Assessment of the efficiency and stability of enzymatic membrane reaction utilizing lipase covalently immobilized on a functionalized hybrid membrane.

Enzyme immobilization in membrane bioreactors has been considered as a practical approach to enhance the stability, reusability, and efficiency of enzymes. In this particular study, a new type of hybrid membrane reactor was created through the phase inversion method, utilizing hybrid of graphene oxide nanosheets (GON) and polyether sulfone (PES) in order to covalently immobilize the Candida rugosa lipase (CRL). The surface of hybrid membrane was initially modified by (3-Aminopropyl) triethoxysilane (APTES), before the use of glutaraldehyde (GLU), as a linker, through the imine bonds. The resulted enzymatic hybrid membrane reactors (EHMRs) were then thoroughly analyzed by using field-emission scanning electron microscopy (FE-SEM), contact angle goniometry, surface free energy analysis, X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, attenuated total reflection (ATR), and energy-dispersive X-ray (EDX) spectroscopy. The study also looked into the impact of factors such as initial CRL concentration, storage conditions, and immobilization time on the EHMR's performance and activity, which were subsequently optimized. The results demonstrated that the CRLs covalently immobilized on the EHMRs displayed enhanced pH and thermal stability compared to those physically immobilized or free. These covalently immobilized CRLs could maintain over 60% of their activity even after 6 reaction cycles spanning 50 days. EHMRs are valuable biocatalysts in developing various industrial, environmental, and analytical processes.

Inclusion of Levodopa into ß-Cyclodextrin: A Comprehensive Computational Study.

This study focused on the inclusion of levodopa (LVDP) into ß-cyclodextrin (BCD) using various computational methods such as quantum mechanics (QM), molecular dynamics/steered molecular dynamics (MD/SMD), and QM/molecular mechanics/Poison-Boltzmann surface area (QM/MM/PBSA). The QM results assigned the most significant charge-transfer atoms and the higher stability of LVDP in the aqueous phase. The MD results indicate the formation of a 1:1 complex with a reasonable estimation of the effective radius of the complex, the significant contribution of hydrogen bonding in the binding energy, and the enhancement of the water solubility of LVDP. By accounting for the water hydrogen bonds and their dipolar effects, QM/MM calculations lead to the more accurate IR spectrum and binding energy of the BCD-LVDP complex. By considering carboxylic and amine functional groups' more precise arrangement, QM/MM assigns stronger hydrogen bonds between LVDP and BCD. While all the methods provide a reasonable estimation of the binding energy, the most accurate value (-4.14 kcal/mol) is obtained from QM/MM/PBSA.

Copper(ii) complexes with tridentate halogen-substituted Schiff base ligands: synthesis, crystal structures and investigating the effect of halogenation, leaving groups and ligand flexibility on antiproliferative activities.

To investigate the effect of different halogen substituents and leaving groups and the flexibility of ligands on the anticancer activity of copper complexes, sixteen copper(ii) complexes with eight different tridentate Schiff-base ligands containing pyridine and 3,5-halogen-substituted phenol moieties were synthesized and characterized by spectroscopic methods. Four of these complexes were also characterized by X-ray crystallography. The cytotoxicity of the complexes was determined in three different tumor cell lines (i.e. the A2780 ovarian, HCT116 colorectal and MCF7 breast cancer cell line) and in a normal primary fibroblast cell line. Complexes were demonstrated to induce a higher loss of cell viability in the ovarian carcinoma cell line (A2780) with respect to the other two tumor cell lines, and therefore the biological mechanisms underlying this loss of viability were further investigated. Complexes with ligand L1 (containing a 2-pycolylamine-type motif) were more cytotoxic than complexes with L2 (containing a 2-(2-pyridyl)ethylamine-type motif). The loss of cell viability in A2780 tumor cells was observed in the order Cu(Cl2-L1)NO3 > Cu(Cl2-L1)Cl > Cu(Br2-L1)Cl > Cu(BrCl-L1)Cl. All complexes were able to induce reactive oxygen species (ROS) that could be related to the loss of cell viability. Complexes Cu(BrCl-L1)Cl and Cu(Cl2-L1)NO3 were able to promote A2780 cell apoptosis and autophagy and for complex Cu(BrCl-L1)Cl the increase in apoptosis was due to the intrinsic pathway. Cu(Cl2-L1)Cl and Cu(Br2-L1)Cl complexes lead to cellular detachment allowing to correlate with the results of loss of cell viability. Despite the ability of the Cu(BrCl-L1)Cl complex to induce programmed cell death in A2780 cells, its therapeutic window turned out to be low making the Cu(Cl2-L1)NO3 complex the most promising candidate for additional biological applications.

Isolation of HLA-G+ cells using MEM-G/9 antibody-conjugated magnetic nanoparticles for prenatal screening: a reliable, fast and efficient method.

The development of an effective and noninvasive early method for obtaining fetal cells is crucial to prenatal screening. Despite proving the presence of fetal cells in the reproductive tract, their use is limited due to their inability to properly isolate them from maternal cells. Magnetic-activated cell sorting (MACS) is a simple technique to separate cells. The present study aimed to develop a MACS-based platform for the isolation of the HLA-G expressing trophoblast cells. For this purpose, first, the triazine functionalized MNPs were synthesized and characterized. Then, MNPs were directly and indirectly conjugated by the MEM-G/9 antibodies targeting HLA-G+ cells. The antibody amount on the surface of the nanoparticles was determined with the Bradford assay. The cell capture efficiency was also investigated. Various characterization methods confirmed the successful nanoparticle synthesis and antibody conjugation. The optimal initial antibody amount for the immobilization was about 20 µg and the optimal time was 3 h. The antibody-nanoparticles by the indirect method had better targeting and capture efficiency than the direct method. The MNPs indirectly conjugated with antibodies are an efficient tool for cell isolation and present considerable potential to be applied in biomedical fields.

Multicomponent Synthesis of Diversified Chromeno[3,2-d]oxazoles.

A practical and efficient synthetic procedure to novel chromeno[3,2-d]oxazoles through a one-pot sequential multistep process is presented. This procedure proceeds efficiently in propylene carbonate (PC) as a green solvent and affords a wide range of the chromenooxazole scaffolds.

Synthesis, characterization, and binding assessment with human serum albumin of three bipyridine lanthanide(III) complexes.

In this work, the terbium(III), dysprosium(III), and ytterbium(III) complexes containing 2, 2'-bipyridine (bpy) ligand have been synthesized and characterized using CHN elemental analysis, FT-IR, UV-Vis and 1H-NMR techniques and their binding behavior with human serum albumin (HSA) was studied by UV-Vis, fluorescence and molecular docking examinations. The experimental data indicated that all three lanthanide complexes have high binding affinity to HSA with effective quenching of HSA fluorescence via static mechanism. The binding parameters, the type of interaction, the value of resonance energy transfer, and the binding distance between complexes and HSA were estimated from the analysis of fluorescence measurements and Förster theory. The thermodynamic parameters suggested that van der Waals interactions and hydrogen bonds play an important role in the binding mechanism. While, the energy transfer from HSA molecules to all these complexes occurs with high probability, the order of binding constants (BpyTb > BpyDy > BpyYb) represents the importance of radius of Ln3+ ion in the complex-HSA interaction. The results of molecular docking calculation and competitive experiments assessed site 3 of HSA, located in subdomain IB, as the most probable binding site for these ligands and also indicated the microenvironment residues around the bound mentioned complexes. The computational results kept in good agreement with experimental data.

The immobilization of Candida rugosa lipase on the modified polyethersulfone with MOF nanoparticles as an excellent performance bioreactor membrane.

In this study, the modified nanocomposite membrane of polyethersulfone (PES) with NH2-MIL-101(Cr) as a metal-organic framework (MOF) is exploited for Candida rugosa lipase (CRL) immobilization. To that end, the various amounts of NH2-MIL-101(Cr) nanoparticles are blended into PES casting solution to fabricate ultrafiltration membrane via phase inversion technique. The incorporation efficiency of NH2-MIL-101(Cr) nanoparticles on the membrane morphology is investigated using various techniques, namely atomic force microscopy (AFM), X-ray diffraction (XRD), and contact angle goniometry. In terms of water pure flux and CRL immobilization efficiency, the best performance is observed for PES-NH2-MIL1% membrane. This bioactive membrane (CRL@GA@PES-NH2-MIL1%) displays an improvement in pH and thermal stability and separation performance that makes it a fruitful candidate for using in bioreactors. The examination of the wet- and dry-storage stability of CRL@GA@PES-NH2-MIL1% demonstrates the high stability for the wet bioactive membrane. The reusability inspection of CRL@GA@PES-NH2-MIL1% represents about 50% conservation of the residual activity after 12 sequential usage cycles.

Biogenic magnetite nanoparticles: A potent and environmentally benign agent for efficient removal of azo dyes and phenolic contaminants from water.

A comprehensive study was conducted toward the green and facile synthesis of biocompatible magnetite nanoparticles for the efficient removal of organic contaminants from water. The nanoparticles were synthesized using a modified co-precipitation method and functionalized by the taxane diterpenoids extracted from Taxus baccata L., and fully characterized using UV-vis spectroscopy, SEM, FTIR, VSM, and XRD. The synthesized monodisperse magnetite nanoparticles, with a narrow size distribution of less than 50 nm, displayed significant and stable magnetic activity without surface oxidation after several months. The batch experiments clearly indicated the efficient iron-catalyzed removal of Nile blue, methylene blue, methylene orange, and 4-nitrophenol for several cycles without significant loss of catalytic activity. The relevant kinetic data of the dyes removal reactions were fitted to a pseudo-first order model. Moreover, in vitro MTT assay revealed high biocompatibility of the nanoparticles with no significant toxicity on different human cell lines. The overall results indicated high potential of green synthesized, biocompatible magnetite nanoparticles for the environmental applications especially wastewater remediation.

Novel folic acid-conjugated doxorubicin loaded ß-lactoglobulin nanoparticles induce apoptosis in breast cancer cells.

Chemotherapy constitutes the main strategy in management of breast cancer (BC). Lack of specificity and high burden of adverse effects of chemotherapeutic agents remain the most important impediments to successful treatment of BC patients. Folate receptor α (FRα) could be very promising for therapeutic targeting in this type of cancer. In this study, ß-lactoglobulin nanoparticles (BNPs) conjugated with folic acid and loaded with doxorubicin (FDBNPs) were prepared. Various characterization techniques were applied to determine the size, polydispersity and doxorubicin loading of prepared FDBNPs in comparison with doxorubicin-loaded BNPs (DBNPs). The results showed that FDBNPs are 109.77 ± 2.80 nm in diameter with well dispersed and spherical shapes. The biodegradation of FDBNPs in the presence of trypsin enzyme and in PBS at different pH (4 and 7) was spectrophotometrically monitored and the results showed that the FDBNPs with encapsulation efficiency of 68.82%±1.76% could deliver doxorubicin at clinically relevant doses. Effects of DBNPs and FDBNPs against MCF-7 and MDA-MB-231, BC and triple negative BC (TNBC) cell lines, respectively, showed significant inhibition of cell proliferation as well as induction of apoptosis. Based on these findings, FDBNPs with facilitated drug release and targeted doxorubicin delivery capacities could have high therapeutic potential for BC and TNBC.

Electrochemiluminescence detection of human breast cancer cells using aptamer modified bipolar electrode mounted into 3D printed microchannel.

In the present manuscript, a closed bipolar electrode system integrated with electrochemiluminescence (ECL) detection has been introduced for sensitive diagnosis of human breast cancer cells (MCF-7). For sensitive and selective detection, the anodic pole of the bipolar electrode was modified with the AS1411 aptamer, a specific aptamer for the nucleolin, and treated by the secondary aptamer modified gold nanoparticles. The electrochemiluminescence of luminol was followed in the presence of hydrogen peroxide on the anode pole of bipolar electrode (BPE) as an analytical signal. Moreover, 3D printed microchannels were used for the fabrication of BPE systems to minimize the required amounts of sample. The present aptasensor offers low cost, sensitive and selective cancer cell detection with two acceptable linear ranges. The first linear section appears within 10-100 cells and the latter is found to be within 100-700 cells. The limit of detection was about 10 cells.

Green synthesis of silver nanoparticles using flower extract of Malva sylvestris and investigation of their antibacterial activity.

High-quality colloidal silver nanoparticles (AgNP) were synthesised via a green approach by using hydroalcoholic extracts of Malva sylvestris. Silver nitrate was used as a substrate ion while the plant extract successfully played the role of reducing and stabilising agents. The synthesised nanoparticles were carefully characterised by using transmission electron microscopy, atomic-force microscopy, energy dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy and UV-vis spectroscopy. The maximum absorption wavelengths of the colloidal solutions synthesised using 70 and 96% ethanol and 100% methanol, as extraction solvents, were 430, 485 and 504 nm, respectively. Interestingly, the size distribution of nanoparticles depended on the used solvent. The best particle size distribution belonged to the nanoparticles synthesised by 70% ethanol extract, which was 20-40 nm. The antibacterial activity of the synthesised nanoparticles was studied on Escherichia coli, Staphylococcus aureus and Streptococcus pyogenes using disk diffusion, minimum inhibitory concentrations and minimum bactericidal concentrations assays. The best antibacterial activity obtained for the AgNPs produced by using 96% ethanolic extract.

New generation of drug delivery systems based on ginsenoside Rh2-, Lysine- and Arginine-treated highly porous graphene for improving anticancer activity.

In this study, Rh2-treated graphene oxide (GO-Rh2), lysine-treated highly porous graphene (Gr-Lys), arginine-treated Gr (Gr-Arg), Rh2-treated Gr-Lys (Gr-Lys-Rh2) and Rh2-treated Gr-Arg (Gr-Arg-Rh2) were synthesized. MTT assay was used for evaluation of cytotoxicity of samples on ovarian cancer (OVCAR3), breast cancer (MDA-MB), Human melanoma (A375) and human mesenchymal stem cells (MSCs) cell lines. The percentage of apoptotic cells was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. The hemolysis and blood coagulation activity of nanostructures were performed. Interestingly, Gr-Arg, Gr-Lys, Gr-Arg-Rh2, and Gr-Lys-Rh2 were more active against cancer cell lines in comparison with their cytotoxic activity against normal cell lines (MSCs) with IC50 values higher than 100 µg/ml. The results of TUNEL assay indicates a significant increase in the rates of TUNEL positive cells by increasing the concentrations of nanomaterials. Results were also shown that aggregation and changes of RBCs morphology were occurred in the presence of GO, GO-Rh2, Gr-Arg, Gr-Lys, Gr-Arg-Rh2, and Gr-Lys-Rh2. Note that all the samples had effect on blood coagulation system, especially on PTT. All nanostrucure act as antitumor drug so that binding of drugs to a nostructures is irresolvable and the whole structure enter to the cell as a drug.

The performance of immobilized Candida rugosa lipase on various surface modified graphene oxide nanosheets.

In this study, we have reported the synthesis of graphene oxide nanosheets (GON) and its functionalization with 2, 4, 6-trichloro-1, 3, 5-triazine (TCT) through two routes, (a) directly reaction of GON with TCT (GON-1), and (b) reaction of GON with pre-functionalized TCT with 3-aminopropyltriethoxysilane (APTS) (GON-2). Subsequently, GON, GON-1 and GON-2 have been used as supports for immobilization of Candida rugosa lipase (CRL). Several techniques such as XRD, SEM, EDS, UV-Vis, CHNS, FTIR and AFM were applied to characterize the nano-structures and success of synthesis, functionalization and CRL immobilization processes. The results corresponding to optimization of immobilization process revealed the following order for values of loading capacity, immobilization yield and leaching of CRL: GON > GON-1 > GON-2, while this order is reversed for, specific activity and recovery activity. The assessment of operational parameters represents the high storage stability and reasonable reusability for all the immobilized CRL while the pH and thermal stability of CRL@GON-2 are higher than two others. It seems the longer linker of GON-2 could more effectively prevent the unfavorable interaction between enzyme-enzyme and enzyme-product that consequently resulted the best catalytic performance, pH and thermal stability. The advantages of these supports make them suitable candidates for practical applications.

Green and Facile Synthesis of Highly Photoluminescent Multicolor Carbon Nanocrystals for Cancer Therapy and Imaging.

Carbon dots (CDs), as a new generation of fluorescent nanoparticles, have been greatly considered for different biomedical applications. In the present study, a one-pot hydrothermal method was developed for the synthesis of a series of carbon dots (CDs) for cancer imaging and therapy. Taxane diterpenoids were utilized as the carbon source, different diamines were used as the nitrogen source, and folic acid was used as a targeting agent. High-quality photostable and multicolor (blue and green) carbon nanocrystals with a hexagonal shape, a narrow size distribution of less than 20 nm, and high fluorescence quantum yield of up to 50.4% were obtained from taxanes in combination with m-phenylenediamine and folic acid to give the best results. The nanoparticles displayed a potent anticancer activity with IC50 values of 31.3 ± 2.7 and 34.1 ± 1.1 µg mL-1 for the human MCF-7 and HeLa cancer cell lines, respectively, and IC50 value of 120.5 ± 3.8 µg mL-1 on the normal human fibroblast cells. The flow cytometry studies determined apoptosis-mediated cell death as the main anticancer mechanism of CDs, and the molecular studies revealed the induction of both extrinsic and intrinsic apoptosis pathways. The overall results indicated the great potential of synthesized CDs for the simultaneous cancer imaging and therapy.

Novel approaches to immobilize Candida rugosa lipase on nanocomposite membranes prepared by covalent attachment of magnetic nanoparticles on poly acrylonitrile membrane.

Novel methods have been developed for lipase immobilization on poly acrylonitrile (PAN) membranes to increase the activity and stability of the immobilized lipase. In this study, poly acrylonitrile (PAN) membranes were aminated and then activated by glutaraldehyde or epichlorohydrine to be used for enzyme immobilization. In the other approach, magnetic nanoparticles (MNPs) which were functionalized with trichlorotriazine (TCT) or glutaraldehyde (GA) were attached to the membrane surface to prepare the nanocomposite membranes named TCT-MNP@PAN & GA-MNP@PAN membranes. Candida rugosa lipase (CRL) was covalently immobilized on this activated nanocomposite membrane. Nanoparticles and nanocomposite membranes were characterized with various techniques such as SEM, TEM, XRD, FTIR, FTIR-ATR, AFM, contact angle goniometry and surface free energy measurement. The evidence of immobilization was also done by FTIR-ATR, enzyme activity, and loading efficiency. It was found that the activity of immobilized lipase on GA and TCT functionalized NCPAN membrane were about 50% and 31% higher than that immobilized on GA-activated PAN membrane. The kinetic parameters of enzymatic membranes showed the better conformation of the lipase enzyme immobilized on the TCT-MNP@PAN membrane. The presented enzymatic nanocomposite membranes are easy to prepare with low cost and are good candidates for use in membrane bioreactors.

Xylanase immobilization on modified superparamagnetic graphene oxide nanocomposite: Effect of PEGylation on activity and stability.

In order to utilize the advantages of immobilization such as improvement of stability, increasing the catalytic activity, ability to recovery and reuse of enzyme from reaction medium, xylanase enzyme was immobilized on superparamagnetic garphene oxide nanosheets (GOMNP). Xylanase, as a hydrolytic enzyme of xylan has widely used in industry. Since the xylan is bulk, for enhance accessibility of active sites of the immobilized xylanase, polyethylene glycol bis amine (PEGA) was used as a spacer for functionalization of GOMNP. The modified GOMNP and immobilized xylanase on PEGA-GOMNP (PEGA-GOMNP/Xy) were characterized through different analysis tools. The results showed that xylanase was attached to the functionalized nanocomposite with a yield of 273mg enzyme per gram PEGA-GOMNP. Thermal stability, pH stability, reusability and storage stability were determined for immobilized enzyme. The free and immobilized xylanase displayed an optimal enzymatic activity at 60°C and pH 6.5 and 7.5, respectively. The immobilized enzyme retained about 40% of the initial activity after 8 cycles with xylan substrate at 60°C. Also immobilized and free enzymes retained 35% and 20% of the initial catalytic activity after 90days storage at 4°C, respectively. Consequently, PEGA- modified GOMNP can be introduced as a biodegradable and suitable support for bioengineering.

Doughnut-shaped bovine serum albumin nanoparticles loaded with doxorubicin for overcoming multidrug-resistant in cancer cells.

Traditional spherical albumin nanoparticles remain as the dominant shape of nano-carriers described in the literature at present, due to their simple desolvation method of synthesis. However, non-spherical shapes also show great promise as cancer drug delivery vectors. In this study, we report a novel synthetic strategy based on dimethyl sulfoxide (DMSO) addition during desolvation step, to produce doughnut-shaped bovine serum albumin nanoparticles (DBSA-NPs), while maintaining narrow size distributions and homogeneity. The characteristics such as size, polydispersity and doxorubicin loading of prepared DBSA-NPs in comparison with spherical ones were determined. The biodegradation of DBSA-NPs loaded with doxorubicin (Dox-DBSA-NPs) in the presence of trypsin enzyme was spectrophotometrically monitored directly based on doxorubicin release profile. The release profile was analyzed with different kinetic models and it was best fitted with Higuchi kinetics model. The anticancer effect of Dox-DBSA-NPs against lymphoblastic leukemia (MOLT-4) and multidrug resistant uterine sarcoma (MES-SA/DX-5) cell lines were also investigated and the results were comparable with doxorubicin loaded spherical BSA nanoparticles. These results showed the potential of Dox-DBSA-NPs as a novel and high potential nano-carrier for management of non-resistance and also multidrug resistant cancer cells.

Thermal stability of pepsin: A predictive thermodynamic model of a multi-domain protein.

Pepsin is generally used in the preparation of F(ab)2 fragments from antibodies. The antibodies that are one of the largest and fastest growing categories of bio- pharmaceutical candidates. Differential scanning calorimetric is principally suitable method to follow the energetics of a multi-domain, fragment to perform a more exhaustive description of the thermodynamics in an associating system. The thermodynamical models of analysis include the construction of a simultaneous fitting of a theoretical expression. The expression depending on the equilibrium unfolding data from multimeric proteins that have a two-state monomer. The aim of the present study is considering the DSC data in connection with pepsin going through reversible thermal denaturation. Afterwards, we calculate the homology modeling identification of pepsin in complex multi-domain families with varied domain architectures. In order to analyze the DSC data, the thermal denaturation of multimer proteins were considered, the "two independent two-state sequential transitions with domains dissociation model" was introduced by using of the effective ΔG concept. The reversible unfolding of the protein description was followed by the two-state transition quantities which is a slower irreversible process of aggregation. The protein unfolding is best described by two non-ideal transitions, suggesting the presence of unfolding intermediates. These evaluations are also applicable for high throughput investigation of protein stability.

Green synthesis of silver nanoparticles using Mentha pulegium and investigation of their antibacterial, antifungal and anticancer activity.

A simple and eco-friendly method for efficient synthesis of stable colloidal silver nanoparticles (AgNPs) using Mentha pulegium extracts is described. A series of reactions was conducted using different types and concentrations of plant extract as well as metal ions to optimize the reaction conditions. AgNPs were characterized by using UV-vis spectroscopy, transmission electron microscopy, atomic force microscopy, dynamic light scattering, zetasizer, energy-dispersive X-ray spectroscopy (EDAX) and Fourier transform infrared spectroscopy (FTIR). At the optimized conditions, plate shaped AgNPs with zeta potential value of -15.7 and plasmon absorption maximum at 450 nm were obtained using high concentration of aqueous extract. Efficient adsorption of organic compounds on the nanoparticles was confirmed by FTIR and EDAX. The biogenic AgNPs displayed promising antibacterial activity on Escherichia coli, Staphylococcus aureus, and Streptococcus pyogenes. The highest antibacterial activity of 25 µg mL-1 was obtained for all the strains using aqueous extract synthesized AgNPs. The aqueous extract synthesised AgNPs also showed considerable antifungal activity against fluconazole resistant Candida albicans. The cytotoxicity assay revealed considerable anticancer activity of AgNPs on HeLa and MCF-7 cancer cells. Overall results indicated high potential of M. pulegium extract to synthesis high quality AgNPs for biomedical applications.