Introduction

An incredible revolution has taken place in the nanotechnology field across the previous two periods [1]. Currently, nanoparticles (NPs) are present in different products and also utilized in scientific laboratories as well as industrial implementations. The peculiar attributes of NPs make them valuable for technology and science [2]. Metallic NPs are principal subjects of substantial and considerable research because of their unique properties and wide range of applications in different fields [1, 3]. Recently, nanobiotechnology has expanded promptly in various biological domains of science, owing to the unique physicochemical properties of the nanoparticles which make them ideal agents for some biomedical phenomena [4,5,6].

Gold NPs are one of the important NPs with peculiar attributes and implementations in biomedical science [5, 7]. These NPs find their utilization in sensors, immune assay [5, 8], vaccine development [9], drug delivery [10], and electrochemical biosensors [11,12,13,14]. Gold NPs (AuNPs) are among the broadly reviewed group of NPs owing to their higher potential in solar cells [15, 16], electronics, catalysis [17, 18], sensor technologies [19, 20], biomedical applications, such as identification of microbes [21], gene and drug delivery [22, 23], and tissue engineering [24]. Surface modification of metallic NPs with suitable molecules or ligands may modify the physicochemical properties of NPs [25, 26], which may take an imperative role in diverse applications as drug carriers [10], biochemical sensors [20, 27,28,29,30], bactericidal agents[31], radiotherapy [32], and cancer therapeutic agents [33,34,35]. Several studies suggested the conjugation of gold and silver NPs by amino acids and proteins make effective bioconjugates for nanomedicine and other biomedical applications [26, 36,37,38,39].

The size of nanoparticles is a principal factor that limits their use for drug delivery to peculiar cells, i.e., simply crossing the cell membrane by translocation [40]. The conjugation of amino acids, ligands, and proteins may enhance or affect the stabilization and biocompatibility of metallic NPs [41, 42]. To functionalize the NPs, surface chemistry is a universal and broad-level employed tool [43]. Surface chemistry not simply modifies the properties of NPs, such as biocompatibility, stability, and dispersion, but also reshapes various available functional groups (–CHO, –COO–, and –NH3+) [44, 45]. The cap** of NPs with compatible amino acids and polymers improves their circulation rate and prevents them to aggregate inside the body. The basic aim of surface functionalization is the binding of a specific ligand or making a persistent monolayer at the surface of AuNPs by employing hydrophobic, chemisorption, and electrostatic forces of interactions. Principally, cap** agents may enhance the compatibility and functionality of NPs and also influence their biochemical interactions and concerning applications [46, 47]. Cap** agents which have been reported in the synthesis of NPs are polysaccharides, dendrimers, small ligands, cyclodextrins, and polymers [48]. Substantial functional groups like guanidine [49], carboxyl [50], amino [30, 40], citrate [51], and methyl [40, 52] are utilized to functionalize NPs.

Amino acids are monomeric molecules and work as building blocks of peptides, polypeptides, and larger proteins. They help in the biosynthesis of different vitamins and biomolecules [53]. Different studies had reported the amino acids-based synthesis of NPs using tyrosine, tryptophan, cysteine, aspartic acids, and lysine [30, 54,55,56,57]. Tyrosine is an important amino acid for signal transduction, enzymatic pathways, and precursor for some important biomolecules, and its phenol group play important role in the synthesis and cap** of NPs with effective biological applications [31, 58]. Various studies have reported the usefulness of tyrosine in mice during fatigue, cold, stress [59], prolonged working, and awakening conditions, as it might reduce the level of stress hormones [60]. While cysteine and glycine are the major constituents of glutathione, a highly important major antioxidant during oxidative stress in the animal body [61]. Oxidation of cysteine yield cystine which provides structural stability to various proteins, under specific condition cystine, reduces back to yield cysteine which plays a crucial role in the synthesis of glutathione [62]. The thiol group of cysteine molecules is highly important for the synthesis of metallothioneins, glutathione, and several other important biological molecules, which play a crucial role during oxidative stress [63, 64].

Antioxidants scavenge reactive oxygen species (ROS) in healthy organisms. Accumulation of reactive oxygen species (ROS) may induce several diseases including neurodegenerative disorders, cancer, and cardiovascular diseases [65]. Burt and co-authors [66] reported bovine serum albumin-coated AuNPs with enhanced surface area for surface functionalization. Kim et al. [67] reported the synthesis of antioxidant AuNPs using biogenic routes. The presence of several biocompatible molecules on the surface of NPs may facilitate the binding or release of specific ligands or drug molecules under different conditions, hence modifying surface properties to achieve specific goals [68, 69].

Functionalized NMs always need to be evaluated in detail for their toxicities and potential before their use in practical applications. These evaluations include in vivo and in vitro methods, indicating possible future issues [70]. Detailed studies are always needed to avoid confusion in scientific matters [71]. Almost no data were reported on the amino acids as cap** agents on the gold NMs for their antioxidant potentials using detailed approaches. Recently, we reported amino acids-capped silver nanoparticles for their altered antioxidant potential in mice [26]. This study was planned to evaluate the antioxidant potential of amino acids-capped gold nanomaterials and to see whether the cap** of different amino acids influences the biomedical properties of NPs differently. Conjugated gold NPs were characterized and evaluated for their possible potential by using in vitro and in vivo procedures.

Material and methods

Reagents and chemicals

All the amino acids including L-tyrosine, L-glycine, and L-cystine were purchased from Merck and employed as obtained. Silver nitrate (AgNO3), sodium borohydride (NaBH4), iron (III) chloride, potassium hexacyanoferrate (III) (K3Fe (CN)6), polyvinylpyrrolidone (PVP) (average molecular weight 40,000), pyrogallol, trisodium citrate (Na3Cit.2H2O), trichloroacetic acid (Cl3CCOOH), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) were obtained from Sigma-Aldrich, USA. Phenyl methyl sulfonyl fluoride (PMSF) (purity > 99%), reduced glutathione (purity ≥ 98.0%), 2-mercapto-ethanol (purity ≥ 99.0%), and all other reagents were obtained from quality vendors and utilized as provided.

Chemical synthesis of gold nanoparticles (AuNPs)

Gold nanoparticles were prepared by using the method of [72] with minor modifications. The AuNPs were synthesized by the reduction of tetrachloroaurate (III) rehydrates solution. The gold solution (0.1 mM, 30 mL) was reduced by the dropwise addition of sodium borohydride (4 mM, 0.5 mL). Furthermore, gold nanoparticles were also stabilized by using PVP or sodium citrate. A stable reddish color was obtained which further changed to a vine red color after proper stirring (200 rpm, 50 °C) for more than 20 min.

Fabrication of amino acids-capped nanoconjugates

The AuNPs were capped with different amino acids including L-tyrosine, L-cysteine, and L-glycine, [57, 73]. Briefly, amino acids-conjugated AuNPs were prepared by conjugating various amino acids on PVP-stabilized AuNPs under constant stirring. A comparable variation in color was used as a key indicator of conjugation. The solution was centrifuged at 13,000 rpm for 15 min to purify NPs and re-suspended in deionized water.

Characterization of nanomaterials

The synthesized products were characterized by employing various techniques. Physiological attributes like stability, size, and shape of AuNCs and NPs were determined by employing different characterization techniques, i.e., scanning electron microscopy (SEM)-JEOL (JSM-6480LV) with 15 kV acceleration voltage, Fourier transform infrared spectroscopy (FTIR) (Shimadzu IR Prestige21), UV–visible (UV–Vis) spectroscopy (AE-S70-1U), transmission electron microscopy (TEM) (JEOL, Japan, JEM-2100), dynamic light scattering (DLS)-based zeta potential analyses (Zeta sizer Nano ZS-90-Malvern Instruments, Malvern, UK), etc.

In vitro antioxidant activity

The DPPH scavenging activity of gold NMs was evaluated with the help of previously published procedures [26, 74, 75]. The final DPPH activity was calculated using Eq. (1). Ferric reducing power (FRP) activity of gold NMs was evaluated by using previously published methods with minor modifications [26, 76]. Finally, the absorbance was measured at a wavelength of 700 nm. Increased absorbance of the reaction mixture demonstrates better and high reducing power [77]. The hydrogen scavenging activity of NMs was determined as per the report of Keshari et al. [78]. The hydrogen peroxide scavenging % was calculated by using Eq. 1.

$$ \% {\text{ of Scavenging}} = ({\text{Pc}} - {\text{Ps}}/{\text{Pc}}) \times {1}00 $$
(1)

where Ps = absorption of NMs and Pc = absorbance of control.

In vivo trials using albino mice

Experimental, 8–10-week-old Swiss albino mice (male and female) with an average body weight of 30–35 g were acquired from Animal House, Department of Zoology, Government College University Lahore, Pakistan. All the experimental conditions like the ventilation system, cooling, and heating (12 h light/dark cycle, 22 ± 2 °C) were maintained properly during the experimental period. For animal use and trial run, ethical approval letter was perceived from the Ethical Committee of GCU Lahore. Before the trial run, all animals were acclimatized for 7 days under regulated conditions. The mice were fed regularly with a standard diet (pellet) and also were delivered with a water supply (ad libitum). Details of the groups are provided in Table 1. Mice were monitored on daily basis. At the end of the experiment, animals were slaughtered, and blood samples and other body organs, i.e., intestine, kidney, and liver, were preserved appropriately for further analysis.

Table 1 Groups of animals for sub-lethal dose study (n = 5) (Test No. 407) (OECD, 2008) [79]. Treatments were administered orally by using oral gavage for 28 days, where Cad; Cadmium metal, C-AuNPs; chemically synthesized gold nanoparticles, Tyr-AuNCs; L-tyrosine-capped gold nanoparticles, Gly-AuNCs; L-glycine-capped gold nanoparticles, Cys-AuNCs; L-cystine-capped gold nanoparticles
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Endogenous antioxidant enzymes

Superoxide dismutase (SOD) was evaluated by employing an already published method using the pyrogallol auto-oxidation process [80]; catalase (CAT) and GST activity was assayed according to the protocol of Javed et al. [81].

Determination of GSH and MTs

The estimation of reduced glutathione (GSH) was analyzed by already reported procedures [26, 82] with minor modifications. The GSH level was analyzed by utilizing a newly formed standard cure, and the final assumption was presented as a milligram (mg) of glutathione per milligram of protein. Metallothioneins (MTs) were assayed by following the previously published reports [26, 83, 84]. The MTs were calculated by using the freshly prepared standard curve of GSH while taking 30 cysteine residues per MTs molecule.

Data analysis

The data were subjected to one-way or two-way ANOVA to estimate the level of significance of individual differences. For the normal dataset, ANOVA was applied with the Tukey test as post hoc, while the Kruskal–Wallis H was applied on skewed data followed by multiple comparisons. The data were analyzed by SPSS software.

Results

Synthesis and characterization of gold nanoconjugates (AuNCs)

UV–visible studies

Gold nanoparticles (AuNPs) were synthesized and conjugated with different amino acids. A prominent change in color was noticed before and after the conjugation. A specific concentration of amino acids (2 mM) was selected due to the formation of more stable conjugates over time; optimization plots are also provided in Fig. 1. A shift in SPR peaks was also recorded for almost all samples after conjugation, which indicated a successful conjugation of the amino acids on AuNPs. Chemically synthesized PVP-stabilized AuNPs showed an SPR peak at 517 nm. However, minute changes in SPR peaks were recorded after conjugation. L-cystine-capped gold nanoconjugates (Cys-AuNCs) showed an SPR peak value of around 522 nm, which further shifted ahead with time. In contrast, in Gly-AuNCs and Tyr-AuNCs, SPR peaks appeared at 517–518 nm, while an additional peak was recorded at 276 nm for Tyr-AuNCs, which is the characteristic peak of tyrosine amino acid (see Fig. 2a).

Figure 1
figure 1

Optimization of amino acids-capped gold nanoparticles at different concentrations of amino acids a Cys-AuNCs, b Gly-AuNCs, c Tyr-AuNCs; inset graphs displaying focused area of SPR change

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Figure 2
figure 2

Characterization of gold NMs; a UV–visible spectroscopy of AuNPs and their nanoconjugates, b Zeta potential, for each sample, n = 3, c TEM micrograph, scale bar = 50 nm, d size distribution map

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Transmission electron microscopy and Zeta potential of gold NMs

Transmission electron microscopy (TEM) revealed the morphology of the samples. Gold nanoparticles also displayed spherical-shaped morphology with an average size of 16.53 ± 1.22 nm (see Figs. 2c and d). Gold NMs displayed values, i.e., −35.89 ± 5.14, −21.46 ± 2.45, −37.69 ± 4.65, and −46.83 ± 3.78 mV for C-AuNPs, Cys-AuNCs, Tyr-AuNCs, and Gly-AuNCs, respectively (Fig. 2b).

Scanning electron microscopy and size distribution of gold NMs

Scanning electron microscopy revealed the synthesis of uniform spherical-shaped C-AuNPs (see Fig. 3a) with 15.20 ± 2.13 nm average size and 14% polydispersion (Fig. 4a). Tyr-AuNCs displayed an average size of 24.79 ± 2.74 nm, with 11% polydispersity, and few particles showed agglomeration and aggregation (see Figs. 3b and 4b).

Figure 3
figure 3

Scanning electron microscopy of; a C-AuNPs, b Tyr-AuNCs, c Gly-AuNCs, and d Cys-AuNCs; scale bar on all SEM images shown is 200 nm

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Figure 4
figure 4

Size distribution maps of; a C-AuNPs, b Tyr-AuNCs, c Gly-AuNCs, d Cys-AuNCs

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Glycine-capped AuNCs showed an average size of 16.48 ± 1.54 nm with irregular surface morphology and 9% polydispersity, somewhere spherical particles were seen prominently in good concentration (see Figs. 3c and 4c). Cys-AuNCs displayed spherical-shaped morphology with a higher level of particles agglomeration. These NPs showed an average size of 22.04 ± 2.76 nm, with 13% polydispersity (see Figs. 3d and 4d).

Fourier transform infrared spectroscopy of gold NMs

The FTIR spectra of gold NMs before and after conjugation are shown in Fig. 5. Several noticeable FTIR peaks were reduced or vanished from FTIR spectra of the amino acids, while a few others showed peak shift in their position and frequency.

Figure 5
figure 5

Fourier transform infrared spectroscopy of; a L-cystine, b Cys-AuNCs, c L-glycine, d Gly-AuNCs, e L-tyrosine, f Tyr-AuNCs

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After synthesis of Cys-AuNCs, various peaks exhibited reduced transmittance, while only two peaks diminished, i.e., 2364 and 3614 cm−1 corresponding to C=N and –OH groups, respectively. After synthesis of Gly-AuNCs, peaks at 1558 cm−1 and 2357 cm−1 diminished, indicating the presence of N–H and C=N, while many other peaks showed a reduced transmittance which showed that these functional groups, somewhere also contributed to the cap** process. After synthesizing Tyr-AuNCs, several peaks at 800, 840, 1043, 2351, 2644, 2933, and 3207 cm−1 correspond to C–H, C–N, C=N, –OH, –N–H, and NH2 were seen to be reduced or completely diminished (see Fig. 5). These results were interpreted from a previously published report [85].

Stability of gold NMs

A slight change in peak absorbance occurred with a redshift in the peak for AuNPs over time. The peak finally disappeared after one year (see Fig. 6a). Redshift in the peaks with a minute reduction in absorbance was also recorded for Gly-AuNCs and Cys-AuNCs (Fig. 6b and c), while in Tyr-AuNCs, the red- or blueshift was not recorded, although the reduction in absorbance was observed (Fig. 6d).

Figure 6
figure 6

UV–visible spectroscopy-based stability study of: a C-AuNPs, b Cys-AuNCs, c Gly-AuNCs, d Tyr-AuNCs

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In vitro antioxidants activity of NCGs

In vitro, the antioxidant activity of all prepared samples was assayed by using DPPH, hydrogen peroxide (HP) scavenging, and ferric-reducing power (FRP) assays.

It was measured by taking absorbance at 517 nm before and after the reaction of the samples with DPPH radical. DPPH changes into yellowish color with the addition of a good antioxidant, while mild antioxidant compounds induce only a minute change in the shade of DPPH, and the absorbance is also decreased. Chemically synthesized gold nanoparticles (C-AuNPs) exhibited a mild antioxidant activity, Gly-AuNCs showed a reduction in the antioxidant potential, while, in Tyr-AuNCs and Cys-AuNCs, DPPH activity increased to some extent; however, it increased more prominently in Cys-AuNCs. Ascorbic acid showed maximum scavenging activity among all the samples under investigation (see Fig. 7a). In a comparison between the gold NMs, Cys-AuNCs exhibited a maximum concentration-dependent DPPH scavenging activity (see Fig. 7b). Among all gold nanoconjugates, Tyr-AuNCs exhibited higher hydrogen peroxide scavenging activity (see Figs. 7c and d). Among all the gold NMs, Gly-AuNCs and Tyr-AuNCs showed good FRP activity. Although Cys-AuNCs also exhibited some FRP activity, it was comparatively less than the other two conjugates. In contrast, L-cystine showed higher FRP activity among pure amino acids than L-tyrosine and L-glycine (see Fig. 8).

Figure 7
figure 7

In vitro antioxidant activity of NMs; a DPPH scavenging activity of different conjugates compared to the ascorbic acid as a standard antioxidant, b gold NMs, the data have shown are Mean ± SD value with n = 6. A statistically significant difference in DPPH scavenging activity was found among different NMs at different concentrations where “*” represent p < 0.05 and “**” represent p < 0.01, two-way ANOVA with post hoc Tukey test was used. c Hydrogen peroxide (HP) scavenging % of gold NMs and ascorbic acid, d HP scavenging % of gold NMs, the data have shown are Mean ± SD values with n = 3. For homogeneity of data, p > 0.05, the difference was found with different concentrations of nanoconjugates in HP scavenging activity

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Figure 8
figure 8

In vitro antioxidant activity of NMs; Ferric reducing power assay of Gold NMs, the data shown are Mean ± SD values with n = 3. For homogeneity of data p > 0.05, different nanoconjugates displayed a difference in FRP (df 8, 30; mean square = 0.01; F = 27.1; partial η2 = 0.88 and p < 0.05); Two-way ANOVA with Tukey test as post hoc was used to the evaluated difference, where “*” denote p < 0.05

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In vivo antioxidant activity of gold nanoconjugates

In vivo, the antioxidant activity of gold nanoparticles (AuNPs) and their nanoconjugates was evaluated by evaluating various parameters and endogenous enzymes, such as reduced glutathione (GSH), metallothioneins (MTs), catalase (CAT), and superoxide dismutase (SOD), etc., in the kidney and liver samples. All results of AuNPs and their conjugates were briefly compared (see Table 2).

Table 2 Comparison of all the results obtained for cadmium metal (Cd), C-AuNPs, and amino acids-capped AuNCs
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Bodyweight

During exposure studies of gold nanoparticles and their nanoconjugates, all groups of experimental mice showed increased body weight except for animals in the G6 group, which showed negligible weight reduction (see Fig. 9a). All animals were stable throughout the study period except for only one animal in the G5 group which developed a shoulder tumor.

Figure 9
figure 9

In vivo animal trial; a Animal body weight, b CAT-activity in liver tissues and serum samples, the data shown are Mean ± SE values in (liver tissues n = 5, serum samples n = 3), for both types of samples homogeneity of data was p > 0.05, so one-way ANOVA with Tukey test as post hoc was applied, where “*” represent p < 0.05 when compared with control, c SOD Activity, the data shown are Mean ± SE values in (liver tissues n = 5, serum samples n = 3), for homogeneity of data (p > 0.05), one-way ANOVA with post hoc Tukey test was applied, where “*” indicates p < 0.05 when compared with control, d For GST activity, the data shown are Mean ± SE values in (liver tissues n = 5, serum samples n = 3); for homogeneity of data (p > 0.05), one-way ANOVA with Tukey test was used, there was no statistically significant difference was found in GST level

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Endogenous enzymes

The CAT activity (in the liver) was decreased in the G2 group (p < 0.05), while increased in the G3 (p < 0.05), G4 (p > 0.05), and G5 (p > 0.05) groups. There was a negligible difference in the G6 group compared to the control group (G1). The CAT activity (in serum) was decreased in the G2 group (p > 0.05), whereas it increased in the G3 (p < 0.05) and G6 (p < 0.05) groups. No significant difference was observed in the G4 and G5 groups when compared to the control group (Fig. 9b). The SOD activity (in the liver) was increased in the G3 group (p > 0.05) but was nonsignificantly reduced in G2, G4, G5, and G6 groups. The SOD activity (in serum) was increased in the G3 group (p > 0.05) and decreased in the G5 group (p < 0.05) and other remaining groups (p > 0.05) with some variations (Fig. 9c).

In the case of liver tissues, GST level was decreased (p > 0.05) in G2, G3, and G4 groups, while a minor difference was noticed in the G5 and G6 groups compared to the control group (G1). The GST level (in serum) was increased in G6 (p > 0.05) but decreased in G2 (p > 0.05) and G3 groups (p > 0.05) in comparison with the control group (G1). A slight change was observed in G4 and G5 groups (Fig. 9d).

Reduced glutathione and MTs in tissues

Reduced glutathione was increased in G2 (p < 0.05), G4, and G5 (p > 0.05) groups, whereas it slightly increased in G3 and G6 mice groups. However, GSH in kidney tissues remained normal in all treatment groups other than in the G4 group, which demonstrated a decreased (p > 0.05) level of GSH (Fig. 10a). Metallothioneins in liver tissues were increased in G2 (p < 0.05) and G5 (p > 0.05) groups, whereas MTs level was reduced (p > 0.05) in G4 and G6 groups. A negligible change was recorded in the G3 group than the control group (G1). MTs’ level in kidney tissues was increased in G2 (p > 0.05) but nonsignificantly decreased in G3, G4, and G6 groups, while a slight difference was noted in the G5 group compared to the G1 group (Fig. 10b).

Figure 10
figure 10

a GSH levels in kidney and liver tissues, the data shown are Mean ± SE values, n = 5; for homogeneity of data (p > 0.05), a significant difference was found in liver GSH and not found in kidney GSH. One-way ANOVA with post hoc Tukey test was performed, where “*” represents p < 0.05 when compared with control, b For MTs in kidneys and liver tissues, the data shown are Mean ± SE values, n = 5; for homogeneity of data (p > 0.05), a statistically significant difference was found in MTs in kidneys and liver tissues. One-way ANOVA with post hoc Tukey test was used, where “*” indicate p < 0.05 when compared with control

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Discussion

Synthesis and characterization of NMs

Polyvinylpyrrolidone (PVP)-stabilized gold nanoparticles (AuNPs) showed an SPR peak at 517 nm with minute changes after conjugation. PVP helped to enhance the biocompatibility and biological stability of the colloids, as it is an inert, nontoxic, and biocompatible excipient and portrays a complex affinity for both hydrophobic and hydrophilic substances or drugs [86,87,88]. Many factors, such as the size of the particles, dielectric constants of the medium, quantity of Au+ ions, and available biomolecules, largely influence the SPR peaks of the AuNPs [89].

Glycine-capped AuNCs and Tyr-AuNCs displayed SPR peaks around 517–518 nm, while an additional peak at 276 nm was recorded for Tyr-AuNCs, which is the characteristic peak of tyrosine amino acid. Aromatic side chains of phenylalanine, tyrosine, and tryptophan residues generally give absorption spectra in the range of 230–300 nm [90, 91].

Another study showed the adsorption of glycine molecules on the surface of AuNPs [92], while Saw et al. [93] emphasized the cystine-capped AuNPs for their size-dependent cellular uptake. Previously, several studies have reported the synthesis of amino acids-capped AuNPs [93,94,95] (see Fig. 11) and silver NPs [26, 47, 56] for different applications.

Figure 11
figure 11

Synthesis of amino acids functionalized-gold nanoparticles

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Tyrosine-capped gold nanoconjugates (AuNCs) were larger than cystine-capped AuNCs and glycine-capped AuNCs. Gold nanoconjugates exhibited agglomeration, which promptly increased the size of synthesized NPs. This phenomenon is common when particles are conjugated or synthesized with biological molecules or plant extract [96]. An increase in size for tyrosine-AuNCs and cystine-AuNCs is directly associated with cap** and interactions of added amino acids with NPs. Rapid nucleation, super-saturation of the solution, and formation of multiple hydroxylated species lower the reduction potential of gold ions at higher pH values. It leads to a quick reduction yielding smaller AuNPs in the presence of small-sized-amino acids, i.e., L-glycine [97, 100, 101]. Surface charges of the nanoparticles mainly depend upon various factors such as the temperature of the reaction, solvent available, ligand terminal chemistry, and ligand length [102, 103]. Lower charge values of Cys-AuNCs might be accredited to the agglomerations seen in SEM images also, as agglomerations of NPs lead to a decreased surface area and charge trap** capability [104]. Furthermore, the surface charges of the NPs largely influence their cellular uptake and distribution in the biological systems which largely influence their biomedical properties and applications [102].

Chemically synthesized AuNPs displayed the least stability compared to AuNCs. Partial oxidation of the gold surfaces in different media may improve the stability of ligand-free AuNPs [105, 106], while different conjugant molecules may also influence the stability, toxicity, and biocompatibility of conjugated gold nanoparticles [106, 107]. Different conjugated molecules or ligands generally form a protective layer on the surface of nanoparticles which improves specificity and stability and prevent aggregation in biological fluids [108]. The pH of the medium and available functional groups may also influence several molecular and electrostatic interactions influencing the stability of NPs [109, 110]. The balance between repulsive and attractive forces helps to assess the stability of NPs [111]. During this study, most of the particles displayed higher negative surface charges portraying higher repulsive forces, hence displaying higher particle stability. Contrary to the results of the present study, another study reported the stability of glycine-capped AuNCs over three months and suggested that these NMs did not show any aggregation or loss of stability. Glycine molecules in an anionic form enhanced the stability of NPs [99]. A plausible explanation of this contradiction is that they evaluated their samples for up to three months, while in the present study, samples were monitored for more than one year. Another study reported the role of glycine in the aggregation control of AuNPs [41].

A few noticeable peaks were reduced or disappeared from the FTIR spectra of the amino acids [85]. After synthesis of Cys-AuNCs, various peaks exhibited reduced transmittance, which showed the consumption of different functional groups in the cap**, while only two peaks, 2364 cm−1, and 3614 cm−1 diminished, corresponding to C=N and –OH, respectively. Many new peaks developed, indicating rearrangements of various atoms and functional groups. Peaks at 1558 cm−1 and 2357 cm−1 diminished after the synthesis of Gly-AuNCs, corresponding to the presence of N–H and C=N, while many other peaks showed a reduced transmittance which showed that these functional groups also contributed to the cap** process. After synthesizing Tyr-AuNCs, several peaks, 800, 840, 1043, 2351, 2644, 2933, and 3207 cm−1, were reduced or diminished completely, corresponding to the presence of C–H, C–N, C = N, –OH, –N–H, and NH2. FTIR interpretation was accomplished using previously reported data [85].

Zeta potential (ZP) is the net charge present on the surface of NMs. Previous studies have shown that the particles with more negative values are more stable than the positively charged NMs; hence, ZP analyses also depict the stability of NPs [111,112,113]. During this study, zeta potential of the NPs was evaluated by dynamic light scattering analyses (DLS). Zeta potential values of gold nanoconjugates showed the lowest negative values for Cys-AuNCs and the highest negative values for Gly-AuNCs. UV–visible studies have shown higher stability of AuNCs. Hamaguchi et al. [99] reported that the anionic form of glycine at higher pH largely affected the stability of AuNCs. The findings of the present study are in agreement with the report of Hamaguchi et al. (2010). Different interactions or effects induce change in the zeta potential (ζ) in the presence of any surfactant or polymer, i.e., displacement of the counterions, shifting of the slip** plane, and the dissociated functional groups from the reactants [114]. Occasionally, these factors influence the ZP of any colloidal solution or conjugates. Dissociated functional groups and their ionic nature or charges; positively charged groups, e.g., –NH2, or negatively charged groups, e.g., –COOH, generally impart positive or negative ZP values, respectively [115].

The lower stability of cystine-capped NMs might be accredited to the positively charged group, i.e., –NH2 on the surface of NMs. The pH also affected ZP values, as pH values influence intermolecular and intramolecular interactions and properties of the molecules [115, 116]. All prepared conjugates were maintained at a pH value of 7.4, more than the pI (isoelectric point) of the concerning amino acids. As pI values are smaller than pH values, the solution contains an excess amount of OH− group, which removes H+ ions from positively charged amine groups of amino acids, resulting in a net negative charge on the carboxylate group, and amino acids-capped NMs showed negative charges, thus displaying negative ZP values [117].

Different interactions of organo-sulfur compounds and bioactive metabolites played a crucial role in determining the size and shape of the synthesized NPs [118]. The AuNPs displayed an average size of 16.53 ± 1.22 nm with an SPR peak at 517 nm, indicating the spherical shape of NPs. The optical properties of NMs are size and shape dependent [119]. A previous study by Mansuriya and Altintas [120] reported the synthesis of AuNPs with an average diameter of 4.7 ± 2 nm with a UV peak at 514 nm. The findings of the present study are in agreement with other studies [119, 121].

In vitro antioxidants activity of NMs

Among all nanoconjugates (NCs) of amino acids, conjugation of cystine and tyrosine generally improved DPPH scavenging activity, whereas glycine reduced it. Chemically synthesized gold NPs displayed mild antioxidant properties. AuNPs demonstrated antioxidant activity [122], which was attributed to the presence of gold atoms in zero-valent form. According to Satnami et al. [123], the chemical composition and nature of ligands attached to the surface of NMs largely influence various properties of NMs. The strong bonds or other types of molecular interactions of L-cystine with AuNPs affected their DPPH scavenging activity. Specific functional groups present in nanoconjugates reduced DPPH free radical into hydrazine molecules by accepting or donating electrons [124]. The higher DPPH activity of Cys-AuNCs might be accredited to the presence of the thiol group which can accept electrons, and oxidize free radical molecules [63, 125] while the amino and hydroxyl groups present in amino acids might also play important role in DPPH scavenging activity [126]. Other studies also reported biomolecule-coated AuNPs and other nanomaterials for their concentration-dependent DPPH scavenging activity [127,128,129].

Gold conjugation with cystine yielded reduced FRP activity. Various groups of amino acids shifted the FRP activity of chemically synthesized gold nanoparticles. Phenolic group on tyrosine and disulfide bond (S–S) of cystine and other functional groups present in different biomolecules or amino acids, influence redox reactions, and metal chelation [130, 131]; thus, these groups might influence FRP activity of NMs. Thiol groups in cysteine show the least reactivity with ferric ions [132, 133], and Cys-AuNCs displayed the least FRP activity. Blue-colored-chelate formation display a concentration-dependent increase in absorbance in the presence of effective antioxidant [134]. This activity measures the ability of molecules to reduce ferric ion (Fe+3) to ferrous ion (Fe+2) by the donation of an electron [135].

Among all the NCs, tyrosine conjugation yielded nanoconjugates with enhanced hydrogen peroxide (HP) scavenging activity. It is mainly because of the availability of different functional groups present on the surface of NMs [136]. Various enzymes or other biochemical pathways generate hydrogen peroxide as a by-product. It generates different free radicals in excess amounts and induces oxidative stress [137]. The appearance and optical properties of the AuNPs did not change even after the reaction with hydrogen peroxide, and the characteristic SPR peaks also remained unchanged. AuNPs generally work as catalysts during H2O2 scavenging activity and form hydroxyl radicals. This reaction occurs under acidic conditions [138]. Previously, several studies had reported the peroxidase-like activity of AuNPs at lower pH values [139, 140].

In vivo antioxidant activity

In exposure studies of AuNCs, an insignificant increase in body weight was observed in all groups other than the G6 group, which displayed a negligible weight reduction. Cadmium ions lead to the activation of pro-survival signaling, which induce toxicity and tumors in the animal body [141,142,143]. Oratic acid is produced in excess amounts during the metabolism of glycine, which might play a role as a tumor promoter [144]. Zhang et al. [145] stated glycine decarboxylase (GLDC)-dependent alteration in the metabolism of serine and glycine, influencing pyrimidine metabolism which further induces tumors in the animal body. The body generates antioxidant enzymes regularly. The liver or kidney clears them off after their specific actions. Hepatocytes clear catalase, while kidneys filter SOD. Superoxide dismutase converts superoxide ions into hydrogen peroxide (HP), and CAT converts HP into oxygen and water. Glutathione-s-transferase (GST) and different xenobiotics make conjugates, which make their disposal more efficient. According to Jamakala and Rani [146], SOD, CAT, and GST activity decreased in cd-intoxicated mice. A change in endogenous enzymes in the body mainly depends upon the chemical and its concentration, exposure duration, synthesis method, and route of exposure. Wu and Zhou [147] reported a dose-dependent decrease in SOD and CAT in the liver tissues of Japanese rice fish (Oryzias latipes) and explained that this decrease was due to the high consumption rate of these enzymes against reactive oxygen species (ROS). These results are in agreement with those of Mehrzadi et al. [148] and Steinebach and Wolterbeek [149]. The shortage or decreased level of CAT was the result of the saturation of all available free CAT molecules in the presence of increased hydrogen peroxide. Surface modification of NPs alters their biological impacts or interactions in living organisms [46]. Several functional groups present in conjugated molecules might help to modify the biological reactions in various ways, influencing endogenous enzymes and other parameters in different ways. Surface functionalization of NPs with L-cystine and L-tyrosine induced insignificant toxicity, and minor alterations in endogenous antioxidant enzymes were detected. These findings are in agreement with the results of Jamakala and Rani [146]. Another study reported an age-related decline in mitochondrial function in response to cysteine amino acid, where cysteine may alter iron homeostasis in vacuole-impaired cells lacking proper vacuolar functions [150]. Another study reported specie-specific high-dose exposure of cysteine to be lethal for a chick not for rats or pigs [151]. World Health Organization (WHO) recommended 4.1 mg/kg of body weight daily intake of L-cysteine, and Shibui et al. reported the no-observed-adverse-effect levels (NOAELs) for L-cysteine to be less than 500 mg/kg/day [152].

Glutathione-s-transferase (GST) plays a substantial physiological role in detoxifying pharmacologically synthesized active compounds and various alkylating agents. Glutathione -SH group attaches with these active agents or compounds during catalysis. Then, the -SH group neutralizes the electrophilic sites of these compounds and makes the by-products more water-soluble. Exposure to NPs generates a higher amount of peroxides and other radical species. Overconsumption of GST decreases its activity [153]. Reduced activity of GST is the result of GSH affinity to NPs. The reduced form of GST is GSH. Glutathione has an excellent attraction for NPs, i.e., substantial glutathione association decreases the level of the immune response [154]. The formation of these complexes leads to glutathione oxidation and the concentration of free radicals increases resulting in a decreased GST activity. Jamakala and Rani [146] reported diminished levels of GST, total antioxidant capacity (TAC), and GSH in mice. These results are in agreement with the study of Rajkumar [155].

Reduced glutathione portrays a higher affinity for different NMs, and it further reduces available GSH. Decreased glutathione is linked with a lowered immune function [154, 156]. Increased levels of GSH in the cadmium-exposed group (G2) might be linked with an elevated immune response. The alteration in the level of MTs referred to metals-induced toxicity in animal tissues.

According to Klaassen et al. [157], toxicity in different organs is generally reduced as a result of MTs chelation with different metals. These proteins also bind with other thiol-group-based molecules, including GSH, and help to regulate ROS in cells. Strong binding sites for different metallic ions are provided by cysteine-rich residues of GSH [158]. Another study reported that different NPs also chelate with other thiol-group-based molecules and MTs [159]. Cadmium (Cd) metal-induced toxicity in animal tissues, while MTs generally make a complex with Cd in the cytosol leading to a reduction in toxicity [160].

According to Nordberg et al. [161], the kidneys work as a target organ for Cd toxicity. Accumulation of Cd-MTs further worsens toxicity. Amino acids-based cap** of NMs somewhere influenced the level of MTs in the liver and kidneys, so different levels of MTs were recorded for all types of nanoconjugates. During the present study, amino acid cap** was performed differently in the living body. Chirality-dependent properties of amino acids also influenced biochemical pathways in different ways [13]. Nanomaterials (NMs) also influenced the regulation of MTs in tissues. Besides, the conjugation of different amino acids also affected this phenomenon differently.

Conclusion

This innovative study reveals the promising and advantageous characteristics of different amino acids as cap** agents on the surface of gold nanoparticles. Amino acids-capped gold NMs exhibited significant biomedical attributes in comparison to uncapped NMs. Chemically synthesized AuNPs are slightly toxic while cap** of amino acids resulted in making comparatively more eco-friendly and less toxic gold NMs for different applications. Tyrosine- and cystine-capped AuNCs displayed comparable antioxidant activity (in vitro and in vivo), i.e., induced insignificant oxidative stress and toxicity compared to C-AuNPs. Moreover, this novel and innovative study also provided evidence of the safety profiling of biomolecule-based cap** of AuNPs. In conclusion, detailed research on biomolecule-based conjugation may produce highly potential and applicable nanoconjugates for various disciplines of sciences and biomedical applications.

Recommendations and future perspectives

Surface functionalization of NPs may produce effective, biocompatible, and eco-friendly NMs. Biomolecules-based surface functionalization would yield more secure and safe NMs for biomedical applications. Various biomolecules might also be explored and investigated for their functionalization potential and biocompatibility. Biomolecules-based nanoconjugates can be explored for their biomimetic and bio-inspired activities. These particles should be tested for their relative potential in different medical applications, i.e., drug delivery, medical imaging, and cancer therapy. Nanoparticles display antibacterial activity against various strains of bacteria, and their functionalization may enhance this property with appropriate biomolecules. L-cystine and L-tyrosine cap** displayed a competent role; therefore, these amino acids should be investigated further for their detailed potential in surface functionalization and modification of the biological properties of NMs. Using NMs with antioxidant properties might provide excellent tools for different biomedical challenges and therapeutics, which may revolutionize healthcare systems in the future.