Generation of a Hydroxyapatite Nanocarrier through Biomineralization

Article Cite This: ACS Appl. Bio Mater. 2019, 2, 2927−2936

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Generation of a Hydroxyapatite Nanocarrier through Biomineralization Using Cell-Free Extract of Lactic Acid Bacteria for Antibiofilm Application Priya Mullick,† Sandipan Mukherjee,† Gopal Das,*,‡ and Aiyagari Ramesh*,† †

Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, India Department of Chemistry, Indian Institute of Technology Guwahati, Guwahati 781039, India

‡

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S Supporting Information *

ABSTRACT: Nanoscale materials hold considerable promise in the mitigation of bacterial infections. In order to exploit nanomaterials as delivery systems in an antibacterial therapeutic paradigm, it is critical to ensure that the generated material is nontoxic. Based on the fundamental principle of biomineralization, we herein report the generation of biocompatible hydroxyapatite nanoparticles (HANPs) in the presence of proteins secreted by the lactic acid bacteria (LAB) Lactobacillus plantarum MTCC 1325, Lactobacillus plantarum CRA52, and Pediococcus pentosaceus CRA51. The biogenic HANPs were characterized by AFM, FETEM, powder XRD, DLS, and FTIR analysis. Interestingly, HANPs could also be synthesized using an ∼20 kDa protein purified from the secreted protein extract obtained from L. plantarum MTCC 1325, which suggested that this lower molecular weight protein fraction was perhaps significantly involved in biomineralization-based generation of HANPs. In order to develop a therapeutic bactericidal nanocomposite, HANPs were loaded with the antibiotic polymyxin B (PB). A Langmuir isotherm model was evident in the studies that measured adsorption of PB onto HANPs. A sustained release profile of PB from the nanocomposite was observed in buffers having varying pH and in simulated body fluid. The nanocomposite (PB−HNC) exhibited bactericidal as well as antibiofilm activity against Pseudomonas aeruginosa MTCC 2488 and was nontoxic to cultured human embryonic kidney cells. KEYWORDS: biomineralization, lactic acid bacteria, hydroxyapatite nanoparticle, antibiotic delivery, antibiofilm

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INTRODUCTION Biogenic nanomaterials have come to the forefront based on their tremendous application potential in healthcare, especially in the domain of diagnosis and treatment of diseases.1−8 Notwithstanding the plethora of chemical synthesis methods available for generating nanomaterials, these methods warrant circumspection owing to the utilization of toxic chemicals. Hence, there is a need to develop facile methods for the synthesis of nanoscale materials in order to leverage their potential in biomedical applications. A case in point is the utilization of microbes for the synthesis of nanomaterials, which is conceived to be a promising approach.9−14 Productions of nanomaterials using enzyme-based methodologies can also yield a safe, eco-friendly, and green route of synthesis. Further, a major benefit of a biogenic approach is that a large number of bacterial species are known to inhabit at ambient conditions of temperature, pressure, and pH. Conceivably, nanomaterials can perhaps be generated using these microbes using facile conditions.15−17 Biomineralization is a ubiquitous phenomenon observed in living beings, starting from the microorganisms to vertebrates.18−20 It essentially encompasses a process where micro© 2019 American Chemical Society

to nanoscale materials are synthesized by organisms. The salient examples of biomaterials that occur in nature include calcium carbonate (CaCO3) in mollusk shells and sponges, hydroxyapatite [Ca10(PO4)6(OH)2], which is prevalent in bones and teeth of vertebrates, amorphous silica (SiO2), known to be present in diatoms and sponges, magnetite (Fe3O4) nanocrystals in magnetotactic bacteria, and chiton teeth.21−23 Inspired by the natural examples of biomineralization, researchers are trying to adopt biomimetic approaches to synthesize inorganic and metal nanomaterials.24−29 Biominerals can be of different forms, sizes, and shape. These minerals can also be formed by using lipid, polysaccharide, and protein-mediated matrix.30−36 In modern healthcare, hydroxyapatite nanoparticles (HANPs) hold special interest, given their application potential in bone implantation and regeneration and anticancer and cell labeling applications.37−40 Although a plethora of established chemical methods are available for synthesizing HANPs,41,42 they warrant circumspection owing to the utilization of toxic Received: April 5, 2019 Accepted: June 3, 2019 Published: June 3, 2019 2927

DOI: 10.1021/acsabm.9b00293 ACS Appl. Bio Mater. 2019, 2, 2927−2936

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ACS Applied Bio Materials

pentosaceus CRA51 amounted to 44.13 μg/mL, 15.63 μg/mL, and 17.15 μg/mL, respectively. Synthesis of HANPs using the LAB protein extracts was essentially carried out by a wet precipitation method.29 Following synthesis, HANPs were recovered by centrifugation at various speeds. Interestingly, HANPs harvested at 10 000 rpm exhibited the least heterogeneity in shape and size and significant crystallinity (Supporting Information, Figure S1). Hence, all the subsequent experiments pertaining to characterization of the synthesized nanoparticles were pursued with HANPs recovered by centrifugation at 10 000 rpm. The two-dimensional topography, amplitude channel, and three-dimensional topography images following AFM analysis revealed that the average height profile of HANPs obtained from the extracts of L. plantarum MTCC 1325, L. plantarum CRA52, and P. pentosaceus CRA51 were approximately 2.38, 3.13, and 4.11 nm, respectively (Figure 1A−1C, Figure S2A−S2F). SAED pattern analysis clearly revealed the crystalline nature of the synthesized HANPs (Figure 1D−1F), while FETEM analysis revealed the morphology of the synthesized HANPs (Figure 1G−1I). FETEM and ImageJ software analysis indicated that the particle size distribution of HANPs obtained from the cell-free protein extract of L. plantarum MTCC 1325 ranged from 75 to 100 nm, while those obtained from the extracts of L. plantarum CRA52 and P. pentosaceus CRA51 were 50−60 nm. It may be mentioned here that the average particle size of HANPs obtained from the LAB extracts compares well with the size of HANPs generated by various routes of synthesis (Table S1). TGA analysis showed a single-step degradation of HANPs from 20 to 400 °C, and the resulting residual mass of HANPs obtained at 1200 °C from the protein extracts of L. plantarum MTCC 1325, L. plantarum CRA52, and P. pentosaceus CRA51 was 53%, 69%, and 61%, respectively (Figure S3A−S3C). The decrease in the mass of HANPs observed in TGA until 1000 °C can perhaps be attributed to an initial loss of weakly entrapped water molecules followed by the loss of lattice water at higher temperature.50−52 FTIR analysis of HANPs revealed the presence of peaks at 3568, 1461, and 1041 cm−1, which correspond to OH−, CO32−, and PO43− stretching frequencies, respectively (Figure S4), while HR-TEM revealed that the synthesized HANPs were oval in shape with the lattice distances being 2.36, 1.9, and 0.9 nm for HANPs obtained from the protein extracts of L. plantarum MTCC 1325, L. plantarum CRA52, and P. pentosaceus CRA51, respectively (Figure S5A−S5C). Evidence for the formation of HANPs from cell-free extract of the LAB strains and their crystalline nature was further substantiated by powder XRD analysis, wherein the sharp peaks observed at around 2θ = 26° corresponding to the (002) lattice plane and 2θ = 31° corresponding to the (211) lattice plane suggested the presence of crystalline hydroxyapatite (Figure S5D−S5F).44,53 It may be mentioned here that the HANPs obtained from the cell-free extracts of the LAB strains compared well with the HANPs obtained by a chemical synthesis method,29 which was also characterized and included as a control sample for comparison (Figure S6). Generation of HANPs with Protein Fractions of LAB Cell-Free Extract. In the context of the biogenic synthesis of HANPs by the cell-free extract from LAB strains, the proteins present in the cell-free extract presumably render the formation of a weak organic scaffold−metal ion complex and a nucleation center for the mineralization to set in.36 However, the cell-free extract of the LAB strains is complex in nature and likely to have a mixture of proteins. The nuances with regard to the role(s) of

reagents. This has kindled a great interest in bringing forth facile methods for the synthesis of HANPs. To this end, a bioinspired mineralization process offers new opportunities for generating hydroxyapatite like material.43−46 In this context, the acidic secreted proteins from lactic acid bacteria (LAB) referred to as SLPs herein can perhaps serve as templates. Further, it is reasonable to conceive that the SLPs from LAB can act as a nucleation center by anchoring the cation (Ca2+) followed by binding of counteranion ([HPO4]2−) (Scheme 1A) and steer the biomineralization-based generation of HANPs (Scheme 1B). Scheme 1. Cartoon Illustrating (A) the Role of Acidic Secreted Proteins from LAB (SLPs) in Anchoring Ca2+ and Capture of Counter-Anion and (B) Hydroxyapatite Nanoparticles (HANPs) Obtained through the SLPMediated Biomineralization Process

The biomineralization-derived synthesis strategy is an emerging tool for generating materials that have potential for translational biomedical applications. However, it is paramount that the proteins or peptide templates used for such bottom-up synthesis not only favor nucleation but also are nontoxic and biocompatible. Importantly, the acidic secreted proteins from LAB are less likely to have toxic implications given the GRAS (generally regarded as safe) status of LAB and their probiotic attributes.47−49 Based on this rationale, herein we report an interesting biogenic approach toward the generation of biocompatible hydroxyapatite nanoparticles (HANPs) in the presence of proteins secreted by LAB and demonstrate their prospect in the development of an antibiotic-loaded nanocomposite having potent antibiofilm activity.

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RESULTS AND DISCUSSION Generation of HANPs from LAB Cell-Free Extract. In the present study, the cell-free extract obtained from the LAB strains Lactobacillus plantarum MTCC 1325, Lactobacillus plantarum CRA52, and Pediococcus pentosaceus CRA51 was used as nucleating agent to synthesize biomineralization-based HANPs. The protein content obtained from the cell-free extract of L. plantarum MTCC 1325, L. plantarum CRA52, and P. 2928


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Figure 1. 2D topography-based AFM images, SAED pattern, and FETEM analysis of HANPs obtained from (A, D, G) L. plantarum MTCC 1325, (B, E, H) L. plantarum CRA52, and (C, F, I) P. pentosaceus CRA51, respectively. The horizontal line in the AFM images represents the regions selected for measuring the height line profile of the samples.

Figure 2. (A) SDS-PAGE analysis of the crude cell-free extract (Lane 1) and the gel-eluted fraction 3 obtained from the cell-free extract of L. plantarum MTCC 1325. (B) HPLC profile for (I) crude cell-free extract and (II) the gel-eluted fraction 3 obtained from the cell-free extract of L. plantarum MTCC 1325.

the individual protein(s) and the mechanistic insights of the process of mineralization for generation of HANPs are perhaps difficult to realize owing to the inherent heterogeneity of the LAB cell-free extracts. Hence, it would be pertinent to unravel the role of the LAB secreted protein(s) in the mineralization process in order to bridge the knowledge gap, identify the key

players, and provide a guideline to select appropriate biomacromolecular templates for nucleation and generation of HANPs. To test the above-mentioned premise, the major protein bands present in the LAB cell-free extract were initially identified. SDS-PAGE analysis revealed that the major proteins 2929


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Figure 3. Characterization HANPs synthesized from purified fraction 3 protein obtained from L. plantarum MTCC 1325. (A) (i) FETEM image analysis. Scale bar is 100 nm. (ii) HR-TEM analysis indicating lattice distance of HANPs and (iii) SAED pattern of HANPs. (B) DLS analysis and (C) powder XRD analysis of HANPs.

present in the cell-free extracts were ∼75 kDa, ∼25 kDa, and ∼20 kDa in size in the case of L. plantarum MTCC 1325 (Figure S7A, lane 1). For L. plantarum CRA52, the major proteins were observed to be ∼25 kDa and ∼20 kDa in size (Figure S7A, lane 2), whereas in the case of P. pentosaceus CRA51, the major proteins were observed to be ∼95 kDa and ∼43 kDa in size (Figure S7A, lane 3). A standard acid−base titration36 experiment suggested that both the crude protein extract as well as the gel-eluted protein fractions obtained from the LAB strains were essentially acidic in nature. In order to ascertain whether the synthesis of HANPs could be accomplished by using separate protein fractions, the three proteins (∼75 kDa, ∼25 kDa, and ∼20 kDa in size) were separately eluted from the gel following SDS-PAGE of the crude cell-free extract of L. plantarum MTCC 1325. The eluted fraction 3 was comprised of the ∼20 kDa protein as evidenced from the single band obtained on the gel (Figure 2A, lane 2). Further, when subjected to HPLC, the eluted fraction 3 revealed a single peak with a retention time of around 11.65 min, which was similar to that obtained for peak 1 in the crude extract (Figure 2B). This suggested that the gel-eluted fraction 3 likely corresponded to the purified ∼20 kDa protein. Following secondary structure analysis, it was evident that the ∼20 kDa protein eluted in fraction 3 was essentially a beta-sheet-rich protein (57%) (Figure S7B). The fraction 3 protein-mediated synthesis of HANPs was subsequently pursued by a wet precipitation method. Interestingly, FTIR analysis of HANPs revealed that the significant peaks of OH − stretching frequency (3568 cm−1) and asymmetric stretching frequencies of CO32− (1461 cm−1) and PO43− (1041 cm−1), respectively, were retained in the HANPs synthesized using the 20 kDa protein fraction also, which suggested that this protein fraction was perhaps significantly involved in biomineralization-based generation of the nanoparticle (Figure S8A). FTIR analysis also suggested that only fraction 3 protein supported biomineralization-based generation

of HANPs as the characteristic peaks with the expected stretching frequencies was not evident in HANPs prepared from either fraction 1 or fraction 2 proteins (Figure S8B). TGA analysis of HANPs generated with the fraction 3 protein revealed a degradation pattern from 20 to 400 °C, and the resulting residual mass of HANPs was 66% (Figure S8C). FETEM analysis suggested that the HANPs obtained from fraction 3 protein of L. plantarum MTCC 1325 were spherical with an average particle size of 40−60 nm (Figure 3A, panel (i)). HR-TEM micrographs showed the presence of a prominent lattice in the nanoparticles, and the lattice distance was 0.37 nm (Figure 3A, panel (ii)). The SAED pattern also supported that the synthesized HANPs were crystalline in nature (Figure 3A, panel (iii)). Solution-based particle size analysis by DLS indicated that the HANPs obtained from purified protein fraction 3 were around 354 nm in size (Figure 3B). Evidence for the crystalline nature of HANPs generated from purified fraction 3 was further acquired by powder XRD analysis, wherein the sharp peaks observed at around 2θ = 26° corresponding to the (002) lattice plane and 2θ = 31° corresponding to the (211) lattice plane suggested the presence of crystalline hydroxyapatite (Figure 3C).44,53 Similarly, protein fractions from P. pentosaceus CRA51 were eluted, and the eluted fractions were used to generate HANPs. Powder XRD analysis and SAED pattern indicated that only the HANPs obtained from the crude extract and the ∼43 kDa protein fraction were crystalline in nature (Figure S9A−C, S9D−F, inset), while HR-TEM analysis revealed that the HANPs were oval with the lattice distances being 0.9, 0.37, and 0.38 nm for HANPs obtained from the crude protein extract, 95 kDa protein, and 43 kDa protein obtained from P. pentosaceus CRA51 (Figure S9D−F). In order to obtain a nuanced understanding of the role of the protein fraction in the biomineralization process, ITC analysis was pursued. ITC studies revealed that the presence of the protein template supported biomineralization, as the binding of the PO43− ion with the Ca2+−protein complex was favorable (K = 5.71 × 105 2930


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Figure 4. (A) Adsorption isotherm of polymyxin B on HANP. (B) Linear regression plot for estimation of adsorption isotherm parameters for polymyxin B. (C) In vitro release kinetics of polymyxin B from PB-HNC incubated in various buffers and simulated body fluid.

Figure 5. (A) Time kill curve of PB-HNC against P. aeruginosa MTCC 2488. (B) FETEM analysis of (i) P. aeruginosa MTCC 2488 cells and (ii) P. aeruginosa MTCC 2488 cells treated with 10 μM PB-HNC. Scale bar is 1.0 μm and 500 nm, respectively. Arrow in 5B (ii) indicates disintegration of P. aeruginosa MTCC 2488 cells treated with PB-HNC. (C) cFDA-SE leakage assay and (D) PI uptake assay on P. aeruginosa MTCC 2488 cells treated with 10 μM PB-HNC. (E) Fluorescence microscope-based live/dead assay with PB-HNC-treated P. aeruginosa MTCC 2488 cells using cFDA-SE and PI. Scale bar for the images is 50 μm.

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Figure 6. MTT-based assay for estimating (A) inhibition and (B) eradication of P. aeruginosa MTCC 2488 biofilm grown in the presence of PB-HNC. (C) Fluorescence microscopic analysis to study inhibition of P. aeruginosa MTCC 2488 biofilm grown in the presence of PB-HNC. Scale bar for the images is 100 μm. (D) MTT assay to determine cytotoxic effect of PB-HNC on HEK 293 cells. Each data point represents mean ± standard deviation from six samples.

M−1) (Figure S10A), which facilitated the subsequent crystal growth process. On the contrary, the binding of the PO43− ion with Ca2+ was not very stable in the absence of the protein template (K = 4.13 × 104 M−1) (Figure S10B), which reiterated the importance of the protein scaffold in the nucleation process. Control experiments were also performed, where the protein extract was titrated with only phosphate ions, but no interaction was recorded, presumably owing to the negatively charged LAB protein fraction as well as PO43− ion (Figure S10C). Loading and in Vitro Release Profile of Antibiotics. In order to leverage the potential of the synthesized HANPs in antibacterial applications, polymyxin B (PB)-loaded hydroxyapatite nanocomposite (PB-HNC) was generated. PB is a membrane-acting cyclic cationic peptide, which primarily targets Gram-negative bacteria.54,55 Pseudomonas aeruginosa is an opportunistic pathogen, which is implicated in a number of serious ailments,56,57 and PB is largely used as an antibiotic to target P. aeruginosa.58,59 Hence to generate a PB-loaded payload and target P. aeruginosa, HANPs were incubated with varying concentrations of PB for 12 h to facilitate adsorption of the antibiotic. Following an NPN assay, a calibration plot was generated with known concentrations of the antibiotic (refer to Supporting Information) in order to quantify adsorption of PB onto HANPs. The adsorption capacity or strength of adsorption (qe) could be correlated to the concentration of PB. The adsorption of PB on HANPs revealed a saturation effect at antibiotic concentrations in excess of 300 mg/L (Figure 4A).

The adsorption isotherm parameters Qm and b were around 500 mg/g and 10965 L/mg, respectively. From the adsorption studies it was evident that the adsorption of the antibiotic on HANPs followed a Langmuir isotherm model (Figure 4B). In the context of antibacterial application, it was critical to ascertain whether the PB-HNC nanocomposite was able to render sustained release of the antibiotic in a physiologically relevant niche. To this end, in an acidic buffer (citrate buffer pH 3.0), the composite rendered rapid release of the antibiotic, with a plateau observed at around 70% release of PB following 48 h of incubation. At physiological pH (HEPES buffer pH 7.4), a sustained release profile of PB was observed with a cumulative release of around 40% following 48 h. Further, a sustained release profile was also observed in the case of simulated body fluid (SBF), and a plateau was observed at around 50% release of the antibiotic (Figure 4C). Antibacterial Activity of PB-HNC. The minimal inhibitory concentration (MIC) and minimal killing concentration (MKC) of PB against P. aeruginosa MTCC 2488 cells were 0.75 μM and 3.125 μM, respectively (Figure S11A-B), while the MIC and MKC of PB-HNC against P. aeruginosa MTCC 2488 cells were 6.25 μM and 25 μM, respectively (Figure S11C,D, Table S2). Time kill curve analysis indicated a notable decrease in the viability of P. aeruginosa cells (up to 2.0 Log10 CFU) upon treatment with PB-HNCs loaded with 10 μM PB, while the viability of the target cells remained unaffected upon treatment with only HANPs (Figure 5A). The bactericidal activity of PB2932


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carboxyfluorescein diacetate succinimidyl ester (cFDA-SE), propidium iodide (PI), congo red (CR), polymyxin B, Dulbecco’s modified eagle medium (DMEM), trypsin-EDTA, 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide (MTT), potassium bromide, glycine, and SDS were procured from Sigma-Aldrich. Bacterial Strains and Growth Conditions. Lactobacillus plantarum MTCC 1325, Lactobacillus plantarum CRA52, and Pediococcus pentosaceus CRA51 were propagated in MRS broth at 37 °C for 18 h in static conditions. Pseudomonas aeruginosa MTCC 2488 was grown in NB at 37 °C and 180 rpm for 12 h. All the bacterial strains were revived from frozen stocks and were subcultured prior to their use in experiments. Preparation of LAB Protein Extract. L. plantarum MTCC 1325, L. plantarum CRA52, and P. pentosaceus CRA51 were grown in 100 mL of MRS broth under static conditions at 37 °C for 18 h. The cell-free supernatant (CFS) was recovered by centrifugation at 8000 rpm for 5 min at 4 °C. Solid ammonium sulfate was added slowly to the CFS solutions at a final concentration of 40% (w/v),60,61 and the mixture was gently stirred at 4 °C until the protein precipitation was visible. The resulting precipitate was subsequently collected by centrifugation at 12 000 rpm for 10 min at 4 °C, suspended in water, and dialyzed using a 12 kDa cutoff dialysis bag (Sigma, USA) over a 24 h period. The amount of protein present in the dialysate was estimated by Bradford reagent (Sigma-Aldrich, USA). The dialysate was lyophilized and stored at −20 °C. Synthesis of Biogenic Hydroxyapatite Nanoparticle (HANP). Preparation of HANPs was accomplished by an in situ precipitation method.29 Initially, a 5.0 M solution of Ca(NO3)2·4H2O was prepared. To 6.0 mL of 5.0 M Ca(NO3)2·4H2O, NH4OH was added dropwise to adjust the pH of the solution to 9.0. A 5.0 mL aliquot of the LAB protein extract solution (20 μg/mL protein) was then slowly added to the pHadjusted calcium salt solution with constant stirring at room temperature. A control experiment was also set up in parallel, using only sterile Milli-Q water instead of LAB protein extracts. Subsequently, in the reaction mixture, 0.018 mol of (NH4)2HPO4 was then added to maintain Ca:P molar ratio of 1.67:1. The pH of the reaction mixture was again adjusted to 9.0 by dropwise addition of NH4OH. The suspension was incubated for 24 h at room temperature in static condition and centrifuged at 10 000 rpm for 5 min, and the supernatant was discarded. The obtained precipitate was then washed twice with Milli-Q water to remove NO3− ions as well as the loosely bound LAB proteins. The pellets were lyophilized followed by calcination at 600 °C for 2 h to obtain HANPS, which were then stored in a freezer at −20 °C. Characterization of HANPs. The synthesized HANPs were characterized by FTIR analysis, powder XRD, FETEM, atomic force microscopy (AFM), dynamic light scattering (DLS), and TGA (refer to Supporting Information). Analysis of Proteins Present in LAB Extract. The dialyzed protein fractions obtained from the LAB strains were analyzed by SDSPAGE.62 Prestained high range protein marker (SRL, India) was run along with the samples. Following electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250 dye for 4 h. The gel was subsequently destained in methanol:acetic solution (25:10 v/v), and its image was captured in a Geldoc system (BioRad, USA). The prominent bands observed on an SDS-PAGE gel were then subjected to a protein elution procedure using the IN-Gel digest protocol (Sigma, USA) but without using trypsin enzyme. The eluted protein fractions were lyophilized, and the amount of the protein was again quantified by Bradford assay. A 20 μL aliquot of the crude extract (20 μg/mL) and the gel-eluted purified fraction (fraction 3, 14 μg/mL) obtained from L. plantarum MTCC 1325 were subjected to reverse-phase HPLC (PerkinElmer Analytical HPLC system, USA) using an analytical RPHPLC column (Agilent Analytical C18 column) with a series 200 HPLC pump and a series 200 UV/vis detector. The solvents used were solvent A (0.1% TFA in Milli-Q water) and solvent B (0.1% TFA in acetonitrile), each at a flow rate of 1.0 mL/min. The run conditions were as follows: 0 to 5 min 5% solvent A, 5 to 60 min a gradient of 5% to 95% solvent B. The eluted fractions were collected from several runs, and the solvent was vacuum evaporated in a SpeedVac concentrator (Eppendorf). The obtained fractions were finally resuspended in sterile

HNC was also captured in FETEM analysis, which indicated significant cellular damage and loss of the typical morphology in nanocomposite-treated P. aeruginosa MTCC 2488 cells (Figure 5B). Treatment of target bacterial cells with the nanocomposite resulted in significant leakage of cFDA-SE dye, indicating extensive membrane perforation (Figure 5C). A PI assay supported the membrane-directed bactericidal activity of the PB-HNC nanocomposite (Figure 5D). Fluorescence microscopy with cFDA-SE and PI also provided additional evidence for the membrane-directed activity of PB-HNC (Figure 5E). Antibiofilm Activity of PB-HNC. A standard MTT assay indicated notable eradication of P. aeruginosa MTCC 2488 biofilm as evident in the reduction of the metabolic activity associated with the biofilm with increasing concentration of PBHNC. The minimal biofilm inhibitory concentration (MBIC50) and minimal biofilm eradication concentration (MBEC50) of PB-HNC against P. aeruginosa MTCC 2488 were observed to be around 75 μM (Figure 6A) and 150 μM (Figure 6B), respectively. Inhibition of P. aeruginosa biofilm growth by PBHNC was also corroborated by crystal violet staining, which indicated a dose-dependent decrease of biofilm biomass (Figure S12). The antibiofilm activity of PB-HNC was also validated by fluorescence microscope analysis with cFDA-SE and congo red, wherein a loss of cell viability and reduction in biofilm matrix upon treatment with 100 μM PB-HNC was evident (Figure 6C). In Vitro Cytotoxic Effect of PB-HNC. In order to determine the cytotoxic effect of PB-HNC on model human cells, a standard MTT assay was performed on HEK 293 cells (human embryonic kidney cells). At concentrations equivalent to its MBIC and MBEC against P. aeruginosa MTCC 2488 (75 μM and 150 μM, respectively), PB was toxic to the cells (Figure 6D). In contrast, HANPs as well as PB-HNC were distinctly nontoxic to HEK 293 cells (Figure 6D). The slow release of PB from the nanocomposite perhaps reduced the local concentration of the antibiotic, which in turn curtailed the toxic effect on HEK 293 cells.

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CONCLUSIONS The present study demonstrated that the secreted protein(s) from LAB serve as templates and provide a facile and a promising conduit for synthesis of hydroxyapatite nanoparticles (HANPs) for biomedical applications. It is envisaged that in the future a more rigorous proteomic approach can perhaps identify appropriate protein templates from LAB for nucleation and mineralization in order to generate safe scaffolds for biomedical application. Interestingly, the HANPs generated in the present study could be leveraged to develop a PB-loaded hydroxyapatite nanocomposite (PB-HNC), which rendered a sustained release of the antibiotic, displayed potent activity against P. aeruginosa biofilm, and was nontoxic to cultured human cells at their bactericidal concentrations. It is envisaged that the present study would provide a rational guideline toward green synthesis and generation of biocompatible materials for translational biomedical applications using beneficial microbes such as LAB.

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EXPERIMENTAL SECTION

Materials. Nutrient broth (NB), Lactobacillus MRS broth (MRS broth), and crystal violet (CV) dye were procured from HiMedia (India). Dimethyl sulfoxide (DMSO), acetic acid glacial, methanol, absolute ethanol, and ammonium sulfate were obtained from Merck (India). N-2-Hydroxyethyl piperazine N-2 ethanesulfonic acid (HEPES buffer), high range protein ladder, and Coomassie brilliant blue R-250 were procured from Sisco Research Laboratories (India). Five (and 6)2933


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ACS Applied Bio Materials Milli-Q water and subjected to further analysis. CD spectra of Fraction 3 protein (14 μg/mL in sterile water) obtained from L. plantarum MTCC 1325 was recorded between 180 and 300 nm in a spectropolarimeter (JASCO, J-815) with a 5.0 mm path length cuvette at 25 °C. The baseline of the instrument was calibrated with sterile Milli-Q water. Measurements were conducted using three independent samples, and for every sample, three scans were recorded and averaged in order to enhance the signal-to-noise ratio. The spectra were expressed in terms of millidegree (mdeg). Isothermal Titration Calorimetry (ITC). ITC measurements were conducted at 37 °C in a VP-ITC device (MicroCal, Northampton, USA). To prevent the formation of air bubbles, the buffers were degassed under vacuum prior to experiment. The crude protein extract from P. pentosaceus CRA 51 (20 μg/mL) and 5.0 mM aqueous solution of Ca(NO3)2·4H2O were taken in Milli-Q water to minimize heats of dilution. To study the binding of ammonified Ca−protein complex and (NH4)2HPO4, the Ca−protein complex was dissolved in Milli-Q-grade water (final protein and Ca concentrations were nearly 17 μg/mL and 0.83 μM, respectively) and loaded onto the cell and titrated against (NH4)2HPO4. The integrated heat effects were ascertained by nonlinear regression using a single site-binding model (Microcal Origin, version 5.0). Synthesis of HANPs with Fractionated Protein. HANPs were also synthesized with the fractionated protein obtained from L. plantarum MTCC 1325 by essentially following the biogenic method described earlier. The synthesized HANPs were characterized by FTIR analysis, FETEM, powder XRD, and TGA analysis. PB-Loaded Hydroxyapatite Nanocarrier (PB-HNC). HANPs (1.0 mg/mL in sterile Milli-Q water) were incubated with varying concentrations of PB (25−800 μM) on a rocker at room temperature overnight. In order to recover PB-HNC, the solution was centrifuged at 10 000 rpm for 5 min, and the pellet was resuspended in sterile Milli-Q water. The amount of PB bound to HANPs was estimated indirectly by measuring the amount of the nonadsorbed antibiotic present in the supernatant by an NPN assay (refer to Supporting Information). Based on quantification of PB adsorbed onto HANPs, the adsorption isotherm model and adsorption capacity of HANPs were also determined. In Vitro Release Kinetics of PB from PB-HNC. In separate sets, PB-HNCs loaded with 300 μM PB were dispersed in 1.0 mL of 10 mM HEPES buffer (pH 7.4), 100 mM citrate buffer (pH 3.0), and simulated body fluid (SBF) (pH 7.4), respectively. The samples were incubated in an orbital shaker at 120 rpm at 37 °C, and at intermittent periods (1, 3, 6, 12, 24, and 48 h) the samples were withdrawn and centrifuged at 10 000 rpm for 5 min. The amount of PB released from PB-HNC was ascertained by measuring the amount of the antibiotic released in the supernatant by an NPN assay and expressed as % cumulative release. Antibacterial Activity of PB-HNC. The minimum inhibitory concentration (MIC) and minimum killing concentration (MKC) of PB-HNC, HANPs, and PB were determined by following a standard protocol (see Supporting Information). The antibacterial activity of PBHNC was also ascertained by (a) time-kill curve, (b) CFDA-PI assay, and (c) microscopic analysis (See Supporting Information). Antibiofilm Activity of PB-HNC. Biofilm of P. aeruginosa MTCC 2488 was grown in a sterile 96-well microtiter plate (refer to Supporting Information) and treated for 24 h in separate sets with various samples of PB-HNC composite wherein the concentration of PB was 25 μM, 50 μM, 75 μM, 100 μM, 150 μM, 200 μM, 300 μM, and 400 μM and the corresponding mass of HANP was 1.0 mg/mL for every concentration of the antibiotic. P. aeruginosa MTCC 2488 biofilm was also treated with PB alone having similar sets of concentrations. Following treatment, the wells were washed with sterile phosphate-buffered saline (PBS), and an MTT and crystal violet-based assay was performed for all the treated samples (see Supporting Information). Cytotoxic Effect of PB-HNC. Cytotoxicity of PB-HNC was assessed in vitro against the human embryonic kidney cell line (HEK 293 cells) by a standard MTT assay. For testing the cytotoxic effect, HEK 293 cells were treated in separate sets with PB-HNC (loaded with PB in the range of 0.325−400 μM). Seeding of HEK 293 cells for the

assay, the treatment method, and the conditions of the MTT assay were essentially based on a previously described method.63

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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/acsabm.9b00293. Additional experimental procedures. FESEM, SAED pattern, AFM, TGA, FTIR, FETEM, powder XRD, and HR-TEM analysis of HANPs. SDS-PAGE analysis of crude protein extracts from LAB, and CD analysis of purified protein fraction from L. plantarum MTCC 1325. ITC analysis for estimating binding isotherms. MIC and MKC plots. Crystal violet assay for biofilm inhibition (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Aiyagari Ramesh). Fax: +91-3612582249. Tel.: +91-361-2582205. *E-mail: [email protected] (Gopal Das). Fax: +91-361-2582349. Tel.: +91-361-2582313. ORCID

Gopal Das: 0000-0003-0043-1372 Aiyagari Ramesh: 0000-0002-4272-298X Funding

A.R. received research grant from the Department of Biotechnology [BT/PR13560/COE/34/44/2015] and G.D. received research grant from the Science & Engineering Research Board [SR/S1/OC-62/2011]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to thank the Department of Biotechnology [BT/PR13560/COE/34/44/2015] and the Science & Engineering Research Board [SR/S1/OC-62/2011] for research grants. We thank Central Instruments Facility, IIT Guwahati for FESEM, FETEM, HR-TEM, TGA, and AFM analysis and Department of Chemistry, IIT Guwahati, for DLS and FTIR analysis. P.M. and S.M. thank IIT Guwahati for a research fellowship.

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REFERENCES

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Generation of a Hydroxyapatite Nanocarrier through Biomineralization

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