The immunosuppressive tumor microenvironment (TME) limits the efficacy of chimeric antigen receptor (CAR) T cells in solid tumors by inducing T cell exhaustion through inhibitory receptors, such as PD1 and TIM3. T memory stem cells (TSCMs) offer superior persistence, and IL15 promotes T cell memory. We engineered MSLN-CAR-T cells with a PD1/IL15Rβ switch receptor to convert PD1/PDL1 inhibitory signals into IL15-mediated STAT5 activation, enhancing T cell function. We developed MSLN-PD1/IL15Rβ-CAR-T cells, incorporating a PD1/IL15Rβ switch receptor, and evaluated their antitumor activity against pancreatic (AsPC-1, PANC-1) and cervical (HeLa) cancer cell lines. Proliferation, cytokine production (IL-2, IFN-γ), exhaustion markers (PD1, TIM3), and memory T cell phenotypes (CD45RO+/CCR7+) were assessed using flow cytometry, ELISA, and western blotting, with or without anti-PD1 antibody (Nivolumab) stimulation. MSLN-PD1/IL15Rβ-CAR-T cells exhibited enhanced STAT5 phosphorylation, significantly increased proliferation, and elevated IL-2 and IFN-γ secretion compared to MSLN-CAR-T cells when co-cultured with mesothelin- and PDL1-positive tumor cells or treated with Nivolumab. These cells exhibited reduced PD1 and TIM3 expression, along with a higher proportion of CD45RO+/CCR7 + memory T cells, suggesting decreased exhaustion and enhanced persistence. The PD1/IL15Rβ switch receptor overcomes PDL1-mediated immunosuppression in MSLN-CAR-T cells by activating STAT5 signaling, improving proliferation, cytokine production, and memory T cell formation while reducing exhaustion. This approach holds promise for enhancing CAR-T cell therapy in mesothelin-expressing solid tumors.

Purpose Cancer testis antigens (CTAs) are a family of proteins typically expressed in male testicles but overexpressed in various cancer cell types. Transmembrane Phosphatase with Tensin homology (TPTE) is expressed only in the testis of healthy individuals and is a member of the family of CTAs. The current study, for the first time, examined the significance of TPTE expression in prostate cancer (PCa) tissues by generating a novel antibody marker targeting TPTE protein. Methods Polyclonal antibodies were prepared for TPTE-p1 and TPTE-p2 peptides, which are derived from the extracellular domains of TPTE. Anti-TPTE-p2 antibody was then used to study the extent and pattern of TPTE expression in 102 PCa and 48 benign prostatic hyperplasia (BPH) tissue samples by immunohistochemistry. The viability of cancer cell lines (PC-3 and MCF-7 cells) was also evaluated in the presence of anti-TPTE-p2 antibody using the MTT test. Results The immunohistochemical analysis demonstrated a significant increase in cytoplasmic and membrane TPTE expression in the PCa samples compared to the BPH group (both P < 0.0001). Cytoplasmic TPTE expression was positively correlated with Gleason score and PSA levels (P = 0.03 and P = 0.001, respectively). Significant correlations were identified between the levels of PSA and perineural invasion and the membrane expression (P = 0.01, P = 0.04, respectively). Moreover, anti-TPTE-p2 antibody inhibited PC-3 and MCF-7 cells proliferation compared to the control group for 24 h (P < 0.001 and P = 0.001, respectively) as well as for 48 h (P = 0.001 and P = 0.001, respectively). Conclusion Our findings indicate that increased TPTE expression is associated with progression of disease. The ability of anti-TPTE-p2 antibody to recognize and target the TPTE protein makes it a potential biomarker to assess and/or target the PCa.

Abstract Background Dendritic cells (DCs) play a crucial role in immunity. Research on monocyte‐derived DCs (Mo‐DCs) cancer vaccines is in progress despite limited success in clinical trials. This study focuses on Mo‐DCs generated from prostate cancer (PCA) patients, comparing them with DCs from healthy donors (HD‐DCs). Methods Mo‐DCs were isolated from PCA patient samples, and their phenotype was compared to HD‐DCs. Key parameters included monocyte count, CD14 expression, and the levels of maturation markers (HLA‐DR, CD80, CD86) were assessed. Results PCA samples exhibited a significantly lower monocyte count and reduced CD14 expression compared to healthy samples (p ⟨ 0.0001). Additionally, PCA‐DCs expressed significantly lower levels of maturation markers, including HLA‐DR, CD80, and CD86, when compared to HD‐DCs (p = 0.123, p = 0.884, and p = 0.309, respectively). Conclusion The limited success of DC vaccines could be attributed to impaired phenotypic characteristics. These observations suggest that suboptimal characteristics of Mo‐DCs generated from cancer patient blood samples might contribute to the limited success of DC vaccines. Consequently, this study underscores the need for alternative strategies to enhance the features of Mo‐DCs for more effective cancer immunotherapies.

Recent advancements in synchronized sensor technologies has introduced an unprecedented level of visibility in power distribution systems. Apart from the Distribution-level Phasor Measurement Units (D-PMUs), a.k.a. micro-PMUs, that have been widely used in recent years, other synchronized sensors have also been developed in this field, including Harmonic Phasor Measurement Units (H-PMUs) and Waveform Measurement Units (WMUs). However, in practice, it is common for these sensors to lose time synchronization over some periods of time and for different reasons. In this paper, we propose new and customized solutions to tackle loss of time synchronization in D-PMUs, H-PMUs, and WMUs, whereby addressing the unique challenges in each case. Our focus is on solving the event location identification problem, for different types of steady-state and transient events. We show that, our methods can maintain high accuracy in event location identification, despite losing time synchronization, whether we use D-PMUs, H-PMUs, or WMUs.

Purpose:L-asparaginase has been widely recognized as a critical component in the treatment of various types of lymphoproliferative disorders, since its introduction in 1960s. However, its use in some cases leads to allergic reactions rendering the continuation of treatment unfeasible. Thus, the development of L-asparaginase from alternative sources or the production of engineered enzymes have always been considered. This study aimed to produce and evaluate a novel enzyme designed based on the sequence of L-asparaginase from Escherichia coli bacteria with Y176F/S241C mutations. Methods:The Y176F/S241C mutant L-asparaginase was successfully expressed as the GST-fusion protein in E. coli, and then was subjected to affinity and size exclusion chromatography. The activity of the purified enzyme was determined based on the released ammonia as the result of substrate hydrolysis using Nessler's reagent. Chemical denaturation experiment in the presence of increasing concentration of guanidinium chloride was applied to determine the folding stability of the purified enzyme. Results:The mutant enzyme was purified with an efficiency of 77-fold but at a low recovery of 0.7%. The determined kinetic parameters Km, Vmax, kcat, specific activity and catalytic efficiency were 13.96 (mM), 2.218 (mM/min), 273.9 (min-1), 237.8 (IU/mg) and 19.62 (mM-1 min-1), respectively. Moreover, unfolding free energy determined by guanidinium chloride induced denaturation for mutated and commercial L-asparaginase enzymes were 8421 J/mol and 5274 J/mol, respectively. Conclusion:The mutant enzyme showed improved stability over the wild-type. Although the expression level and recovery were low, the mutant L-asparaginase demonstrated promising activity and stability, with potential clinical and industrial applications.

Background: More than 3 y into the coronavirus 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to undergo mutations. In this context, the Receptor Binding Domain (RBD) is the most antigenic region among the SARS-CoV-2 Spike protein and has emerged as a promising candidate for immunological development. We designed an IgG-based indirect enzyme-linked immunoassay (ELISA) kit based on recombinant RBD, which was produced from the laboratory to 10 L industry scales in Pichia pastoris.Methods: A recombinant-RBD comprising 283 residues (31 kDa) was constructed after epitope analyses. The target gene was initially cloned into an Escherichia coli TOP10 genotype and transformed into Pichia pastoris CBS7435 muts for protein production. Production was scaled up in a 10 L fermenter after a 1 L shake-flask cultivation. The product was ultrafiltered and purified using ion-exchange chromatography. IgG-positive human sera for SARS-CoV-2 were employed by an ELISA test to evaluate the antigenicity and specific binding of the produced protein.Results: Bioreactor cultivation yielded 4 g/l of the target protein after 160 h of fermentation, and ion-exchange chromatography indicated a purity > 95%. A human serum ELISA test was performed in 4 parts, and the ROC area under the curve (AUC) was > 0.96 for each part. The mean specificity and sensitivity of each part was 100% and 91.5%, respectively.Conclusion: A highly specific and sensitive IgG-based serologic kit was developed for improved diagnostic pur-poses in patients with COVID-19 after generating an RBD antigen in Pichia pastoris at laboratory and 10 L fermentation scales.

BACKGROUND:Prostate cancer is one of the most widespread cancers in the world. Early diagnosis is the most important factor in treatment efficiency. Furthermore, new methods for early diagnosis and treatment play an important role. In this study, we designed targeted conjugation of antibodies with iron nanoparticles and evaluated the binding properties of antibodies to prostate cancers and benign tissues. This method in addition to having a lower cost has high sensitivity and specificity.METHODS:Anti- PSCA antibodies were purified and conjugated to super magnetic oxide nanoparticles (SPION). Then, iron staining on prostate adenocarcinoma tissues was performed. At the same time, immunohistochemically staining was performed on similar tissues to compare the results. In addition, benign prostatic hyperplasia (BPH) samples were used as a control sample.RESULTS:In adenocarcinoma tissues with iron staining, many blue spots are seen compared to benign tissues, and the number of these spots increases with increasing tumor grade.CONCLUSION:These findings indicate the characteristic of iron staining as a conjugate antibody to iron can be an appropriate approach to specific staining of tumor markers in cancer tissues and can be used to diagnose prostate cancer due to its safety, low cost, sensitivity, and specificity.

Purpose:Medical usage of L-asparaginase (ASNase), the first-line of acute lymphoblastic leukemia treatment, is linked to allergic responses and toxicities, which necessitates the development of new bio-better ASNases. The aim of the current study was in silico design of a novel ASNase with predicted improved enzymatic properties using strategies encompassing sequence-function analysis of known ASNase mutants. Additionally, current study aimed to show that the new enzyme is active.Methods:Based on 21 experimentally reported mutations for ASNase, a virtual library of mutated enzymes with all 7546 possible combinations of up to 4 mutations was generated. Three-dimensional models of proposed mutant enzymes were built and their in silico stabilities were calculated. The most promising mutant was selected for preparing a genetic construct suitable for expression of the designed ASNase in bacterial cells.Results:Computational study predicted that Y176F/S241C double mutation of Escherichia coli ASNase may increase its folding stability. The designed ASNase was expressed in two different E. coli strains (Origami B(DE3) and BL21(DE3)pLysS) and then the soluble fractions prepared from the cell lysates of the host cells were used in enzyme activity assay. Results showed that enzyme activity of soluble fraction from Origami (95.4 ± 7.5 IU/0.1 mL) was four times higher than that of soluble fraction from pLysS (25.8 ± 2.5 IU/0.1 mL).Conclusion:A novel functional double mutant ASNase with predicted improved enzymatic properties was designed and produced in E. coli. The results of the current study suggest a great commercial potential for the identified enzyme in pharmaceutical and industrial applications.

Background Prostate cancer (PCa) is the second leading cause of cancer-related deaths among men worldwide. Immunotherapy is an emerging treatment modality for cancers that harnesses the immune system’s ability to eliminate tumor cells. In particular, dendritic cell (DC) vaccines, have demonstrated promise in eliciting a tumor-specific immune response. In this study, we investigated the potential of using DCs loaded with the MAGE-A2 long peptide to activate T cell cytotoxicity toward PCa cell lines. Methods Here, we generated DCs from monocytes and thoroughly characterized their phenotypic and functional properties. Then, DCs were pulsed with MAGE-A2 long peptide (LP) as an antigen source, and monitored for their transition from immature to mature DCs by assessing the expression levels of several costimulatory and maturation molecules like CD14, HLA-DR, CD40, CD11c, CD80, CD83, CD86, and CCR7. Furthermore, the ability of MAGE-A2 -LP pulsed DCs to stimulate T cell proliferation in a mixed lymphocyte reaction (MLR) setting and induction of cytotoxic T cells (CTLs) in coculture with autologous T cells were examined. Finally, CTLs were evaluated for their capacity to produce interferon-gamma (IFN-γ) and kill PCa cell lines (PC3 and LNCaP). Results The results demonstrated that the antigen-pulsed DCs exhibited a strong ability to stimulate the expansion of T cells. Moreover, the induced CTLs displayed substantial cytotoxicity against the target cells and exhibited increased IFN-γ production during activation compared to the controls. Conclusions Overall, this innovative approach proved efficacious in targeting PCa cell lines, showcasing its potential as a foundation for the development and improved PCa cancer immunotherapy.

Prior studies have shown that most phasor measurement units (PMUs) in practice suffer from some level of time synchronization loss at least once every day. We address this issue in the context of distribution-level PMUs, i.e., micro-PMUs, and with focus on the application of micro-PMUs in event location identification in situational awareness. We show that a state-of-the-art method that is highly successful in identifying the correct event location when the micro-PMUs are synchronized, fails when time synchronization is lost among the micro-PMUs. An alternative method is proposed to identify the location of events not only when the micro-PMUs are time synchronized but also when micro-PMUs lose time synchronization. The proposed model works for different scenarios for losing time synchronization among some or all micro-PMUs.

Abstract With more than two years in COVID-19 pandemic, the underlying virus is mutating which indicates it is not over yet hence SARS-CoV-2 Spike RBD is a potent candidate for immunological development purposes. Here, we produced a recombinant-RBD protein from micro- to macro-scale production by Pichia pastoris with high purity that was assessed by immunological tests. A recombinant-RBD compromising 283 residues (31kDa) was constructed after epitope analyses. The target gene was initially cloned into Escherichia coli Top10 genotype and transformed into Pichia pastoris CBS7435 muts for protein production. The production was scaled-up in a 10L fermenter after 1L shake-flask cultivation. The product was ultrafiltered and purified using ION-Exchange chromatography. IgG-positive human sera for SARS-CoV-2 were employed by ELISA test to evaluate the antigenicity and specific binding of the produced protein. Bioreactor cultivation yielded 4g/L of target protein after 160 hours fermentation, and ion-exchange chromatography indicated a purity of > 95%. Human serum ELISA test performed in four parts (1–4) and ROC curve area under curve (AUC) was > 0.96 for each part. The mean specificity and sensitivity of each part was (100, 91.5) respectively. In conclusion, the recombinant-RBD can be used for IgG-based serologic kit and preventive purposes for patients with COVID-19 infection.

Immunotoxins have represented a great potency in targeted therapeutics to encounter tumors. They consist of a protein toxin conjugated to a targeting moiety, which recognizes a specific antigen on surface of cancer cells and accordingly induces cell death by toxin segment. The targeting part could be a nanobody, which is a group of antibodies composed of an only functional single variable heavy chain (VHH).Therefore, this study was done to produce an immunotoxin (VGRNb-DT) by chemical conjugation of a truncated diphtheria toxin moiety to an anti-vascular endothelial growth factor receptor 2(VEGFR-2) nanobody, and to identify effectiveness of immunotoxin in recognizing the VEGFR-2- positive cancer cells and inhibiting cell growth and survival. Diphtheria toxin was expressed and purified by nickel affinity chromatography, and accordingly, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis confirmed its expression. Function of heterobifunctional crosslinkers, Sulfo-SMCC (sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate), and SATP (N-succinimidyl-S- acetylthiopropionate) for bioconjugation purposes was acknowledged by cation exchange high-performance liquid chromatography (HPLC). Cytotoxicity of immunotoxin was evaluated on the VEGFR-2 positive PC-3 cell line by MTT assay. Overexpression of VEGFR-2 in the PC-3 cell line allowed immunotoxin to recognize them by anti-VEGFR-2 nanobodies. The concentrations above 5 μg/ml represented a significant decrease in cell survival rate in PC-3 cells compared to HEK293 cells (VEGFR-2 negative cells) as controls.VGRNb-DT demonstrated a successful bioconjugation; furthermore, variable concentrations were correlated with cell death in prostate cancer PC-3 cells.

Given multiple treatment strategies for prostate cancer, its mortality rate is still high; therefore, novel treatment strategies seem necessary. G2013 or α‐L‐guluronic acid is a new patented drug with immunomodulatory and anti‐inflammatory properties. This study aimed to evaluate the property of G2013 on inflammatory molecules involved in tumorigenesis of prostate cancer. MTT assay was used to assess the effect of the drug on the proliferation of PC‐3 cells. Expression of interleukin 8 (IL‐8), Nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB), myeloid differentiation factor 88 (MYD‐88), cyclooxygenase 2 (COX‐2), matrix metalloproteinase‐2 (MMP‐2), and MMP‐9 genes were studied in the PC‐3 cells treated with 25 (low dose) or 50 (high dose) µg/mL of G2013 for 24 h using quantitative real‐time polymerase chain reaction (qRT‐PCR) technique. Protein expression of NF‐κB and protein activities of MMP‐2 and MMP‐9 were assayed using flow cytometry and gelatin zymography, respectively. The expression of COX‐2 (p = 0.007 at low dose), MMP‐2 (p = 0.023 at low dose, p = 0.002 at high dose), NF‐κB (p = 0.004 at low dose) and IL‐8 (p < 0.0001 in both doses) genes, NF‐κB protein (p < 0.0001 in both doses), and MMP‐2 activity (p < 0.0001 in both doses) were significantly reduced in the presence of G2013 as compared to the control group. Cancer cell proliferation was also inhibited under 10–500 µg/mL G2013 treatment. Our results revealed that G2013 has the potential to inhibit PC‐3 cell proliferation and reduce the expression of tumour‐promoting mediators, COX‐2, MMP‐2, NF‐κB, and IL‐8 involved in the progression and metastasis of prostate cancer.

The recent advent of distribution-level phasor measurement units (D-PMUs), a.k.a., micro-PMUs, has introduced a wide range of new applications in power distribution systems. A sub-class of such emerging applications are called event-based methods. These methods focus on the analysis of events in the stream of micro-PMU measurements to achieve situational awareness, enhance load modeling, integrate distributed energy resources, etc. In this article, we explore a scenario, where a cyberattack compromises the micro-PMU measurements during an event. Such a targeted attack could be limited in scope but result in a major impact on the operation of the power grid by highly deviating the outcome of the event-based methods. First, we investigate and model two types of such attacks, event-unsynchronized (basic) attacks and event-synchronized (advanced) attacks. We then conduct a geometric analysis to understand each attack type, in a setting where the events are represented in the phasor domain in a differential mode. Next, we introduce a novel method to detect the presence of the attack and then identify which micro-PMUs are compromised so as to discard the compromised measurements as a defense mechanism. The proposed approach makes critical use of magnitude as well as phase angle measurements from micro-PMUs. The method is tested on the IEEE 33-bus power distribution test system.

Objectives: To develop a two-antigenic ELISA for combined screening for HIV-1 and HCV. High rate of HCV/HIV co-infection rate have become a global concern in recent years. Likewise, in Iran due to Injection Drug Use (IDU), the dominant transmission pattern, this rate is increasingly on the rise standing at approximately 70 percent. To reduce screening costs, an ELISA with a new solid phase system for simultaneous detection of HCV and HIV-1 infections was explored. Study Design and Methods: Sera samples from patients infected with HIV-1, HCV, and negative controls were tested. In the new ELISA, wells were primarily coated with Streptavidin overnight followed by blocking with bovine serum albumin. Then biotinylated gp 41 (HIV-1 antigens) and recombinant core and NS4 antigens (HCV antigens) were added to wells either separately or simultaneously. Then, the alkaline phosphatase (AP)-conjugated anti-human IgG secondary antibodies and para-nitro phenyl phosphate (pNPP) substrates were added to wells followed by reading ODs at 450 nm. Results: Both single and combined assays showed high diagnostic sensitivity and specificity of about 99 percent and 97 percent respectively. Conclusions: Due to differences in physicochemical properties, antigens require various coating conditions. However, by using this method, multi antigens could be coated on a well surface to obtain an efficient, inexpensive and accurate detection.

Background:: Prostate Stem Cell Antigen (PSCA) is a small cell surface protein, overexpressed in 90% of prostate cancers. Determination of epitopes that elicit an appropriate response to the antibody generation is vital for diagnostic and immunotherapeutic purposes for prostate cancer treatment. Presently, bioinformatics B-cell prediction tools can predict the location of epitopes, which is uncomplicated, faster, and more cost-effective than experimental methods. Objective:: We aimed to predict a novel linear peptide for Prostate Stem Cell Antigen (PSCA) protein in order to generate anti-PSCA-peptide (p) antibody and to investigate its effect on prostate cancer cells. Methods:: In the current study, a novel linear peptide for PSCA was predicted using in silico methods that utilize a set of linear B-cell epitope prediction tools. Polyclonal antibody (anti-PSCA-p antibody “Patent No. 99318”) against PSCA peptide was generated. The antibody reactivity was determined by the Enzyme-Linked Immunosorbent Assay (ELISA) and its specificity by immunocytochemistry (ICC), immunohistochemistry (IHC), and Western Blotting (WB) assays. The effect of the anti-PSCA-p antibody on PSCA-expressing prostate cancer cell line was assessed by Methylthiazolyldiphenyl- Tetrazolium bromide (MTT) assay. Results:: New peptide-fragment of PSCA sequence as “N-CVDDSQDYYVGKKN-C” (PSCA-p) was selected and synthesized. The anti-PSCA-p antibody against the PSCA-p showed immunoreactivity with PSCA-p specifically bound to PC-3 cells. Also, the anti-PSCA-p antibody strongly stained the prostate cancer tissues as compared to Benign Prostatic Hyperplasia (BPH) and normal tissues (P < 0.001). As the degree of malignancy increased, the staining intensity was also elevated in prostate cancer tissue (P < 0.001). Interestingly, the anti-PSCA-p antibody showed anti-proliferative effects on PC-3 cells (31%) with no growth inhibition effect on PSCA-negative cells. Conclusion:: In this study, we developed a new peptide sequence (PSCA-p) of PSCA. The PSCA-p targeting by anti-PSCA-p antibody inhibited the proliferation of prostate cancer cells, suggesting the potential of PSCA-p immunotherapy for future prostate cancer studies.

BACKGROUND:The unique expression pattern of prostate stem cell antigen (PSCA) in a number of prevalent neoplasms has made the antigen a great target for cancer researches, and many clinical methods have been developed based on the application of this tumor marker. Hence, optimal PSCA laboratory production can be considered a hallmark for many researchers.OBJECTIVE:An analytical study was designed to improve the quality and quantity of PSCA production.MATERIALS AND METHODS:The effects of different compositions of lysis buffers and some ultrasound durations were assessed by calculation of the protein recovery followed by PSCA specific blotting experiments. Then, based on the results of the web-based characterization, interference removal, followed by re-solubilization of the protein in various buffers, was designed, applied, and assessed.RESULTS:Since the selection of an appropriate methodology depends merely on the research purposes, we tried to discuss the pros and cons of the investigated methods according to the hydrophobic nature of PSCA as well as its dramatic tendency to aggregate in the form of inclusion bodies in the expression hosts.CONCLUSIONS:We introduced a newly designed method to fit the delicate immunological surveys and overcome some limiting factors in PSCA production.

Topology identification (TI) in distribution networks is a challenging task due to the limited measurement resources and therefore the inevitable need to use pseudo-measurements that are often inaccurate. To address this issue, a new method is proposed in this paper to integrate harmonic synchrophasors into the TI problem in order to enhance TI accuracy in distribution networks. In this method, topology identification is done jointly based on both fundamental synchrophasor measurements and harmonic synchrophasor measurements. This is done by formulating and then solving a mixed-integer linear programming (MILP) problem. Furthermore, an analysis is provided to capture the number of and the location of harmonic sources and sensors that are needed to ensure full observability. The benefits of the proposed TI scheme are compared against those of the traditional scheme that utilizes only the fundamental measurements. Finally, through numerical simulations on the IEEE 33-Bus power system, it is shown that the proposed scheme is considerably accurate compared to the traditional scheme in topology identification.

INTRODUCTION:This study aimed to evaluate the antioxidant property of Silymarin (SM) extracted from the seed of Silybum marianum and its anticancer activity on KB and A549 cell lines following 24, 48, and 72 h of treatment.METHODS:Ten grams of powdered S. marianum seeds were defatted using n-hexane for 6 hours and then extracted by methanol. The Silymarin extracted of extraction components. The extracted components of Silymarin were measured by spectrophotometric assay and HPLC analysis. 2, 2- diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, phenol content, total flavonoid content, and total antioxidant capacity were measured to detect the antioxidant properties of SM. The anticancer activity of the SM on cell lines evaluated by MTT.RESULTS:In HPLC analysis, more than 50% of the peaks were related to silybin A and B. SM was reduced DPPH (the stable free radical) with a 50% inhibitory concentration (IC50) of 6.56 μg/ ml in comparison with butylated hydroxyl toluene (BHT), which indicated an IC50 of ~3.9 μg/ ml. The cytotoxicity effect of SM on the cell lines was studied by MTT assay. The cytotoxicity effect of the extracted Silymarin on KB and A549 cell lines was observed up to 80 and 70% at 156 and 78 μg/ml, respectively. The IC50 value of the extracted SM on KB and A549 cell lines after 24 hours of treatment was seen at 555 and 511 μg/ml, respectively.CONCLUSION:Due to the good antioxidant and anticancer properties of the isolated Silymarin, its use as an anticancer drug is suggested.

Objective(s): Silymarin (SM) is a natural antioxidant compound with good anti-inflammatory effects, but its poor water solubility restricts its usage. Today, nanomaterial compounds (such as PLGA Poly D, L-lactic-co-glycolic acid) can provide a proper drug delivery system and help improve the accessibility of bioactive compounds to cells and tissues. Materials and Methods: In this study, PLGA nanoparticles (NPs) containing SM (SM-PLGA) were synthesized and characterized and their biological effects were evaluated on M2 macrophage polarization to regulate inflammation. SM-PLGA NPs were fabricated by the oil in water emulsion (O/W) method. Macrophages (MQs) were isolated from mouse peritoneum by the cold RPMI lavage protocol. Primary mouse MQ cells were treated by SM and SM-PLGA NPs and then stimulated with lipopolysaccharide (LPS). M2 polarization was evaluated by measurements of cytokine secretion levels (TNF-α, IL1-β, and IL-10), flow cytometry markers (F4/80, CD11b, CD38, and CD206), and the expression of specific proteins (M2 Ym1 and Fizz1).Results: SM-PLGA characterization showed that NPs were fabricated in the desired form. SM and SM-PLGA decreased pro-inflammatory cytokines (TNF-α and IL1-β) and increased IL10 as an anti-inflammatory cytokine. On the other hand, the M2-associated markers and proteins increased following treatment with SM and SM-PLGA. Post-hoc analysis indicated that these changes were more pronounced in the SM-PLGA group.Conclusion: This study revealed that SM-PLGA could markedly promote M2 polarization, thereby providing a valuable medical approach against sepsis and septic shock.