Characterization, biological activity, and anticancer effect of green-synthesized gold nanoparticles using Nasturtium officinale L.

BACKGROUND: Nanostructured materials used have unique properties and many uses in nanotechnology. The most striking of these is using herbal compounds for the green synthesis of nanoparticles. Among the nanoparticle types used for green synthesis, gold nanoparticles (AuNPs) are used for cancer therapy due to their stable structure and non-cytotoxic. Lung cancer is the most common and most dangerous cancer worldwide in terms of survival and prognosis. In this study, Nasturtium officinale (L.) extract (NO), which contains biomolecules with antioxidant and anticancer effects, was used to biosynthesize AuNPs, and after their characterization, the effect of the green-synthesized AuNPs against lung cancer was evaluated in vitro. METHODS: Ultraviolet‒visible (UV‒Vis) spectrophotometry, scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDS), multiple analysis platform (MAP), and Fourier transform infrared (FT-IR) spectroscopy analyses were performed to characterize the AuNPs prepared from the N. officinale plant extract. Moreover, the antioxidant activity, total phenolic and flavonoid contents and DNA interactions were examined. Additionally, A549 lung cancer cells were treated with 2-48 µg/mL Nasturtium officinale gold nanoparticles (NOAuNPs) for 24 and 48 h to determine the effects on cell viability. The toxicity of the synthesized NOAuNPs to lung cancer cells was determined by the 3-(4,5-dimethylthiazol-2-il)-2,5-diphenyltetrazolium bromide (MTT) assay, and the anticancer effect of the NOAuNPs was evaluated by apoptosis and cell cycle analyses using flow cytometry. RESULTS: The average size of the NPs was 56.4 nm. The intensities of the Au peaks from EDS analysis indicated that the AuNPs were synthesized successfully. Moreover, the in vitro antioxidant activities of the NO and NOAuNPs were evaluated; these materials gave values of 31.78 ± 1.71% and 31.62 ± 0.46%, respectively, in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay at 200 g/mL and values of 25.89 ± 1.90% and 33.81 ± 0.62%, respectively, in the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. The NO and NOAuNPs gave values of 0.389 ± 0.027 and 0.308 ± 0.005, respectively, in the ferrous ion reducing antioxidant capacity (FRAP) assay and values of 0.078 ± 0.009 and 0.172 ± 0.027, respectively, in the copper ion reducing antioxidant capacity (CUPRAC) assay. When the DNA cleavage activities of NO and the NOAuNPs were evaluated via hydrolysis, both samples cleaved DNA starting at a concentration of 25 g/mL in the cell culture analysis, while the nanoformulation of the NO components gave greater therapeutic and anticancer effects. We determined that the Au nanoparticles were not toxic to A549 cells. Moreover, after treatment with the half-maximal inhibitory concentration (IC50), determined by the MTT assay with A549 cells, we found that at 24 and 48 h, while the necrosis rates were high in cells treated with NO, the rates of apoptosis were greater in cells treated with NOAuNPs. Notably, for anticancer treatment, activating apoptotic pathways that do not cause inflammation is preferred. We believe that these results will pave the way for the use of NOAuNPs in in vitro studies of other types of cancer. CONCLUSION: In this study, AuNPs were successfully synthesized from N. officinale extract. The biosynthesized AuNPs exhibited toxicity to and apoptotic effects on A549 lung cancer cells. Based on these findings, we suggest that green-synthesized AuNPs are promising new therapeutic agents for lung cancer treatment. However, since this was an in vitro study, further research should be performed in in vivo lung cancer models to support our findings and to explain the mechanism of action at the molecular level.

Chitosan Nanoparticles Loaded with Quercetin and Valproic Acid: A Novel Approach for Enhancing Antioxidant Activity against Oxidative Stress in the SH-SY5Y Human Neuroblastoma Cell Line.

BACKGROUND: Multiple drug-delivery systems obtained by loading nanoparticles (NPs) with different drugs that have different physicochemical properties present a promising strategy to achieve synergistic effects between drugs or overcome undesired effects. This study aims to develop a new NP by loading quercetin (Que) and valproic acid (VPA) into chitosan. In this context, our study investigated the antioxidant activities of chitosan NPs loaded with single and dual drugs containing Que against oxidative stress. METHOD: The synthesis of chitosan NPs loaded with a single (Que or VPA) and dual drug (Que and VPA), the characterization of the NPs, the conducting of in vitro antioxidant activity studies, and the analysis of the cytotoxicity and antioxidant activity of the NPs in human neuroblastoma SH-SY5Y cell lines were performed. RESULT: The NP applications that protected cell viability to the greatest extent against H2O2-induced cell damage were, in order, 96 µg/mL of Que-loaded chitosan NP (77.30%, 48 h), 2 µg/mL of VPA-loaded chitosan NP (70.06%, 24 h), 96 µg/mL of blank chitosan NP (68.31%, 48 h), and 2 µg/mL of Que- and VPA-loaded chitosan NP (66.03%, 24 h). CONCLUSION: Our study establishes a successful paradigm for developing drug-loaded NPs with a uniform and homogeneous distribution of drugs into NPs. Chitosan NPs loaded with both single and dual drugs possessing antioxidant activity were successfully developed. The capability of chitosan NPs developed at the nanometer scale to sustain cell viability in SH-SY5Y cell lines implies the potential of intranasal administration of chitosan NPs for future studies, offering protective effects in central nervous system diseases.

Determination of High-Resolution HLA-DQB1 Suballeles and IL-17 Polymorphisms in Turkish Pediatric Patients.

Celiac disease (CD) is an autoimmune enteropathy in the small intestine caused by gluten intolerance of the patients. The most important genetic disease-related factor is human leukocyte antigen (HLA)-DQ polymorphism. Association between interleukin (IL)-17A expression of CD4 + T cells and various autoimmune diseases has been reported. The aim of this study was to investigate the relationship between single nucleotide polymorphism (rs2275913) IL-17A and HLA-DQ polymorphisms in Turkish pediatric celiac patients. Study group included 125 pediatric celiac patients with CD and 100 healthy pediatric controls. Deoxyribonucleic acid was isolated from peripheral blood samples. IL-17A polymorphism (rs2275913) was analyzed by polymerase chain reaction-restriction fragment polymorphism method. IL-17A polymorphism and low-/high-resolution HLA-DQ results of patients were evaluated. GG and GA genotype frequencies of IL-17A (rs2275913) polymorphism were significantly higher ( p < 0.05) in the CD patients than the control group. HLA-DQB1*02 and HLA-DQA1*05 alleles were detected in patients, while HLA-DQB1*03 and HLA-DQA1*01 alleles in the control group. Also, when we compared the patient and control groups in terms of HLA-DQ-DR haplotypes, HLA-DQB1*02-DQA1*05-DRB1*03 was found with the relative risk of 42.5 ( p < 0.05). As a result of high-resolution HLA-DQB1 typing, DQB1*02:01 and DQB1*03:02 were at high frequency ( p < 0.05; in 25 patient group). IL-17A (rs2275913) polymorphism genotype frequency was found to be significant in the patient group compared with the control group. The most common HLA-DQB1 suballele was observed as DQB1*02:01.

The changes in the expression levels of ß-catenin gene in pre- and post- Kidney Transplants.

PURPOSE: Wnt signaling is an important pathway in kidney development and disease. We aimed to establish the levels of ß-catenin expression in CD4+ T cells before and after renal transplantation and to associate it with the form of transplant type, rejection, and graft dysfunction. METHODS: CD4+ T cells were isolated from patients before and after kidney transplantation and their purity was confirmed by flow cytometer. RNA isolation and cDNA synthesis were carried out from these cells. The expression changes of the ß-catenin were investigated by real-time polymerase chain reaction (RT-PCR). Changes in the ß-catenin protein levels were determined by the western blot analysis. RESULTS: The increasing expression levels of ß-catenin were detected in 60.8% of the patients 6 months after transplantation when compared to patients before transplantation result. 12 of these 14 patients had no graft rejection. It was observed that 11 of 14 patients with increased ß-catenin expression had not graft dysfunction after the transplantation. CONCLUSION: According to our results, the increased levels of ß-catenin expression after transplantation may have a protective function for kidney survival. To understand this protective mechanism, further analysis of this signaling pathway is necessary.

Consideration of IL-2, IFN-γ and IL-4 expression and methylation levels in CD4+ T cells as a predictor of rejection in kidney transplant.

Kidney transplantation is the certain treatment for the end-stage-kidney disease patients. However after transplantation, allograft rejection or graft dysfunction are serious problems which the patients can be encountered. In several studies new biomarkers to predict rejection episodes tried to be evaluated and cytokines are thought to be one of these biomarkers. Additionally, epigenetic regulation of the cytokine genes can be an opportunity to detect the graft survival or dysfunction that lead to rejection. In this study, we aimed to detect the expression levels and methylation profile of cytokines IL-2, IL-4 and IFN-γ to follow the clinical situation of the patients. 25 kidney transplant patients were included in our study group and peripheral blood samples were collected before and 6 months after transplantation. CD4+ T cells were separated by using magnetic separation system and expression levels are detected by qPCR while methylation profile analysis was performed by pyrosequencing. According to our study we noticed that all of the patients with allograft rejection have increased expression levels of IFN-γ. When methylation profile of the CpGs in the promotor region of IFN-γ is evaluated, +128CpG was found as methylated when compared with +122. In conclusion, epigenetic mechanisms can effect several processed in renal transplantation and further studies with higher numbers of patients are needed to detect new biomarkers for prediction of allograft rejection.

Impact of Subclinical Rejection on Kidney Graft Function.

BACKGROUND: In kidney transplant recipients with borderline infiltration, protocol biopsy results demonstrated the relationship with chronic injury. The purpose of this study was to evaluate the effect of subclinical rejection (SCR) on 6-month protocol biopsy results in long-term renal function in renal transplant recipients with stable graft function. MATERIAL AND METHODS: Transplant protocol biopsies performed in 45 patients with stable renal function were included in this study at 6 months. Biopsy specimens were evaluated for SCR. Study groups were divided into patients with and without SCR. Renal functions were compared with pathologic evaluation. The effect of immunosuppressive regimens on renal function were evaluated in patients with SCR RESULT: The median age of patients was 32 years (range, 18-64 years). The median follow-up was 56 months (range, 24-84 months). According to the 6-month protocol biopsy results, 20 of 45 patients (44.4%) met SCR criteria based on Banff 07 parameters. There was not a statistically significant difference in renal function with SCR. CONCLUSION: The presence of SCR on the 6-month protocol biopsy results in renal transplant recipients with a stable graft function does not cause deterioration in the long-term graft function.

Long-Term Effects of Antibodies against Human Leukocyte Antigens Detected by Flow Cytometry in the First Year after Renal Transplantation.

OBJECTIVE: In this study, we aimed to investigate the incidence, dynamics and profiles of human leukocyte antigen (HLA)-directed antibodies developed after transplantation and their impact on graft rejection and outcome in kidney recipients. STUDY DESIGN: Prospective follow-up study. MATERIAL AND METHODS: A total of 56 kidney recipients were monitored at 1(st), 6(th) and 12(th) months for the development of anti-HLA antibodies using bead based flow-cytometry assays (Flow PRA tests). RESULTS: In 21 (37.5%) patients, panel reactive antibodies (PRA) was positive after transplantation, however, in 35 (62.5%) patients PRA was found negative. Twelve (57.1%) patients with post-transplantation HLA-reactive antibodies [PRA (+)] and 8 (22.9%) patients with no detectable alloantibodies [PRA (-)] were developed allograft rejection (p=0.010). In the PRA positive patient group the rates of early period infection and delayed graft function (DGF) were higher than the PRA negative patient group. Serum creatinine levels of PRA positive group at 6. and 12. months after transplantation were significantly higher than the PRA negative group (p=0.015 and p=0.048, respectively). The rejection rates of patients who had class I and II HLA antibodies were significantly higher than the patients who had either class I or II HLA antibodies (p=0.011). Acute rejection rates were significantly higher in patients who had class I and II HLA antibodies at the first month (p=0.007). CONCLUSION: Higher occurrence of rejection episodes in PRA positive group may show the importance of anti-HLA antibody monitoring using Flow-PRA after renal transplantation as a prognostic marker in terms of graft survival.

Lipid parameters, doses and blood levels of calcineurin inhibitors in renal transplant patients.

The calcineurin inhibitors (CNIs) [cyclosporin A (CsA) and tacrolimus (Tac)] are currently the most widely prescribed drugs for maintenance of immunosuppression after renal transplantation. These immunosuppressants are associated with side effects such as hyperlipidemia. We evaluated the differential effects of different CNIs on serum lipid parameters in renal transplant patients. Moreover, the aim of this study is to investigate the relationships between doses and blood levels of CNIs, and blood levels of CNIs and lipid parameters retrospectively. Two groups of 98 non-diabetic renal transplant patients, each treated with different CNIs, were studied: group A (n = 50, mean age: 31 ± 10 years), CsA, mycophenolate mofetil/azathioprin, steroid; group B (I = 48, mean age: 34 ± 12 years), Tac, mycophenolate mofetil/azathioprin, steroid. In renal transplant patients, CNIs blood levels and doses were examined at 1, 3, 6, 9, and 12 months after transplantation. Biochemical laboratory parameters including plasma lipids [total-cholesterol (CHOL), low-density lipoprotein (LDL)-CHOL, high-density lipoprotein (HDL)-CHOL, and triglycerides (TG)], CNI levels and doses were examined at 1, 3, 6, 9, and 12 months after transplantation. None of the patients received anti-lipidemic drugs during the study period. Blood levels of CNIs were detectable in all whole-blood samples by Cloned- Enzyme-Donor Immunoassay (CEDIA). The relationship between CNIs blood levels and CHOL, (LDL)-CHOL, HDL-CHOL, TG were evaluated. The mean serum CHOL levels and LDL-CHOL levels of patients in group A were found significantly higher than the patients in group B during the 12 month of follow up (p < 0.05). There was no significant difference in TG and HDL-CHOL plasma levels between group A and group B (p > 0.005). In group A the daily dose of CsA was significantly correlated with the mean blood levels of CsA at the 1st and 3rd months (r = 0.387, p = 0.005; r = 0.386, p = 0.006), respectively. In group A, the daily dose of CsA was significantly correlated with the mean serum TG levels during the 12 month of follow up (r = 0.420, p = 0.003). In group B, the daily dose of Tac was significantly correlated with the mean blood level of Tac (r = 0.335, p = 0.020) at the 1st month. No correlation was found between mean Tac blood levels and lipid parameters during the 12-month of follow up (p > 0.05). Significant positive correlation was observed between the CsA blood levels and LDL-CHOL levels (r = 0.338, p = 0.027) at the 3rd month. In the renal transplant patients with well functioning grafts, CsA therapy is associated with increased CHOL and LDL-CHOL ratio which represents an increased atherogenic risk tended to be associated with CsA. Serum LDL-CHOL levels may be effected by blood CsA levels.