite upon the codelivery with vancomycin. Although ampicillin and erythromycin produced an effect only in the presence of CP nanoparticles, MRSA is sensitive to vancomycin per se and the presence of CP nanoparticles could not increase the effect of the drug but rather decreased the susceptibility of MRSA to it. MDR P. aeruginosa is sensitive to erythromycin alone and, again, CP nanoparticles negatively interfered with its action, but the

Figure 10. Agar assays showing the detectable presence of the antibacterial effect of ACP nanoparticles and vancomycin against P. aeruginosa only when they are delivered together.

After the synergy was observed between CP nanoparticles and antibiotics against P. aeruginosa, we wanted to check if this synergy would be present in another species that exhibited resistance to antibiotics. Thus, we picked a common Gramnegative pathogen, E. coli. Although the lab strain of this bacterium (K-12) is more sensitive to antibiotics than P. aeruginosa, the bacterium showed resistance to three antibiotics: clindamycin, vancomycin, and rifampicin (Figure 12a). As seen in Figure 12b, for each of these three antibiotics, a significant synergistic effect with CP nanoparticles was detected in the entire concentration range tested, from 1 to 20 mg/mL. Antibiotic-only controls had no effect on the bacterial viability, whereas the bacterial concentration dropped significantly when antibiotics were codelivered with ACP at all of the four concentrations tested (Figure 12b) and the effect was just slightly less intense for HAp nanoparticles (Figure 12c). For example, at the lowest nanoparticle concentration tested, 1 mg/mL, all ACP/antibiotic combinations significantly reduced the bacterial colony count (Figure 11b), but this was not the case for HAp, which needed concentrations of 5 mg/ mL or higher to produce a significant synergistic effect (Figure 11c). The synergy between antibiotics and CP nanoparticles was further corroborated in an experiment comparing the activity of the concomitant administration of the two and the administration of the antibiotic-loaded CP nanoparticles. As shown in Figure 13, the intimate interaction between the antibiotics and the nanoparticles in cases when the former is delivered as adsorbed on the surface of the latter augments the activity of the antibiotic compared to the codelivery of the nanoparticles and an equivalent dose of the antibiotic simultaneously but as separate reactants. For antibiotics such as vancomycin and erythromycin, the activity significantly increased when they were delivered as loaded onto the CP nanoparticles surface and the effect was somewhat more pronounced for ACP than for HAp. This result was expected in view of the observed synergy between the two. Namely, if a synergy between two components exists, it should be more pronounced when they are delivered with a tighter contact between one another. 34021


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Figure 11. Broth concentrations of colony-forming units of P. aeruginosa treated synergistically with 5 mg/mL of either ACP (a,c,e) or HAp (b,d,f) nanoparticles and different low, clinically relevant concentration ranges of antibiotics in the 1−10 μg/mL range, including ampicillin (a,b), erythromycin (c,d), and vancomycin (e,f). Data points represent averages (n = 3), and error bars represent the standard deviation. Data points statistically significantly lower (p < 0.05) compared to the untreated control broth are marked with an asterisk. Data points statistically insignificantly different (p > 0.05) compared to the untreated control broth are marked with “n.s.”.

positive synergy was observed for vancomycin. Although this antibiotic produced no effect when delivered alone, in combination with ACP and particularly HAp at certain nanoparticle concentrations, the synergetic effect on the bacterial growth was pronounced. Because of the biofilmforming nature of MDR P. aeruginosa, the trend of an increased bacterial growth at higher nanoparticle concentrations was more pronounced for this bacterium than for MRSA. Likewise, as was the case with regular strains, HAp was more prone to exhibit this effect than ACP, indicating its greater propensity for acting as a biofilm substrate, presumably because of the less diffusive and volatile surface than that typifying ACP. Another possibility is that the combination of HAp and vancomycin

imposed a greater stress on the bacterium, resulting in more abundant biofilm formation and resistance to the treatment. Compared to the regular strains, against which CP nanoparticles were synergistically effective at low, therapeutically relevant concentrations of the antibiotics, such an effect was not present for either MRSA or MRD P. aeruginosa. Specifically, the intense synergetic effect observed for the combination of both forms of CP with erythromycin at a higher antibiotic dose against the MDR P. aeruginosa (20 μg/ mL, Figure 15d) was not observed for the lower one (10 μg/ mL, Figure S2a). Also, MRSA was resistant to all of the antibiotics administered at low concentrations (10 μg/mL) in combination with 5 mg/mL CP nanoparticles (Figure S2b). 34022


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Figure 12. Broth concentrations of colony-forming units of E. coli treated with fixed doses of antibiotics (A) adjusted to their activity: 50 μg/mL kanamycin, 100 μg/mL ampicillin, 5 μg/mL oxacillin, 5 μg/mL minocycline, 20 μg/mL erythromycin, 5 μg/mL linezolid, 5 μg/mL clindamycin, 100 μg/mL vancomycin, and 20 μg/mL rifampicin (a). Data points statistically significantly lower (p < 0.05) compared to the untreated control broth are marked with an asterisk, and these antibiotics are treated as effective against E. coli. Data points statistically insignificantly different (p > 0.05) compared to the untreated control broth are marked with “n.s.” and E. coli is considered resistant to these antibiotics. Broth concentrations of colony-forming units of E. coli treated synergistically with different concentrations of ACP (b) and HAp (c) nanoparticles and fixed doses of E. coliinsensitive antibiotics adjusted to their activity: 5 μg/mL clindamycin, 100 μg/mL vancomycin, and 20 μg/mL rifampicin. All of the data points in the ACP-treated group were statistically significantly lower (p < 0.05) with respect to both the untreated control broth and broths treated with antibiotics only and no ACP nanoparticles. Statistically significant difference is marked with an asterisk. Data points in all graphs represent averages (n = 3), and error bars represent the standard deviation.

discovery by the metallurgist-pharmacist team of Ghan and Scheele,36 it has fallen out of favor in the nanoparticle world, being considered too simple and limited in its functionality.37 However, it is this simplicity that can be the backbone of its strength in a postcolonial milieu, making it a material that is easy and economically feasible to synthesize even in laboratories with the most modest of resources, without the need for expensive and specialized equipment. A century of research into the potentials of CP as a biomaterial38 has established it as a standard for stable, biocompatible, nontoxic materials, possessing excellent bioactivity and capability of inducing an overwhelmingly positive response by the bone cells.39 However, to date, its antimicrobial effects have not been explored in a sufficient detail. Presently, CP nanoparticles are either supplemented with antibiotics40,41 or doped with heavy metal ions42,43 to obtain an antimicrobial effect. In this study, we show that CP nanoparticles have an intrinsic antimicrobial effect as well as the ability to synergistically interact with antibiotics in a manner that can overcome the bacterial resistance to these antibiotics. Here, we examine the antibacterial properties of two CP phases: HAp and ACP. Although the MIC50 of CP nanoparticles in this iteration, being in the mg/mL range rather than the μg/mL range, cannot be compared to that of silver nanoparticles,44−46 the CP nanoparticles exhibit superior antibacterial activity over zinc oxide,47 another material commonly cited as antimicrobial. The fact that both HAp and ACP exhibited an antibacterial effect against S. aureus, the bacterium responsible for 90% of bone infections,48 and P. aeruginosa, the bacterium forming the biofilm wherein the resistance of microorganisms is ∼103 times higher than that in their planktonic form,49 is promising from

Figure 13. Broth concentrations of colony-forming units of P. aeruginosa treated with 5 mg/mL of ACP or HAp nanoparticles and vancomycin, ampicillin, or erythromycin either concomitantly or by dosing the broths with the antibiotic-loaded nanoparticles. Data points statistically significantly lower (p < 0.05) with respect to the comparative data points are marked with an asterisk. Data points in all graphs represent averages (n = 3), and error bars represent the standard deviation.

4. DISCUSSION CP is a pervasive and well-known material, simplistic in design and composition. Two hundred and fifty years after its 34023


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Figure 14. Broth concentrations of colony-forming units of different clinical, MDR bacterial strains, including ESBL(+) E. coli (a,b), MDR P. aeruginosa (c,d), MDR K. pneumoniae (e,f), and MDR S. aureus (g,h), after the 24 treatment with 5 mg/mL of either ACP (a,c,e,g) or HAp (b,d,f,h) alone, with different antibiotics alone and with 5 mg/mL antibiotic-loaded ACP/HAp nanoparticles. Doses of antibiotics were adjusted to the amounts loaded onto 5 mg/mL of ACP/HAp nanoparticles: 1.46 mg/mL ampicillin, 34.8 μg/mL erythromycin, and 152 μg/mL vancomycin for HAp and 4 mg/mL ampicillin, 48 μg/mL erythromycin, and 0.7 mg/mL vancomycin for ACP. Data points statistically significantly lower (p < 0.05) compared to the untreated control broth are marked with an asterisk, and these antibiotics are treated as effective against E. coli. Data points statistically insignificantly different (p > 0.05) compared to other data points are marked with “n.s.”. Data points in all graphs represent averages (n = 3), and error bars represent the standard deviation. 34024


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Figure 15. Broth concentrations of colony-forming units of the clinical, MDR strain of P. aeruginosa treated synergistically with different concentrations of ACP (a,c) or HAp (b,d) nanoparticles, ranging from 1 to 20 mg/mL, and fixed doses of antibiotics (A) adjusted to their activity: 100 μg/mL ampicillin, 20 μg/mL erythromycin, and 100 μg/mL vancomycin. Data points represent averages (n = 3), and error bars represent the standard deviation. Data points statistically significantly lower (p < 0.05) compared to the only control, untreated broth are marked with an asterisk.

and penicillin-binding proteins of the cell wall for oxacillin. All of the antibiotics assayed were drugs to which the bacterium either showed a complete resistance or slight sensitivity. The synergetic interaction with vancomycin, in particular, is striking because vancomycin is not an effective treatment for Gramnegative bacterial infections given that it functions through inhibition of the peptidoglycan matrix, the main component of Gram-positive bacterium cell walls. Although a Gram-negative bacterium does have a peptidoglycan layer, it is protected by the outer membrane and thus is inaccessible to vancomycin, which is hydrophilic (log P = −3.1) and too large (Mw = 1449.3 Da) to cross the outer membrane. The synergy with vancomycin is also different from the synergy seen with other antibiotics. In general, if there is a synergy with an antibiotic, it will be seen for both HAp and ACP, although one phase may be more effective than the other. With vancomycin, however, the synergy is seen only with ACP, but not HAp. This has suggested a different antibacterial mechanism of the action of these two phases, which we will explore in the further parts of this study. These findings are important in view of the widespread dissemination of the drug-resistant strain of P. aeruginosa, a commonly found bacterium that is a serious cause of illness in hospital patients and/or those with weakened immune systems in disease processes such as cystic fibrosis and chronic obstructive pulmonary disease (COPD).51 With the

the clinical standpoint. Interestingly, HAp shows a greater effect against Gram-negative bacteria, whereas ACP is much more effective against Gram-positive ones. The higher antibacterial activity of ACP than HAp against Gram-positive S. aureus perhaps explains why this phase has been mostly used in commercial dental formulations (Staphylococcus mutans is also a Gram-positive bacterium) and also why renal calculus rich in ACP is typically noninfectious.50 For HAp to exert an inhibitory effect on Gram-positive species, it requires a much higher concentration of the nanoparticles. However, when HAp and ACP were tested in the treatment of MDR clinical isolates, MDR P. aeruginosa, and MRSA, the results were reversed, with HAp becoming more effective than ACP against the Gram-positive MRSA. A large reduction in the amount of HAp required to obtain the MIC50 compared to nonclinical strains was also observed. When antibiotics were codelivered with CP nanoparticles in doses as small as 0.5 mg/mL, the growth inhibition was induced in a number of bacterial organisms, including a fourfold reduction of P. aeruginosa growth in combination with vancomycin as well as other antibiotics. The targets of action of antibiotics engaging in this synergy with CP nanoparticles greatly varied, including the peptidoglycan matrix for vancomycin, ribosomal subunits for erythromycin, kanamycin, minocycline, and clindamycin; transpeptidase for ampicillin; 34025


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ACS Applied Materials & Interfaces advent of MDR P. aeruginosa, the need to find new antibiotics effective against this pathogen is urgent. Our findings show that combining CP nanoparticles with antibiotics that normally have no effect on P. aeruginosa can create a synergistic effect in which antibiotics that have never been effective against P. aeruginosa are suddenly able to kill the bacterium. Of note as well is that in laboratory strains of P. aeruginosa, an antimicrobial effect is seen when 5 mg/mL of nanoparticles are combined with low doses of antibiotics mimicking the concentrations present in the blood plasma levels of patients. Although the synergy between CP nanoparticles and clinical isolates is less pronounced, the effect is present both when the antibiotics are loaded onto the nanoparticles and when they are added concomitantly. The advent of MDR bacterial strains is a growing global health crisis and an issue of great concern. Although the need to discover new antibiotics to stem the tide of resistance is necessary, it is also clear that there is a need to find other, nondrug options that have good antimicrobial properties and that can help reduce the dependency on antibiotics. Here, we show that two different phases of CP nanoparticles have intrinsic antimicrobial effects against both laboratory strains of bacteria as well as their MDR clinical analogues. The nanoparticles can also act in synergy with antibiotics that the bacteria are resistant to, bypass that resistance, and in combination decrease the bacterial viability. A previous study52 showed no effect of ACP with a similar particle size as that utilized in this study against Gram-positive S. mutans, but the results of this study contradict these earlier findings. One reason why the effect was absent in this earlier work is that the material was in a compact form, as opposed to nanoparticles able to diffuse through inoculated broths in this study. Another reason is that the material was tested against a mixed population of Gram-positive biofilm-forming organisms. As demonstrated here, the effectiveness of CPs varies depending on the organism in question. In addition, biofilms are composed of a heterogeneous population of organisms and how each responds to ACP can vary. During this present investigation, we did not focus on how CPs affect the biofilm community of the biofilm-forming organisms, but we did note that the organisms that did form biofilm continued to form it in the presence of CPs, although we did not quantitate the exact amount of the biofilm. The next step will be to determine the effect of CPs on biofilm and derive the means by which CPs can be formulated to be effective in disrupting the biofilm that contributes to chronic infections. Understanding the effect of polymicrobial interactions and mechanisms by which multispecies communities would respond to the nanoparticle treatment by using appropriate microbiome in vitro models presents an equally important step in bringing CPs closer to real-life antibacterial applications. In a longer run, these findings may pave the way for the formulation of ACP and HAp that would be useful in a clinical setting. ACP and HAp can be formulated into bone or dental cements to prevent infection in surgical implants or at fracture sites.53 In one such application, ACP would be particularly useful against Gram-positive bacterial infections, whereas HAp would be useful as a carrier of antibiotics given its increased ability to overcome the bacterial resistance to certain antibiotics. The different specificities in the activity of these two CPs corroborate the complexity of the chemistry of CPs and their biological effects. They also open the way for the design of a more complex combinatorial therapies based on

this material through the control of its chemistry and physical structure at multiple scales.

5. CONCLUSIONS In this study, we report on the intriguing antibacterial effect of CP nanoparticles, displayed both intrinsically and synergistically with a number of antibiotics against a number of laboratory and clinical bacterial species and strains. These findings lead to a number of questions in need of answering before we are able to understand the phenomena at hand, including: (a) the intrinsic antibacterial effect of CP nanoparticles; (b) the qualitatively selective effect of an amorphous and a crystalline CP phase; (c) the reversal of this selective effect when clinical isolates are used instead of laboratory strains; and (d) the greater effect of HAp nanoparticles against the clinical strains compared to their regular, laboratory analogues. Hoping that these interesting phenomena may present the basis for delineating the mechanism of action of these nanoparticles, in the immediate follow-ups to this study, we embark on the voyage to understand (i) which of the nanoparticle properties play a key role in causing these effects by digging the tunnel through a mountain starting from the materials science side and (ii) which biological disturbances are imposed onto the bacterial cell by the nanoparticles by digging the tunnel starting from the cell biology side. Whether we have succeeded in this dual effort and come up with a treasure hidden in the heart of a black mountain will be revealed in these ensuing parts of this study.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b12784. Antibacterial assays against P. aeruginosa and S. aureus treated with pure antibiotics (including ampicillin, kanamycin, oxacillin, vancomycin, minocycline, erythromycin, linezolid, cephalexin, clindamycin, and rifampicin) and against MRD P. aeruginosa and MRSA treated concomitantly with the different antibiotics and ACP/ HAp nanoparticles (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Vuk Uskoković: 0000-0003-3256-1606 Author Contributions

V.M.W.data acquisition, formal analysis, investigation, methodology, supervision, writing. S.T.data acquisition. V.U.formal analysis, funding acquisition, investigation, resources, supervision, writing. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS National Institutes of Health grant R00-DE021416 is acknowledged for its support. We thank Smilja Marković from the Institute of Technical Sciences of the Serbian Academy of Sciences and Arts for technical assistance with TGA/DTA measurements and Caroline Sun from Chapman University for assistance with the bacterial stock preparation. 34026


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Calcium Phosphate Nanoparticles as Intrinsic Inorganic Antimicrobials

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