Novel Nanocombinations of L-tryptophan and L-cysteine: Preparation, Characterization and Their Applications for Dye Decolorization, Antimicrobial and Anticancer Activities

doi:10.21203/rs.3.rs-650889/v1

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Novel Nanocombinations of L-tryptophan and L-cysteine: Preparation, Characterization and Their Applications for Dye Decolorization, Antimicrobial and Anticancer Activities

https://doi.org/10.21203/rs.3.rs-650889/v1

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Tungsten oxide WO₃ nanoparticles (NPs) were prepared in a form of nanosheets with a homogeneous size and dimensions in one step through acid precipitation using a cation exchange column. The resulting WO₃ nanosheets surface was decorated with one of the two amino acids (AAs) L-tryptophan (Trp) or L-cysteine (Cys) for their dye removal, antimicrobial, and antitumor activities. A noticeable improvement in the biological activity of WO₃ NPs was detected upon amino acid modification compared to the original WO₃. The prepared WO₃-Trp and WO₃-Cys exhibited strong dye removal activity toward methylene blue and safranin dyes with complete dye removal (100%) after 6 h. WO₃-Cys NPs and WO₃-Trp NPs revealed higher broad-spectrum antibacterial activity toward both G-ve and G+ve bacteria with strong antifungal activity toward Candida albicans. Anticancer results of the modified WO₃-Cys and WO₃-Trp NPs against various kinds of cancer cells including MCF-7, caco-2, and HepG-2 cells indicated that they have a potent effect in a dose-dependent manner with high selectivity to cancer cells and safety against normal cells. The expression levels of E2F2, Bcl-2 genes were found to be suppressed after treatment with both WO₃-Cys and WO₃-Trp NPs more than 5-FU-treated cells. While expression level of the p53 gene in all tested cells was evidently up-regulated after treatment by more than 5-8 folds as compared to untreated cells. The docking results confirmed the ability of both NPs to bind to P53 gene with relevant potency in binding to other tested gens and participation of cysteine SH-functional group in such interaction.

Cancer Biology

nanoparticles

two amino acids (AAs) L-tryptophan (Trp) or L-cysteine

Candida albicans

Tungsten oxide (WO₃) is considered as one of the most promising transition metal oxides, for their regard performance in various applications including electrochromic devices ^1,2, dye-sensitized solar cells ^3,4, photocatalytic applications ⁵, sensing applications ^6,7, field-emission applications ⁸, high-temperature superconductors ⁹, optical recording devices ¹⁰ and adsorbent ¹¹. The wide application for WO₃couldbe attributed to its high chemical stability and remarkable electric conductivity in addition to the ability to reverse-redox process ¹². Moreover, tungsten oxide could be prepared in different morphologies involved: nanorods ¹³, nanotubes ¹⁴, nanosheets ⁷, nanowires ¹⁵, and nanobelts ¹⁶. Several techniques were investigated for the preparation of WO₃ using either physical or chemical approaches; thermal evaporation ¹⁷, spray pyrolysis ¹⁸, sol-gels ¹⁹, templating method ²⁰, hydrothermal ¹³, electrochemical anodization ²¹, electrodeposition ¹², microwave-assisted method ⁵. however, these methods are time-consuming, and relatively complicated processes with a limitation in controlling the size and shape of the resulted nanoparticles ^5,22. One of the simplest and cost-effective methods for preparing tungsten trioxide nanoparticles is acid precipitation ²³. Great attention is recently directed toward increasing the nanometals implementation in various industrial sectors attributed to its tiny size and large surface-to-mass ratio, besides it’s a remarkable stability toward many harsh conditions ^24,25. Nonetheless, the high biotoxicity of some well-known NPs (Ag, Cu, Ti, etc.) restricted their wide commercial usage ^26,27. To overcome the toxicity issue, attention was directed toward NP-oxides as a more safer and effective alternative ²⁸. In the same regard, NPs surface modification was also proposed to increase the NPs applicability range with lower side toxicity ^29,30. Recently, growing interest in nanoparticles surface functionalization with different functional groups and bio-macules as amino acids, short peptide, and proteins toward new applications was intensively reported ^29,31. Owing to the large surface-to-mass ratio, NPs adsorb these biomolecules on their large surface area in corona-like structure through Van der Waals forces revealing new biological activity ³².

Nowadays, surface-functionalized NPs are efficiently applied in many fields including environmental and medical ones. With growing dye-dependent industries, synthetic dye effluents represent great environmental and public health challenges attributed to their significant toxicity ^25,33–35. Though many approaches were proposed for dye remediation ³³, the lower efficiency and high cost involved limiting their commercial applications ³⁶. Biotechnological applications of various NPs in hazardous chemicals remediation is an emerging field ²⁵, with advantages of surface modification that greatly enhance their dye-removal capacity ³⁷. Cancer is recently seen as a global health threat and it consider the world's second leading cause of death, as well as it is expected to become the leading cause of death in the future years ^38,39. Cancer treatment usually resulted in immunosuppression for treated patients, a condition that renders them at high risk for severe microbial infections ⁴⁰. Developing a new anticancer agent with consolidated antimicrobial activity is a pressing need. To this end, inorganic nanometals represent one step forward, with their high antitumor and antimicrobial activity with minor bacterial-resistance evolution ability ⁴¹. Moreover, the high stability of NPs toward harsh conditions including temperature and pH intensify the efforts for its successful medical applications ⁴². Short peptides and amino acids also attracted attention as promising antimicrobial agents ⁴³. Currently, a wide interest in the individual AAs’ interaction with different NPs for better understanding and elucidation of corona-like structure and give a deeper insight for the new resulting biological activity ^44,45. Cysteine and tryptophan-rich short peptides revealed potent antimicrobial activity against many human pathogens ^46,47. The current study concerning the preparation and functionalization of WO₃ NPs with tryptophan or cysteine amino acids and evaluating the efficacy of the resulted NPs-AAs in versatile applications including synthetic dye removal, antimicrobial, and antitumor activities.

Characterization of WO₃ NPs and WO₃-amino acid (WO₃-AAs) complex

The WO₃ NPs was prepared from Na₂WO₄ solution through strong HCl acid type of cation exchange resin. The yielded yellow solution was precipitated and dried at 70°C. The surface of dried NPs was functionalized with Trp or Cys (Fig. 1). The appearance of synthesized WO₃·H₂O and modified WO₃-AAs at various magnification is explored by SEM (Fig. 2). The SEM photos presented WO₃·H₂O in separated nanosheets morphology with a homogeneous size and dimensions (Fig. 2). The functionalized WO₃-AAs complexes under the SEM microscope presented as coagulated structure formed from of the WO₃·H₂O sheets linked with each other by the amino acid to form agglomerated WO₃.H₂O nanosheets/AAs complexes (Fig. 2). In a similar work, individual amino acids, especially for histidine, were proposed for better understanding the NPs and protein interaction ⁵¹.Though several mechanisms were proposed for interaction of protein and amino acids on the surface of NPs, the exact mechanism is not fully understood ³⁰. The crona-like structure resulting upon the proteins and NPs interaction is well stablished theory for elucidating the different biological behavior of NPs in the biological media ^51,52. It importance to note that, the WO₃·H₂O still keep on its sheet morphology in the resulted WO3-AAs complexes.

Energy-dispersive X-ray spectroscopy (EDS) was applied for revealing the elemental analysis of the materials. The analysis demonstrated that the synthesized WO₃.H₂O is free form impurities (Fig. 2). The analyzed powder is shown to be composed of %At (W = 42.10 and O = 57.90). After modification of WO₃NPswith Trp or Cys, the EDS analysis of the complexes (WO₃-Trp & WO₃-Cys) showed the presence of carbon, as revealed in Fig. 2, denoting the carbons comes from amino acid.

The FT-IR spectra was used to identify the structural functional groups of WO₃.H₂O and WO₃-AAs complexes (Fig. 3a). The IR spectrum of WO₃.H₂O (Fig. 3a) revealed a broad band at 3387 cm^-1 corresponding to the stretching motion of (O-H), the band at 1624 cm^-1 is due to in-plane bending δ(H-O-H) of the water molecule. Moreover, the week band at 938 cm^-1 characterized the stretching of (W=O_t) (where O_t the terminal oxygen). The intense sharp band at 666 cm^-1 revealed the stretching (W-O). Upon reacting with Trp or Cys, the peaks related to the stretching motion of (O-H) and become more intense, broader and shifted to 3428 and 3442, respectively (Fig. 3a). Furthermore, the bending δ(H-O-H) of the water molecule band become more intense and shifted to 1650 cm^-1 with Trp and to 1644 cm^-1 in case of Cys. Moreover, a new bands appears at 1434, 1272, 1115, 1063, 984, 888, 627, 525 cm^-1 for WO₃-Trp complex and 1423, 1272, 1114, 1061, 983, 888, 654, 532 cm^-1 for WO₃-Cys complex where the intense sharp band at 666 cm^-1 disappear (Fig. 3a). These results clearly confirm the successful interaction between the two amino acids and WO₃NPs.

The thermal stability of WO₃.H₂O and WO₃-AAs complexes were tested using TGA analysis as seen in Fig. 3b. The WO₃.H₂O present two main decomposition stages 64-90^oC (2.5%) related to evaporation of adsorbed water 197-249^oC (4.5%) corresponding to release of intermolecular water (Fig. 3b). Attributed to modification of the WO₃ NPs with tryptophan (WO₃-Trp) or cysteine (WO₃-Cys), the TGA properties of the produced composites highly alter from the original WO₃(Fig. 3b). For the both composites, desorption of solvent, physically bonded water and surface hydroxyl groups was assessed through the loss in the mass in the region below 200°C. at temperature (≥ 250°C) the two composites presented smoothly decrease in their masses. This is related to the degradation of the organic materials on the substrate of WO₃NPs (Fig. 3b). This decomposition behavior presented suitable thermal stability for the organic modifier confirming the interaction and linking of the amino acids molecules to the surface of WO₃NPs.

Dye decolorization activity of prepared WO₃and modified WO₃-AAs NPs

The ability of modified WO₃-Trp and WO₃-Cys NPs toward synthetic dyes remediation was evaluated against some synthetic dyes solution (100 ppm/final conc.) and compared to plain WO3 NPs and separate AAs. Among the tested dye solutions, WO₃-AAs revealed high dye removal capacity against methylene blue and safranin dyes (Fig. 4) compared to WO₃ NPs and separate AAs. The decolorization rate of the two dyes was further studied through following up dye decolorization for 6 h, where samples were withdrawn and measured using UV-visible spectrophotometer with 1 h interval (Fig. 4). The results indicated great enhancement in the dye removal capacity for WO₃-Trp and WO₃-Cys modified NPs toward methylene blue and safranin dyes with complete dye removal (100%) after 6 h compared to plain WO₃ revealed moderate potential toward two dyes with 47 and 70% of dye decolorization for methylene blue safranin, respectively. The surface modification of WO₃ NP was reported to enhance its dye removal activity ⁵³. Dinari and his colleagues reported enhancement in methylene blue dye removal by a photocatalytic modified multi-walled carbon nanotube/WO₃compared to WO₃ NP ⁵⁴. The two separate AAs revealed no detectable decolorization activity toward both dyes solution (Fig. 4). Though decolorization rate of WO₃-Trp for both dyes was faster than WO₃-Cys, the time required for complete degradation of methylene blue and safranin dyes was the same (6 h).

Antimicrobial activity of prepared NPs and modified amino acid-NPs

The wide spread for multi-resistance microbial pathogens, implies a necessity for efficient broad spectrum antimicrobial molecules with lower microbial resistance induction capacity. Antimicrobial activity of the WO₃-AAs NPs was investigated against three pathogenic strains including Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853), and Candida albicans (ATCC 10231) along with plain WO₃ and AAs as controls, refer Table 1. The results (Table 1) indicated that pure WO₃ has no effect on the applied pathogens which in constant to other’s findings ⁵³ reported the inability of pure WO₃ to inactivate pathogenic bacterial cells even after under visible light illumination for 6 h. Separate Cys and Trp revealed considerable antimicrobial activity compared to that of Trp and WO₃ NPs. The results also indicated a synergistic enhancement in the antimicrobial activity of WO₃ NPs via modification with the applied amino acids as WO₃-Cys revealed higher broad-spectrum antibacterial activity toward Staphylococcus aureus and Pseudomonas aeruginosa compared to MIC values of Cys (85.82 and 112.7 µg/ml), with limited antifungal activity toward Candida albicans (105.9 µg/ml) compared to that of Cys (115 µg/ml). The results are in accordance with other’s findings stated the potency of Cys and Cys-rich peptides as promising antibacterial agents ^46,55. Recently, short peptides and amino acids-rich biomolecules rise as promising bacteriostatic agents that most likely targeting outer bacterial membrane rather than intracellular bacterial structures ^46,56. The thiol side chain in cysteine is highly reactive and hypothetically initiate the first interaction toward bacterial cell membranes ^46,57. On the other hand, WO₃-Trp also revealed broad-spectrum antibacterial activity toward Staphylococcus aureus (MIC 72.5 µg/ml) and Pseudomonas aeruginosa (MIC 104.7 µg/ml) with strong antifungal activity toward Candida albicans (42.8 µg/ml). The role of Trp as a hydrophobic AAs in antimicrobial-peptides was asserted ^47,58 through initiating the first and strong anchoring to the bacterial membrane and affect the interface region of lipid bilayer leading to cell membrane destruction.

Anticancer activity of the WO₃ and surface functionalized WO₃-AAs NPs

MTT assay used to determine cell metabolic activity for testing the cytotoxicity of the certain compound or drug. Here in, we evaluated the anticancer effect of the modified WO₃-Trp and WO₃-Cys and NPs against various kinds of cancer cells including MCF-7 caco-2 and HepG-2 cell lines in comparison with free forms of Cys and Trp AAs as well as original WO₃ NPs. Also, we investigated the effect of all of these compounds on WISH cells as a normal cell line. Our results indicated that the highest values of IC₅₀ were found to be for normal cells which refer to the highest safety of the applied modified NPs. As presented in table 2, IC₅₀ values for of the modified WO₃-Cys and WO₃-Trp NPs against WISH cells were be near of those free forms of Cys, Trp and WO₃ NPs which highlighting their safety.

Furthermore, MCF-7 cells are the most sensitive cells to treatment with both modified NPs. The modified WO₃-Cys NPs had an anticancer activity against MCF-7, Caco-2 and HepG-2 cells after treatment for 48 h at IC₅₀ values evaluated to be 92.53±5.2, 162.9±7.5 and 184.5±12.4 μg/ml, respectively, with SI values of 22.01±0.01, 12.49±0.02 and 11.04±0.01, respectively. While after treatment with WO₃-AAs NPs for 48 h, values of IC₅₀ were calculated to be 147.5±9.7, 176.3±10.1 and 202.6±15.3 μg/ml against the same cell lines, respectively, with SI values of 14.98±0.03, 12.54±0.02 and 10.91±0.17, respectively. Collectively, the results (Table 2) indicate that WO₃-Cys NPs displayed an anticancer activity higher than WO₃-trp NPs against all tested cells. The interaction of bare NPs to cellular components is indirect ³², where L-cysteine is known to improve the cellular uptake ⁵⁹, hence, the high activity of the WO₃-Cys NPs may be attributed to the high protonation effect of its SH group to many intracellular proteins ⁶⁰. The results are in accordance to ⁶¹, demonstrated the role of L-cysteine in suppressing of cancer development through regulating the excess activity of kinases that enhance cancer progression. In addition, a significant increase in the anticancer activity of the modified WO₃-Cys and WO₃-Trp NPs against all tested cancer cells in dose dependent manner with high selectivity to cancer cells and high safety against normal cells (Fig. 5). Fig. 5 also confirms the apoptotic effect of the modified WO₃-AAs NPs and reveals the proportional changes in HepG-2 morphology using nuclear stain method. HepG-2 cells were observed to lose their normal shape after treatment with both modified WO3 NPs for 48 h as compared to reference cells. Fig. 6 also indicates more fragmented HepG-2 chromatin with condensed nuclei in dose dependent manner. Furthermore, all observed chromatin fragmentation, shedding of apoptotic bodies and nuclear condensation were considered the major properties of the apoptotic pathway which became more noticeable with increasing the dose of treatment than untreated cells. Fig. 7 also confirms the effect of the modified WO₃-Cys and WO₃-Trp NPs on expression levels of E2F2, p53 and Bcl-2 genes in MCF-7, Caco-2 and HepG-2 cells as evaluated by real time PCR in comparison with untreated cells as control and 5-fluorouracile (5-FU) treated cells as a standard drug. Fig. 7 indicates that levels of expression of E2F2, Bcl-2 genes were found to be suppressed and down-regulated after treatment with both modified NPs more than 5-FU-treated cells. Furthermore, expression level of Bcl-2 gene was obviously reduced in the treated cancer cells by around 2 folds more than control cells. While expression level of p53 gene in all tested cells was evidently increased and up-regulated after treatment by more than 5-8 folds as compared to untreated control cells. Collectively, figures 1, 2, and 3 indicated the superiority of modified WO₃-Cys NPs toward all tested cells over that of the modified WO₃-Trp NPs.

Molecular docking studies

Molecular docking studies were carried out for the three compounds into three proteins namely; Bcl-2, P53 and E2F4 which are of crucial roles in cancer development ^62–65. This is to get insights into the proposed targets of the studied compounds and explore to what extent the modified NPs can accommodate the pocket and form interactions with the essential amino acid residues. The crystal structures of the three aforementioned proteins were downloaded from protein data bank (PDB) web site with ID 2W3L, 3ZME and 5TUU, respectively.

For Bcl-2 gene, the obtained results showed the significance of the binding of WO₃-Trp to Bcl-2 through an arene proton interaction with the essential amino acid residue Tyr67 in a manner similar to that of the ligand. Further an arene proton interaction was observed with the side chain of Met74. Additionally, it showed two hydrogen bonds (2.08 and 2.21 Å) with guanidine moiety of Arg105 (Fig. 8a). At the same time WO₃-Cys NPs showed three hydrogen bonds with guanidine moiety of Arg88 (distances, 2.15, 2.16 and 2.40 Å). Moreover, an electrostatic interaction was observed between thiol group of cysteine and alpha proton of Met74 (Fig. 8b). While WO₃ NPs showed four hydrogen bonds with Bcl-2 protein. Two of which were with side chain carboxylate of Glu95 (1.97 and 2.13 Å). The other two were of distances of 2.21 and 3.23 Å with backbone oxygen and mercapto-group of Met74, respectively. With respect to P53 protein, the ligand was found to interact with three residues Asp228, Cys229 and Thr230. The most significant binding pattern with P53 was that of WO₃-Cys NPs which accommodated the pocket showing interactions with the essential residues Cys229 and Thr230 as shown in Fig. 8c. Electrostatic interaction with the beta carbon of Cys229 was observed. Two hydrogen bonds of 1.83 and 2.50 Å length were formed with side chain OH and backbone NH of Thr230, respectively. Additionally, it showed electrostatic interaction with alkyl side chain of Leu145. The second most promising interaction with P53 was that of WO₃ NPs showing a hydrogen bond (distance: 2.26 Å) with the side chain OH of the essential residue Thr230. Moreover, electrostatic interaction with the backbone oxygen of the effective residue Asp228 was observed. Further hydrogen bond (distance: 2.46 Å) was formed with the backbone oxygen of Val147 (Fig. 8d). On the other side the modified WO₃-Trp was unable to accommodate the pocket of P53, showing different binding mode rather than the ligand. However, it formed one hydrogen bond with Thr150 and another with Pro151. It also showed arene proton interactions with Thr150, Pro222 and Pro223.

Finally, E2F4 protein was reported to have four domains; DNA binding domain, a dimerization domain, a transactivation domain, and a pocket protein binding domain ⁶⁶. The amino acid residues from 86 to 195 construct dimerization domain. These residues are responsible for formation of heterodimers with a DP family protein ⁶⁶. 5TUU at PDB web site is the crystal structure of E2F4-DP1 coiled coil. Docking results into this protein showed three hydrogen bonds for WO₃-Trp NPs as illustrated in Fig. 8e. The first (distance: 2.65 Å) was with guanidine moiety of Arg158. The second was of 2.21 Å length with the backbone oxygen of Ser135. The third one (distance: 2.28 Å) was observed with the backbone oxygen of Thr129. Moreover, an arene proton interaction was formed with Ala138. Regarding WO₃-Cys composite, Fig. 8f shows four hydrogen bonds, two of which were of 2.07 and 2.17 Å lengths with guanidine moiety of Arg158. Another hydrogen bond (2.19 Å length) can be observed with the backbone oxygen of Ala159. The fourth one was of 3.14 Å length with the backbone NH of Ala159. WO₃ NPs showed one hydrogen bond (2.01 Å length) with side chain OH of Thr163. It also showed another hydrogen bond of 2.01 Å length with the backbone oxygen of Ala159.

Preparation of WO₃ NPs

The WO₃ NPs was prepared by passing 10 ml (0.5M) of Na₂WO₄ (Sigma-Aldrich > 98%) solution through Ion exchanger Dowex® 50 WX 4 (strongly acidic cation exchanger), H⁺ form, (obtained from Merck) packed column. The yielded yellow solution was collected from the column using elution process in a blue capped bottle. Thereafter, the bottle placed in oven at 50°C over a period 24 h (Scheme 1: A). The produced precipitate separated by filtration, washed several times with water, dried at 70°C and finally, stored in dark bottle for further use.

Surface functionalized of WO₃NPs withTryptophan or cysteine

Surface functionalization of prepared WO₃ was conducted through mixing equal volumes of the prepared WO3 NPs (0.2 mg/ml) with tryptophan or cysteine (0.05 mg/ml) in 0.1 M phosphate pH 7.0 with stirring for 6 h. The mixtures were then centrifuged to eliminate the unbound amino acids, and the resulting NP-AA pellets were used during following experiments (Scheme 1: B).

Characterizations of WO₃ NPs and surface functionalized WO₃-AAs

The pristine WO₃ and surface functionalized WO₃-AAs were characterized using scanning electron microscopy (SEM; JSM 6360LA, Japan), Fourier transform infrared (FT-IR) analysis using Shimadzu FTIR (Model, FTIR8400, Japan), Thermal gravimetric analysis (TGA) (Shimadzu Thermal Gravimetric Analysis (TGA)—50), Japan), and the elemental EDS unit related to SEM.

Application of WO₃ NPs and functionalized WO₃-AAs in synthetic dye removal

The ability of modified WO₃-AAs in synthetic dyes remediation was evaluated against some synthetic dyes solution and compared to plain WO₃NPs and separate amino acids. Dye solution (100 ppm/final conc.) were prepared by dissolving methylene blue and safranin in 0.1 M phosphate buffer, pH 7.0. Dye decolorization test was performed in a final volume of 5.0 ml by separately adding 0.4 mg/ml of NPs, WO3-AAs, or AAs to each dye solution. The reaction mixtures were incubated at room temperature for 6 h. Samples were withdrawn from each tube every 1 h and measured using UV-vis spectrophotometer at wavelength of 662 nm and 495 nm for methylene blue and safranin, respectively.

Antimicrobial efficacy of the surface functionalized WO₃-AA NPs

Antimicrobial activity of the prepared WO₃-AA NPs was investigated against three pathogenic strains including Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853), and Candida albicans (ATCC 10231) along with plain WO₃ and separate AAs as controls. Microplate reader assay was applied in this test where, pre-culture was prepared by overnight cultivation of the three organisms on nutrient broth medium at 37°C. Flat-bottom 96 well tissue culture plate wells were inoculated separately with 100 µl of diluted tested organism (10⁶CFU/ml), and serially diluted compounds (5-80 µg) to a final volume of 200 µl. The microtiter plate was incubated overnight at 37°C, and measured at 600 nm with microplate reader. Antimicrobial results were expressed as minimum inhibitory concentration (MIC), defined as the lowest concentration that completely eliminated all cells.

In vitro anticancer studies; Cell culture and media

Human normal amnion (WISH), human cells derived from breast adenocarcinoma (MCF-7), human epithelial derived from colorectal adenocarcinoma (Caco-2), and human hepatocyte carcinoma (HepG-2) cell lines were obtained from (VACSERA, Cairo, Egypt). DMEM and RPMI-1640 media were obtained from (Lonza Lmd. Co., USA). Human WISH and Caco-2 cell lines were cultured in DMEM media supplemented with 10% FBS (Fatal bovine serum). However, MCF-7 and HepG-2 cell lines were cultured in RPMI-1640 media supplemented with 10% FBS.

Cytotoxicity evaluation using MTT assay

The anticancer effect of the functionalized WO₃-AAs NPs was evaluated through MTT (3-[4, 5-Dimethylthiazol]-2, 5-Diphenyltetrazolium bromide) assay protocol using normal and cancerous cells as described previously by El-Fakharany et al. (2020). Briefly, both normal and cancer cells (1.0×10⁴/well) were seeded in four sterile 96-well microplates and incubated for 24 h with complete culture media. Serially diluted functionalized WO₃-AAs NPs at concentrations of 0.1-0.5 mg/ml was added to the cultured cells in triplicates and incubated for 48 h in 5% CO₂ incubator. Then, cells were washed 3 times with fresh media and 200 μl of MTT solution (0.5 mg/ml) was added to each well for 3-5 h. After incubation period, MTT solution was substituted with 200 μl of DMSO for dissolving formazan crystals. The optical density was measured at 590 nm using a micro-plate reader. The IC₅₀ value (half maximal inhibitory concentration) of each WO₃-AAs NPs that cause killing of 50% cells was determined by the Graphpad Instate software 6.0 and values of selectivity index (SI) which defined as the ratio of IC₅₀ of normal cells versus cancer cells was estimated also as described previously by Abu-Serie and El-Fakharany (2017).

Nuclear staining

The anticancer and apoptotic effect of the WO₃-Cys and WO₃-Trp NPs against HepG-2 cells was studied by fluorescent nuclear stain method using propidium iodide (PI) dye in comparison with untreated reference cells. HepG-2 cells were incubated in triplicates and treated with different NPs at different concentrations of 0.1, 0.2, and 0.3 mg/ml as aforementioned described. Both untreated and treated cells were washed 3 times with fresh media for removing debris and dead cells and then fixed with 4% paraformaldehyde for 10 min at room temperature. After fixing step, permeabilization the cells with 0.5% Triton X-100 and 3% paraformaldehyde was performed for 1 min. The PI dye at concentration of 10 μg/ml was added for staining the cells 20 min. The cells were observed and imaged by a fluorescence phase contrast microscope (Olympus, Japan) using an excitation filter of 480/30 nm ⁴⁹.

Effect of the prepared WO₃-AA NPs on gene expression

The effect of the prepared WO₃ and WO₃-AA NPs on the expression level of some tumor genes including transcription factor 2 gene (E2F2), tumor suppressor gene (p53) and oncogene (Bcl-2) was determined in human MCF-7, Caco-2 and HepG-2 cells using qPCR method. After treatment the cancer cells with the modified WO₃-Cys and WO₃-Trp NPs at IC₅₀ concentrations for each modified NPs as described above, total RNAs of each untreated and treated cancer cell line were extracted using protocol of Gene JET RNA Purification Kit (Thermo Scientific, USA). Real time PCR of each cDNA was carried out by master mix of SYBR green kit using specific primers (Forward/Reverse) as follow: 5’-GCATCCAGTGGAAGGGTGTG-3′/ 5’-ACGTTCCGGATGCTCTGCT-3′ for E2F2 gene, 5′-TAACAGTTCCTGCATGGGCGGC-3′/ 5′-AGGACAGGCACAAACACGCACC-3′ for p53 gene, and 5′-TCCGATCAGGAAGGCTAGAGTT-3′/ 5′-TCGGTCTCCTAAAAGCAGGC-3′ for Bcl-2 gene. The change of the gene expression level for each cell line after and before treatment was determined by using equation of 2-ΔΔCT.

Docking studies

Molecular docking studies were carried out for simulating the interaction of WO₃ NPs and WO₃-AAs into three important cancer developing proteins namely; Bcl-2, P53 and E2F4. The crystal structures of the three aforementioned proteins were downloaded from protein data bank (PDB) web site with ID 2W3L, 3ZME and 5TUU, respectively. Molecular operating environment (MOE) software was used to conduct molecular docking. The downloaded protein was prepared by 3D protonation, deletion of water molecules and unwanted peptide chains and energy minimization. The pocket was obtained by isolation the molecular surface around the binding site (within 4.5 Å near the ligand atom). Validation was carried out by redocking the crystallized ligand. Rigid protocol docking (default protocol) was selected. The docking protocol is considered to be valid when RMSD of the docking pose compared to the co crystal ligand position is about 1.5 Å or less. To prepare WO₃ NPs and modified WO₃-AAs for docking the structure of the studied compound was built on MOE. 3D protonation was selected then energy minimization (force field: MMFF94x) was applied. The prepared compounds were added to the created database which was then selected for docking according to the default protocol in which the bond rotation method generated ligand conformations. The conformers are placed in the site with the Triangle Matcher method ⁵⁰.

Statistical Analysis

All data were expressed as mean±SEM (standard error of the mean) of three different experiments measurements. The statistical significance was assessed by the multiple comparisons Tukey post-hoc analysis of variance (ANOVA) using SPSS16 program. Differences of the results were considered statistically significant at (p)-values < 0.05.

Tungsten oxide (WO₃) represents one of the most interesting metal oxide with various potentials industrial application. In this work WO₃ nanosheets were prepared in one single step through cation exchange column and characterized through different methods. The surface modification of the prepared WO₃NPs with either L-tryptophan or L-cysteine greatly enhanced their biological activities. The prepared WO₃-Trp and WO₃-Cys exhibited strong dye removal activity toward methylene blue and safranin dyes with complete dye removal (100%) after 6 h. The antimicrobial activity assessment revealed broad-spectrum antimicrobial activity toward the three applied organisms Staphylococcus aureus and Pseudomonas aeruginosa, and Candida albicans with higher antibacterial activity from WO₃-Cys and potent antifungal activity through WO3+Trp. The results also indicated a remarkable antitumor activity in a dose-dependent manner through WO₃-Trp and WO₃-Cys toward all applied tumor cell lines (MCF-7, Caco-2, and HepG-2) attributed to induction of apoptotic effects. The highest antitumor activity was against the breast cancer tumor cell MCF-7. WO₃-Cys revealed the superior antitumor activity which could attributed to high activity of the thiol group as indicated in the molecular docking studies. Collectively, the surface modification of WO₃NPs with amino acids specially that of L-tryptophan or L-cysteine greatly enhanced their biological activities and may expand their industrial applications.

Funding

This study did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Author Contributions

All authors have equally contributed to designing and accomplishing the experiments, analyzing and interpreting the results as well as writing and revising the manuscript.

Ethical statement

This research did not include any human subjects and animal experiments.

Competing Interests

The authors declare that they have no competing interests.

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Table 1. The antimicrobial activity (MIC values) of the modified WO₃-Cys and WO₃-Trp NPs against three pathogenic microorganisms in comparison with WO₃ NPs and free amino acids (Cys and Trp).

Test organism	WO₃	WO₃-Trp	WO₃-Cys	Trp	Cys
Staphylococcus aureus	452.90	72.53	53.14	454.00	85.82
Pseudomonas aeruginosa	307.70	104.70	60.52	199.50	112.70
Candida albicans	324.90	42.81	105.90	199.50	115.00

Table 2. EC₁₀₀, IC₅₀ (μg/mL) and SI values of the modified WO₃-Cys and WO₃-Trp NPs against WISH, MCF-7, Caco-2 and HepG-2 cell lines after treatment for 48 h in comparison with WO₃ NPs and free amino acids (Cys and Trp).

Sample		WISH	MCF-7	Caco-2	HepG-2
WO₃	IC₅₀	2315±224.3	2805±196.6	2238±210.4	2517±232.5
WO₃	SI	-	0.83±0.08	1.03±0.09	0.92±0.1
Cys	IC₅₀	2221±159.8	2365±159.7	2635±118.9	2936±102.1
Cys	SI	-	0.94±0.07	0.84±0.05	0.76±0.04
Try	IC₅₀	2064±197.5	2210±156.9	2084±188.4	2958±134.7
Try	SI	-	0.93±0.07	0.99±0.09	0.69±0.06
WO₃-Cys	IC₅₀	2036±193.2	92.53±5.2	162.9±7.5	184.5±12.4
WO₃-Cys	SI	-	22.01±0.01	12.49±0.02	11.04±0.01
WO₃-Trp	IC₅₀	2210±166.8	147.5±9.7	176.3±10.1	202.6±15.3
WO₃-Trp	SI	-	14.98±0.03	12.54±0.02	10.91±0.17

All values were expressed as mean ± SEM.

No competing interests reported.

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Novel Nanocombinations of L-tryptophan and L-cysteine: Preparation, Characterization and Their Applications for Dye Decolorization, Antimicrobial and Anticancer Activities

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