Annealing dependent synthesis of cyto-compatible nano-silver/calcium hydroxyapatite composite for antimicrobial activities

2.1 Isolation and screening of ureolytic bacteria

The samples were collected from the toilet dropping from public toilet for isolation of ureolytic bacteria. Sterilized swab was dipped in sterile distilled water immediately and carried out to the laboratory within 6 h. To isolate bacterial colonies from the sample a pour plate technique was used. Urea base agar and nutrient agar medium was used for bacterial isolation and purification was followed from the sample details in the supplementary data as shown in Fig. S1.

2.2 Preparation of synthetic urine

Synthetic urine was prepared as outlined by (Webster et al., 2000). Synthetic urine is made up of the following ingredients (g/L) purchased from Sigma Aldrich, Mumbai: 0.651 g calcium chloride, 0.651 g magnesium chloride, 4.6 g sodium chloride, 2.8 g potassium dihydrogen phosphate, 2.3 g sodium sulphate, 1.6 g potassium chloride, 1.0 g ammonium chloride, 1.1 g creatinine, 10 g trypticase soya broth (or) tryptone in 1000 mL water followed by sterilization then 25 g of urea was added in Fig. S2.

2.3 Estimation of biogenic ammonia production

A total of 100 mL of synthetic urine was prepared, seeded with ureolytic bacterial culture, and kept at room temperature for three days. The pH of the medium was measured, and the enrichment of bacterial culture was checked for ammonia generation using the Indo phenol method (Miernik et al., 1991). The synthetic urine was collected and spun at 6000 rpm for 30 min (Dhandapani et al., 2020 Jun 1), and the supernatant was filtered through a membrane filter (0.2 M). The presence of ammonia in the filtrate solution was determined using the indophenol technique. The resulting sample was kept at room temperature for 30 min, and the absorbance was read at 625 nm using UV–vis (Thermo Scientific Evolution 201) to determine the ammonia concentration as shown in the supplementary Fig. S2.

2.4 Determination of pH

The ureolytic bacteria were introduced into synthetic urine and kept for 7 days at room temperature. The change in pH during ammonia production was measured using digital pH meter (Eutech Instruments pH 510 - as shown in the supplementary Fig. S3 (Brundavanam et al., 2015).

2.5 Chemical oxygen demand (COD)

Digester solution was prepared by adding 1.02 g K₂Cr₂O₇ was added in 16.8 mL of concentrated H₂SO₄ which was diluted in 50 mL of Millipore water containing 3.34 g of HgSO₄, finally total volume was made up to 100 mL. Catalyst solution was made by dissolving 1.34 g of Ag₂SO₄ in 250 mL of concentrated H₂SO₄ and stirring constantly until a clear solution was obtained. The 1.5 mL of above said digester solution and catalysis solution 3.5 mL was mixed with 2.5 mL of sample. For comparative study of COD, three different samples were analyzed (Urea broth, Synthetic urine, and Human urine) and the mixture were kept in the COD digester for 2 h at 150 °C. Later the samples were analyzed in COD meter at 420 nm as shown in the supplementary Fig. S4 (Reeta Mary et al., 2018).

2.6 Estimation of carbonate and bicarbonate (Alkalinity reaction)

The acid-base titration is suitable for alkalinity solution where the alkalinity is neutralized to acid pH by using 0.5 N HCl as burette solution. At acidic pH, the solution contains carbonate, bicarbonate, and hydroxide. These weak bases were volumetrically calculated using acid-base titrations using strong acids such as diluted H₂SO₄ and phenolphthalein (P) and methyl orange (MO) as an indicator. In the burette, standard acid was taken, and the sample was taken in the Erlenmeyer flask. The P indicator was introduced and titrated, and the reading was recorded as “P” when it reached the end point. Then MO indicator was added to the Erlenmeyer flask, and the titration was maintained until the end point was reached, which was labeled as “M”. The nature and amount of bases present in the solution may be easily determined based on the titrated values of “P” and “M” (Tian et al., 2016). The result was calculated by using the following formula:

$\mathrm{B}\mathrm{a}\mathrm{s}\mathrm{e}=\frac{\mathit{PorMvalue}\times NormalityofHCl\times mol.wt.ofcarbonateandbicarbonate}{\mathit{Volumeofsample}}$ (mg/L)

2.7 Biogenic ammonia mediated synthesis of silver doped hydroxyapatite

Calcium acetate, biologically activated ammonia, disodium hydrogen phosphate and silver nitrate are used as base material for the synthesis of hydroxyapatite (Won et al., 2010). The filtered biogenic ammonia from synthetic urine of about 250 mL was taken and mixed with disodium hydrogen phosphate. The mixture was continuously stirred for 1 h and 0.1 mM silver nitrate was added and stirred for 1 h. Calcium acetate (4.72 g) was dissolved thoroughly in 20 mL of sterile distilled water before being added drop by drop to the solution. The mixture was then mixed and heated at 70 °C for 6 h, then exposed to sunlight for silver photo-reduction, before being cooled to room temperature then the mixture was spun for 30 min at 6000 rpm. To eliminate excess ammonia, calcium, and phosphate (CP) ions, the precipitate was rinsed with distilled water. CP-50 °C, CP-400 °C, and CP-800 °C were used to calcinate the precipitate at varied annealing temperatures of the sample.

2.8 Characterization studies

2.9 Statistical analysis

Student's t-test was used to examine the statistical significance of the experiments. For three corresponding measurements, all the results were represented as mean ± SD.

3.1 Identification of bacteria

Urease test was used to classify organisms based on their ability to hydrolyze urea with Urease enzyme to identify ureolytic bacteria in a series of isolated single colony. The cultures were cultivated in urea broth medium with phenol red as a pH indicator. The positive bacteria used urea as a substrate and formed an alkaline environment in the presence of ammonia, which turned the phenol red into a deep pink color. The presence of urease enzyme and biochemical studies confirmed that the isolated microorganism as ureolytic Bacillus sp. (Fig. S1).

3.2 Production of ammonia

The isolated ureolytic Bacillus sp. was incubated in synthetic urine for 5 days at room temperature. Then the sample was withdrawn at regular time interval from the synthetic urine. The samples were filtered, centrifuged and the presence of ammonia was estimated by Indophenol blue method in UV spectrophotometer (Thermo Scientific Evolution 201) at 625 nm. The resultant production of ammonia was 9000 ppm on 3rd day of cultures in the medium shown in supplementary data (Fig. S2). The isolated ureolytic bacteria were inoculated into synthetic urine at initial pH 6.0. The production of ammonia was observed as the pH of the bacterium changed during its growth. The result of changes in pH was shown in supplementary data Fig. S3. The pH was gradually increased from pH 9.0 to pH 12.0 within 48 h. The COD was estimated in urea broth, synthetic urine and human urine using standard procedure. The level of COD in synthetic urine was greater than the urea broth but less than the human urine, almost the level of COD in synthetic urine was approximately equal to human urine and the details are provided in supplementary data (Fig.S4). The cultured synthetic urine by titration. It was estimated that bacteria produced 12 mg/L carbonate and of 19.75 mg/L bicarbonate in synthetic urine as shown in the supplementary data’s Table S1.

The isolated bacterial culture was introduced into the urease medium, which contains peptone and glucose, two necessary nutrients for a wide variety of bacteria. Potassium phosphate is a moderate buffer that prevents the medium from becoming alkaline due to peptone metabolism. When the media was below pH 8.4, phenol red indicated yellowish orange, and when the media was in the alkaline range, it signified red or pink. (pH 9–12). The urea hydrolysis of urea to ammonia by urease positive organisms changed the medium pH resulted in orange to pink. Urease catalyzes the hydrolysis of urea to produce ammonia and carbonate, which then hydrolyzes automatically to produce carbonic acid and another molecule of ammonia. The ureolytic bacterium which was isolated from toilet dropping was confirmed as Bacillus sp by biochemical test and it was inoculated in the synthetic urine to produce the ammonia. It was observed that 9000 ppm of ammonia was produced on third day which was in dissolved state in synthetic urine and combined with the carbonate and bicarbonate that was estimated by the alkalinity reaction.

The culture filtrate was rich in biogenic ammonia used for the synthesis of hydroxyapatite. Because of their chemical and structural similarities to the mineral phase of bone and tooth, calcium phosphate compounds have been explored for biomedical uses. Synthetic apatite's composition, physico-chemical characteristics, crystal size, and shape are particularly sensitive to preparative conditions, and non-stoichiometric calcium deficient hydroxyapatite (HA) powder has occasionally produced. Silver is doped on the surface of calcium hydroxyapatite due to electrostatic contact. The previous research of (Supraja et al., 2018) states the roughness of the calcium hydroxyapatite is increased due to the deposition of the silver particles which result in enhanced cell proliferation.

3.3 Characterizations of silver deposited hydroxyapatite

The UV–vis absorption spectrum of the synthesized silver deposited hydroxyapatite sample at different temperatures is given in supplementary data Fig. S5. There is a maximum absorption peak at 470 nm was observed for the samples prepared at 50 °C, 400 °C and 800 °C. The adsorption peak was gradually increased by increasing the temperature from 400 °C to 800 °C. This is an indication that AgNO₃ is reduced to Ag at higher temperature without organic or chemical reducing agents. The synthesized particle of ureolytic Bacillus sp. mediated hydroxyapatite was analyzed by XRD to know the nature of the particle. At 50 °C the particle was in amorphous state and at the 800 °C the particle was crystalline in nature and the peak at 26°, 29°, 31°, 32°, 33°, 34°, 37° and 40° theta showed the presence of hydroxyapatite Fig. 1. At 50 °C, only the broad peak at 32° appeared and then at 400 °C the peak at 26⁰, 29⁰, 37⁰ and 40⁰ got raised and all the linear peaks appeared at 800 °C which confirms the presence of the hydroxyapatite in crystalline nature. XRD of silver particles with one strong distinguishing peak at 2θ = 37.2 °C corresponds to the Braggs crystal faces of (1 1 1) and (2 0 0) (JCPDS # 04-0783) corroborating the characteristic of face-centered cubic structure. The zeta potential is a crucial characteristic in determining the hydrosol's dispersion state (nanoparticulate solution). It denotes that the overall charge that a particle obtains in each medium, as well as the charge present on the nanoparticles' surfaces, are responsible for the repulsive interactions that exist between them. If the minimum charge around the nanoparticles is −30 mV, the nanoparticles are said to be stable. At 50 °C, 400 °C, and 800 °C the silver doped hydroxyapatite had a zeta potential of 7.7 mV, -7.8 mV, and 11.1 mV respectively. An anionic charge on the surface of a composite material is shown in Fig. 2(a-c).

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Fig. 1

X-ray diffraction pattern of silver doped hydroxyapatite at different temperatures (A-50 °C, B-400 °C and C-800 °C).

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Fig. 2

Zeta potential study of silver doped hydroxyapatite at different temperatures (A-50 °C, B-400 °C and C-800 °C).

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The existence of the PO₄³⁻ group is shown by the primary peak being elevated at 961 cm⁻¹ in the laser Raman spectrum. The intensity of the peak at 50 °C was significantly lower than at 400 °C and 800 °C, which is attributable to the removal of the hydroxide and carbonate groups. At 800 °C, however, the disturbance of the peak was decreased by raising the temperature (Fig. 3). The size and shape of silver deposited hydroxyapatite nanoparticles were analyzed and shown by the TEM micrograph (Fig. 4). Individual silver particles as well as several aggregates are observed in the micrograph, that are spherical in shape and have an average particle size of 0.2 m.

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Fig. 3

Laser Raman spectral analysis of silver doped hydroxyapatite at different temperatures (A-50 °C, B-400 °C and C-800 °C).

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Fig. 4

Transmission electron microscopic analysis of silver doped hydroxyapatite at different temperatures (A-50 °C, B-400 °C and C-800 °C).

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The XRF spectrum of silver doped hydroxyapatite is shown in Fig. 5. The elemental maps for calcium (Ca), phosphor (P) and silver (Ag) was observed in which the highest peak was observed at Ca region followed by silver and P, respectively. FTIR spectra of silver doped hydroxyapatite is given in Fig. 6. The peaks at 3443 cm⁻¹ and 2361 cm⁻¹ were responsible to the asymmetrical stretching mode of O-H and C = O; an asymmetrical stretching of CH₂ was observed at 1450 cm⁻¹; the peaks at 1049 cm⁻¹, 866 cm⁻¹ and 563 cm⁻¹ were liable for to the asymmetrical stretching mode of O-H, C-C and C-H respectively. The peaks of 3443 cm⁻¹ and 2361 cm⁻¹ were present at 50 °C whereas these two peaks were not seen in 400 °C and 800 °C, which is due to the presence of H₂O absorption in the samples. The FTIR spectra obtained clearly showed the presence of calcium phosphate and carbonate groups, as well as hydroxyapatite.

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Fig. 5

X-ray fluorescence microscopic investigation of silver doped hydroxyapatite at 800 °C.

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Fig. 6

FTIR analysis of silver doped hydroxyapatite at A-50 °C, B-400 °C and C-800 °C.

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3.4 Antibacterial activity

The study of antibacterial activity suggested that silver doped hydroxyapatite could inhibit the growth of bacteria 6 and the zone was formed in Sample A (50 °C), B (400 °C) and C (800 °C) and the zone was not formed in the control and A (Fig. 7). As a result, the nanoparticles of silver doped hydroxyapatite samples are placed closer to the bacteria, resulting in direct interaction between the bacteria and the silver nanoparticles. This is owing to the calcium particles' hydrophilic groups, which aid in the dispersion of the silver nanocomposite in aqueous solution. Bacteria (E. coli) easily interact with hydrophilic surfaces; as a result, bacteria may easily attach to the hydrophilic surface of the nanocomposite, where the silver particles of the composite could kill the bacteria (Supraja et al., 2015; Bauer et al., 19661966; Saritha et al., 2019; Bindu et al., 2019). Our results of antibacterial study clearly show that infection of the implant materials will be decreased.

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Fig. 7

Antibacterial efficacy of silver doped hydroxyapatite at different concentrations (Control-CHAP, A-50 °C, B-400 °C and C-800 °C). The different letters on each column indicates the significant difference.

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3.5 Anticancer activity

The in-vitro cytotoxicity of the silver doped hydroxyapatite was evaluated against HeLa cell lines at different concentrations (Control and 10–50 µg/mL). The samples cytotoxicity analysis revealed a direct temperature response relationship, with cytotoxicity increasing as temperature rose (Fig. 8). The results demonstrated a considerable cytotoxicity against the HeLa cell lines. Silver deposited hydroxyapatite inhibited HeLa cell proliferation significantly with an IC₅₀ value of 30.9155 (124.6%) μg/mL of the concentration at 50 °C, whereas the IC₅₀ value of 40.3786 (1947%) μg/mL of the concentration at 400 °C and IC₅₀ value of 42.7488 (1014%) μg/mL at 800 °C respectively. Silver doped hydroxyapatite inhibited the proliferation of the cell line by less than 30% at the lowest concentration tested (10 g/mL). The existence of 50 g/mL of silver doped hydroxyapatite, drastically slowed the cell line's development (>75%). When compared to the temperature, the 800 °C shown highest cytotoxicity effect. Nanoparticles deplete intracellular antioxidants causing cancer cell death according to a previous study (Supraja et al., 2020; Anand et al., 2017; Renuka et al., 2020).

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Fig. 8

Anticancer efficacy of silver doped hydroxyapatite at different temperatures (Control, 50 °C, 400 °C and 800 °C).

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3.6 Brine shrimp assay

The larvicidal activity of the silver doped hydroxyapatite tested on brine shrimp was weak. The test sample concentration was inversely proportional to the degree of lethality (Paramanantham et al., 2019; Paramanantham et al., 2019; Sairengpuii et al., 2020). The highest mortality of 40% was observed at 50 °C while, 400 °C and 800 °C does not show any mortality (Fig. 9). The lethality of silver doped hydroxyapatite is very moderately toxic (active) if the LC50 value is less than 1000 mg/mL, but non-toxic (inactive) if the LC50 value is higher than 1000 mg/mL, indicating the existence of significant cytotoxicity (Bauer et al., 19661966). The ureolytic bacterial species are converted ammonia and carbonic acids from synthetic urine by urease and ammonia production as described by our previous report (Dhandapani et al., 2020). The culture filtrate was rich in biogenic ammonia used for the synthesis of hydroxyapatite and chemical ammonia was also used for the synthesis for comparative study. Calcium phosphate compounds have been studied for biomedical applications due to chemical and structural similarity to the mineral phase of bone and tooth. The composition, physico-chemical properties, crystal size and morphology of synthetic apatite are extremely sensitive to preparative conditions and sometimes it resulted into non-stoichiometric calcium deficient hydroxyapatite (HA) powder (Nagasundari et al., 2021; Mani et al., 2021). In this study, ureolytic bacteria were isolated and developed ureolytic bacteria mediated hydroxyapatite and doped with silver nanoparticles. The silver doped hydroxyapatite was characterized and its antibacterial activity against the pathogenic microorganism of E. coli was evaluated (Badineni et al., 2021). The present results (UV, FTIR, XRD, DLS, TEM) was in good in accordance with previous reports regarding silver doped HAp material preparation by various methods (Shi et al., 2015; Stanić et al., 2011; Vukomanović et al., 2015). The antibacterial activity of the silver doped hydroxyapatite was found to be substantially higher in 20 mg concentration than 10 mg the characterization studies were carried on 20 mg concentration of silver doped hydroxyapatite these doped hydroxyapatite can be used for bone grafting and Biofertilizers purposes the previous investigators have also proposed several mechanisms on antibacterial activity by AgNPs [30, 34 & 39]. The present result was indicated that our synthesized material has good cell viability for HeLa cell lines. Besides, the antibacterial activity exhibited by our material was also found to prevent bacterial infection (Mani et al., 2021; Mani et al., 2021).

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Fig. 9

Probit analysis brine shrimp assay of silver doped hydroxyapatite.

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