Insights into additional lactone-based signaling circuits in Streptomyces: existence of acyl-homoserine lactones and LuxI/LuxR homologs in six Streptomyces species.

Acyl-homoserine lactones (AHLs), mediating pivotal physiological activities through quorum sensing (QS), have conventionally been considered limited to Gram-negative bacteria. However, few reports on the existence of AHLs in Gram-positive bacteria have questioned this conception. Streptomyces, as Gram-positive bacteria already utilizing a lactone-based QS molecule (i.e., gamma-butyrolactones), are yet to be explored for producing AHLs, considering their metabolic capacity and physiological distinction. In this regard, our study examined the potential production of AHLs within Streptomyces by deploying HPLC-MS/MS methods, which resulted in the discovery of multiple AHL productions by S. griseus, S. lavendulae FRI-5, S. clavuligerus, S. nodosus, S. lividans, and S. coelicolor A3(2). Each of these Streptomyces species possesses a combination of AHLs of different size ranges, possibly due to their distinct properties and regulatory roles. In light of additional lactone molecules, we further confirm that AHL- and GBL-synthases (i.e., LuxI and AfsA enzyme families, respectively) and their receptors (i.e., LuxR and ArpA) are evolutionarily distinct. To this end, we searched for the components of the AHL signaling circuit, i.e., AHL synthases and receptors, in the Streptomyces genus, and we have identified multiple potential LuxI and LuxR homologs in all 2,336 Streptomyces species included in this study. The 6 Streptomyces of interest in this study also had at least 4 LuxI homologs and 97 LuxR homologs. In conclusion, AHLs and associated gene regulatory systems could be more widespread within the prokaryotic realm than previously believed, potentially contributing to the control of secondary metabolites (e.g., antibiotics) and their complex life cycle, which leads to substantial industrial and clinical applications.

A novel co-culture assay to evaluate the effects of sympathetic innervation on vascular smooth muscle differentiation.

Dedifferentiation of vascular smooth muscle cells (VSMCs) from a functional phenotype to an inverse synthetic phenotype is a symptom of cardiovascular disorders, such as atherosclerosis and hypertension. The sympathetic nervous system (SNS) is an essential regulator of the differentiation of vascular smooth muscle cells (VSMCs). In addition, numerous studies suggest that SNS also stimulates VSMCs to retain their contractile phenotype. However, the molecular mechanisms for this stimulation have not been thoroughly studied. In this study, we used a novel in vitro co-culture method to evaluate the effective cellular interactions and stimulatory effects of sympathetic neurons on the differentiation of VSMCs. We co-cultured rat neural-like pheochromocytoma cells (PC12) and rat aortic VSMCs with this method. Expression of VSMCs contractile genes, including smooth muscle actin (acta2), myosin heavy chain (myh11), elastin (eln), and smoothelin (smtn), were determined by quantitative real-time-PCR analysis as an indicator of VSMCs differentiation. Fold changes for specific contractile genes in VSMCs grown in vitro for seven days in the presence (innervated) and absence (non-innervated) of sympathetic neurons were 3.5 for acta2, 6.5 for myh11, 4.19 for eln, and 4 for smtn (normalized to Tata Binding Protein (TBP)). As a result, these data suggest that sympathetic innervation promotes VSMCs' contractile gene expression and also maintains VSMCs' functional phenotype.

The effect of Nano-liposomal sodium nitrite on smooth muscle cell growth in a tissue-engineered small-diameter vascular graft.

BACKGROUND: Nitric oxide is a chemical agent produced by endothelial cells in a healthy blood vessel, inhibiting the overgrowth of vascular smooth muscle cells and regulating vessel tone. Liposomes are biocompatible and biodegradable drug carriers with a similar structure to cell bilayer phospholipid membrane that can be used as useful nitric oxide carriers in vascular grafts. METHOD: Using a custom-designed apparatus, the sheep carotid arteries were decellularized while still maintaining important components of the vascular extracellular matrix (ECM), allowing them to be used as small-diameter vascular grafts. A chemical signal of sodium nitrite was applied to control smooth muscle cells' behavior under static and dynamic cell culture conditions. The thin film hydration approach was used to create nano-liposomes, which were then used as sodium nitrite carriers to control the drug release rate and enhance the amount of drug loaded into the liposomes. RESULTS: The ratio of 80:20:2 for DPPC: Cholesterol: PEG was determined as the optimum formulation of the liposome structure with high drug encapsulation efficiency (98%) and optimum drug release rate (the drug release rate was 40%, 65%, and 83% after 24, 48, and 72 h, respectively). MTT assay results showed an improvement in endothelial cell proliferation in the presence of nano-liposomal sodium nitrite (LNS) at the concentration of 0.5 µg/mL. Using a suitable concentration of liposomal sodium nitrite (0.5 µg/mL) put onto the constructed scaffold resulted in the controllable development of smooth muscle cells in the experiment. The culture of smooth muscle cells in a pulsatile perfusion bioreactor indicated that in the presence of synthesized liposomal sodium nitrite, the overgrowth of smooth muscle cells was inhibited in dynamic cell culture conditions. The mechanical properties of ECM graft were measured, and a multi-scale model with an accuracy of 83% was proposed to predict mechanical properties successfully. CONCLUSION: The liposomal drug-loaded small-diameter vascular graft can prevent the overgrowth of SMCs and the formation of intimal hyperplasia in the graft. Aside from that, the effect of LNS on endothelial has the potential to stimulate endothelial cell proliferation and re-endothelialization.

Fungal Infected Adipose Stem Cells: The Effects of Novel Lipo-Niosome Nanoparticles Loaded with Amphotericin B and Thymus Essential Oil.

OBJECTIVE: In this study, we aimed to develop new Lipo-niosomes based nanoparticles loaded with Amphotericin B (AmB) and Thymus Essential Oil (TEO) and test their effectiveness in the treatment of fungal-infected human adipose stem cells (hASCs). MATERIALS AND METHODS: In this experimental study, optimal formulation of AmB and TEO loaded lipo-niosome (based on lipid-surfactant thin-film hydration method) was chemically, and biologically characterized. Therefore, encapsulation capacity, drug release, size, and the survival rate of cells with different concentrations of free and encapsulated AmB/ TEO were evaluated using the MTT method, and its antifungal activity was compared with conventional AmB. RESULTS: Lipo-Niosome containing Tween 60 surfactant: cholesterol: Dipalmitoyl phosphatidylcholine (DPPC): Polyethylene glycol (PEG) with a ratio of 20:40:60:3 were chosen as optimal formulation. Lipo-Niosomes entrapment efficiency was 94.15%. The drug release rate after 24 hours was 52%, 54%, and 48% for Lipo-AmB, Lipo-TEO, and Lipo-AmB/TEO, respectively. Physical and chemical characteristics of the Lipo-Niosomes particles indicated size of 200 nm and a dispersion index of 0.32 with a Zeta potential of -24.56 mv. Furthermore, no chemical interaction between drugs and nano-carriers was observed. The cell viability of adipose mesenchymal stem cells exposed to 50 µg/ml of free AmB, free TEO, and free AmB/TEO was 13.4, 58, and 36.9%, respectively. Whereas the toxicity of the encapsulated formulas of these drugs was 48.9, 70.8, and 58.3% respectively. The toxicity of nanoparticles was very low (8.5%) at this concentration. Fluorescence microscopic images showed that the antifungal activity of Lipo-AmB/ TEO was significantly higher than free formulas of AmB, TEO, and AmB/TEO. CONCLUSION: In this study, we investigated the efficacy of the TEO/AmB combination, in both free and encapsulatedniosomal form, on the growth of fungal infected-hASCs. The results showed that the AmB/TEO-loaded Lipo-Niosomes can be suggested as a new efficient anti-fungal nano-system for patients treated with hASCs.

Osteogenic and Angiogenic Synergy of Human Adipose Stem Cells and Human Umbilical Vein Endothelial Cells Cocultured in a Modified Perfusion Bioreactor.

Synergistic promotion of angiogenesis and osteogenesis in bone tissue-engineered constructs remains a crucial clinical challenge, which might be overcome by simultaneous employment of superior techniques including coculture systems, differentiation-stimulated factors, combinatorial scaffolds and bioreactors.Current study investigated the effect of flow perfusion along with coculture of human adipose stem cells (hASCs) and human umbilical vein endothelial cells (HUVECs) on osteogenic and angiogenic differentiation.Pre-treated hASCs with 1,25-dihydroxyvitamin D3 were seeded onto poly(lactic-co-glycolic acid)/ß-tricalcium phosphate/polycaprolactone (PLGA/ß-TCP/PCL) scaffold with/without HUVECs, and cultured for 14 days within a flask or modified perfusion bioreactor. Analysis of osteogenic and angiogenic gene expression, alkaline phosphatase (ALP) activity and ALP staining indicates a synergistic effect of perfusion flow and coculture system on osteogenic and angiogenic differentiation. The advantage of modified perfusion bioreactor is its five-branch flow distributor which directly connect to the porous PCL hollow fibers embedded in the 3D scaffold to improve flow and flow-induced shear stress uniformity.Dynamic coculture increased VEGF165 by 6-fold, VEGF189 by 2-fold, and Endothelin-1 by 4-fold, relative to dynamic monoculture. Static coculture enhanced osteogenic and angiogenic differentiation, compared with static monoculture. Although dynamic coculture is in preference to static coculture due to significant increase in ALP activity and promoted angiogenic marker expression. Our finding is the first to indicate that the modified perfusion bioreactor combined with the beneficial cell-cell crosstalk in pre-treated hASC/HUVEC cocultures provides a synergy between osteogenic and angiogenic differentiation of the accumulation of cells, suggesting that it represents a promising approach for regeneration of critical-sized bone defects.

Preclinical studies of acellular extracellular matrices as small-caliber vascular grafts.

Due to morbidity and mortality of cardiovascular diseases around the globe, there has been an unmet clinical need for small caliber vascular grafts. Autologous vessels are still the gold standard for small caliber vascular grafts (<6 mm). In an attempt to develop a tissue-engineered vascular graft, several approaches have been pursued. One of the promising techniques is the use of acellular matrices offering a prospect of being able to meet the demand for small caliber vessels. Acellular matrices can ideally preserve the vascular wall complexity, biochemical properties, and bioactivity required for tissue regeneration and function. Various strategies have emerged to increase long term patency of acellular matrices including surface modification and pre-implantation cell seeding. This article reviews the most recent and relevant in vivo studies on acellular small caliber vascular grafts, which provides an outlook toward the preclinical potential of acellular extracellular matrices in vascular tissue engineering.

Comparison of two different antioxidants in a nano lecithin-based extender for bull sperm cryopreservation.

The objective of the present study was to assess the effect of two different antioxidants, enzymatic compared with non-enzymatic, in a nano lecithin-based extender on post-thaw bull sperm quality. Semen samples (n = 36) were collected from six bulls. In the first experiment, 11 different extenders were prepared by adding five quantities of vitamin E (α-tocopherol) as a non-enzymatic antioxidant (VE: 0.1, 0.2, 0.4, 0.6 and 1.0 mM), or four quantities of glutathione peroxidase (GPx) as an enzymatic antioxidant (GPx: 0.5, 1, 2 and 3 mM) to the extender. Other extenders were a Control 1 (C1: Extender with ethanol) and Control 2 (C2: Extender without ethanol). Sperm motility (CASA), plasma membrane functionality test (HOST) and lipid peroxidation (MDA) were assessed to determine the optimal treatment in the first experiment. In the second experiment, the optimally supplemented group from the first experiment (GPx-1) was compared to C2 group. Apoptotic-like changes (Annexin staining), mitochondrial activity (Rhodamine-123 staining), acrosome integrity (PSA staining), DNA fragmentation (SCSA test) and in vitro embryo production capacity were evaluated. In the first experiment, there were the greatest percentages of plasma membrane functionality and least MDA (P ≤ 0.05) in sperm diluted GPx-1 group. In the second experiment, percentage of live sperm, blastocyst formation and hatching rate were greater (P ≤ 0.05) in the GPx-1 group compared with C2 group. In conclusion, data indicate adding 1.0 mM GPx as an enzymatic antioxidant to the nano lecithin-based extender can improve post-thaw quality and in vitro fertility of bull sperm.

Carboxymethyl kappa carrageenan-modified decellularized small-diameter vascular grafts improving thromboresistance properties.

The development of decellularized small-diameter vascular grafts is a potential solution for patients requiring vascular reconstructive procedures. However, there is a limitation for acellular scaffolds due to incomplete recellularization and exposure of extracellular matrix components to whole blood resulting in platelet adhesion. To address this issue, a perfusion decellularization method was developed using a custom-designed set up which completely removed cell nuclei and preserved three-dimensional structure and mechanical properties of native tissue (sheep carotid arteries). Afterwards, carboxymethyl kappa carrageenan (CKC) was introduced as a novel anticoagulant in vascular tissue engineering which can inhibit thrombosis formation. The method enabled uniform immobilization of CKC on decellularized arteries as a result of interaction between amine functional groups of decellularized arteries and carboxyl groups of CKC. The CKC modified graft significantly reduced platelet adhesion from 44.53 ± 2.05% (control) to 19.57 ± 1.37% (modified) and supported endothelial cells viability, proliferation, and nitric oxide production. Overall, the novel CKC modified scaffold provides a promising solution for thrombosis formation of small-diameter vessels and could be a potent graft for future in vivo applications in vascular bypass procedures. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1690-1701, 2019.

Integrated optical and electrochemical detection of Cu2+ ions in water using a sandwich amino acid-gold nanoparticle-based nano-biosensor consisting of a transparent-conductive platform.

In this paper, an optical-electrochemical nano-biosensor was introduced for measuring Cu2+ ion concentrations in water. A multi-step procedure was used to fabricate the transparent-conductive biosensor platform consisting of an l-cysteine-gold nanoparticle-based sandwich structure. First, colloidal gold nanoparticles (GNPs) were synthesized according to the Turkevich-Frens method with some modifications and then functionalized with l-cysteine molecules (GNP/l-cys). Then, cyclic voltammetry was preformed in buffered solutions containing HAuCl4·3H2O for gold nanoparticle electrodeposition on cleaned ITO glasses. The GNP-electrodeposited ITO glasses (ITO/GNPs) were thermally treated in air atmosphere for 1 hour at a temperature of 300 °C. Following the procedure, the gold nanoparticles on ITO/GNPs substrates were functionalized with l-cysteine to prepare ITO/GNPs/l-cys substrates. Finally, the sandwich-type substrates of ITO/GNPs/l-cys⋯Cu2+⋯l-cys/GNPs were fabricated by accumulation of Cu2+ ions using an open circuit technique performed in copper ion buffer solutions in the presence of previously produced colloidal GNP/l-cys nanoparticles. The effective parameters including GNP/l-cys solution volume, pre-concentration pH and pre-concentration time on the LSPR and SWV responses were investigated and optimized. The fabricated transparent-conductive platforms were successfully assessed as a nano-biosensor for detection of copper ions using two different methods of square wave voltammetry (SWV) and localized surface plasmon resonance (LSPR). As a result, the proposed biosensor showed a high sensitivity, selectivity and a wide detectable concentration range to copper ions. The total linear range and the limit of detection (LOD) of the nano-biosensor were 10-100 000 nM (0.6-6354.6 ppb) and below 5 nM (0.3 ppb), respectively. The results demonstrated the potential of combining two different optical and electrochemical methods for quantitation of the single analyte on the same biosensor platform and obtaining richer data. Also, these results indicated that the developed LSPR-SWV biosensor was superior to many other copper biosensors presented in the literature in terms of linear range and LOD. The developed nano-biosensor was successfully applied in the determination of trace Cu2+ concentration in actual tap water samples.

Codelivery of doxorubicin and JIP1 siRNA with novel EphA2-targeted PEGylated cationic nanoliposomes to overcome osteosarcoma multidrug resistance.

PURPOSE: Osteosarcoma (OS) mostly affects children and young adults, and has only a 20%-30% 5-year survival rate when metastasized. We aimed to create dual-targeted (extracellular against EphA2 and intracellular against JNK-interacting protein 1 [JIP1]), doxorubicin (DOX)-loaded liposomes to treat OS metastatic disease. MATERIALS AND METHODS: Cationic liposomes contained N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP), cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and distearoyl-phosphatidylethanolamine-methyl-poly(ethylene glycol) (DSPE-mPEG) conjugate. EphA2 targeting was accomplished by conjugating YSA peptide to DSPE-mPEG. Vesicles were subsequently loaded with DOX and JIP1 siRNA. RESULTS: Characteristics assessment showed that 1) size of the bilayered particles was 109 nm; 2) DOX loading efficiency was 87%; 3) siRNA could be successfully loaded at a liposome:siRNA ratio of >24:1; and 4) the zeta potential was 18.47 mV. Tumor-mimicking pH conditions exhibited 80% siRNA and 50.7% DOX sustained release from the particles. Stability studies ensured the protection of siRNA against degradation in serum. OS cell lines showed increased and more pericellular/nuclear localizations when using targeted vesicles. Nontargeted and targeted codelivery caused 70.5% and 78.6% cytotoxicity in OS cells, respectively (free DOX: 50%). Targeted codelivery resulted in 42% reduction in the siRNA target, JIP1 mRNA, and 46% decrease in JIP1 levels. CONCLUSION: Our dual-targeted, DOX-loaded liposomes enhance toxicity toward OS cells and may be effective for the treatment of metastatic OS.

Preparation of PEGylated cationic nanoliposome-siRNA complexes for cancer therapy.

Cationic liposomes have been investigated as non-viral vectors for gene delivery for more than a decade to overcome challenges associated with viral gene delivery. However, due to instability of liposomes, siRNA delivery is still a serious problem. In this study, we developed stealth PEGylated liposome formulations and focused on the effects of PEGylated liposomes on parameters related to size, zeta potential, polydispersity index, siRNA-loading efficiency and long-term stability of the siRNA-liposome complex. We were able to generate siRNA lipoplexes that could be very efficiently loaded, did not aggregate, could be stored at 4 °C for at least 6 months with only marginal release (1-5%) of siRNA and enhanced intracellular delivery of siRNA. Moreover, we could demonstrate that PEGylation positively contributed to all these parameters compared to liposomes, which were not PEGylated. The prepared lipoplex was successfully silenced J1P1 expression in MG-63 osteosarcoma cell line. In conclusion, our novel PEGylated liposomes have high potential for systemic delivery of siRNA and can improve in vivo stability of free siRNA and also siRNA lipoplexes.

A comprehensive mathematical model of drug release kinetics from nano-liposomes, derived from optimization studies of cationic PEGylated liposomal doxorubicin formulations for drug-gene delivery.

This study focuses on the development of a universal mathematical model for drug release kinetics from liposomes to allow in silico prediction of optimal conditions for fine-tuned controlled drug release. As a prelude for combined siRNA-drug delivery, nanoliposome formulations were optimized using various mole percentages of a cationic lipid (1,2-dioleoyl-3-trimethylammonium-propane, DOTAP) in the presence or absence of 3 mol% distearoyl phosphoethanolamine, polyethylene glycol (PEG-2000mDSPE). Outcome parameters were particle size, zeta potential, entrapment efficiency, in vitro drug release, and tumor cell kill efficiency. The optimized formula (containing 20% DOTAP with 3% DSPE-mPEG(2000) was found to be stable for six months, with round-shaped particles without aggregate formation, an average diameter of 71 nm, a suitable positive charge, and 89% drug encapsulation efficiency (EE). The 41% drug release during 6 h confirmed controlled release. Furthermore, the release profiles as functions of pH and temperature were investigated and the kinetics of the drug release could adequately be fitted to Korsmeyer-Peppas' model by multiple regression analysis. The statistical parameters confirmed good conformity of final models. Functionality of the novel cationic liposome formulations (± DOX) was tested on osteosarcoma (OS) cell lines. Increased OS cell toxicity (1.3-fold) was observed by the DOX-loaded vs. the free DOX. A feasibility pilot showed that siRNA could be loaded efficiently as well. In conclusion, we have established a predictive mathematical model for the fine-tuning of controlled drug release from liposomal formulations, while creating functional drug-delivery liposomes with potential for siRNA co-delivery to increase specificity and efficacy.

EphA2 Targeted Doxorubicin-Nanoliposomes for Osteosarcoma Treatment.

PURPOSE: To employ Doxorubicin-loaded liposomes, modified with YSA-peptide to target EphA2, to reduce adverse effects against primary bone cells and maximize toxicity against Saos-2 osteosarcoma cells. METHODS: PEGylated liposomes were prepared by thin film method using Dipalmitoylphosphatidylcholine (DPPC), cholesterol and distearylphosphatidylethanolamine-polyethyleneglycol conjugate (DSPE-mPEG) in 67.9:29.1:3 M ratios, and loaded with DOX (L-DOX) by pH-gradient method. Targeted liposomes (YSA-L-DOX), were prepared by conjugating YSA-peptide to DSPE-mPEG. Liposomes were physicochemically characterized and tested in cellular toxicity assays. RESULTS: YSA conjugation efficiency was >98%. Size and polydispersity index of both L-DOX and YSA-L-DOX were around 88 nm and 0.188, respectively. Both had similar zeta potential, and 85% DOX loading efficiencies. DOX release kinetics followed the Korsmeyer-Peppa model, and showed comparable release for both formulations from 1-8 h, and a plateau of 29% after 48 h. Both formulations could be stably stored for ≥6 months at 4°C in the dark. Toxicity assays showed a significant 1.91-fold higher cytotoxicity compared to free DOX in the Saos-2 cells, and 2-fold lesser toxicity in primary bone cells compared to the Saos-2 cells. Cellular uptake studies showed higher and more nuclear uptake in YSA-L-DOX compared to L-DOX treated cells. CONCLUSIONS: YSA-L-DOX vesicles might be effective for targeted treatment of osteosarcoma.

A Novel Approach on Drug Delivery: Investigation of A New Nano-Formulation of Liposomal Doxorubicin and Biological Evaluation of Entrapped Doxorubicin on Various Osteosarcoma Cell Lines.

OBJECTIVE: In this study we prepared a novel formulation of liposomal doxorubicin (L- DOX). The drug dose was optimized by analyses of cellular uptake and cell viability of osteosarcoma (OS) cell lines upon exposure to nanoliposomes that contained varying DOX concentrations. We intended to reduce the cytotoxicity of DOX and improve characteristics of the nanosystems. MATERIALS AND METHODS: In this experimental study, we prepared liposomes by the pH gradient hydration method. Various characterization tests that included dynamic light scattering (DLS), cryogenic transmission electron microscopy (Cryo-TEM) imaging, and UV- Vis spectrophotometry were employed to evaluate the quality of the nanocarriers. In addition, the CyQUANT® assay and fluorescence microscope imaging were used on various OS cell lines (MG-63, U2-OS, SaOS-2, SaOS-LM7) and Human primary osteoblasts cells, as novel methods to determine cell viability and in vitro transfection efficacy. RESULTS: We observed an entrapment efficiency of 84% for DOX within the optimized liposomal formulation (L-DOX) that had a liposomal diameter of 96 nm. Less than 37% of DOX released after 48 hours and L-DOX could be stored stably for 14 days. L-DOX increased DOX toxicity by 1.8-4.6 times for the OS cell lines and only 1.3 times for Human primary osteoblasts cells compared to free DOX, which confirmed a higher sensitivity of the OS cell lines versus Human primary osteoblasts cells for L-DOX. We deduced that L- DOX passed more freely through the cell membrane compared to free DOX. CONCLUSION: We successfully synthesized a stealth L-DOX that contained natural phospholipid by the pH gradient method, which could encapsulate DOX with 84% efficiency. The resulting nanoparticles were round, with a suitable particle size, and stable for 14 days. These nanoparticles allowed for adequately controlled DOX release, increased cell permeability compared to free DOX, and increased tumor cell death. L-DOX provided a novel, more effective therapy for OS treatment.

New liposomal doxorubicin nanoformulation for osteosarcoma: Drug release kinetic study based on thermo and pH sensitivity.

A novel approach was developed for the preparation of stealth controlled-release liposomal doxorubicin. Various liposomal formulations were prepared by employing both thin film and pH gradient hydration techniques. The optimum formulation contained phospholipid and cholesterol in 1:0.43 molar ratios in the presence of 3% DSPE-mPEG (2000). The liposomal formulation was evaluated by determining mean size of vesicle, encapsulation efficiency, polydispersity index, zeta potentials, carrier's functionalization, and surface morphology. The vesicle size, encapsulation efficiency, polydispersity index, and zeta potentials of purposed formula were 93.61 nm, 82.8%, 0.14, and -23, respectively. Vesicles were round-shaped and smooth-surfaced entities with sharp boundaries. In addition, two colorimetric methods for cytotoxicity assay were compared and the IC50 (the half maximal inhibitory concentration) of both methods for encapsulated doxorubicin was determined to be 0.1 µg/ml. The results of kinetic drug release were investigated at several different temperatures and pH levels, which showed that purposed formulation was thermo and pH sensitive.

In Vitro Co-Delivery Evaluation of Novel Pegylated Nano-Liposomal Herbal Drugs of Silibinin and Glycyrrhizic Acid (Nano-Phytosome) to Hepatocellular Carcinoma Cells.

OBJECTIVE: This study aimed to evaluate a co-encapsulated pegylated nano-liposome system based on two herbal anti-tumor drugs, silibinin and glycyrrhizic acid, for delivery to a hepatocellular carcinoma (HCC) cell line (HepG2). MATERIALS AND METHODS: In this experimental study, co-encapsulated nano-liposomes by the thin layer film hydration method with HEPES buffer and sonication at 60% amplitude. Liposomes that co-encapsulated silibinin and glycyrrhizic acid were prepared with a specified molar ratio of dipalmitoylphosphatidylcholine (DPPC), cholesterol (CHOL), and methoxy-polyethylene glycol 2000 (PEG2000)-derived distearoyl phosphatidylethanolamine (mPEG2000-DSPE). We used the MTT technique to assess cytotoxicity for various concentrations of co-encapsulated nano-liposomes, free silibinin (25% w/v) and glycyrrhizic acid (75% w/v) on HepG2 and fibroblast cell lines over a 48-hour period. RESULTS: Formulation of pegylated nano-liposomes showed a narrow size distribution with an average diameter of 46.3 nm. The encapsulation efficiency (EE) for silibinin was 24.37%, whereas for glycyrrhizic acid it was 68.78%. Results of in vitro cytotoxicity showed significantly greater co-encapsulated nano-liposomes on the HepG2 cell line compared to the fibroblast cell line. The half maximal inhibitory concentration (IC50) for co-encapsulated pegylated nanoliposomal herbal drugs was 48.68 µg/ml and free silibinin with glycyrrhizic acid was 485.45 µg/ml on the HepG2 cell line. CONCLUSION: This in vitro study showed that nano-liposome encapsulation of silibinin with glycyrrhizic acid increased the biological activity of free drugs, increased the stability of silibinin, and synergized the therapeutic effect of silibinin with glycyrrhizic acid. The IC50 of the co-encapsulated nano-liposomes was lower than the combination of free silibinin and glycyrrhizic acid on the HepG2 cell line.

An Optically-Transparent Aptamer-Based Detection System for Colon Cancer Applications Using Gold Nanoparticles Electrodeposited on Indium Tin Oxide.

In this paper, a label-free aptamer based detection system (apta-DS) was investigated for detecting colon cancer cells. For this purpose, we employed an aptamer specific to colon cancer cells like HCT116 expressing carcinoembryonic antigen (CEA) on their surfaces. Capture aptamers were covalently immobilized on the surface of gold nanoparticles (GNPs) through self-assembly monolayer of 11-mercaptoundecanoic acid (11-MUA) activated with EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide)/N-hydroxysuccinimide (NHS). The cyclic voltammetry (CV) and chronopotentiometry (CP) methods were used for electrodeposition of GNPs on the surface of indium tin oxide (ITO). In this work, the CV method was also used to demonstrate the conjugation of GNPs and aptamers and identify the cancer cell capturing events. Additionally, Field Emission Scanning Electron Microscopy (FE-SEM) confirmed the deposition of GNPs on ITO and the immobilization of aptamer on the apta-DS. The electrodeposited GNPs played the role of nanoprobes for cancer cell targeting without losing the optical transparency of the ITO substrate. A conventional optical microscope also verified the detection of captured cancer cells. Based on this study's results relying on electrochemical and optical microscopic methods, the proposed apta-DS is reliable and high sensitive with a LOD equal to 6 cell/mL for colon cancer detection.

An Apta-Biosensor for Colon Cancer Diagnostics.

This paper reports the design and implementation of an aptasensor using a modified KCHA10a aptamer. This aptasensor consists of a functionalized electrodes using various materials including 11-mercaptoandecanoic acid (11-MUA) and modified KCHA10a aptamer. The HCT 116, HT 29 and HEp-2 cell lines are used in this study to demonstrate the functionality of aptasensor for colon cancer detection purposes. Flow cytometry, fluorescence microscopy and electrochemical cyclic voltammetry are used to verify the binding between the target cells and aptamer. The limit of detection (LOD) of this aptasensor is equal to seven cancer cells. Based on the experimental results, the proposed sensor can be employed for point-of-care cancer disease diagnostics.

Simulation of blood oxygenation in capillary membrane oxygenators using modified sulfite solution.

Blood oxygenation is the main performance characteristic of capillary membrane oxygenators (CMOs). Handling of natural blood in in vitro investigations of CMOs is quite complex and time-consuming. Since the conventional blood analog fluids (e.g. water/glycerol) lack a substance with an affinity to capture oxygen comparable to hemoglobin's affinity, in this study a novel approach using modified sulfite solution is proposed to address this challenge. The solution comprises sodium sulfite as a component, simulating the role of hemoglobin in blood oxygenation. This approach is validated by OTR (oxygen transfer rate) measured using native porcine blood, in two types of commercially available CMOs. Consequently, the number of complicated natural blood investigations in the evolution procedure of newly developed oxygenators would considerably decrease. Moreover, the reassessing of failed devices, in clinics, would be performed more precisely using a modified sulfite solution than simple water/glycerol testing.

Surface modification of polypropylene membrane by polyethylene glycol graft polymerization.

Polypropylene hollow fiber microporous membranes have been used in a wide range of applications, including blood oxygenator. The hydrophobic feature of the polypropylene surface causes membrane fouling. To minimize fouling, a modification consisting of three steps: surface activation in H2 and O2 plasma, membrane immersion in polyethylene glycol (PEG) and plasma graft polymerization was performed. The membranes were characterized by contact angle measurement, Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), tensile test, scanning electron microscopy (SEM) and atomic force microscopy (AFM). Oxygen transfer of modified membranes was also tested. The stability of grafted PEG was measured in water and in phosphate buffer saline (PBS) at 37°C. Blood compatibility of modified surfaces was evaluated by the platelet adhesion method. Water contact angel reduction from 110° to 72° demonstrates the enhanced hydrophilicity, and XPS results verify the presence of oxygenated functional groups due to the peak existence in 286 eV as a result of PEG grafting. The results clearly indicate that plasma graft-polymerization of PEG is an effective way for antifouling improvement of polypropylene membranes. Also, the results show that oxygen transfer changes in PEG grafted membranes are not significant.