Facile synthesis of hair-extract-capped gold and silver nanoparticles and their biological applications

Abstract

Hair is a waste keratinous biomaterial. In tanneries, hair burning is a common practice, resulting in a residue containing a large amount of COD, BOD, and TDS, which are environmental hazards. Meanwhile, microorganisms have developed resistance against various drugs. Herein, with the aim to utilise this waste hair material, silver (AgHr) and gold (AuHr) nanoparticles were synthesised using NaBH₄ as a reducing agent and hair extract as a stabilizing material. The formation and structure of the nanoparticles were scrutinised by different analytical techniques, including UV-Vis, TEM, and AFM. As a result, spherical shaped silver (AgHr) and gold (AuHr) nanoparticles of various sizes, ranging from 4–30 nm, were prepared. The stabilities of AgHr and AuHr toward acidity, alkalinity, salinity, and temperature were also investigated, which showed that they remained stable for more than six months. Moreover, the hair-extract-stabilised silver and gold nanoparticles underwent bioactivity evaluations. The nanoparticles showed promise in multiple applications, including as enzyme inhibitors and bactericidal agents.

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Introduction

The exploitation of nanoscale materials has become a vast field of scientific interest. Furthermore, bacterial resistance towards antimicrobial agents has resulted from the unnecessary overuse of antimicrobial agents, with an increasing number of microbial strains developing resistance through permutations and horizontal gene transfer, affecting the treatment of infectious diseases.¹

The antibacterial and antifungal applications of noble and semi-noble metals, including silver, gold, copper, and palladium, are effective from from anciant period.^2–4 Silver and gold nanoparticles are promising enzyme inhibitors, stable long-term, and able to stop drug resistant bacterial growth and impede larvicidal and fungicidal activities. The urease enzyme secreted by bacteria causes kidney stones, photogenic urolithiasis, pyelonephritis, ammonia production, hepatic coma, hepatic encephalopathy, urinary catheter escalation, and Helicobacter pylori, which leads to cancer.^5–7 This enzyme has become difficult to control because many enzyme-producing bacteria have developed drug resistance. However, nanoparticles are effective against the urease enzyme.⁸ Bacterial cell membranes possess copious amounts of sulfur-containing proteins. Ag/AuNPs interact with sulfur-containing protein amino acids both inside and outside the cell membrane, generating aggregates of metals ions and sulfur ions, which affect the cell chemistry. Noble-metal ions discharged from nanoparticles can react with phosphorus in DNA, stopping replication, deactivating proteins, affecting permeability, creating gaps between the cytoplasm and cell wall, and imbalancing discharge of respiratory enzymes, leading to cell death.^9,10 The urease enzyme contains nickel metal at its active site. The enzyme is produced by plant, bacteria, and fungi, and is well known for catalysing the hydrolysis of urea into ammonia and carbamate. Excessive ureolytic activity results in ammonia discharge into the atmosphere, causing ammonia toxicity and increasing soil pH, resulting in economic losses.^11,12

Hair is an alpha keratinous biomaterial, with applications in cosmetics, drug coating, wound healing, antibacterial activity, blood clotting, trauma, tissue and nerve regeneration, and treating lethal liver injuries.^13–17 Hair acquired as a by-product from tanneries is thought to comprise 5% of recovered dry hair, but in many tanneries hair burning is still a common practice, contributing large amounts of COD, BOD, TDS, and more, to waste discharge and causing environmental pollution.¹⁴ Human hair was first used as a support for gold and silver nanoparticles in a nanocatalyst for cycloaddition and aza-Michael reactions.¹⁸ Since then, quantum dots have been prepared in a one-step pyrolysis process using hair as a precursor,¹⁹ gold nanoparticles have been synthesised inside human hair by reacting tetrachloroauric acid with human hair,²⁰ and the shape and distribution of lead sulfide nanocrystals have been shown within hair during dying.²¹

In this research, we have synthesised silver (AgHr) and gold (AuHr) nanoparticles using hair extract as a stabilizing agent, and estimated their enzyme inhibition and antibacterial activities, with the aim to utilise waste hair to produce a useful material and, thus, reduce pollution contributed by hair.

Experimental

Materials and measurements

Tetrachloroauric acid trihydrate (HAuCl₄·3H₂O), a yellow salt, was purchased from Merck, sodium borohydride (NaBH₄) was purchased from Fisher Chemicals UK, and sodium hydroxide, was purchased from Scharlau Chemie S.A. Human hair was collected from the Kohat District, Khyber Pakhtunkhwa, Pakistan. Double distilled water was used throughout practical procedures for the synthesis of gold and silver nanoparticles stabilised with hair extract, and for subsequent analysis. Nanoparticle syntheses were confirmed by UV-Vis spectra, recorded on a UV-Vis spectrophotometer (Shimadzu 1800), using water as solvent.

Dissolution of hair

Hair (7.5 g), well washed with locally available surfactant and deionised water, was dissolved in NaOH solution (250 mL, 1 M) for 24 h. The solution was filtered, and 10 mL of filtrate was further diluted to 1000 mL and stored as the stock solution. Hair extract has also been obtained using chemicals such as thiourea, urea, and mercaptoethanol, but we adopted simple methods to dissolve hair and synthesise nanoparticles with hair extract due to the facilities available in our laboratory.

Synthesis of silver (AgHr) and gold (AuHr) nanoparticles

Silver (AgHr) and gold (AuHr) nanoparticles were synthesised separately by reducing a solution of AgNO₃ or HAuCl₄·3H₂O (1 mmol), respectively, with NaBH₄ solution (10 mmol) and hair extract from the stock solution.

Reactions were carried out using different volumes of the hair extract solution and the AgNO₃ or HAuCl₄ solutions. The reaction mixtures with different ratios were stirred for 30 min, and then a few drops of freshly prepared 10 mM NaBH₄ solution were added, followed by stirring for 3 h. Changes in colour were constantly monitored, with a range of colours observed over time, from light yellow to dark black for AgHr, and light pink to dark red for AuHr.

Nanoparticle preparation was confirmed by UV-Vis spectrophotometry. The nanoparticles were then freeze-dried for onward analyses using AFM, TEM, XRD, and bioassays.

Urease inhibition protocol

Reaction mixtures contained jack bean urease enzyme (25 μL) and a buffer solution (55 μL) containing 100 mM urea and 2 μL of hair extract. Hair-extract-stabilised AgHr (0.5 mM) and AuHr (0.5 mM) were incubated in the mixtures for 15 min at 30 °C in 96 well plates. Ammonia production was assessed using indophenol method to determine urease activity via Weatherburn method. In short, 45 μL of each phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprusside), and alkaline reagent (70 μL, 0.5% w/v NaOH and 0.1% active chloride NaOCl) were also added to each well. After 50 min, an increase in absorbance was observed at 630 nm using a microplate reader (Molecular Devices, USA). All reactions were carried out in triplicate, using 200 μL as the final volume. The results (variation in per min absorbance) were processed with SoftMax Pro software (Molecular Devices, USA). Comprehensive assessments were carried out at pH 6.8. Percentage inhibition was calculated using the formula 100 − (OD test well/OD control) × 100, using thiourea as the standard for urease inhibition.²²

Protocol for xanthine oxidase assay and inhibition

Xanthine oxidase (XO) is a crucial enzyme that catalyses the oxidation process of oxypurines (hypoxanthine and xanthine) to uric acid in the purine metabolic pathway. The XO inhibitory activities of hair extract, AgHr, and AuHr (test compounds) were examined by calculating the rate of substrate (xanthine) hydroxylation and, consequently, the appearance of colourless end-product uric acid, which exhibited absorption at 295 nm. The solution containing pure sample (10 μL, 1 mmol L⁻¹) dissolved in dimethyl sulfoxide (DMSO), and phosphate buffer (150 μL, 0.05 mol L⁻¹, pH 7.4), and xanthine oxidase (0.003 units dissolved in 20 μL buffer). For enzyme xanthine oxidase, 20 μL of 0.1 mmol L⁻¹ of substrate (xanthine) was added to the reaction mixture and incubated at room temperature for 10 min. UV spectra (λ_max = 295 nm) were prerecorded as the substrate was added to the reaction mixture, and then recorded for 15 min at 1 min intervals (Spectra MAX-340). The percentage inhibition caused by the samples was investigated against a DMSO blank and calculated using the following formula: inhibition (%) = 100 − [(OD test compound/OD control) × 100]. The IC₅₀ values of the extracts, products, and standard compounds were calculated using EZ-Fit software (Perrella Scientific Inc., Amherst, USA). Reactions were performed in triplicate for each compound using allopurinol as standard.²³

Antibacterial assessment

The antibacterial activities of AgHr and AuHr were evaluated using a slightly modified disk diffusion method. The bacterial strains were cultured and refreshed by inoculating each strain on nutrient broth media at 37 °C for 1 day. Nutrient agar media (28 g) was primed by dissolving in freshly prepared double distilled water (1 L). The media, cotton buds, Petri dishes, borers, and other necessary instruments were autoclaved at 121 °C for 15 min, increasing the pressure to 15 psi. The cold media (25 mL) was transferred to each plate and solidified in a laminar flow chamber to avoid contamination by different microbes. Three holes were created in each plate, with an 8 mm borer, and 1 mL of sample was introduced into each hole. Levofloxacin was used as a standard, and the plates were kept in an incubator at 37 °C. Inhibitory zones were measured after 24 and 48 h. The entire process was carried out in triplicate.

Characterization

The reduction of Ag and Au salts and formation of Ag/Au nanoparticles were scrutinised using visual colour changes in the solutions, i.e., yellow for AgHr, and red for AuHr. UV-Vis spectra were recorded in the range 200–800 nm to confirm Ag/Au nanoparticle formation. Transmission electron microscopy (TEM, JEOL Ltd. Tokyo, Japan), functioning at an accelerated voltage of 200 kV, was used to determine the sizes and shapes of AgHr and AuHr, while atomic force microscopy (AFM) was used to determine the morphologies and sizes of the synthesised AuHr and AgHr nanoparticles.

Results and discussions

The highly basic medium used to prepare the hair extract would surely destroy the proteins in the hair. However, the aim our study was convert hair (biowaste) into a useful product using the simplest possible method. First separating proteins from the hair, and then utilizing each protein to prepare useful product, is another possibility. The likely outcome using this method, is the breakage of S–S to bonds in hairs, leading to the formation of SH bonds, which are highly susceptible to stabilise nanoparticles in colloidal solutions^9,24,25. Keratine, a fibrous structural protein in hair, contains 18 different amino acids involved in the structural morphology of hair. Cysteine (the major component), threonine, aspartic acid, glutamic acid, serine, proline, alanine, glycine, trace valine, methionine, leucine, isoleucine, tyrosine, cystic acid, phenyl alanine, histidine, lysine, arginine, and citrulline are the main amino acids of hair. Amine and thiol groups in the extract, such as in cysteine, are responsible for nanoparticle stability.^26,27

AgHr were synthesised using an optimised 3 : 1 ratio of AgNO₃ (1 mM solution) and hair extract (stock solution), and a few drops of NaBH₄ as the reducing agent. UV-Vis spectra in the range 400–480 nm indicated the presence of silver nanoparticles.

Therefore, the absorbance peak around 400 nm confirmed the synthesis of spherical silver nanoparticles, as evidenced by the colour and UV-Vis spectrum (Fig. 1). Controlling the amount of reducing agent played a significant role in the synthesis of uniformly shaped and sized nanoparticles. The as-prepared AgHr were further used in different analyses and applications.

Fig. 1 UV-Vis spectra of hair-extract-stabilised AgNPs reduced with NaBH₄.

Gold nanoparticles stabilised with hair extract were synthesised using an optimised 4 : 1 ratio of HAuCl₄·3H₂O (1 mM solution) and hair extract (stock solution), using a few drops of NaBH₄ as the reducing agent. The colour appearance, and sharpest peak at around 513 nm, confirmed the synthesis of spherical gold nanoparticles. UV-Vis spectra in range 500–580 nm also showed the presence of gold nanoparticles (Fig. 2). The TEM images of AgHr show that the nanoparticles are spherical in shapes with variable sizes ranging from 10 to 32 nm (Fig. 3). The as-prepared AuHr were used in subsequent analyses and applications.

Fig. 2 UV-Vis data for AuHr stabilised with hair extract.

Fig. 3 TEM image and histogram of AgHr.

TEM images of AuHr showed that the nanoparticles were spherical and of varying sizes, ranging from 9 to 30 nm (Fig. S1†). AFM images also confirmed AuHr growth, showing spherical shapes with sizes varying from 15 to 30 nm (Fig. S2†).

AgHr stability was assessed using various physiochemical parameters, showing that AgHr were stable in basic media, and in neutral and slight acidic media (pH 5–6), for several months. However, AgHr was unstable in strong acidic media (Fig. S3†). The stability in basic media might be due to the presence of amino acids containing amine and thiol groups.

The salt stability of AgHr (3 mL) was confirmed using 1 M NaCl solution (Fig. S4†). The temperature stability of AgHr nanoparticles was verified by heating at various temperatures (50–100 °C at 10 °C intervals), each for 30 min, which showed that hair-extract-stabilised AgNPs were stable up to 100 °C (Fig. 4). The stability of AuHr was also investigated. The pH stability of AuHr was tested by changing the solution pH, which showed that AuHr were stable for a long period in basic, neutral, and slightly acidic (pH 5–0) media. However, AuHr were unstable in strong acidic media. Again, the stability of these nanoparticles in basic media might be due to the presence of amine and thiol groups (Fig. S5 and S6†). Moreover, the salt stability of hair-extract-stabilised AuNPs was scrutinised, finding that 3 mL of AuHr was stable to 800 μL of 1 M NaCl solution (Fig. S7 and S8†). The temperature stability of AuHr was verified by heating at various temperatures (50–100 °C at 10 °C intervals), each for 30 min, which showed that they were stable up to 100 °C (Fig. S9†).

Fig. 4 Heat stability of AgHr.

Urease enzyme inhibition of AgHr and AuHr

Urease is known to cause several infections in humans. Its reducing activity is the focus of research towards different types of antibiotics effective against microbes. Hair extract and hair-extract-stabilised silver and gold nanoparticles were investigated in jack bean urease enzyme inhibition analysis. Hair extract, AgHr, and AuHr were all found to be active against urease, giving the following respective percentage inhibition and IC₅₀ values: hair extract, 99.2% and 32.1 ± 0.57 μg mL⁻¹; AgHr, 96.5% and 58.0 ± 1.03 μg mL⁻¹; and AuHr, 65.0% and 119.3 ± 1.01 μg mL⁻¹ (Fig. 5 and Table S1†).

Fig. 5 Urease and xanthine enzyme inhibitions of AgHr and AuHr.

Xanthine oxidase enzyme inhibition

Xanthine oxidase, like urease, is also known to cause several infections in humans, and has a reducing property that is the focus of research towards developing different types of antibiotics effective against microbes. AuHr and AgHr were investigated for xanthine oxidase enzyme inhibition, while the hair extracts were shown to be inactive against xanthine oxidase.

Results are shown in (Table S2†). The enzyme inhibitory mechanism might be noncompetitive, where substrate and inhibitor are both attached to enzyme, and the loss in enzymatic action could be either due to the nanoparticles blocking the enzyme active sites or a conformational change in the protein. The small Au/AgNPs could provide more active binding centres to immobilise the enzyme than a monolayer bound to large colloids.¹²

Antibacterial evaluation of hair-extract-stabilised Ag and Au nanoparticles

Freshly synthesised gold and silver nanoparticles stabilised with hair extract were tested against four bacterial strains, namely, P. aeruginosa, S. aureus, K. pneumoniae, and E. coli. Levofloxacin was also used as the standard drug. The highest zone of inhibition (80%) was achieved by AuHr against P. aeruginosa. Details of antibacterial activities are presented in Fig. 6 and Table S3.†

Fig. 6 Antibacterial activities of AgHr and AuHr.

Usually silver and silver nanoparticles have better antibiotic properties than gold and gold nanoparticles. However, in the case of P. aeruginosa, gold nanoparticles showed the better antibiotic activity. This might be because the AuNPs were smaller than the AgNPs, causing the AuNPs to have higher hindrance activities than the AgNPs. Another possible reason is that the smaller AuNPs could provide more active binding centres for bacteria immobilization than a monolayer bound to large-size colloids. AuNPs present in high concentrations cannot have significant effects, but, in low concentrations, they form small aggregates within biofilms, which are more effective against Gram negative bacteria due to the thin peptidoglycan layer in the cell wall. The high affinity of silver towards sulfur and phosphorus is key to its antimicrobial effect. Due to the abundance of sulfur-containing proteins in the bacterial cell membrane, silver nanoparticles can react with sulfur-containing amino acids inside or outside the cell membrane, generating electron-dense granules of silver and sulfur ions, which in turn affects bacterial cell viability. It has also been suggested that silver ions (particularly Ag⁺) released from silver nanoparticles can interact with phosphorus moieties in DNA to weaken replication, deactivate proteins involved bacterial growth, affect permeability and gap creation between the cytoplasm and cell wall, and imbalance the release of respiratory enzymes, resulting in cell death.^28–30

Conclusion

The burning of hair, a waste keratinous biomaterial, in tanneries produces a large amount of COD, BOD, and TDS, contributing to hazardous levels of environmental pollution. Herein, we presented the synthesis of silver and gold nanoparticles utilising hair extract, which contains cysteine, an amino acid having amine and thiol functional groups, as a major component, as a capping agent. The synthesised AgHr and AuHr nanoparticles were stable to various physicochemical parameters, including heat, pH, and salinity. The spherical AgHr and AuHr showed good bactericidal and enzyme inhibitor properties where most antibiotic and enzymes inhibitors have failed to be effective due to drug resistance in bacteria. This research work may inspire scientists to develop hair-extract-capped noble-metal nanoparticles applications in various fields.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra21455j

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