Abstract
Selenium (Se) is an important trace element that is involved in controlling oxidative stress and inflammatory disorders. Gouty arthritis is the inflammation and pain within the joints and tissues caused due to the accumulation of monosodium urate (MSU) crystals. This study aimed to investigate the antigout, antioxidant, anticoagulant, and thrombolytic potential of ascorbic acid-mediated Se nanoparticles (A-SeNPs). Different analytical techniques were used to investigate the formation of A-SeNPs. The antigout potential of the nanoparticles was carried out using MSU crystal dissolution, uric acid (UA) degradation assay, and xanthine oxidase inhibition (XOI). A-SeNPs exhibited excellent antihyperurecemic activity in a concentration-dependent manner. It was observed that at the tested concentration of 20 mg·mL−1, the A-SeNPs demonstrated significant breakage and dissolution of MSU crystals and resulted in UA degradation of 67.76%. Similarly, A-SeNPs resulted in 76% XOI with an excellent IC50 of 140 µg·mL−1. Furthermore, considerable antioxidant activity was noted for the A-SeNPs as evaluated with multiple antioxidant assays. Finally, the NPs were found to have significant anticoagulant and thrombolytic potential. Thus, it was concluded that A-SeNPs have potent antihyperuricemic, antioxidant, anticoagulant, and thrombolytic activities, making them an ideal choice for future biomedical applications.
Graphical abstract
1 Introduction
Nanotechnology has attracted the most attention in recent years due to the development of innovative types of materials with distinct physical and chemical properties that are efficiently used in many different areas of research [1,2]. Because of their greater surface-to-volume ratio and higher surface energy, nano-sized particles have distinct characteristics [3,4]. Selenium is an essential element that the human body needs and has shown promising results in the treatment and diagnosis of numerous illnesses [5]. Selenium is a micronutrient in plants, animals, and humans and plays key biological functions [6]. Selenium is a component of proteins and enzymes, such as glutathione peroxidase (GPx), selenoprotein N, thyroxine 5-deiodinase, selenoprotein, and P selenoprotein K [7]. The trace element is required for the biosynthesis of the amino acid selenocysteine and acts as a key cofactor in selenoenzymes that protects the human body against free radical species. Lower Se levels in the human body have been associated with weak immunity, cognitive decline, and high risks of mortality. Recently, there has been growing interest in the preparation and study of SeNPs, because of their excellent mechanical characteristics, optoelectronic and magnetic properties, as well as a plethora of applications in nano-medicines, sensors, and catalysis to name a few [8,9]. SeNPs have also been found to exhibit potential biological activities, reduced toxicities, and significant bioavailability. The NPs are found to be involved in the antioxidant defense system of cells and protect against oxidative stress [10,11]. Elemental Se in the form of NPs also exhibits antimicrobial activities and has shown to possess excellent growth inhibition potential against Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa, the pathogens that are associated with hospital and medical devices-acquired infections. Some studies even show that SeNPs exhibit reduced toxicities and are more effective as compared to AgNPs [12,13]. Se is a trace mineral that is essential for maintaining human health. Adults need between 40 and 300 mg of Se daily as a dietary supplement, and it has been linked to more than 40 diseases in humans. SeNPs are naturally broken down by the body, and the resulting Se nutritional supply is harmless to humans [14,15]. SeNPs have been employed in the treatment of cancer [16], drug delivery [17], antibacterial agents [18], antiviral [19], antifungal [9], antioxidant [20], and fertilizers [21]. To date, different fabrication approaches such as physical, chemical, and biological methods have been used to prepare SeNPs with varied morphologies and physicochemical characteristics [22]. However, these approaches particularly chemical and physical methods are considered disadvantageous because of high energy consumption, use of hazardous chemicals, and the non-ecofriendly nature of the methods [23,24]. El Saied et al. successfully investigated and synthesized SeNPs using cell-free extract of the microalgae, Spirulina platensis. The innovative SeNPs used were recommended to generate effectual bioactive agents to control hazardous bacterial species [25]. Srivastava and Mukhopadhyay used the non-pathogenic bacterium Zooglea ramigera to biosynthesize SeNPs with sizes ranging from 30 to 150 nm. Se oxyanions are employed in Zooglea ramigera growth media, and bacterial proteins and enzymes are responsible for Se oxyanions reduction [26]. Similarly, the bacterium Pantoea agglomerans strain UC32 was employed for selenite [Se(iv)] bioreduction to manufacture SeNPs smaller than 100 nm [27]. Wadhwani et al. reported the manufacture of SeNPs from Acinetobacter sp. SW30 cell suspensions and complete cell protein by decreasing Na2SeO3 [28]. The bacterium Klebsiella pneumoniae was used to synthesize SeNPs with a size range of 100–550 nm for the reduction of Se chloride (Se2Cl2) [29].
Besides, SeNPs prepared from naturally existing substances such as ascorbic acid (AA) are comparatively less toxic than SeNPs produced from other physical and chemical routes [30]. Basic calcium phosphate, MSU crystal, and calcium pyrophosphate are identified to produce arthropathies. It is developing proof that these crystals are responsible for the spread of synovitis, cartilage injury, and the development of gout and joint damage [31]. Gout is the main example of arthritis related to MSU crystal deposition in joints and is characterized by hyperuricemia. Inflammatory arthritis gout occurs due to the formation of MSU that results from increased UA levels in the blood [32]. Increased UA levels not only cause inflammatory arthritis but have also been linked to risk factors for various other diseases [33]. To treat gout, anti-inflammatory drugs such as colchicine are prescribed as the first-line treatment option [34]. However, such drugs have a little healing frame and may result in noxious effects [35]. Likewise, urate-lowering drugs (xanthine oxidase inhibitors) such as Allopurinol are also suggested to patients; however, prolonged use of the drug has been associated with severe skin reactions, painful or bloody urination, and liver problems. Thus, safe and effective treatment of hyperuricemia is still unsatisfactory and in need of urgent development [36,37].
The current study aims to use a simple and green route for the synthesis of SeNPs for multiple biomedical applications, including antioxidants, antigout, and anticoagulant studies. The A-SeNPs were synthesized by an eco-friendly approach via the reduction of sodium selenite with L-AA. Then, the synthesized NPs were characterized by X-ray diffraction (XRD) analysis, Scanning electron microscopy (SEM), and Fourier transform infrared (FTIR) spectroscopy, and after characterization, the NPs were explored for multiple biological properties.
2 Experimental
2.1 Materials and methods
The chemicals used were L-Ascorbic acid 99.7% and trichloroacetic acid 99.5% (VWR Chemicals, AnalaR Normapur), sodium selenite Na2SeO3 (Sigma Aldrich, Germany), sodium hydroxide 98% (Daejung Chemicals Siheung-si, South Korea), 2,2-diphenyl-1-picrylhydrazyl (Sigma Aldrich, Germany), uric acid 98% (Avonchem Macclecfield, UK), sodium phosphate 98% (Icon Chemicals) and sulfuric acid 96% (Daejung Chemicals Siheung-si, South Korea). All the chemicals and reagents are used as such without further purification.
2.2 Synthesis of SeNPs
For the synthesis of SeNPs, 2 g of L-AA was dissolved in 100 mL of distilled water (dH2O). In a separate flask, a 10 mmol precursor salt solution of sodium selenite (Na2SeO3-Sigma Aldrich) was prepared in 100 mL of dH2O at 70℃. The prepared L-AA solution was added dropwise to the salt solution, with continuous stirring at 70℃. The transparent sodium selenite solution slowly changed to a reddish color, which indicated the successful synthesis of SeNPs. Afterward, the obtained product was collected and washed three times with dH2O via centrifugation at 4,500 rpm for 15 min and oven-dried at 80℃ for 7 h. The well-dried material was then ground into a fine powder using pastor and mortar, labeled as ascorbic acid-mediated Se nanoparticles (A-SeNPs), and utilized further for characterization and bioassays.
2.3 Characterization
The prepared A-SeNPs were subjected to various characterizations that included FTIR spectroscopy, XRD, SEM, and energy-dispersive X-ray spectroscopy (EDX). For the identification of functional groups and to monitor the surface chemistry of SeNPs, FTIR spectroscopy in the wave number range of 4,000–400 cm−1 was carried out. To determine the material and phase structure of A-SeNPs, XRD was employed in the 2θ (10–80°) using Panalytical X-pert pro MPD XRD. Furthermore, to examine the particle morphology and size, an SEM was used with various magnifications. The morphology and elemental compositional were determined by using Hitachi SU6600 SEM and EDX. Before the characterization, the sample was dried and pasted on carbon tape and then sputtered through gold.
2.4 Biological applications
2.4.1 In vitro anti-gout study
The in vitro anti-gout study was carried out via MSU crystals degradation assay, UA degradation test, and XOI potential of the NPs.
2.5 MSU crystals degradation assay
2.5.1 Preparation of MSU crystal
MSU crystals were prepared using a previously reported method with minor alterations. Briefly, 0.001 M of sodium hydroxide (NaOH) solution was prepared and heated at 70°C. When the temperature reached 70°C, 1.68 g UA dissolved in 50 mL was added dropwise to the NaOH solution. The pH of the mixture was maintained at 7.2. When the dissolution was ensured, the mixture was allowed to cool down at room temperature for 24 h. The supernatant was discarded and the suspension was washed with dH2O thrice. After drying the suspensions, needle-shaped MSU crystals were observed under a microscope and were used for further studies [38].
2.5.2 Preparation of positive control
Two solutions were prepared; A1 is the buffer solution containing 50 mmol·L−1 phosphate (pH 7.4) and 4 mmol·L−1 2-4 dichlorophenol sulfonate (DCPS) was prepared. A2 is the enzyme solution, which was prepared that contained 60 µ·L−1 uricase, 660 µ·L−1 peroxidase (POD), 200 µ·L−1 ascorbate oxidase, and 1 mmol·L−1 4-aminophenazone (4-AP). Both the A1 and A2 mixtures were mixed in 1:1 and were used as a positive control.
2.5.3 Preparation of negative control
A 4 mg·mL−1 MSU crystal in dH2O was used as a negative control.
2.5.4 Effects of SeNPs on crystal dissolution
Four different concentrations of 20, 10, 5, and 2.5 mg·mL−1 of SeNPs were taken into four different falcon tubes and the NPs were well sonicated. After that, 0.5 mg MSU crystals were added into each tube and gently mixed to avoid physical damage to the crystals [39]. The test tubes were maintained on a shaker at a slow speed of 37℃. Consequently, small aliquots (drops) were taken from each tube after 2, 12, and 24 h and were put on a glass slide to observe the dissolution of crystals under the inverted microscope at different time intervals. Multiple micrographs were taken at 40×. In the assay, MSU crystals and dH2O solution were used as a negative control. Enzymes working solution was used as a positive control.
2.5.5 UA degradation test
The UA degradation potential of the NPs was also investigated using the SPINREACT kit. For the preparation of working reagents, working solutions R1 and R2 were prepared according to the SPINREACT kit protocol.
2.5.6 Preparation of working reagents
For the UA degradation test, R1 (buffer solution) and R2 (enzyme solution) were used as working reagents. The working reagent R1 constituted 50 mmol·L−1 phosphate buffer with PH 7.4 and 4 mmol·L−1 DCPS and R2 consisted of 60 U·L−1 uricase, 660 U·L−1 POD, 200 U·L−1 ascorbate oxidase, and 1 mmol·L−1 4-AP. According to the protocol, both the working reagents (R1 and R2) were mixed in 1:1 (v/v). A 6 mg·dL−1 UA aqueous primary standard is ready-made and is provided within the kit.
2.5.7 UA degradation assay
The assay was carried out by mixing 1 mLWR reagents, 25 µL MSU crystal solution (available in the kit) with 25 µL A-SeNPs with varied concentrations, i.e., 2.5–20 mg·mL−1 and the test tubes were incubated for 5 min at 37℃ followed by absorbance recording at 520 nm. In the study, a blank was also used that contained 1 mL working reagents R1 and R2 only. A standard containing 1 mL working reagents (WL) and 25 µL UA aqueous primary standard (6 mg·dL−1) was also employed in the assay. The % UA degradation was calculated using the formula:
2.6 XOI assay
The colorimetric XOI assay that is based on the formation of UA from xanthine was employed as the reference method for the XO inhibition potential of A-SeNPs [40]. In brief, 30 µL of the test sample with different concentrations (25–200 µg·mL−1) was mixed with 210 µL of the phosphate buffer in a test tube. Subsequently, 180 µL of the freshly prepared XO was gently added to the reaction mixture followed by an incubation period of 20 min at 25℃. After the brief incubation period, 960 µL of xanthine was added to the reaction mixture, and the mixture was re-incubated at 25℃ for 20 min, followed by absorbance measurement at 293 nm and calculation of % XOI as
where A depicts enzymatic activity without the presence of sample/positive control and B represents Blank, that is the optical density of the reaction mixture without xanthine oxidase (XO) or the test samples/positive control. C is the enzymatic activity in the presence of sample or positive control and D is the optical density of sample/positive control without the presence of XO.
2.7 Antioxidant study
2.7.1 Total antioxidant capacity
The different concentration of NPs was evaluated for total antioxidant capacity (TAC) by using a phosphomolybdenum-based analysis technique according to the previously reported protocol in the literature with some modifications [41]. In brief, 100 µL aliquot of the sample was carefully added in an Eppendorf tube by using a micropipette and mixed with 900 mL of TAC reagent (which collectively contains 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate, in 50 mL dH2O. The mixture was followed by water bath incubation for 45 min, at 80°C, and allowed to cool. After that, the mixture was subjected to the measurement of absorption at the wavelength of 630 nm by using a microplate reader. The TAC was expressed as µg AA equivalent per mg of the NP weights (µg AAE·mg−1).
2.7.2 Total reducing power
The different concentrations of NPs were analyzed for total reducing power (TRP) which was carried out by using the ferric reducing analysis technique in a way of well-optimized reported protocol in the literature with some modifications [42]. Briefly, in an Eppendorf tube, 100 µL aliquot of the sample, 400 µL phosphate buffer (pH 6.6), and 100 µL of potassium ferric cyanide (1% w/v) were added and mixed. The reaction mixture was then incubated by using a water bath for 30 min at 55°C. After that, 200 µL of trichloroacetic acid (10% w/v) was added to the reaction mixture and mixed gently. It was followed by centrifugation at 4,000 rpm for 12 min. Then, an aliquot of 140 µL was taken from the supernatant and poured carefully into the corresponding well in the 96-well plates in which 60 µL of the ferric cyanide solution (0.1% w/v) was already added. After that, the reaction mixture was followed by a measurement of absorption at the wavelength of 630 nm by using a microplate reader. The TRP was calculated as µg AAE·mg−1.
2.7.3 Free radical scavenging assay (FRSA)
NPs were evaluated for 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity which was done by way of a well-optimized protocol that was reported in the literature, by using a standard bioassay in multiple concentrations that range from 12.5 to 400 µL [43]. The free radical scavenging activity was determined based on the discoloration of the purple color of the DPPH solution. Briefly, in the experiment, an aliquot of 10 µL of the sample was added to the wells of 96-well plates, and a DPPH reagent of 190 µL was mixed with it. After that, it was followed by dark incubation for 1 h at 37°C, and absorbance was measured at the wavelength of 525 nm by using a microplate reader. Free radical scavenging activity was measured as percent (%) inhibition which was calculated by using the given equation:
where Abs indicates absorbance.
The ability of A-SeNPs to scavenge free radicals was also assessed using the ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical cation bleaching assay [44]. Briefly, ABTS + cationic radical solution was prepared by mixing equal amounts of 2.45 mM potassium persulfate and 7 mM ABTS solution (dH2O), (1:1) and then kept on dark incubation 4℃ 12 h before use. The assay was performed on 96-well plates containing 10 µL of the test sample (4 mg·mL−1, DMSO) and 190 µL of ABTS + solution in each well. Abs was measured at 730 nm and % ABTS was calculated using Eq. 3.
2.8 Anticoagulant and thrombolytic study
Anticoagulant activity of the synthesized SeNPs was evaluated using in vitro co-incubation of the sample with freshly isolated human blood from a healthy person. The isolated blood was co-incubated instantly with varied concentrations of NPs (2.5, 5, 10, and 20 mg·mL−1) in plastic vials (1–8 mL) at room temperature. In the experiment, 100 µL of the sample and 900 µL of the fresh blood were gently mixed, followed by visual observation at different time intervals (10 min, 30 min, 2 h, and 6 h) at room temperature for clot formation. The thrombolytic potential of the A-SeNPs was also determined, by observing the clot dissolution property of A-SeNPs. In the experiment setup, 2–3 drops of freshly isolated human blood were poured onto a sterile clean glass slide and allowed the clot formation. When the clot was ensured, 500 µL of A-SeNPs (20 mg·mL−1) was poured onto the clot. The clot was then observed visually for 1 h to gain an insight into the various stages of thrombolysis [45].
3 Results
3.1 Synthesis of SeNPs
AA was used as a reducing and stabilizing agent during the safe synthesis of A-SeNPs. The AA solution was combined with the stock solution of sodium selenite. The reaction mixture was initially bright; later, the color started to alter, and eventually, brick brick-red color developed, confirming the synthesis of A-SeNPs. It is crucial to be able to produce A-SeNPs from AA since doing so requires less processing afterward. The color of Na2SeO3 solution changed from translucent to brick red during the reduction reaction, indicating the formation of NPs [46]. In our work, the color of the Na2SeO3 solution went from colorless to brick red. The aforementioned findings demonstrate that L-AA can be used as both a reducing agent and a stabilizing agent in the reduction of Se2+ to produce well-dispersed A-SeNPs. L-ascorbic acid is strongly polar and highly water soluble. The electrons in the double bond, the hydroxyl group lone pair, and the carbonyl double bond on the lactone ring form a conjugated system, causing it to behave as a vinylogous carboxylic acid [47]. As a result, L-AA’s structural features provide sufficient reducibility for the creation of Se(0) NPs from Se2+ ions. Scheme 1 can be used to express the redox equation involving selenium ions and L-AA.
The equation for the reduction and formation of A-SeNPs using AA.
3.2 Characterization
3.2.1 FTIR analysis
A-SeNPs were characterized through FTIR, to examine the chemical bonding on the surface of NPs through several vibrational modes produced during FTIR as shown in Figure 1. The dangling bonds are formed on the surface which makes the NPs chemically more reactive due to the large aspect ratio as compared to the bulk counterpart. To examine the chemical bonding on the surface of NPs, FTIR spectroscopy in the spectral range from 400 to 4,000 cm−1 was used. The spectrum confirmed the absorption peaks at 978, 1,106, 1,375, 1,626, 2,330, 2,360, and 3,386 cm−1.
FTIR analysis of A-SeNPs.
3.2.2 Structural analysis
The phase composition and crystal structure of A-SeNPs were determined through XRD. The pattern of the sample (SeNPs) was monocrystalline with a hexagonal shape that was also confirmed through the XRD technique. The average crystallite size of the SeNPs was examined using the Scherrer formula. Figure 2 shows that the sample was examined in the range of 2θ = 10–80°. The intensity of diffraction peaks was observed at 2θ = 23.9, 29.8, 43.7, 45.7, 51.9, and 68.4° with miller indices that correspond to (100), (101), (102), (111), (201) and (211) planes respectively shown [48]. The XRD pattern of SeNPs was confirmed through JCPDS No: 06-0362.
XRD analysis in A-SeNPs.
3.2.3 Morphology analysis
The SEM displays the morphological conformation of the synthesized A-SeNPs as shown in Figure 3a. From the apparent surface of the SeNPs seem to be approximately hexagonal, circular with agglomerated in shape. The SEM image (Figure 3a) also confirmed the average size of the A-SeNPs, in the range of 30–40 nm. The compositional analysis was confirmed through energy EDX spectra shown in Figure 3b. Such typical spectra of EDX give conformation about the purity of the SeNPs. Similarly, other researchers also studied the morphological results of SeNPs [49].
(a) SEM micrograph of A-SeNPs and (b) EDX spectra of A-SeNPs.
(a) Zeta potential measurement of prepared SeNPs and (b) DLS analysis of SeNPs; size: 70–140 nm; average size: 110.3 nm.
3.2.4 Zeta potential and dynamic light scattering (DLS) analysis
It was discovered that the pattern of distribution of the particle size range from 70 to 140 nm, with an average size of 110.3 nm, was obtained using DLS distributing histograms (Figure 4b). The stability of the dispersion of colloidal NPs is assessed using the zeta potential, which measures the ability of NPs to electrostatically repel one another. It controls the interplay between particles in colloidal dispersion. Moreover, the sign value indicates whether the particle’s surface is dominated by positive or negative forces. The monodispersed dispersion of the NPs was found to be more stable, as evidenced by the zeta potential value of −13.9 mV for SeNPs (Figure 4a). The synthesized SeNPs’ zeta potentials revealed that they were mostly negatively charged. Because the reducing agent AA exposes the presence of gross electrostatic forces with the produced SeNPs, the value of the negative charge potential can be determined [50]. There would be a slight tendency for the particles to stick together if every particle in suspension had a positive or negative zeta potential. Otherwise, the particles would tend to repel one another [51]. The great stability of SeNPs in the absence of aggregation formation was likely caused by the negative charge on the particles (Figures 4 and 5).
3.3 Biological applications
3.3.1 Effect of A-SeNPs on MSU crystals
Gout is a collective term, used to represent certain metabolic conditions that arise from the enhanced production and deposition of MSU crystals, in different connective tissues and joints. It is thus made imperative to grow, characterize, and study the effect of therapeutic substances in vitro and in vivo on the MSU crystals [52]. In the study, a concentration-dependent eminent effect was observed on the morphology and desolation of crystals. For instance, A-SeNPs with different concentrations, i.e., 2.5–20 mg·mL−1, were used to investigate the effect of NPs on MSU crystals as a function of incubation time. In general, A-SeNPs resulted not only in breakage, deformity, and reduction of the size of the MSU crystals but also in desolution. For instance, as can be seen in the figure, a significant effect was observed at 20 mg·mL−1 where the A-SeNPs led to the breaking of maximum crystals, rearrangement, and changing the crystals into small, irregular black and transparent dots. The effect was noticed to be more significant after 12 and 24 h. A similar, but comparatively less effective response was also induced in MSU crystals at 10 mg·mL−1. The effect was found to be alleviated when the concentration of the NPs was decreased as can be observed in Figure 5.
Pictorial presentation of micrographs (40×) showing the effect of A-SeNPs on MSU at varying concentrations. PC denotes a positive control and NC represents a negative control.
Contrary to the positive control and NPs, the MSU crystals in the negative control setup retained their needle-shaped triclinic structure even after 24 h. Our study thus concludes that A-SeNPs, affect the structural integrity and result in the desolation of MSU crystals in a dose-dependent pattern.
3.3.2 UA degradation activity
UA in the human body is commonly produced as an end product of purine metabolism. Elevated levels of UA are associated with different complications such as urinary stones and gout arthritis [53]. In the current A-SeNPs with various concentrations were investigated for in vitro quantitative degradation of UA using a SPINREACT (Spain) kit. The results are summarized in Figure 6. In general, it was observed that the UA degrading ability of SeNPs is concentration-depended. For instance, at 20 mg·mL−1, A-SeNPs showed maximum UA degradation of 60.76%. However, the % degradation decreased by reducing the NP concentration, and at the lowest tested concentration of 2.5 mg·mL−1, the UA was degraded up to 39.52%.
Percent degradation of UA by A-SeNPs.
3.3.3 XOI study
XO or xanthine dehydrogenase serves as an important enzyme that is involved in the conversion of xanthine into UA, leading to hyperuricemia and thus resulting in UA deposition in the joints [54]. In the current study, A-SeNPs were investigated against XOI activity with multiple concentrations ranging from 25 to 200 µg·mL−1. In general, a significant but concentration-dependent XO inhibition activity of the NPs was observed as illustrated in Figure 7. For instance, at the highest tested concentration of 200 µg·mL−1, the NPs resulted in 76% XO inhibition activity as compared to Allopurinol which resulted in 93.2% XO inhibition. When the concentration of A-SeNPs was decreased the inhibitory activity of the NPs also decreased, and at a concentration of 25 µg·mL−1 of NP, only 9.7% inhibition activity was calculated. The IC50 as calculated for the A-SeNPs was found to be 64 µg·mL−1.
% XO inhibition activity of A-SeNPs.
3.3.4 Antioxidant study
Antioxidants are substances that both scavenge and prevent the synthesis of free radicals, which are created during metabolic events in plants and animals. Cellular damage is caused by greater concentrations of reaction intermediates such as superoxide and hydrogen peroxide [55]. SeNPs regulate reactive oxygen species (ROS) and GPx, which helps reduce free radicals and shield cells from damage. Numerous academic works exist that delineate the antioxidant properties of SeNPs [56,57]. Multiple antioxidant assays, i.e., DPPH and ABTS radical scavenging assays (DPPH-FRSA and ABTS-FRSA), TAC, and ferric-reducing antioxidant power (FRAP), were executed to quantify the in vitro antioxidant potential of A-SeNPs as summarized in Figure 8. ABTS-FRSA (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and DPPH-FRSA (2,2-diphenyl-1-picrylhydrazyl) depend on neutralization of the stable colored radicals of DPPH˙ + (purple) and ABTS + (green) by the antioxidant sample in question signifying the potential free radical scavenging ability of the sample [58]. SeNPs stabilized with chitosan showed antioxidant action by enhancing and preserving GPx and inhibiting the production of lipofuscin in mice, according to a study by Zhai et al. [59]. Likewise, phytofabricated SeNPs derived from Emblica officinalis fruit extract showed antioxidant efficacy in DPPH and ABTS experiments [46]. When tested for hydroxyl radical and DPPH radical scavenging, gum-Arabic-stabilized SeNPs showed more antioxidant efficacy than gum-Arabic-hydrolyzed alkali [60].
Antioxidant potential of A-SeNPs.
In the present study, the considerable concentration-dependent free radical scavenging potential of the A-SeNPs was confirmed. For instance, at the maximum tested concentration of 400 µg·mL−1, ABTS-FRSA and DPPH-FRSA were noted as 50.58% ± 1.20 (IC50: 380 µg·mL−1) and 33.97% ± 1.01 (IC50 > 400 µg·mL−1) respectively. The free radical scavenging ability of the NPs was further supported by FRAP and phosphomolybdate-based TAC assays. The mechanism of FRAP or TRP is based on the formation of the ferric–ferrous chromatic complex by the antioxidant sample which can be quantified spectrophotometer at 700 nm while the phosphomolybdenum-based assay depends on the spectrophotometric detection of molybdenum(v) which is formed as reduction of molybdenum(vi) in an acidic environment by the test sample [61]. At the tested concentration of 400 µg·mL−1, maximum, FRAP and TAC activities were found to be 29.66 µg AAE·mg−1 of NPs and 31.27 µg AAE·mg−1 of NPs, respectively. However, the antioxidant potential significantly decreased by reducing the concentration of NPs, thus affirming the concentration-dependent antioxidant capacity of the A-SeNPs.
3.3.5 Anticoagulant and thrombolytic properties
Anticoagulant activity of the A-SeNPs was tested by co-incubation of samples with freshly isolated blood and the clots were visualized as a function of storage time as displayed in Figure 9. The blank containing only fresh blood (NC) began to coagulate soon after the initiation of the experiment and the blood sample, finally formed a thick blood clot till 10 min. On the other hand, the A-SeNPs, with different concentrations, showed excellent anticoagulant activity and the blood samples did not undergo significant changes till 3 h as can be visualized in Figures 9a and b.
Anticoagulant (a) and thrombolytic (b) results of A-SeNPs as a function of time.
The best anticoagulant results were visualized at 20 mg·mL−1 where negligible or no clot can be seen. However, as the concentration of the NPs was reduced, the anticoagulant ability of the test samples was slightly reduced as little or considerable clots can be observed in other test samples, especially after 3 h. Furthermore, the positive control containing ethylenediaminetetraacetic acid (EDTA) also resulted in no clot formation till 3 h, thus affirming and supporting the anticoagulant property of A-SeNPs as observed in the experiment. For the determination of potential thrombolytic properties, dH2O dispersion of A-SeNPs (20 mg·mL−1) was added to a pre-formed blood clot on a clean sterile glass slide, and visual observations were made. With time, eminent clot lysis was observed as shown in Figure 9b.
4 Discussion
There has been growing interest in the preparation and study of SeNPs because of their excellent mechanical characteristics, optoelectronic and magnetic properties, as well as their plethora of applications in nano-medicines, sensors, xerography, and catalysis to name a few [62].
Conventionally, chemical and physical methods have been used to prepare nanoscale Se particles with different morphologies and physicochemical characteristics [63]. However, these approaches, in particular chemical and physical methods, are considered disadvantageous because of high energy consumption, use of hazardous chemicals, and the non-ecofriendly nature of the methods. It has also been reported that the SeNPs synthesized from phytochemicals such as AA is a less toxic and environmentally friendly process than the SeNPs prepared from the chemical and physical process [63,64].
Recent studies show that Se at a nano-scale exhibits far better biocompatibility and lower toxicities than inorganic and organic Se compounds. Such benefits of the SeNPs make them potential nanomaterials to be explored as therapeutic and theranostic agents [65]. In the present study, we planned to investigate the A-SeNPs for potential antigout (MSU crystals desolation, UA degradation, XOI, antioxidant, anticoagulant, and thrombolytic properties.
After successful synthesis, the ascorbic-mediated synthesized SeNPs were characterized using XRD, FTIR, SEM, and EDX.
XRD patterns confirmed the diffraction peaks at 2θ = 23.9, 29.8, 43.7, 45.7, 51.9, and 68.4° with miller indices that correspond to (100), (101), (102), (111), (201), and (211) planes respectively (JCPDS No: 06-0362) confirming the hexagonal structure [66]. The size of the prepared synthesized A-SeNPs was calculated by Scherrer’s equation [67] as given;
where D represents the size of the NP, k is a constant equal to 0.9, λ used as radiation source from CuKα 0.15432 nm, β1/2 is the full width at half maximum, and 2θ is the diffraction angle. The calculated crystallite size of A-SeNPs was 78 nm. Similar XRD patterns and crystallite dimensions of the nanoscale Se were also reported previously in the literature [66,68].
The surface interaction between the A-SeNPs and stabilizing agent was investigated using FTIR analysis. The FTIR spectra of A-SeNPs confirm the variety of bands present in AA (Figure 1). The broadband at 3,550–3,190 cm−1 corresponds to the O–H hydroxyl group of the AA present on the surface of NPs sample (A-SeNPs) [69,70,71,72]. The two sharp peaks at 2,330 and 2,360 cm−1 correspond to the molecules of CO2 that may exist in the air during the characterization of the SeNPs. The bond at 1,626 cm−1 represents the symmetric C═C double bond stretching motion of dehydroascorbic acid [73]. The bond in a region 1,375 cm−1 is attributed to O–H bend or ring deformation [74]. Figure 1 confirms that peaks at 978 and 1,106 cm−1 are attributed to aromatic in-plane C–H bending and secondary OH, respectively.
Similarly, SEM micrographs revealed the hexagonal, spherical, and slightly agglomerated morphology of the prepared A-SeNPs with a mean size of 30–40 nm. Almost similar morphology of SeNPs was also presented in other studies [75,76]. For instance, in a study, stable, uniform, hexagonal phase and spherical SeNPs with an average size of 30–40 nm were prepared using a green and economical approach. Such SeNPs were found to be highly potent against bacterial and fungal strains [76]. Finally, EDX affirmed the elemental Se within A-SeNPs. After physiochemical, morphological, and elemental confirmation, the A-SeNPs were explored in multiple biological studies including anti-gout, antioxidant, and anticoagulant.
Gout is caused by hyperuricemia, a metabolic disorder characterized by elevated UA in the body due to an imbalance of UA excretion and production [77]. Currently, throughout the world, the prevalence of hyperuricemia has continuously increased manifolds [78]. Presently, a limited number of medicines are available, for treatment and most of these are labeled with side effects. For instance, in European countries since 2003, the uricosuric agent benzbromarone was discontinued due to severe hepatotoxicity reports [79]. While XO inhibitor allopurinol is primarily used for the treatment of hyperuricemia, this drug has severe cutaneous side effects [80]. Hence, a lot of previous research has been conducted to develop safe therapeutic agents for the treatment of elevated UA level-related disorders [79].
In this study, we mainly targeted MSU crystals that are formed through the accumulation of UA in the body leading to gout and UA causal agent of hyperuricemia. Interestingly, A-SeNPs showed remarkable crystal dissolution activity and after 24 h inhibited the reformation of MSU crystal. Few considerations such as extremes of pH and high temperature could be argued to influence the dissolution of MSU crystals. However, the pH of A-SeNPs dispersion was noted as 5.56, which is in fact near neutral, not extremely acidic. Similarly, high temperature may facilitate MSU crystal dissolution; however, the current experiment was executed at the physiological body temperature, i.e., 37°C [81], which thus leads to the only possibility that A-SeNPs played a potential role in the crystal dissolution process.
Recently, various enzymes-NPs that mimic natural enzymes have been explored that may be used as an alternative to uricase (UOX) or in combination with uricase to catalyze the degradation of UA. For instance, Jung and Kwon found that UOX-AuNP successfully degraded UA, five times more rapidly than uricase alone [82]. Similarly, in another study, researchers demonstrated that platinum NPs (PtNPs) with sizes 5–55 nm, mimic the natural uricase, and effectively carry out the oxidative degradation of UA. The authors also concluded that the degradation mechanism was pH-independent and was affected little by the experimented size range of the NPs [83]. In our study, the A-SeNPs were also screened for the UOX mimic characteristic to degrade the UA. We found a concentration-dependent degradation ability of A-SeNPs as it was noted to be a maximum of 20 mg·mL−1 and a minimum of 2.5 mg·mL−1. However, a detailed study is needed to be formulated to investigate the exact mechanism involved in the degradation process of UA. The A-SeNPs were also tested for the XOI potential. XO or xanthine dehydrogenase is a key enzyme in the pathogenesis of gout as it catalyzes the conversion of xanthine into UA, leading to hyperuricemia and thus resulting in UA deposition in the joints. The search for novel potential molecules with XO inhibition activity has grown in the recent past, because of the toxic effects that are being associated with approved drugs like allopurinol and febuxostat [84]. It is now a well-established fact that the interaction of proteins with NPs results in the formation of “protein corona” that may in turn affect the physicochemical properties and nature of both the interacting NPs and proteins. This also forms based on NP bio-reactivity with specific protein molecules [85]. In the study, an excellent but concentration-dependent inhibition activity of A-SeNPs was measured at 400 µg·mL−1 resulting in 76% XO inhibition activity, but the activity decreased considerably at the lowest tested concentration of 50 µg·mL−1 to only 9.7%. Because of the ease of production and low cost, metallic NPs may be considered as a potential alternative to conventional enzyme inhibitors, once their biosafe nature is ensured and thus may prove to be useful tools to treat different pathological conditions such as gout.
It is now a fact that different abiotic stresses build up highly toxic ROS in plant and animal cells that in turn denature key proteins, lipids, carbohydrates, and DNA and ultimately result in oxidative stress [86,87]. Several different diseases may thus arise due to the buildup of oxidative stress. Such oxidative stress-related diseases can be prevented by the intake of antioxidants that function to neutralize the effects of ROS [88]. In the presented study, DPPH and ABTS radical scavenging assays (DPPH-FRSA, ABTS-FRSA), TAC, and FRAP were performed to evaluate the antioxidant capacity of A-SeNPs. The considerable, concentration-dependent free radical scavenging ability of the A-SeNPs was detected as maximum ABTS-FRSA and DPPH-FRSA at 400 µg·mL−1 was noticed as 50.58% ± 1.20 (IC50: 380 µg·mL−1) and 33.97% ± 1.0 (IC50: 400 µg·mL−1), respectively.
As blood comes into contact with subendothelial surfaces, it clots rapidly and stays fluid inside the vasculature. Coagulation / fbrinolysis balance prevents thrombosis and bleeding in normal circumstances. Any imbalance promotes coagulation, resulting in thrombosis, platelet aggregation, fibrin formation, and stuck red blood cells in arteries or veins. Several antithrombotic medications are available on the market to treat thrombosis. Antiplatelet drugs reduce platelet activation or aggregation, while anticoagulants prevent fibrin formation; however, fibrinolytic treatments break down fibrin synthesis [89]. It has recently been shown that using naturally produced AgNPs instead of selective active molecules has effective anticoagulant effects and requires less than active molecules [90]. Using a different strategy, red algae-derived bioinspired cobalt NPs have effectively stopped blood clot formation in vitro [91]. Previous studies on metal oxide NP, especially on gold and silver NP, have helped to clarify the likely biochemical mechanism, even though the mechanisms underlying SeNPs’ anticoagulant activity have not been theoretically deciphered. These investigations showed that these NPs prevent prothrombin from becoming thrombin, which is an essential stage in the creation of insoluble fibrin strands and the catalysis of other clotting factors [92,93]. However limited material in the literature is available about the use of metallic NPs as anticoagulant and thrombolytic agents, especially regarding SeNPs. Therefore, we explored the greenly synthesized SeNPs for anticoagulant and thrombolytic potential. The considerable anticoagulant activity of the NPs was visualized which was mainly found to be concentration-dependent as even till 3 h no eminent clot was observed in the blood sample at 20 mg·mL−1. The findings obtained in the current study, are comparable to the thrombolytic behavior of AgNPs, AuNPs, and bimetallic Au-Ag-NPs [82,94,95]. Our investigation thus revealed the effectiveness of A-SeNPs as potential anticoagulant and thrombolytic agents. The exact mechanism of the thrombolytic activity of a metallic nanostructure has not yet been interpreted, but the probable biochemical mechanism can be explained via the thrombolysis process. The A-SeNPs might have resulted in the inhibition of clot-forming enzymes or prevention of prothrombin transformation into thrombin, which in turn catalyzes the formation of insoluble fibrin and other clotting factors [92]. Traditional antithrombotic drugs such as streptokinase come with a couple of drawbacks including low shelf life, neutralization of foreign materials, and the prospect of unnecessary bleeding [96]. So, the potential anticoagulant behavior of A-SeNPs may have some beneficial medical applications to control thrombosis and other related illnesses. Antihyperuricemic, antioxidant, anticoagulant, and thrombolytic activities of A-SeNPs and their comparison with the recent literature is presented in (Table 1).
Antihyperuricemic, antioxidant, anticoagulant, and thrombolytic activities of A-SeNPs and their comparison with the recent literature
| S. no | Materials used | Shape and size | Applications | Ref |
|---|---|---|---|---|
| 1 | Bacillus endophyticus-mediated SeNPs | Rod-shaped with an average size from 10 to 20 nm | Ba-SeNpMo had 67% thrombolytic activities, compared to 82% for the positive control and 4.0% for the negative control | [97] |
| 2 | CTAB-SeNPs | Irregular with an average size of 32.53 nm | DPPH radical scavenging activity of 23.72 ± 008% | [98] |
| SDS-SeNPs, | Irregular with an average size of 48.04 nm | DPPH radical scavenging activity of 25.14 ± 0.01% | ||
| Brij-58-SeNPs | Spherical with average size of 37.78 nm | DPPH radical scavenging activity of 24.46 ± 0.04% | ||
| 3 | Chitosan-stabilized SeNPs | Spherical with an average size of 103 nm | SeNPs have a higher ABTS scavenging ability that the value could reach up to 87.45 ± 7.63%. | [59] |
| 4 | Penicillium expansum-mediated SeNPs | Spherical with an average size 4–12.7 nm | Concentrations above 30 μg·L−1 of SeNPs have antioxidant activity above 50% | [99] |
| 5 | Crocus caspius-mediated SeNPs | Spherical with a size of 79 nm | The IC50 of SeNPs has obtained 123.9 ± 2.5 mg·mL−1. The standard compound was Butylated Hydroxyanisole with an IC50 value of 53.96 mg·mL−1 | [100] |
| 6 | Diospyros Montana-mediated SeNPs | Spherical shape with an average size of 4–16 nm | SeNPs showed 61.12% of DPPH scavenging activity at the concentration of 200 μg·mL−1 compared to the AA (99.84%) | [68] |
| 7 | Tinospora cordifolia-mediated SeNPs | Spherical shape with a size range of 20–200 nm | SeNPs showed DPPH scavenging activity of 78.03% at 100 μg·mL−1 than aqueous extract of T. cordifolia which showed 50.83% scavenging activity | [101] |
| 8 | Withania somnifera-mediated SeNPs | Spherical with average size of 45–90 nm | SeNPs were found to possess significant antioxidant activity (IC50 – 14.81 μg·mg−1) | [102] |
| 9 | Solanum lycopersicum-mediated SeNPs | Irregular with an average size of 1,155 nm | Solanum lycopersicum-mediated SeNPs have DPPH antioxidant activity with an IC50 value of 20.7398 mg·mL−1 | [103] |
| 10 | Citric acid-mediated SeNPs | Spherical with size from 10.5 to 20 nm | Citric acid-mediated SeNPs exhibit higher anticoagulant properties with increased SeNPs dose but still less than heparin which is the standard anticoagulant drug | [93] |
| 11 | AA-mediated SeNPs | Hexagonal with particle size range from 70 to 140 nm, with an average size of 110.3 nm | DPPH 50.58 ± 1.20% (IC50 of 380 µg·mL−1 and ABTS 33.97 ± 1.01% (IC50 > 400 µg·mL−1 | Present work |
| At a concentration of 400 µg·mL−1 showed 76% XO inhibition activity and 9.7% at a concentration of 50 µg·mL−1 | ||||
| Prepared A-SeNPs, showed excellent anticoagulant activity and the blood samples did not undergo significant changes till 3 h |
5 Conclusion
In the current study, A-SeNPs were synthesized through a facile precipitation technique by using L-AA as a reducing and stabilizing agent. The L-AA played an important role as a reducing agent by controlling the size of particles. The technique used in the study is simple, non-toxic, inexpensive, and environmentally friendly. Characterization techniques such as FTIR, XRD, EDX, and SEM support the structure, size, and crystallinity of selenium NPs. In the current study, A-SeNPs were very effective against MSU crystal dissolution and UA degradation. After a 2–24 h experiment, it was noted that A-SeNPs considerably dissolved and inhibited the regrowth of MSU crystal at concentrations of 20 and 10 mg·mL−1. Further, the synthesized NPs showed potent inhibition against XO. According to these results, it can be concluded that A-SeNPs have the potency to treat gout and hyperuricemia-related diseases. However, further in vivo studies need to be conducted regarding the efficacy of A-SeNPs to treat such conditions in an in vivo environment. Moreover, the A-SeNPs showed considerable antioxidant potential as evaluated by multiple antioxidant assays. Most importantly, A-SeNPs were found to have good anticoagulant and thrombolytic activities. Our study thus extends the knowledge of biological applications of SeNPs and opens up new opportunities in the area of biomedical research.
Acknowledgments
This article is part of the Ph.D. thesis of Mr. Muhammad Aamir Ramzan Siddique. The authors are very thankful to the Department of Biological Sciences, International Islamic University Islamabad, and HEC Pakistan for providing an opportunity for an IRSIP fellowship for important research to the University of Alberta Canada to complete this research work.
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Funding information: Authors state no funding involved.
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Author contributions: M.A.R. Siddique and S.A.I. Bokhari perceived the idea, carried out experimental work, and wrote the manuscript. M.A. Khan, K. Ahmed, and H.A. Haseeb assisted in experimental work and manuscript preparation, M. M. Kayani and N. Zahid, assisted in experimental work and characterizations of samples, S. Khan, M. Ismail, S. B. Khan, proofread and assisted the nano work, and S. A. I. Bokhari supervised the project. All authors read and approved the final manuscript.
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Conflict of interest: Authors state no conflict of interest.
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Data availability statement: All data generated or analyzed during this study are included in this published article.
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