Posljednjih je godina mnogo pažnje posvećeno samosloživim strukturama temeljenim na dipeptidima, a posebno samoregulaciji tih struktura s različitim nizovima aminokiselina. U ovome smo ispitivanju htjeli ustanoviti djelovanje različitih otopina na slaganje struktura dipeptida glicin-histidin (GH). Prvo smo utvrdili morfološka svojstva tih GH filmova odležanih u različitim otapalima pomoću pretražne (skenirajuće) elektronske mikroskopije, a zatim i njihova strukturna svojstva pomoću infracrvene spektroskopije s Fourierovom transformacijom (FTIR). Zatim smo utvrdili djelovanje tih struktura u filmu na raspoređivanje srebrnih nanočestica (AgNP) te antioksidacijska i citotoksična svojstva filmova obogaćenih AgNP-ovima. Osim toga, ocijenili smo antibakterijsko djelovanje filmova, s Ag česticama i bez njih na Gram negativne bakterije
- antibacterial surface
- antibakterijska površina
Many biomolecules and other components form supramolecular structures in biological systems, most notably amino acids, peptides, and proteins in human metabolism (1, 2, 3). Particularly unique are peptide-based structures with amino acid groups connected by peptide bonds (4). However, the self-assembly processes of high molecular mass peptides and more complex protein structures is still not entirely clear (5, 6), as there are too many functional groups that interact in self-assembly. At this point, however, much can be learned from dipeptides as the simplest peptide structures (7). For example, many studies have reported diphenylalanine (Phe) dipeptide, the core recognition motif of amyloid plaques found in Alzheimer’s disease and type II diabetes (8). Furthermore, many dipeptides can form different structures through their interactions and be used in various applications as bio-inspired materials (6, 9). It is therefore important to study their activities and toxic effects and try to elucidate the self-assembly processes, including the solvent effects, co-arrangement, nanoparticle effects, and different architectural and morphological properties (10) to reveal the driving forces and interactions behind spontaneous arrangement of dipeptide structures. It is also important to study their beneficial effects, as a number of biopeptides, such as glutathione (γ-Glu-Cys-Gly), carnosine (β-alanyl-L-histidine), homocarnosine (γ-aminobutyryl-L-histidine), and anserine (β-alanyl-
Thanks to their self-assembly, dipeptide structures are currently used in drug release, bio-sensing studies, architecture studies with surface-enhanced Raman spectroscopy (SERS), and catalysis studies, often in combination with metallic nanoparticles, gold and silver in particular, because of their antibacterial activity (14, 15, 16, 17, 18). So far, polymer, lipid-based, and metallic nanoparticles (NPs) have shown antibacterial, antifungal, and antiviral activities against different microorganisms (19), and silver nanoparticles (AgNPs) are the most studied for antibacterial activity (20).
Considering that glycine-histidine (Gly-His) based dipeptides can have remarkable structures and that Gly-His films formed in the solvent environment can have different useful applications, the aim of this study was to investigate how different solvents affect dipeptide self-assembly and how the obtained structures combine with AgNPs. To this end, we studied the self-assembly of Gly-His films in AgNO3 solution and the antioxidant and antibacterial effects of AgNPs formed on dipeptide structures. We also investigated the cytotoxic effects of Gly-His films in murine fibroblasts (L929).
The chemicals used in the study were purchased from the following suppliers: Dulbecco’s Modified Eagle Medium (DMEM) and Dulbecco’s Phosphate-Buffered Saline (PBS) from Wisent (Saint-Jean-Baptiste, QC, Canada); trypsin-EDTA from Biological Industries (Beit-Haemek, Israel); foetal bovine serum (FBS) and penicillin-streptomycin from Capricorn Scientific (Ebsdorfergrund, Germany); and dimethyl sulphoxide (DMSO) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) from Sigma-Aldrich Chemicals (Munich, Germany).
Gly-His dipeptides were dissolved in one of the following solvents: 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), ethanol, methanol, chloroform, tetrahydrofuran (THF), water, or acetone in the final concentration of 2 mg/mL and then dropped onto silicon discs. The solvent was evaporated at room temperature.
Gly-His dipeptides dropped onto the silicon disc were kept in 1 mmol/L AgNO3 medium for 3, 6, 12, and 24 h to self-assemble. The discs were then removed from the silver medium, immersed in deionised water to remove residual ions, and then taken out to dry in room conditions.
Antioxidant activity of Gly-His dipeptides and AgNP-decorated Gly-His films was determined with the DPPH assay as described elsewhere (21). Briefly, DPPH solution was prepared by dissolving 6.0 mg of the compound in 100 mL of methanol. Then, various concentrations (0.5 mg/mL, 1 mg/mL, 2 mg/mL, 4 mg/mL, or 6 mg/mL) of 50 μL solutions of Gly-His dipeptide films with or without AgNPs were added to 150 μL of the prepared DPPH solution in 96-well plate. Ascorbic acid was used as standard and DPPH in methanol as blank. The plate was shaken and incubated in the dark for 30 min. After incubation, the absorbances of the solutions were measured with a microplate reader (BioTek Instruments, Winooski, VT, USA). Their scavenging activities were calculated as follows:
Scavenging activity = 100[(Ac-As)/Ac] [Equation 1] where Ac is the absorbance of control and As the absorbance of the standard or sample.
L929 cells (kindly provided by Ankara University Faculty of Pharmacy, Department of Pharmaceutical Toxicology, Ankara, Turkey) were seeded in 25 cm2 flasks with 7 mL of DMEM supplemented with 10 % FBS and 1 % penicillin-streptomycin and then grown in an incubator (Sanyo, Japan) at 37 °C in an atmosphere supplemented with 5 % CO2 for 24 h.
Cytotoxicity was determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described earlier by Bacanli et al. (22). Following cell disaggregation with trypsin/EDTA and resuspension in medium, a total of 105 cells/well were plated in 96-well tissue-culture plates. The cells were incubated in full medium with various concentrations (0.5, 1, 2, 4, and 6 mg/mL) of AgNP-decorated Gly-His films at 37 °C in 5 % CO2 in air for 24, 48, and 72 h. Full medium was used as negative control. After exposure, the medium was aspirated, cells washed with PBS, 10 μL of MTT (5 mg/mL of stock solution with PBS) added to 100 μL of cell suspension per well, and cells incubated for another 4 h. The MTT dye was then carefully removed, and 100 μL of DMSO added to each well. The absorbance of each well was measured with a microplate reader (BioTek Instruments) at 570 nm.
Gly-His dipeptide dropped onto Oxoid™ blank antimicrobial susceptibility discs, 6 mm in diameter (Thermo Scientific, Waltham, MA USA) in the final concentration of 2 mg/mL were kept in 1 mmol/L AgNO3 medium for 3, 6, 12, and 24 h to self-assemble. The discs were then removed from the silver medium and immersed in deionised water to remove residual ions and then taken out to dry in room conditions.
The activity of AgNP-decorated Gly-His dipeptide films against
To characterise all dipeptide structures we coated the samples with a 5 nm Au-Pd layer and used a Quanta 400F Field-Emission Scanning Electron Microscope (FE-SEM, Thermo Fisher Scientific, Altrincham, UK) with an acceleration voltage of 20 kV and attenuated total reflection Fourier Transform Infrared (FTIR) spectrometer (Perkin Elmer, Waltham, MA, USA). Crystallographic features were also determined with the X-ray diffraction (XRD) (Ultima IV, Rigaku, Tokyo, Japan). Measurements were performed at Cu Kα radiation (40 kV and 30 mA). The scan rate was 2°/min and range 2.5–80°.
Figure 1 shows SEM images of self-assembled Gly-His dipeptides dissolved in HFIP, water, ethanol, methanol, acetone, and THF. Most dipeptide moieties easily solved in all solvents. They generally assumed a film-like morphology, probably owing to hydrogen bonding between the N-H (NH2 group) and O-H (COOH group) terminals of dipeptide molecules (24). However, their structures differ: dipeptides solved in HFIP self-assembled in a spherical structure (Figure 1a), and those dissolved in chloroform assumed square structure (Figure 1e). In addition, the film layer of dipeptides dissolved in acetone formed pores (Figure 1d). Continuous film layers were preserved in HFIP, water, and ethanol, which suggests that they have potential application as film biomaterials.
An earlier report suggests that polarity, hydrogen bonding capacity, and dielectric constants of solvents may be the driving forces in dipeptide self-assembly (6). In Gly-His dipeptides, the driving force are inter- and intra-molecular hydrogen bonds. The latter are possible due to the carboxylic acid and amine head groups, and they often take the form of a film. Weaker film formation is particularly common in solvents with low hydrogen-bonding capacity, such as chloroform and acetone. In this case, the effective driving force is thought to be the interaction between the hydroxyl groups of the amine and carboxylic acid (6).
Figure 2 shows similar FTIR spectra of the film-like Gly-His dipeptide structures, regardless of solvent. The primary amide band in the 1600–1700 cm-1 frequency range usually takes the α-helix, β-sheet, β-turn, or random-coil structure after self-assembly and is independent of the amino acid sequence, hydrophilic or hydrophobic properties of the system, or the skeletal structure and charge of the amino acid. In the β-sheet conformation, two prominent peaks arise around 1630 cm-1 and 1690 cm-1. In the α-helix structure, primary amide band usually appears around 1650 cm-1 and in random-coil structure around 1648 cm-1. The secondary amide band with the α-helix structure usually appears around 1545 cm-1 together with the primary band. Sharp peaks around 3250 cm-1 correspond to N-H stretching. In contrast, β-turned structure can be seen between 1660 and 1680 cm-1. Broad peaks, especially around 3000–3500 cm-1, are believed to originate from -OH and -NH groups (25, 26). The band around 1700 cm-1 is considered to be the C=O band. One of bands that explains the role of hydrogen bond interactions is the C=O band. The fact that these bands have remained similar in Figure 2 may indicate that self-assembly is owed to ionic and other weak interactions. Here, to examine the secondary morphology of these structures, the primary amide frequency range between 1600 and 1700 cm-1 should be examined, but this part cannot be seen in detail because the tight polymer-like film structure formed by the arrangement of Gly-His dipeptides gives a spectrum in the form of broad bands with behaviour specific to polymer structures.
Another feature of amino acid and dipeptide structures is that they can be decorated with different nano and microstructures by reducing or oxidising metal cations with a specific reduction potential. Several studies have investigated the capacity of Gly and His amino acids to form complexes with metal ions (27, 28, 29, 30, 31). Metal chelation, which often takes place, determines the antioxidant profile exhibited by several histidine-containing dipeptides (32). For this purpose, we prepared Gly-His dipeptide films in ethanol to examine their behaviour in the presence of the Ag ion and their antioxidant properties. Interestingly, their morphological properties changed, as can be seen in SEM images in Figure 3. The most notable is the rearrangement of fibres in the presence of AgNO3 solution.
Figure 3b also shows the EDX spectra of Gly-His film kept in the AgNO3 solution, in which the peaks point to C and O in the dipeptide structure. In addition, the rugged Si peak is caused by the Si wafer, which is the substrate of retaining.
Figure 4 shows time-related morphological changes of Gly-His dipeptide films left in the AgNO3 medium for 0, 3, 6, 12, or 24 h. At 0 h, AgNPs take globular structure. At 3 and 6 h, AgNPs randomly scatter across the surface, still retaining their globular form. At 12 and 24 h, they deteriorate into needle-like and cube-like forms. These structures are formed at the end of the cooldown and may be collapsed AgNPs.
Figure 5 shows the FTIR spectra of AgNP-decorated Gly-His structures. The bands obtained around 1300–1500 cm-1 are more clearly separated. The peaks around 1700 cm-1 correspond to the COO- groups. The spectrum’s peaks at 3000–3500 cm-1 correspond to the -OH and -NH groups.
Figure 6 shows the XRD patterns of AgNP-decorated Gly-His film at the four time points. At the angle range of 2θ (2.5−80°), Gly-His dipeptide film has an amorphous structure without distinct diffraction pattern, but in the presence of AgNO3 it has transited to a needle and fibre-like structure. Sharp diffraction peaks are visible at 7.22, 10.65, 14.14, 16.40, 19.23, 27.86, 32.24, and 46.30 °. The d spacing values corresponding to these peaks are 12.24, 8.26, and 6.30, 5.39, 4.62, 3.20 2.77, and 1.96 Å, respectively. The same peaks at 27.86, 32.24, and 46.32 ° have already been reported for Ag2O nanoparticles on a dipeptide surface by Babu et al. (33).
There are various studies about the cytotoxic effects of AgNPs, but ours is the first to evaluate the cytotoxic effects of the Gly-His dipeptide, with and without AgNPs (Figure 8). The Gly-His dipeptide without AgNPs did not significantly lower L929 cell viability at hour 24, 48, and 72 of incubation, but the one with AgNPs did. The IC50 values of AgNP-decorated Gly-His films at 24, 48, and 72 h of exposure were 3.679 μg/mL, 3.251 μg/mL, and 3.096 μg/mL.
Antibacterial activity of AgNP-decorated Gyl-His dipeptide films turned out to be the most effective against
Our study has shown that the Gly-His dipeptides self-assemble as film-like structures through hydrogen-bond-induced rearrangement resulting in an amorphous flat layer. This film-like structure has potential, especially for topical applications, as it can coat wide surfaces. Pairing it with AgNPs can increase antibacterial activity of the film, without being toxic to other cells at concentrations below 2 mg/mL. However, further