Facile synthesis and characterization of beta lactoglobulin–copper nanocomposites having antibacterial applications

Abstract

The synthesis of Cu⁰ nanoparticles and Cu–protein nanocomposites is a great challenge. Here we describe a simple and convenient method for the synthesis of Cu–β-lactoglobulin nanocomposites using very cheap CuSO₄·5H₂O and the retinol binding model protein bovine β-lactoglobulin (β-lg) at pH 10.0 in ammoniacal medium. Then addition of hydrazine hydrates in the reaction mixture and heating the solution at 55 °C for 2 h resulted in the formation of hexagonal Cu–β-lg nanocomposite (average size 0.5 μm) containing the embedded Cu-nanoparticles as revealed from SEM and TEM analysis. The important feature of this method is that the highly stable Cu-nanoparticle present in the composites were synthesized without employing any inert atmosphere; decomposition of hydrazine hydrate generated the nitrogen in situ which produced the inert atmosphere for this reaction. Synthesis of this nanocomposite is justified by a docking study. The synthesized nanocomposite exhibits potential antibacterial activity against both Gram positive and Gram negative bacterial strains. Thus it can be employed in different medical applications and also in the preparation of various nanomedicines.

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Introduction

The synthesis and development of metallic nanoparticles (NP) have received a great deal of attention during the last few years. NPs not only act as carriers for various drugs, but they themselves can be effectively used as sources for biosensing, bioimaging, luminescence tagging, immunoassay and inhibiting the formation of amyloid aggregates of protein.^1–5 NPs of certain inorganic materials also possess antimicrobial and healing properties. Particularly Cu-NPs have drawn significant attention because of their unique properties such as electrical conductivity, catalytic activity, antimicrobial activity, drug delivery, biosensing and chemical stability.^6–10 In the last few years huge efforts have been made to invent new synthetic routes to produce Cu-NPs, such as a radiation method,¹¹ thermal decomposition,¹² and microemulsion.^13,14

However, since copper is redox-active, it is usually difficult to prepare metallic copper via reduction of simple copper salts in aqueous solution in contrast with noble metals, such as Au, Ag, Pt. The reason being, even though zero valence copper is initially formed, it gets easily oxidized in the solvents with high dipolar moments under ambient conditions.¹⁵ The stabilization as well as the control of size and growth rate of nanoparticles can be achieved in polymeric matrices resulting in the formation of nanocomposite, an advanced functional material composed of nanoparticles dispersed inside the polymeric matrix and coated by polymer.^16–18 The resulting nanocomposites material combines the suitable properties of both partners thus have enhanced functions as compared to individual components. Thus increasing attention has been paid to the fabrication of bi-functional nanostructures consisting of discrete domains of two materials.¹⁹ Various methods are available for the preparation of copper nanocomposites in various media and with a variety of agents.^20–22 Cu nanocomposite is now used to remove nitrate from ground water where it shows both catalytic reduction and chemical reduction.^23,24 Cu nanocomposite has good antibacterial activity such as on Staphylococcus aureus and Escherichia coli as pathogen microorganisms and it exhibited good antibacterial potential against both Gram positive and Gram negative bacterial strains.²⁵ Graphene–copper nanocomposite can be used as antifriction additive.²⁶ In our present work we have synthesized the Cu–β-lactoglobulin nanocomposite (a Cu–protein nanocomposite) having good antibacterial property by a simple chemical reduction method where metallic Cu-NPs were formed without the using of any inert environment. β-lg is a most widely studied whey protein and a member of the lipocalin family. This protein contains 162 amino acids with a molecular mass of ∼18 400 Da, featuring an eight-stranded β-barrel (strands A-H) succeeded by a three-turn a-helix and a final β-strand (strand I) that forms part of the dimerization interface.^27,28 It contain two disulfide bonds (Cys66–Cys160) and a free thiol group (Cys121). The free thiol (Cys121) and disulphide linkage present in β-lg help to generate Cu-NPs by reducing Cu²⁺ tetramindiaqua Cu(II) complex.

Experimental

Materials and methods

Preparation of Cu β-lg nanocomposite. Beta-lactoglobulin (β-lg), copper sulfate (CuSO₄·5H₂O), hydrazine hydrate (N₂H₄·2H₂O) and ammonium hydroxide (NH₄OH) solutions were used for preparation of this nanoparticle. Bovine β-lg was isolated and purified from cow milk as described earlier.²⁹ Since extinction coefficient of β-lg (0.96 mg⁻¹ mL⁻¹ cm⁻¹ at 280 nm) is known, different concentration of protein samples were prepared by dissolving protein samples in milli-Q-water and then measuring the O.D. at 280 nm. Other reagents for this experiment were purchased from Merk (India). Ammoniacal solution of 15 mM copper sulphate (pH 10) was first stirred by using a small magnetic bar for 15 min maintaining the temperature at 55 °C. Then 0.04% freshly prepared β-lg was poured to copper sulphate solution and stirred for 1 h followed by the addition of 120 mM hydrazine hydrate in a tightly closed glass vial containing the reaction mixture. After 2 h the colour changes to dark red indicating the formation of Cu-NPs. It is observed that the colour remain unchanged up to 7 days.

UV-vis spectroscopy. At room temperature surface plasmon resonance (SPR) of the colloidal solution containing copper nanocomposite (Cu-nanocomposite) was measured by using Shimadzu TCC 240A UV-Vis Spetrophotometer equipped with 1 cm path length quartz cell.

X-ray diffraction (XRD). XRD measurement was done with a Bruker AXS (Model D8, WI, USA) powder diffractometer set up with Cu Kα radiation (1.5409 Å) and scan speed of 5° min⁻¹ in the scanning range from 30 to 80 (2θ) running at 40 kV and 30 mA in the room temperature. Small amount of dry Cu–β-lg nanocomposite was used to prepare the sample.

Fourier transform infrared spectroscopy (FTIR). IR spectrum was recorded in the region 500–4000 cm⁻¹ on Brucker Optics Alpha-T spectrophotometer with samples as KBr disks. Samples were prepared by prepared by centrifuging the synthesized colloid at 10 000 rpm for 10 min and drying the pellet under vacuum. The dried sample powder was then used to make KBr pellet for further analysis. About 0.2 g of KBr (spectroscopy grade) was thoroughly mixed with sample powder (1 wt% of KBr) and then made into disks by uniaxial pressing.

Field emission scanning electron microscope (FESEM). The morphology and size study of synthesized Cu–β-lg were done with the help of FE-SEM by (Hitachi S-4800, JAPAN) operating with a voltage of 20 kV. The fine power was diluted with ethanol to make a film on carbon tape and then kept under vacuum desiccator for evaporation and finally the dried samples were gold coated for FESEM.

Transmission electron microscopy (TEM). The morphological study of synthesized Cu–β-lg nanocomposite was also investigated by high resolution transmission electron microscopy (Jeol-HRTEM-2011, Tokyo, Japan) with an accelerating voltage of 80–85 kV in different magnifications. The sample solutions were diluted 50 times. A droplet of the diluted sample was put on a carbon coated copper grid of mesh size 300C (Pro Sci Tech). After 20 s the droplet was removed with a filter paper followed by a droplet of 2% uranyl acetate (Sigma, Steinheim, Germany) solution put on the grid and finally removed after 15 s and left for air dry and used for imaging purpose. Before taking the image all the samples were incubated 6 h.

Docking study. The structure of tetramindiaqua Cu(II) complex was optimized using Density Funtional Theory. This optimized structure was used for docking with β-lg protein (PDB ID: 2BLG) to find the probable mode of binding and strength of complex with beta-lg using Auto Dock 4.2. The data obtained from docking was examined by Discovery Studio 4.1 Client and Chimera 1.10.1rc.

Antibacterial activity studies. The antibacterial activity of the synthesized Cu nanocomposite material was tested by plate count technique on Gram negative Escherichia coli (ATCC 25922) and Gram positive Staphylococcus aureus (ATCC 25923) bacteria.³⁰ Each of these two freshly prepared overnight (log phase) cultured bacteria containing 10⁶ CFU mL⁻¹ was taken into different 5 mL LB medium (10% tryptone, 0.5% yeast extract, 0.5% NaCl of pH 7.0). Two plates were considered as control (l and n, untreated normal culture) and the other two (m and o) contained nanocomposite having concentrations 0.005 gm mL⁻¹. These were incubated for 16 h keeping the temperature at 37 °C. After incubation, a 10 μL aliquot of bacterial suspension was plated uniformly on the surface of different LB agar plates (LB, 1.5% agar powder, pH 7). Finally these plates were incubated for overnight at 37 °C.

Cytocompatibility study. For the cytotoxicity test, the fibroblast cell line (L929) was obtained from the National Centre for Cell Science, Pune, India. The cell was cultured in DMEM (Dulbecco's Modified Eagle's Medium) medium at 37 °C.³¹ The cells were washed with sterile phosphate buffer saline (pH 7.4), trypsinized and released into suspension to suitable cell density for further studies. Cells without any treatment served as control. The different concentrations (1–5 mg mL⁻¹) of the Cu–β-lg nanocomposite were taken for this investigation. Initially, 96-well plate (containing cell concentration of 10³ cells per well) were seeded and allowed to adhere. The cell mono-layers were then treated with Cu–β-lg nanocomposite (1–5 mg mL⁻¹) up to 24 h. After that, 50 μL of freshly prepared MTT solution (5 mg mL⁻¹ PBS; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added into each well and incubated for 6 h at 37 °C in culture hood. After that 100 μL MTT solvent (4 mM HCl, 0.1% Nondet P-40 (NP40) all in isopropanol) was added to the cells and agitated on orbital shaker for 15 min. The absorbance of the solution was measured at 595 nm using a micro-plate reader (Bio-Rad, USA).³²

Results and discussion

UV-vis spectroscopy

The UV-vis absorption characteristics of the ammoniacal copper sulfate solution is shown in Fig. 1. The mixture of CuSO₄ and NH₄OH shows absorption peak at 612 nm (curve ‘a’) with the formation of deep blue coloured solution at pH 10. Curve ‘b’ is the spectrum of copper ammonium sulfate solution in the presence of β-lg showing a light bluish color after 15 min of stirring. A clear shift of absorption maxima from 612 nm to 574 nm (curve ‘c’) occurs when the mixture of CuSO₄ and NH₄OH was continuously stirred with 0.04% β-lg solution at 55 °C followed by the addition of hydrazine hydrate solution. The colloidal solution of the same mixture revealed a dark red colour (inset Fig. 1) after 2 h. This curve shows a strong absorption peak at 574 nm which confirms the generation of Cu nanoparticles.²¹ A hump near 280 nm signifies the presence of the protein, β-lg. Thus the formation of NPs was established from the presence of the surface plasmon resonance (SPR) band in UV-vis absorption spectra.

Fig. 1 UV visible spectra of CuSO₄ + NH₄OH (a), CuSO₄ + NH₄OH + 0.04% β-lg after 15 min (b), CuSO₄ + NH₄OH + 0.04% β-lg + hydrazine after 2 h (c).

X-ray diffraction

X-ray diffraction (XRD) pattern of the synthesised Cu–β-lg nanocomposite absorption versus 2θ values is shown in the Fig. 2. The diffraction peaks at 2θ values were 43.5, 50.9 and 75 due to the presence of (111), (200) and (220) planes of metallic copper and thus confirming the presence of crystalline Cu particles in the colloids.³³ The XRD pattern also shows the absence of impurities like copper oxide (CuO) and cuprous oxide (Cu₂O) indicating the high purity of the final product.

Fig. 2 XRD pattern of synthesized Cu-nano particles at 25 °C showing three types of planes (100), (200) and (220).

Fourier transform infrared spectroscopy

Fourier transform infrared (FTIR) spectroscopy is an important technique for the structural characterization of proteins. This experiment is carried out to identify and estimate the different secondary structures of the native protein and subsequent change in both secondary and tertiary structures due to its interactions with other molecules. Major information of protein secondary structure can be obtained from the study of amide I band of FT-IR spectra.³⁴ The FTIR spectra of native β-lg and Cu–β-lg nanocomposite are shown in Fig. 3. The IR bands near 1635 cm⁻¹ confirm the presence of amide I band in both the native β-lg (curve a) and in Cu β-lg nanocomposite (curve b). But the peak at 1480 cm⁻¹ in native protein shifts to 1516 cm⁻¹ in the Cu–β-lg nanocomposite indicating more N–H bending of amide II in case of the latter. The above two results give a clear idea of presence of amide I and amide II of protein around the synthesized Cu nanoparticles. The 1516 cm⁻¹ peak in synthesized nanocomposite may also be due to the absorption of tyrosine residues.³⁵ This would suggest that there may a perturbation of environment of tyrosine residue and it causes the shift of absorption maximum. The sharp peak at 1400 cm⁻¹ and a hump near at 2930 cm⁻¹ in the nanocomposite correspond to the conformational change in β-lg after its conjugation with copper and it supports the formation of Cu nanoparticles.³⁶ The broad band near 3500 cm⁻¹ is due to the –OH stretching vibrations of water in both the β-lg and Cu β-lg nanocomposite.

Fig. 3 FTIR spectra of pure β-lactoglobulin [curve (a)] and Cu–β-lg nanocomposite [curve (b)].

Scanning electron microscopy (SEM)

The morphological characteristics of the synthesized Cu–β-lg nanocomposite are shown in the SEM images (Fig. 4a–c). The SEM study reveals the formation of nanocomposite of distinct morphology. The Fig. 4a shows that the morphology of synthesized Cu β-lg nanocomposite is hexagonal and the average size of this aggregated nanocomposite is 0.5 μm.

Fig. 4 Scanning electron microscopy images of Cu–β-lg nano-composite in different magnification (a–c).

Transmission electron microscopy (TEM)

TEM image also supports the hexagonal morphology of synthesized Cu–β-lg nanocomposite. The size of this nanocomposite having hexagonal morphology was 200 nm (Fig. 5h). The bright SAED pattern of these particles shows the metallic nature of the particles (inset of Fig. 5e).³⁷ The zoomed out picture of 100 nm, 50 nm and 20 nm confirmed the formation of β-lg nanocomposite of different sizes (Fig. 5e–g). Careful observation of the TEM images revealed the presence of uniformly distributed round shaped Cu nanoparticles (Fig. 5g and h) embedded in the matrix of the synthesized β-lg nanocomposite.

Fig. 5 TEM micrograph of Cu–β-lg nanocomposite (h). 100 nm, 50 nm and 20 nm zoomed out pictures of the same composite particles (g, e and f). SAED pattern of nanocomposite particles is in inset of (e).

Docking study

Molecular docking, a very useful theoretical technique, is effective to understand characteristics of interactions of small molecules into the binding site of the target specific region of the macromolecules like protein, DNA etc.³⁸ To the best of our knowledge, for the first time the docking is used to predict the most probable mechanism for the synthesis of cupper nanoparticles. The structure of tetramindiaqua Cu(II) complex was optimised using Density Functional Theory. This optimised structure was used for docking with the β-lg protein (PDB ID: 2BLG) to find the probable mode of binding and binding strength of complex with β-lg (Fig. 6). It has been observed that the small molecules gain stability into the protein binding sites due to intermolecular H-bonds and stacking interactions with hydrophobic parts of the proteins. In this case, the hydrogen bonding between the tetramindiaqua Cu(II) complex and β-lg protein is the key interaction mode. β-lg protein has three pockets to interact, first is inside the β-barrel, second is between the β-barrel and the helix and the final pocket is at the region near Trp19-Arg124.³⁹ The energetically most prosperous docking positions were prognosticated. Tetramindiaqua Cu(II) complex prefers the second pocket described above. In this pocket the complex interact with Q5, D96 and K135 (GLN5, ASP96 and LYS135) amino acid residues through H-bonding. The docking pose also support the mechanism mentioned above. Here, inside the pocket the free SH group of C121 (Cys121) may involve in reduction of Cu²⁺ to Cu⁺ as mentioned Pradhan and co-workers.⁴⁰ The change of Gibbs free energy of docked structure for tetramindiaqua Cu(II) complex was found to be −2.60 kcal mol⁻¹. The low value of the Gibbs free energy indicates that a weak interaction between the complex and β-lg. It is very essential in the hydrazinehydrate reduction step of Cu⁺ to metallic Cu⁰ in the nanoparticle formation because a strong interaction inhibit the separation process between tetramindiaqua Cu(II) complex and protein in this step.

Fig. 6 (i) Protein surface and environment around cupper complex, (j) Docking pose showing the tetramindiaqua Cu(II) complex in between the β-barrel and the helix pocket, (k) mode of interaction of complex with amino acid residues.

Possible mechanism of synthesis of Cu–β-lg nanocomposite

In the Biuret reaction, Cu²⁺ ion in alkaline medium forms a blue coloured chelate complex with the proteins or peptides containing three or more amino acids.⁴¹ When β-lg is incubated with alkaline copper sulphate solution at room temperature; it forms a tetradentate complex with four peptide bonds and one atom of copper resulting the partial reduction of Cu²⁺ to Cu⁺ (Fig. 7). The Cu⁺ is further reduced by the addition of hydrazine hydrate to yield the metallic Cu⁰. In details, when an ammoniacal copper sulphate solution (pH range 9.8–10.2) is allowed to react with β-lg then the tetramindiaqua Cu(II) complex⁴² is introduced into the protein cavity where the free thiol group of Cys121 of β-lg play a vital role in reduction of Cu²⁺ to Cu⁺ as itself remains negatively charged at this alkaline pH. Under these conditions β-lg binds with multiple Cu²⁺ and these Cu²⁺ can serve as docking sites for multiple β-lg adsorptions as shown in the docking study. The Cu⁺ on further reduction by hydrazine hydrate turns to metallic Cu⁰. These types of interactions cause the formation of composite of Cu nanoparticle and β-lg. The result of FTIR study indicates the gross change of secondary structures of β-lg in the region 1600–1700 cm⁻¹.

Fig. 7 Possible mechanism of synthesis of Cu β-lg-nanocomposite.

These Cu⁰ once formed may be surrounded by the free thiols which inhibit it to oxidise further.⁴⁰ Strong intermolecular forces like van der Walls interaction and magnetic dipole–dipole interaction causes aggregation of Cu–β-lg nanocomposite. Finally this agglomerated structure undergoes a structural change to form a hexagonal geometry (Scheme 1). The reorganization of agglomerated structures into hexagons may be due to change in both secondary and tertiary structures of β-lg in the alkaline condition.

Scheme 1 Synthesis Cu–β-lactoglobulin nanocomposite.

Antibacterial activity study

The antibacterial activity of Cu–β-lg nanocomposite tested on Gram negative Escherichia coli (ATCC 25922) and Gram positive Staphylococcus aureus (ATCC 25923) bacteria showed positive results after 16 h of incubation. Fig. 8 shows the colonies of controls (controlled Gram negative bacteria and Gram positive bacteria) were large number in agar plate (l & n) whereas Cu nanocomposite treated plates (m and n) had a very few numbers of bacterial colonies. Due to interaction of this newly synthesized Cu nanocomposite, the bacteria could not survive at that time period. It clearly reveals that Cu–β-lg nanocomposite has good antibacterial property and this activity was prominent in case of Gram negative as well as Gram positive bacteria.

Fig. 8 Colonies of cultured bacterias E. Coli (l) and S. Aureus (n) are controls whereas nanocomposite treated (m and o).

Cytocompatibility study

Cytotoxicity plays an important role in assessing the antimicrobial activity of the synthesized nanocomposite. To be useful for clinical applications, excellent activity against bacteria with minimal effect on mammalian cells is considered as one of the major requirements of anantimicrobial agent. Here, the cytotoxicity of the Cu–β-lg nanocomposite on fibroblast as well as mammalian cells has been studied. The MTT assay, carried out by different higher concentration of nanocomposite, exhibits a reduction in cellular viability (Fig. 9).

Fig. 9 Cell viability study (MTT assay) with varying concentration of Cu–β-lg nanocomposite for fibroblast cell line (L929).

Conclusions

Briefly this method describes the preparation Cu–β-lg nanocomposite utilizing the retinol binding protein β-lg and economically very cheap CuSO₄ without using any external inert medium. The composite material consisted of pure metallic copper nanoparticles free from oxides. The synthesis of hexagonal Cu β-lg nanocomposite is evidenced by SEM, TEM, XRD, FTIR, UV and other techniques used in this experiment. The synthesized nanocomposite has excellent antibacterial property which can be employed further as a potential antibacterial agent in different medical applications and also in the preparation of various nanomedicine.

Acknowledgements

Financial supports from DST-PURSE-II (Govt. of India) and CAS-II Programe of the Department of Chemistry, Jadavpur University, Kolkata are greatly acknowledged. The authors also wish to acknowledge Prof. P. S. Dastidar of Organic Chemistry Division, Indian Association for Cultivation of Scince (IACS), Kolkata for providing the TEM instrumental facility.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra14162e

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