Developing Strategy for a Successful Antioxidant, Anticancer Activity via an Improved Method Prepared to Porous Silicon Nanoparticles

*Corresponding Author: Butheina A. Hasson 100235@uotechnology.edu.iq Abstract Porous silicon nanoparticles, or PSNPs, are one of the most common NPS with distinct characteristics. PSNPs were created using an enhanced approach known as the electrochemical etching process. The nanoparticles were treated with a post-laser to obtain the nanoparticles, which were subsequently analyzed using (SEM), (UV-Vis), and (XRD). The size refers to porous silicon with a nanostructure. Because of the differences in interaction between the HF electrolyzed and silicon and crystal structure, PS (100) offers better NP properties than directional silicon (111). PSNPs' antioxidant activity was measured using the DPPH test, while cytotoxicity was measured using the MTT assay on Hella cells. PSNPs have an inhibitory impact on cancer cell growth and antioxidants, according to the findings. PSNPs did not have any toxicological effects on the skin, lungs, or spleen after injection.


Introduction
PS is a porous semiconductor with a high reactivity because of its huge surface area and surface chemistry. On its surface, it is possible to create a precise chemical composition or molecule adsorption [1]. PS is also a good biomaterial since it is biodegradable and biocompatible [2]. These PS bio-characterizations are large-scale productions that rely on their oxidation susceptibility. Body fluids dissolve the silicon oxide [3]. Later, it is excreted in the urine as silicic acid, which is non-toxic [4]. PS has also been mixed with other materials, injected into its pores, or coated on its surface to create composites. [5] That can broaden or deepen its range of uses and personality, including the bio [3,6]. PS research in medical and non-medical applications is still increasing in this regard since it is a viable biomaterial for commercial use. Silicon, as well as the substrate on which it is manufactured, is a low-cost commodity that is suitable for use in the high-tech electronics sector. Drug delivery and cell scaffolds are two of the most common uses of PS as a biomaterial. PSi-based devices geared toward these applications are becoming more common [7,8]. PSi became a potential material for optoelectronic applications as a result of these properties, and papers devoted to PSi increased dramatically in the 1990s [9]. Optoelectronics [10], Bragg reflectors [11], solar cells [12], and photodetectors [13] are among the most PSi applications in lightemitting devices (LEDs) and optical waveguides [14].
The goals of this work are to synthesize colloidal silicon NPS utilizing electrochemical etching and laser in liquid techniques, as well as to investigate the influence of silicon NPs on antioxidant and anticancer activities in vitro and in vivo models.

PSNPs production
Porous silicon (PS) samples were created by electrochemical etching silicon wafers (100) doped with orientated boron and having a thickness of (55050) nm (Diameter 7.6 cm). (Resistivity 1.5-4.cm) and (Resistivity 1.5-4.cm) (central circular of 5.5cm 2 ). He employed a 1:1 (v/v) electrolyte of hydrofluoric acid (24%) and ethanol (99.9 percent). After that, the NPs were dissolved in deionized water and placed in an ultrasound instrument for four hours. The NPS was then created using a filter that allowed NPs with a size of up to 0.22 m to pass through [15]. Finally, NPs were generated using a post-laser with a wavelength of 1064 nm and laser intensity of 350 mJ.

UV-Vis, SEM, XRD
PSNPs were optically analysed using UV-Vis within the spectral range of 200-900 nm. SEM was utilized to generate pictures from the surface of a sample, and XRD analysis was performed to validate the structure. The PSNPs chart by UV-Vis, XRD, and SEM were done in the service centre University of Baghdad.

Antioxidant Activity: DPPH Assay
Antioxidant activity of PSNPs wear calculated using DPPH assay with minor adjustments according to [16,17]. Extract was applied to investigate the scavenging activity. Every sample were mix up with DPPH solution and then completes the quantity to one mL using ethanol. Absorbance was calculated at 517 nm. Scavenging activity computed corresponding to the equation formula:

Cytotoxicity Assays
The MTT test was used to assess cytotoxic effects. Cell lines were cultured in a well of (1640 cells). The monolayer was reached after 24 h, and the cells were treated with the tested drug. After 72 h, the viability of the cells was determined by extracting the medium and incubating the cells for 1.5 h. at 37 °C with 28 µL of 2 mg/mL MTT solution. After that the MTT stain was eliminated and solubilized the rock crystal that staying in the wells by the add-on (130 μl) of DMSO followed by keep at 37 °C for 15 min [18]. The absorbency was determined on a micro plate reader at 492 nm. The percentage was determination as the following calculation:

In-Vivo Model Experiment
The Biotechnology Research Centre provided albino male mice (Al-Nahrain University). They were 8-10 weeks old when the trials began, and their average weight was 35-45 grams. They were divided into five groups, each of which had three mice that were housed in their own plastic cage (details of these groups are given in the section of experimental design). The animals were kept at room temperature and given access to unlimited food and water. In this experiment, 1 mg/kg was the dose of PSNPs, and assess the toxicity effects of this dose, the mice were distributed into five groups, and each group contains three animals (total: 15 mice): [19,20].
Group I: Mice were administrated with physiological saline (Negative controls.) Group II: (Injection PS NPs), Killing after a week.
End of the experiment, mices were killed for study Histological, sections.

Histological, Sections, Preparation
The tissues (lung, spleen, and skin) were collected from mice in the control and PSNP-treated groups. These organs were preserved in 10% formalin [21], and their slices were performed in the central lab. Baghdad Hospital-Iraq.

Statistical Analysis
A p-value of ˂ 0.05 was considered significant. Unpaired t test using comparison between groups was made [15].

UV-Vis Spectroscopy
Figure (1) depicts the colors of PSNPs colloidal NPs used to reduce the size of silicon nanoparticles in double deionized water at various NP concentrations of (750, 500, and 250) g/ml. The degree of color relies on the concentration of PSNPs and increasing the concentration of NPs and the number of laser pulses will increase particle concentration. From light orange to dark gray, the color NPs are ordered. These are dependent on the NPs' form and size, as well as D.W.'s dielectric constant [22]. UV-Vis. is the measurement of the absorption of PSNPs within the wavelength range of 200-1000 nm. UV-Vis. for PSNPs indicated that the top peak could be seen at 420 nm, In Figure (2) curves 1, 2, and 3 at concerns. (750, 500 and 250) μg/ml respectively with absorption intensities of (0.98, 0.70, and 0.35) the wavelength (448) nm has the maximum absorption intensity. As a result, we may deduce that the concen. rose as absorbance increased. PSNPs have a high absorption factor at short wavelengths, as seen in the graph. This suggests that the optical absorption is caused by a direct spectral transition [23].   Figure (3) demonstrates an increase in peak intensity with increasing concentration at 750 g/ml. In Table, there is also less displacement at the diffraction angle (4.3). This implies that the PSNPs of the FCC type have levels (111) and (100) of 2 =28.5 and 70, respectively. At a concentration of 500 g/ml, the porous structure causes reduced breading and intensity, as well as a decrease in size, resulting in breading of the plan (111) and (100) at 2=28.5 and 70, respectively. A broadening of the diffraction peak was found at concentration 250 g/ml, with the broader of the peak suggesting a drop in nanosize, and at 2=28.5 and 70 with the levels (111) and (100) showing a decrease in particle size than the other concentrations. Figure (4) shows the SEM findings of the PSNPs, with particle sizes of 35.5, 65, and 75 nm, respectively. These results suggest that the increase in concentration was accompanied by an increase in nanosize. At a concentration of 250 g/ml, the typical particle size is 18-70 nm. At concen. 500 g/ml, the average particle size is (35-70) nm, and the particle rate is 10-160 nm, whereas at concen. 750 g/ml, the average particle size is 35-70 nm and the particle rate is 10-160 nm. Image J. software was used to evaluate the size of the NPS, which is dependent on the concen. Value, with the higher the concen. the greater the NPS size.

Antioxidant Action of PSNPs
The DPPH was shifted exactly proportionate to the rise in concentration, according to the results of my research. When antioxidant chemicals returned a proton and an electron, the hue shifted to yellow [16]. Concern. of (25,50, and 75) g/ml is provided to free radicals at (42.56, 50.94, and 57.22) respectively, as compared to control positive, as shown in figure (5). The high concentration of PSNPs of Si-O-Si might be to blame for the increase in displaced free radicals. The findings revealed that the high content SiO stretching in (O-SiO), CH3 symmetric stretching, CO binding, CH2 symmetric stretching, and OH stretching (SiOH) are all responsible for antioxidant action [24,25,26,27,31]. Enzymatic antioxidants have a role in cellular defense against ROS [28], and they have been hypothesized to serve as antioxidants.

Anticancer action of PSNPs
PSNPs' impact on tumor cell proliferation for 48 hours, Hella cells were treated with various doses of PSNPs (25, 50, 75 g/ml). The cytotoxicity test resulted in a statistically significant impact. The concerns. of (75, 50, and 25) µg/ml had a greater impact than the concentration of 25 g/ml, indicating that PSNPs can suppress cancer cell proliferation ( Figure 1). (6). PS NPs with a size of 39 nm, on the other hand, demonstrate anticancer activity against kidney tumors at a concentration of (400) g/ml [31].

Evolution the Effect of PSNPs in Vivo
To study the effect of PSNPs in tissues and body weight.

Measurement of Body Weight
The body weight findings, as shown in Figure 7, revealed no significant changes in body weight between the treatment groups

Evolution Histopathology of PSNPs
PSNPs were injected into the lung, spleen, and skin of mice for four weeks to determine their impact. There are no changes in histology results between the treatment and control groups. figures (8,9 and 10). As indicated in figure 8, there was no change in the surface of the red pulp or the size of the lymphoid follicles in the spleen. Figure 9 depicts the typical anatomy of the lung, including alveoli and alveolar space, as well as the existence of bronchials. In figure10, skin tissue depicts the typical structure of the skin.   Biocompatibility and biodegradability of PSNPs in vivo were examined. The PSNPs preparation is relatively nontoxic in vivo within the examination concentration range. This result compared with the slow removed normally observed for other kinds of inorganic nanoparticle [29]. By terminating a period of 4 weeks. In vivo studies, PSNPs (1 μg/ kg) were injected intraperitoneal into mice. However, the PS NPs collected in the organs are noticeably removed from the body within a period of first week and totally removed in four weeks. The mice of porous silicon nanoparticle internalization have been shown to be determined by particle size [30]. Some factors are thought to have a profound effect on the development of mediated toxicity nanoparticle. The key factor is nanoparticle diameter, which, at minimum for colloidal nanoparticle, inversely associated with the surface area. Special toxicological effects of PSNPs in mice were more established by histopathological investigation. Figure (8,9,10) shows that no atypical in histopathological findings were shown in lung, spleen, skin after 4-week injection of PSNPs. This result clearly shows that PSNPs did not cause toxic effects. Pathological damage associated to the test materials was not shown in every treated group. Porous silicon nanoparticle used in the present study did no source severe toxicity in mice. On contrast study, the results indicate that the PS NPs had not cause toxic effects at all examined [31].

Conclusions
PS reasonably biocompatible nanomaterials. Toxicity of PSNPs was assessed in mice and in vivo after 4-weeks. Results injection that PSNPs did not because cytotoxic effects after 4-weeks, with added PSNP have potential antioxidant and anticancer activity.