Doxycycline hyclate mediated silver–silver chloride nanoparticles and their antibacterial activity

Materials

Silver nitrate (AgNO ₃ ) was purchased from Fisher Scientific (New Jersey, USA). Doxycycline hyclate (DX), 1,1,3,3-tetramethylguanidine (TMG), and iodonitrotetrazolium chloride (INT) were purchased from Sigma-Aldrich (USA). Mueller–Hinton Broth (MHB) and Mueller–Hinton Agar (MHA) were obtained from BD Diagnostic, France. The 96-well plate was supplied by SPL Life science, Gyeonggi, Korea.

Synthesis of DX-Ag–AgCl NPs

AgNO ₃ (1 mM), DX (50 mg mL ⁻¹ ) and TMG (50 mg mL ⁻¹ ) solutions were prepared in triple distilled water. In a 10 mL of AgNO ₃ solution, 20 µL of DX and 40 µL of TMG were added, and the reaction was performed at 80 °C for 5 min with continuous stirring. Immediately after the reaction, the solution was cooled under running tap water. Separation of prepared NPs was performed by high-speed centrifugation (12,000 rpm, 30 min) and unbounded materials were removed by washing with distilled water twice. For characterization, powder form of DX-Ag–AgCl NPs obtained by lyophilization.

Characterization of DX-Ag–AgCl NPs

DX-Ag–AgCl NPs were observed visually, while the absorbance spectra were recorded by Jasco V-670 UV–Vis spectrophotometer (Japan) in the range of 300–800 nm during all reactions. Characterization of synthesized NPs was performed as per procedure reported previously [ ²⁷ , ²⁸ ]. In brief, the morphological features such as the size, shape, and distribution of DX-Ag–AgCl NPs were observed through TEM, and, for the sample preparation, colloidal suspension was dropped on a copper disk and then dried at room temperature. TEM (Hitachi, H7500, Tokyo, Japan) images captured at 120 kV. Lyophilized DX-Ag–AgCl NPs were used to determine the elemental composition and crystalline nature by EDX spectroscopy and XRD, respectively. XRD (Philips X’Pert-MPD Diffractometer, Netherland) was operated at 40 kV and 30 mA using Cu Kα radiations. Fourier transform infra-red (FT-IR) spectra were obtained in transmission mode on powdered DX and lyophilized DX-Ag–AgCl NPs that were mixed with dry KBr (1:100) and compressed into a thin pellet film. The NPs zeta potential and particle-size analysis (PSA) were analyzed by electrophoretic light scattering spectrophotometer (ELS-8000, OTSUKA Elec., Osaka, Japan). Samples for zeta potential and PSA analysis were prepared/diluted in tripled distilled water [ ²³ ]. FT-IR (Nicolet iS10, Thermo Electron Scientific Inst. LLC, Fitchburg, WI, USA) spectra in the range of 4000–500 cm ⁻¹ were obtained to determine the involvement of functional groups [ ²⁸ ].

Antibacterial activity

The pathogenic Gram-positive ( B. cereus KCCM 11773, B. subtilis KCCM 11316, and S. aureus KCCM 40050) and Gram-negative ( E. coli KCCM 11234, K. pneumoniae KCCM 11418, and P. vulgaris KCCM 40211) bacterial strains used in this study were purchased from the Korean Culture Center of Microorganisms (KCCM), Republic of Korea. Agar well-diffusion method, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) for DX and DX-Ag–AgCl NPs were performed as per previously published report [ ²⁸ ].

Agar well-diffusion method

A single colony of bacterial strain was inoculated in 20 mL of the MHB broth and grown at 37 °C overnight. An overnight culture, 0.5 McFarland-standard bacterial suspension (1 × 10 ⁸  CFU mL ⁻¹ ), was prepared by adjusting optical density (OD) to around 0.08–0.1, and then used for agar well-diffusion method. OD ₆₀₀ was obtained using a spectrophotometer (Libra S22, Biochrom Ltd, Cambridge, England). 100 µL of bacterial suspension spreaded over MHA plate with the help of sterile glass spreader, while the wells were prepared using 8 mm cork borer. 100 µL of DX and DX-Ag–AgCl NPs stock solutions (1 mg mL ⁻¹ ) were dropped in wells, and plates were incubated at 37 °C for 24 h. After incubation, the zone of inhibition (ZOI) diameter was measured in mm. These experiments were performed in triplicate.

Determination of MIC and MBC

The MIC and MBC were performed by broth microdilution and spread plate method, respectively. A 0.5 McFarland-standard adjusted bacterial suspension (1 × 10 ⁸  CFU mL ⁻¹ ) was diluted with 1:100 ratios in MHB to obtain 1 × 10 ⁶  CFU mL ⁻¹ . In broth microdilution method, each well of 96-well plate contained 50 µL of DX and DX-Ag–AgCl NPs with different concentrations (0.5, 1, 2, 4, 6, 8, 10, 12, 14, and 16 µg mL ⁻¹ ) and 50 µL of each bacterial strain suspension (1 × 10 ⁶ CFU mL ⁻¹ ) were added separately. Two sets of 96-well plate were prepared: one for MIC and another for MBC determination. Negative control was prepared, which contained only media; while the positive control contained bacterial strains without DX or DX-Ag–AgCl NPs. Plates were incubated for 24 h at 37 °C. After that, in one 96-well plate, 40 µL of INT (0.2 mg mL ⁻¹ ) solution was added to each well. The plate was further incubated for 30 min, to interaction of INT with enzyme dehydrogenase. Pink color formation can be seen in the presence of live cells, while there will be no color change in dead cells. The lowest dilution without color change will be indicated as the MIC. From the second plate, 100 µL of MIC concentrations was plated on MHA plate and spread with sterile glass spreader. These plates were incubated for 24 h at 37 °C. After incubation, MBC will be recorded as the lowest concentration of DX or DX-Ag–AgCl NPs that resulted in no bacterial growth.

Characterization of DX-Ag–AgCl NPs

UV–visible spectroscopy

The reactions were carried out at boiling temperature to promote AgNO ₃ reduction. A typical reaction solution contained 10 mL of AgNO ₃ (1 mM), 20 µL of DX (50 mg mL ⁻¹ ), and 40 µL of TMG (50 mg mL ⁻¹ ) solution. Reaction was performed at 80 °C for 5 min. During reaction, the formation of DX-Ag–AgCl NPs was observed by change in the color of the solution. After reaction, brown color formation was observed and characteristic sharp peak at 407 nm in UV–visible spectroscopy was observed, as shown in Fig.  ¹ . AgNPs shows characteristic peak, and the surface plasmon resonance (SPR) absorption band is strongly depending on the NPs size, surrounding chemicals and dielectric medium [ ²⁹ , ³⁰ ]. The SPR peak towards shorter wavelength indicates decreased size of NPs [ ²⁹ ]. Our findings are in good agreement with these reports.

Fig. 1

Elemental and crystalline nature of nanoparticles

The DX-Ag–AgCl NPs synthesized by addition of 40 µL of TMG and 20 µL of DX in 10 mL of 1 mM AgNO ₃ solution, and reaction was performed at 80 °C for 5 min with continuous stirring; TMG and DX solutions were prepared in distilled water with concentration of 50 mg mL ⁻¹ . Synthesized NPs was lyophilized for further characterization.

Figure  ² indicates the EDX spectrum, obtained from lyophilized NPs by EDX on FE-SEM. The EDX analysis confirms the presence of elements in the synthesized DX-Ag–AgCl NPs. The EDX spectrum shows two signals at 2.7 and 3.0 keV, which correspond to the Cl and Ag, respectively. The characteristic presence of element, Cl and Ag, in EDX spectrum confirms the composition of the Ag–AgCl NPs as being consistent with the previous reports [ ²³ , ²⁸ ].

Fig. 2

Figure  ³ illustrates the XRD diffraction patterns for DX-Ag–AgCl NPs. Nine peaks were detected on the XRD pattern, with five peaks corresponding to AgCl and four to AgNPs. The diffraction peaks at 2 θ  = 27.96°, 32.35°, 46.26°, 54.97°, and 57.61° correspond to the diffractions from {111}, {200}, {220}, {311}, and {222} planes of a face-centered cubic (FCC) AgCl, respectively. Furthermore, diffraction peaks at 2 θ  = 38.29°, 44.39°, 64.55°, and 77.69° correspond to the diffractions from {111}, {200}, {220}, and {311} planes of a FCC Ag, respectively. The bars (FCC planes) in the graph are from the Joint Committee on Powder Diffraction Standards (JCPDS) reference diffraction files for Ag (#87-0720) and AgCl (#01-1013). The crystalline nature of DX-Ag–AgCl NPs was confirmed by XRD.

Fig. 3

The crystalline size of DX-Ag–AgCl NPs was calculated by Debye–Scherrer’s equation as follows:

D=Kλβcosθ,

where D is the crystalline size in nm, K is the Scherrer’s constant ( K  = 0.94), λ is the wavelength of incident X-ray, β is the full width at half maximum (FWHM) of the diffraction peak, and 2 θ is the diffraction angle. The major five diffraction peaks, viz, 32.35, 38.29, 46.26, 64.55, and 77.69 corresponding to (200), (111), (220), (200), and (311) hkl planes, were considered for the determination of average particle size of NPs [ ³¹ ]. XRD spectra revealed highly crystallinity, and the average crystalline particle size of DX-Ag–AgCl NPs was found 8.25 nm which was calculated by Debye–Scherrer’s formula. The plausible reaction mechanism for formation of AgCl NPs is presented in Scheme  ¹ . These results are compatible with the previous reports of Ag and AgCl NPs synthesis using biological methods [ ²³ , ²⁸ ] and chemical method [ ³² ].

Scheme 1

Morphology of nanoparticles

The shape, size, and distribution of the DX-Ag–AgCl NPs were observed by TEM. In Fig.  ⁴ , the TEM image reveals the synthesized NPs mostly spherical, and polydisperse. In addition, the particle size was found to be in the range of 10–40 nm for DX-Ag–AgCl NPs. The polydisperse nature of NPs is due to the formation of Ag and AgCl NPs. These patterns were found to be well in agreement with those observed from XRD peaks and demonstrate the crystalline nature.

Fig. 4

Zeta potential and particle size of nanoparticles

Zeta potential analysis was applied to determine the surface overall charges on NPs [ ²³ ]. The zeta potential value of synthesized DX-Ag–AgCl NPs is − 5.01 mV (shown in Fig.  ⁵ ), which indicates negative charges on the NPs. Distilled water were used as a dispersion medium for zeta potential and PSA analysis. Figure  ⁶ indicates the histogram of synthesized DX-Ag–AgCl NPs’ particle-size distribution. From PSA analysis, it is observed that maximum NPs are in range of 5–20 nm. This observation is compatible with NPs’ size observed in TEM image.

Fig. 5

Fig. 6

Nanoparticle surface study by Fourier transform infra-red (FT-IR) spectroscopy

The FT-IR measurement was carried out to determine the presence of biomolecules on the nanoparticles copping layer. In Fig.  ⁷ , the FT-IR spectra of DX and DX-Ag–AgCl NPs suggested extensive similarities between the samples. From the FT-IR studies, the characteristic bands for functional groups of DX were identified, including O–H group at 3318 cm ⁻¹ , C=O group at 1683 cm ⁻¹ , and N–H group at 1573 cm ⁻¹ . In particular, the characteristic bands of DX at 1650 cm ⁻¹ region related to the NH ₂ group, which is known to interact with HCl, cause a decrease in transmittance intensity. On the other hand, the DX-Ag–AgCl NPs sample had a more intense peak at 1650 cm ⁻¹ which corroborate the fact that HCl has been removed from the molecular complex and DX transformed to doxycycline monohydrate [ ³³ ]. Formation of the doxycycline monohydrate from DX is presented in Scheme  ¹ . The present study results are compatible with these results.

Fig. 7

Plausible mechanism of DX-Ag–AgCl NPs formation

The plausible formation of AgCl NPs can be best explained on the basis of Scheme  ¹ . Here, the DX is the source of H ⁺ (via HCl) which, under the reaction conditions, can react with the TMG to produce the Lewis adduct and chloride anion. The chloride anion thus formed reacts with the free Ag ⁺ in aqueous medium forming the AgCl NPs. However, the formation of Ag NPs can be best explained on the basis of hypothetical mechanism reported previously, by the functional groups (–OH) similarity of the DX with quercetin in the leaf extract [ ³⁴ ].

Antibacterial activity of DX-Ag–AgCl NPs

As a potential application, the DX-functionalized Ag–AgCl NPs were tested as a bactericidal. Antibacterial activity was performed by agar well-diffusion method against Gram-positive ( B. cereus KCCM 11773, B. subtilis KCCM 11316, and S. aureus KCCM 40050) and Gram-negative ( E. coli KCCM 11234, K. pneumoniae KCCM 11418, and P. vulgaris KCCM 40211) pathogenic bacteria. The antibacterial activity of DX and DX-Ag–AgCl NPs was observed after 24 h. Figure  ⁸ shows the antibacterial activity of synthesized DX-Ag–AgCl NPs compared with pure DX. The variation in the ZOI was noticed with respect to the type of bacterium, which might be due to the difference in the bacterial surface characteristics. Gram-positive and Gram-negative bacteria differ in several ways when looking at the cell wall. Gram-positive bacteria have a thick, multi-layered peptidoglycan (negatively charged), virtually none of lipopolysaccharide and presence of teichoic acid; Gram-negative bacteria have a thin single-layered peptidoglycan, which contains high lipopolysaccharides and absence of teichoic acid. All these different cell surface compositions make differences in the sensitivity of bacteria to NPs and antibiotics [ ³⁵ , ³⁶ ]. Table  ¹ shows the MIC and MBC of DX-Ag–AgCl NPs and DX, which was obtained against Gram-positive and Gram-negative pathogens. From these results, it is observed that the growth inhibition of Gram-positive ( B. cereus KCCM 11773) bacteria inhibited at lowest concentration of DX-Ag–AgCl NPs (MBC, 1 µg mL ⁻¹ ), while Gram-negative ( E. coli KCCM 11234) bacteria inhibited at higher concentration of DX-Ag–AgCl NPs (MBC, 12 µg mL ⁻¹ ). When the antibacterial effect of the DX-functionalized NPs is compared with that of DX alone, the amount of DX required is equal or considerably lower. These findings are in good agreement with the reported studies [ ¹⁶ , ³⁷ , ³⁸ ]. AgNPs are effective antibacterial agent, which cause adverse toxic effect on bacterial cells [ ⁸ ]. It is well known that the antibacterial activity of AgNPs is dependent on their size and shape; smaller AgNPs shows more bactericidal activity due to their more surface-to-volume ratio [ ¹¹ , ³⁹ ].

Fig. 8