Assessment of mechanism involved in the apoptotic and anti-cancer activity of Quercetin and Quercetin-loaded chitosan nanoparticles

In prior studies, Quercetin was revealed to exhibit anti-cancer features in a variety of cancer cell lines. However, the impact of Quercetin on neuroblastoma is unknown. This study looked into the potential cytotoxic effects of Quercetin and Quercetin-loaded chitosan nanoparticles (NPs) on the SH-SY5Y cell line. In this study, NPs containing Quercetin was prepared and characterization studies were performed. The vitality of the cells was measured using the XTT test after 24 h of treatment with various concentrations of Quercetin (0.5, 1, 2, 4, and 8 µg/mL). ELISA kits were used to detect the amounts of cleaved PARP, BCL-2, 8-Hydroxy-deoxyguanosine (8-oxo-dG), cleaved caspase 3, Bax, total oxidant status, and total antioxidant status in the cells. The results of the chitosan NPs characterization investigation revealed that the particle size, encapsulation effectiveness, and drug release profile of NPs were all appropriate for cell culture studies. Quercetin and Quercetin-loaded chitosan NPs significantly reduced cell viability in SH-SY5Y cells at different concentrations (**p < 0.05). 2 µg/mL Quercetin and Quercetin-loaded chitosan NPs significantly enhanced the levels of 8-oxo-dG, cleaved caspase 3, Bax, cleaved PARP, and total oxidant in ELISA testing. However, treatment with 2 µg/mL of Quercetin and Quercetin-loaded chitosan NPs did not affect the amount of BCL-2 protein. Overall, Quercetin and Quercetin-loaded chitosan NPs caused significant cytotoxicity in SH-SY5Y cells via producing oxidative stress, DNA damage, and eventually apoptosis.


Introduction
Cancer is a significant health problem and is the second leading cause of death. Cancer is both a disease and includes a variety of illnesses with every system and organ growing a various set of ailments [1,2]. About one-third of cancer deaths are related to smoking, lifestyle, or dietetic practices. In addition, several types of cancers are avoidable by modifying unhealthy lifestyle habits [3,4].
Surgery, radiation therapy, chemotherapy, immunotherapy, targeted therapy, and hormone therapy are some of the traditional therapeutic approaches and equipment used in cancer treatment [5,6]. These approaches are frequently associated with adverse effects and a significant risk of recurrence. Neuropathies, bone marrow suppression, skin, and gastrointestinal issues, and hair loss are some of the most prevalent side effects. The fact that traditional treatment methods used in cancer treatment have the stated side effects has paved the way for researching new treatment methods [7,8]. Nanotechnology-based drug delivery systems are promising in the treatment of cancer and pioneering these studies [9,10]. Nanoparticular delivery systems provide significant benefits, with biocompatibility, multi-functional encapsulation of active substances, high bioavailability, easy tumor targeting, sustained blood circulation time, active and passive targeting, and reduced or eliminated side effects [11,12].
Nanomedicine with anti-cancer features can provide anticancer efficacy mainly via increasing cytotoxic drug quantity in tumor area hence stimulating anti-cancer activity [13,14]; decreasing its clearance and lowering side effects in the normal organs thus reducing undesired toxicity, and delivering various anti-cancer active substances within the same process [15][16][17].
Chitosan is a deacetylated (DA) chitin product that is widely employed in the medicinal and pharmaceutical 1 3 176 Page 2 of 9 industries due to its biodegradability, biocompatibility, and low toxicity [18,19]. It can also be used for medication transport, packaging, antibacterial agents, tissue engineering, antiaging agents, antibody response enhancers, and the treatment of diseases such as cancer treatment [20,21]. It's commonly used in nanoapplications including medicine delivery [22].
Quercetin is an antioxidant flavonoid that can be found in a variety of fruits and vegetables. It has anti-cancer qualities, according to research. Quercetin has been extensively researched as a chemopreventive drug displaying antiinflammatory and antioxidant effects (mostly by scavenging reactive oxygen/nitrogen species) in a variety of in vitro and in vivo cancer models. Furthermore, Quercetin has been shown to inhibit cell proliferation, differentiation, and survival in specific types of cancer cells by targeting critical chemicals involved in tumor cell growth [23,24]. Due to the anti-proliferative and antioxidant activity of Quercetin, this material was chosen in the study.
In this assay, quercetin was encased in nano-sized particles of chitosan, a biologically friendly and bio-degradable cationic polymer. In XTT and bioactivity experiments, the effects of quercetin and quercetin nanoparticles were investigated on NIH 3T3 and SH-SY5Y cell lines. In this study, the human ELISA kits of 8-Hydroxy-Desoxyguanosine (8-oxo-dG), cleaved caspase 3, BCL-2, cleaved PARP and Bax were used to assess the amounts of cleaved caspase 3, cleaved PARP, 8-oxo-dG, BCL-2 and Bax in Quercetin and Quercetin-loaded NPs treated and untreated SH-SY5Y cells. In addition, necessary characterization studies on nanoparticles were performed in terms of the suitability and usability of quercetin-loaded nanoparticles in XTT and bioactivity studies.

Preparation of chitosan NPs
Ionic gelation method was used to prepare NPs including Quercetin. The determined amount of chitosan was dissolved in acetic acid (0.5% v/v) at 1000 rpm under magnetic stirring. To obtain high encapsulation and loading capacity of NPs, the pH value of the chitosan solution should be between 4 and 5. The pH value of the chitosan solution was adjusted to 4.4 using 5 M sodium hydroxide solution [26,27]. PVP was dissolved in sterile deionized water at a determined concentration (0.25% w/v). PVP solution including Quercetin dropped into chitosan solution (0.5% w/v). NP suspension was centrifuged at 10,000 rpm for 30 min. The supernatant was removed and 1 mL sample was separated from the supernatant to calculate the encapsulation efficiency (EE) and loading capacity (LC) of the NPs. Then, the pellet was washed with deionized water. Deionized water (30 mL) was added to the pellet and centrifuged at 10,000 rpm for 15 min. This process was repeated twice. Then NPs were lyophilized and stored at + 4 °C.

EE and LC studies of NPs
Ultraviolet-visible (UV-vis) spectrophotometer was used to measure the EE % and LC % of Quercetin in NPs [28]. By reading the absorbance of Quercetin at various concentrations at a wavelength of 380 nm, the standard calibration curve of Quercetin was established and a spectral line equation was obtained. The amount of Quercetin in the supernatant was calculated from the line equation. The following Eqs. (2 and 3) were used to determine the encapsulation efficiency and loading capacity of the NPs: where mo is the initial mass of Quercetin and ms mass of Quercetin in the supernatant and wnp = total weight of Quercetin of NPs [29,30]. All measurements were performed in triplicate and were reported as mean ± SEM (n = 3).

Measurement of particle size and zeta (ζ) potential
The size and ζ potential measurements of NPs were evaluated via a Zetasizer Nano ZS instrument. In this study, NPs were suspended in PBS (pH 7.4) and measured.

In vitro release study of Quercetin-loaded NPs
In vitro release of Quercetin from chitosan NPs in PBS (pH 7.4) were performed according to methods with slight modifications of the release assay [30,31]. Initially, a determined amount of Quercetin-loaded NPs was dispersed in 2 mL of buffer solution and maintained in a shaking water bath at 25 °C. At predetermined time intervals, samples were centrifuged at 10,000 rpm for 10 min at 25 °C. 800 μl of the supernatant was withdrawn for analysis, and was replaced with an equivalent volume of fresh buffer to maintain the total volume. The amount of released Quercetin at a specific time was determined using UV-vis spectrophotometer.

Cell viability assay
Cytotoxic activity of Quercetin and Quercetin-loaded NP were evaluated using the XTT assay on the SH-SY5Y and NIH 3T3 cells. Initially, cells were seeded in 96-well plates with DMEM (100 μL, 10% FBS) and incubated overnight [25,32]. After that, the various concentrations (0

Total antioxidant status (TAS) and Total oxidant status (TOS) assessment in Quercetin and Quercetin-loaded NP treated and untreated SH-SY5Y cells
The total antioxidant status assay kit (Rel Assay Diagnostics, Turkey) and total oxidant status assay kit (Rel Assay Diagnostics, Turkey) were used to evaluate TAS and TOS quantities in Quercetin and Quercetin-loaded NP treated and untreated SH-SY5Y cells, respectively [33]. SH-SY5Y cells were treated with 2 μg/mL Quercetin and Quercetin-loaded NP for 24 h. For TAS and TOS, the data were expressed as mmol Trolox Equiv./L and mol H 2 O 2 Equiv./L, respectively.

Statistical analysis
The findings were evaluated using one-way ANOVA and repeated measures ANOVA followed by a Tukey post hoc test (SPSS 14.0 for Windows) for multiple comparisons between groups. All results are presented as a mean ± SEM. The significance level was determined as **p ˂ 0.05, ***p < 0.01 and *p < 0.1.

Characterization of chitosan NPs
Particle size, ζ potential, polydispersity index (PDI) of NPs were assessed, and the results are indicated in

In vitro release kinetics study result of Quercetin-loaded NP
The release profile of Quercetin from NP was investigated at 37 °C over a period of 240 h (Fig. 1). In this study, 0.1 M PBS was used in accordance with physiological conditions. The release results of Quercetin were shown a controlled release characterized by a fast initial release (50%) during the first 24 h, followed by a continuous and slower release (75%) till 120 h. Almost all of the Quercetin (99.58%) in the NP was released in 240 h. This type of continuous and slow release has been experienced in a study for acetylsalicylic acid (34). Diffusion and molecular matrix degradation of the Quercetin play an important role in the release of the Quercetin from the NP. Since the size of Quercetin is lower than the particle, Quercetin can easily diffuse from the surface or pores of the NP [28,34].

Assessment of cytotoxic activity results
To calculate the IC 50 values of Quercetin and NP including Quercetin at determined concentrations were treated to SH-SY5Y and NIH 3T3 cell lines. According to the results of the XTT study (Fig. 2)   According to the concentration-dependent XTT assay results in the NIH 3T3 cell line (Fig. 3), the Quercetin and the NP including Quercetin were treated with NIH 3T3 cells at 0.5 µg/mL concentration, the cell viability was calculated as 90.08 ± 0.53% and 95.58 ± 0.47%, respectively. Quercetin and the Quercetin-loaded NP were administered with NIH 3T3 cells at 4 µg/mL concentration, and the cell viability was calculated as 86.30 ± 0.11% and 90.86 ± 0.08%, respectively. Quercetin and the Quercetin-loaded NP were treated with NIH 3T3 cells at the greatest concentration (16 µg/ mL), the cell viability was calculated as 80.97 ± 0.29% and 86.10 ± 0.33%, respectively. According to the results IC 50 values of the Quercetin and NP containing the Quercetin at the determined concentrations could not be calculated.

The effect of Quercetin and Quercetin-loaded NPs on TAS and TOS quantities in SH-SY5Y cells
The effect of Quercetin and Quercetin-loaded NP on TAS and TOS quantities in SH-SY5Y cells was investigated using a TAS and TOS assay kit. Both Quercetin and Quercetin-loaded NP had no effect on TAS quantity (p > 0.05). In addition, the Quercetin and Quercetin-loaded NP increased in TOS quantity in SH-SY5Y cells (p < 0.05). TAS value of control, Quercetin and Quercetin-loaded NP was measured as 0.666 ± 0.018 μg/mL, 0.647 ± 0.016 μg/ mL, and 0.682 ± 0.011 μg/mL, respectively. Additionally, Fig. 4 At a dosage of 2 µg/mL, Quercetin and Quercetin-loaded NP increased apoptosis in SH-SY5Y cells. A ELISA kits were used to calculate the BAX quantity. ***p < 0.01 when compared to the control groups. B At a dosage of 2 µg/mL, Quercetin and Quercetinloaded NP increased apoptosis in SH-SY5Y cells. ELISA kits were used to calculate the amounts of cleaved Caspase 3. **p < 0.05 when compared to the control groups. C BCL-2 quantities were calculated using the ELISA kits. Each results are represented as mean ± SEM. Quercetin and Quercetin-loaded NP have not potential effect on antiapoptotic BCL-2 expression Fig. 5 At a dosage of 2 µg/mL, Quercetin and Quercetin-loaded NP increased apoptosis in SH-SY5Y cells. A ELISA kits were used to calculate the amounts of cleaved PARP. The results are shown as mean ± SEM. **p < 0.05 when compared to the control groups. B At a dosage of 2 g/mL, Quercetin and Quercetin-loaded NP increased DNA damage in SH-SY5Y cells. The ELISA kit was used to calculate the 8-oxo-dG level. The results are shown as mean ± SEM, with a **p < 0.05 significance level when compared to the control groups TOS value of control, Quercetin and Quercetin-loaded NP was measured as 36.946 ± 0.281 μg/mL, 45.336 ± 0.550 μg/ mL, and 52.276 ± 0.589 μg/mL, respectively (Fig. 6).

Discussion
Drug resistance, recurrence, and metastasis have hampered neuroblastoma treatment in many developing and developed countries around the world, despite the use of sophisticated chemotherapy and radiotherapy in cancer treatment. Antioxidant, anti-inflammatory, and anti-cancer characteristics are among the bioactive qualities of quercetin. On SH-SY5Y cells, Quercetin and Quercetin-loaded NP demonstrated a concentration-dependent cytotoxic impact. After 24 h, it dramatically reduced SH-SY5Y cell reproduction in a concentration-dependent manner, with an IC 50 values of Quercetin and NP of 2.08 ± 0.03 µg/mL and 1.67 ± 0.02 µg/mL on SH-SY5Y cell line. In particular, NP containing Quercetin showed a significantly more potent cytotoxic effect on SH-SY5Y cells compared to Quercetin and the control group. In this context, the cytotoxic effects of Quercetin and Quercetin-loaded NP on healthy NIH 3T3 cells were investigated, and the results revealed that Quercetin and NP had no significant cytotoxic effects on NIH 3T3 cells.
Apoptosis is widely known to play a function in tumor cellular development and to influence the outcome of pharmacological therapies [35]. To confirm the apoptotic effect of Quercetin on SH-SY5Y cells, the levels of cleaved PARP, cleaved caspase 3, Bax, and BCL-2 were evaluated by ELISA. The onset of cell apoptosis, which is a key focus of cancer treatment [36], is one of the defense mechanisms against tumor growth and development. The BCL-2 protein family contains both pro-apoptotic and anti-apoptotic proteins, and the ratio of pro-apoptotic-to-anti-apoptotic proteins is commonly utilized to determine cell fate.
The apoptosome complex is formed when the apoptosisinducing protein Bax disrupts the mitochondrial membrane, releasing cytochrome c. Apoptosis is triggered by this complex, which activates effector caspases. BCL-2, on the other hand, preserves membrane stability, suppresses cytochrome c release, and prevents apoptosis [37].
Caspases are a family of enzymes that are critical apoptosis effectors, and their activation is a key feature of the process. PARP is also an important component of DNA repair pathways, particularly in the repair of base excisions, and its cleavage or suppression induces cell death by exploiting a DNA repair deficiency [38].
In this study 2 µg/mL Quercetin and Quercetin-loaded NP significantly increased Bax, cleaved caspase 3, and PARP protein expressions, but no potential effect on anti-apoptotic BCL-2 expression. When compared to Quercetin alone and the control group, Quercetin-loaded NP samples dramatically enhanced Bax, cleaved caspase 3, and PARP amount. To test if the cytotoxic impact of Quercetin and Quercetinloaded NP samples is connected with DNA damage, the 8-oxo-dG ELISA technique was used to investigate DNA fragmentation in SH-SY5Y cells after 24 h of Quercetin and Quercetin-loaded NP samples administration. 8-oxo-dG is a well-known DNA oxidative damage indicator [38][39][40]. The amount of 8-oxo-dG in SH-SY5Y cells was significantly increased after treatment with Quercetin and Quercetinloaded NP at 2 g/mL, suggesting that Quercetin and Quercetin-loaded NP had cytotoxic and apoptotic effects.
The majority of anti-cancer drugs act by creating oxidative stress in tumor cells, which is regarded to be the root of most macromolecular changes in the cell. The total oxidant status is one of the numerous measures used to estimate oxidative stress and is thus frequently used to evaluate the overall oxidative condition of cells. The overall antioxidant status of cells is also assessed using the total antioxidant status. We wanted to see whether Quercetin and Quercetin-loaded NP could cause cytotoxicity by increasing total oxidant status levels. Exposure to Quercetin and Quercetin-loaded NP for 24 h raised total oxidant status levels in comparison to untreated cells, but there was no significant difference in total antioxidant status levels. According to the results treatment with Quercetin and Quercetin-loaded NP increased total oxidant status while having no effect on total antioxidant status, indicating that oxidative stress was produced in the Quercetin and Quercetin-loaded NP treated SH-SY5Y cells. Furthermore, the intrinsic apoptotic pathway can be triggered by disruption to the mitochondrial membrane and an excessive rise in reactive oxygen species [40,41].

Conclusions
Quercetin and Quercetin-loaded NP significantly reduced SH-SY5Y cell reproduction in a concentration-dependent manner while generating no cytotoxicity in NIH 3T3 cells. The expression of pro-apoptotic Bax, cleaved caspase 3, and cleaved PARP proteins was significantly increased by Quercetin and Quercetin-loaded NP. In SH-SY5Y cells, Quercetin and Quercetin-loaded NP dramatically increased 8-oxo-dG levels, suggesting that the cytotoxic action of Quercetin and Quercetin-loaded NP is linked to DNA damage. The cytotoxic effects of Quercetin and Quercetin-loaded NP were supported by an increase in TOS. Because of its encouraging results in cancer cell suppression and lack of toxicity in healthy cells, Quercetin and Quercetin-loaded NP have the potential to be a new and effective anti-cancer approach.