Modares Journal of Biotechnology

Abstract Cancer is one of the causes of death in human societies, and the main reason for failure in its treatment is late diagnosis and involvement of other body organs. Biomarkers measurement of body fluids can be one of the most important methods of cancer screening and diagnosis in the early stages. Circulating microRNAs have been proposed as new biomarkers for cancer diagnosis and prognosis. The use of these molecules, in addition to early and timely diagnosis before metastasis, will reduce the damage to the patient due to the possibility of non-invasive access. Therefore, developing a method to identify, reveal and quantify it is a necessity. In this study, a biosensor was designed to isolate and identify circulating blood microRNAs in a hydrogel platform. In our work, microRNA was isolated using a single-stranded DNA receptor probe and fixed in a platform containing hydrogels. By attaching the microRNA to the probe, the second probe, which complements the biotinylated DNA at the top of the microRNA, forms a sandwich structure. Finally, microRNA trapped between the two probes was detected by FITC-bound streptavidin in the hydrogel platform. In this research, different concentrations of microRNA-9 from 1000 picomolar to 1 femtomol were used to estimate the limit of detection (LOD) of the designed biosensor, and 50 femtomol was measured as the detection limit.

Abstract Hepatocellular carcinoma, predominant form of liver cancer, is the main cause of death in patients with liver cirrhosis. Podophyllotoxin, a natural anticancer compound, has ideal anti-tumor properties. However, its use is limited due to poor solubility and bioavailability. Finding a suitable drug delivery system have great importance in improving the bioavailability of podophyllotoxin. In this study, mPEG-PCL nanoparticles have been used for delivery of podophyllotoxin to liver cancer cells. mPEG-PCL copolymers were synthesized and characterized by DLS, FTIR and NMR analyses methods. The critical micellization concentration was 0.055 µg/ml. The z-average and surface charge of micelle was 186 ± 12 nm and -5.13 mV, respectively. podophyllotoxin was loaded in micelles in different w/w ratios of drug: copolymer. The size of the nanodrug was 214 ± 20 nm and the weight ratio of 1:1 with encapsulation efficiency of 77.36 ± 1.23 % was selected as the optimal ratio. The drug release results showed a significant difference between the rapid release of free podophyllotoxin and the more stable release of the loaded drug. At 37°C, drug release was higher, which was attributed to the destruction of polymersome structure at this temperature. According to the cytotoxicity study, the IC50 value for nanodrug (8.64 μg/ml) was lower than the IC50 value for the free drug (12.79 μg/ml), which showed the effect of improved cytotoxicity of nanodrug compared to the free drug. The results proved the polymersome can be potential carriers for delivery, controllable release and improve the toxicity effect of podophyllotoxin in cancer chemotherapy.

Abstract In recent years, targeted drug delivery systems have emerged as a promising approach to increase the efficacy and minimize side effects of therapeutic agents. Cerasomes are a special type of liposomes with covalent siloxane networks on the surface that provide exceptional morphological stability while retaining all the beneficial properties of liposomes. Cerosomes provide a unique platform for drug encapsulation and delivery due to their biocompatibility, stability, controllable release, and long-term storage. In this research, an attempt has been made to engineer the surface of cerosomes to increase the selectivity and efficiency of drug delivery. In such a way that the Herceptin antibody is placed on the surface of the serosa and allows the precise targeting of HER²⁺ cells. Then, the physicochemical characteristics of antibody-functionalized cerosomes, including size and surface charge 229±15.6 nm and 13.5±1.2 mV were respectively obtained. The results of IR and fluorescence spectrum showed that the antibody was successfully attached to the surface of cerasome with a binding efficiency of 64%. These results prove the basic mechanisms governing the synthesis of immunocerasomes and provide a valuable approach for future developments in targeted drug delivery systems.

Abstract Curcumin, a potent antioxidant extracted from the turmeric plant, is known for its ability to suppress reactive oxygen species (ROS) through activation of antioxidant response elements. However, its clinical utility is limited by poor solubility and rapid elimination. In this study, we aimed to enhance the solubility and bioavailability of curcumin by encapsulating it in fifth-generation polyamidoamine dendrimer nanoparticles. UV-Vis and fluorescence spectroscopy confirmed successful encapsulation, with an average nanoparticle size of 275 nm and a positive surface charge of 8 mV. The nanocarrier exhibited a loading capacity of approximately curcumin6 mol per dendrimer 1mol and achieved complete drug release within 72 hours. Cellular uptake assays using a live/dead assay revealed superior uptake of the nanocarrier compared to free curcumin, leading to a significant reduction in cellular ROS levels and apoptosis. Furthermore, in a model of induced oxidative stress using H2O2, curcumin-loaded dendrimer nanoparticles significantly reduced cellular ROS levels compared to free curcumin. These findings highlight the potential of this nanocarrier as an effective and intelligent system for delivering water-insoluble drugs, offering a promising approach for improving the therapeutic efficacy of curcumin.

Abstract Rapid and sensitive diagnosis of breast cancer, especially in the early stages of its formation, is very important. One of the methods of detecting cancer cells is the use of electrochemical sensors. Here, a new nanocomposite including an organic metal framework and silver nanoclusters are used. The resulting nanocomposite can be used as a scaffold to attach antibodies for the detection of HER2-positive cells. In the final nanocomposite structure, silver nanoclusters are placed in the internal cavities of the metal-organic framework, leading to strong electron transport, good biocompatibility, and high electrochemical activity. Our results showed that the designed electrochemical sensor has a high sensitivity in identifying HER2 positive cells, with a detection limit of 3 cells and a linear range of 100 to 5000 cells/ml. Also, the investigations showed that the introduced sensor has stability, good selectivity and acceptable application. The proposed strategy for the development of sensors based on metal-organic frameworks provides a promising approach for early detection of cancer markers and living cancer cells.

Abstract Matrix metalloproteinases (MMPs) are a zinc endopeptidase family that increases the metastatic behavior of human malignant tumors. Epigallocatechin gallate (EGCG) is a major component of green tea polyphenols and is used as an MMP inhibitor in cancer treatment. This study aims to develop and optimize the loading of EGCG in the liposomal delivery system in an experimental/ computational way. In this study, nanoliposomes were prepared by passive loading and thin-film hydration method. Size, zeta potential, stability, encapsulation efficiency, and nanoliposome drug release profile were investigated. Cytotoxicity of nanoliposomes was evaluated on three breast cancer cell lines using an MTT viability assay. To investigate the EGCG-Liposome interaction, coarse-grained Molecular Dynamic simulations were carried out. The mean diameter of liposome was 73.6±6.9 nm, the surface charge was -14.6 mV and the encapsulation efficiency was 78.5±7.3%. The encapsulation of EGCG into the liposome caused a continuous release of the drug after 72 h, which also increased the potency of the drug. Due to the EGCG hydrophobic properties, the major distribution is located at the hydrophobic part of the membrane. The energy and radial distribution function results indicate the stability of liposomes. Simulation results demonstrate that the majority of the drug is surrounded by liposomes, which indicates high encapsulation efficiency and confirms the developed synthesis method. Due to the low solubility of the drug, it seems that the use of liposomal carriers to deliver and release EGCG is a suitable solution to increase the efficiency of the drug.

Abstract The COVID-19 pandemic has created a global health crisis, and developing effective treatments is essential to prevent the spread of the disease and save millions of lives. One of the key proteins involved in the replication cycle of SARS-CoV-2, the virus that causes COVID-19, is the main protease enzyme, 3CL^pro. Due to its high importance, this enzyme is the subject of molecular, structural, and clinical investigations, and efforts have been made to develop drugs that can inhibit its activity. One such drug is the chemical compound N3, which has been found to have a high inhibitory effect against 3CL^pro. However, traditional medicine perspectives on this issue have been less explored. In this research, molecular docking interaction simulation and all-atom molecular dynamics (MD) simulation were conducted to study the potential inhibitory capability of generally available 21 plant-extracted compounds against the 3CL^pro enzyme. Three compounds with the highest inhibition probability were selected from the molecular docking results and subjected to 100 ns of MD simulation to investigate their stability and structural-dynamic-energetic features. Beside the complexes stability, the results from the simulation demonstrated that, all our selected three compounds induce N3 comparable structural-dynamics characteristics to 3CL^pro and, therefore, are expected to have a similar inhibitory ability against this enzyme. Compound number 5 was found to have the most favorable binding energy and was proposed as the best plant substitute for N3. The results from this research can be directly used to design experimental research for 3CLpro enzyme inhibition, saving the time-financial cost.

Abstract Vitamins D and E are two common medicines for diabetes treatment. Among the main issues in this field is the release of insulin into the circulatory system. Increasing the stability of insulin hexamer is an evolving strategy in improving insulin secretion efficiency. Insulin protein is commonly found in three forms: monomer, dimer, and hexamer. In this study, for the first time, computational approaches were used to investigate the effect of vitamins D3 and E on the stability of insulin hexamer. The molecular docking results indicate six specific binding sites for these vitamins. These bind to the hydrophobic sites of insulin subunits due to their structural rings and hydrophobic properties. The G-mmpbsa analysis indicates the stabilizing role of both vitamins. The binding of these vitamins to the hexamer has significantly increased the binding energy between insulin subunits. Also, the number of hydrogen bonds between monomeric subunits of each insulin homodimer increased in the presence of the vitamins. It also significantly increases the number of internal hydrogen bonds of hexamer protein. Accordingly, vitamins D3 and E bind to and stabilize the insulin hexamer, resulting in a slower and more balanced insulin release as well as a longer half-life for the dimer in the bloodstream. These findings will pave the way to design a new strategy to regulate insulin release and increase its half-life in the blood for type II diabetes treatment. Besides, hexamer stabilization can be an effective treatment strategy for type I diabetes through slow release from an implanted biosensor system.

Abstract Inflammatory bowel disease is a chronic inflammatory disease of the gastrointestinal tract. Despite numerous endeavors over the past few years, as well as an increase within the number of patients with the disease, there are currently limited medications available to manage intestinal inflammation. Designing a new biological treatment using natural bioactive medications with fewer side effects and more secure transmission than chemical compounds could be advantageous. In this study, a new strategy for the controlled release of Gallic acid as a bioactive polyphenol with anti-inflammatory impacts was proposed. This bioactive compound was loaded on a Cerosome nanocarrier and its stability was investigated. Cerosome-forming lipid (CFL) was synthesized through a two-step chemical reaction and then the Cerosomes were prepared by thin layer hydration by distinctive proportions of DPPC: CFL mole ratio. Cerosome with a mean diameter of 335 nm and zeta potential of -23 mV were homogeneous. The optimal formulation of the Cerosomal gallic acid system shows 34% loading and controlled release of the medication in gastrointestinal fluid environments. Structural stability was systematically evaluated by physicochemical characterization methods, and Cerasomes showed greater stability than liposomes and could be present longer in the bloodstream. These results indicate that Cerasomes can be a better medication delivery system for long-term storage and controllable release of gallic acid and have remarkable applications as carriers of intestinal inflammation drug delivery.

Abstract Stem cells are characterized by their capacity for self-renewal and their ability to differentiate into specific cell types under the influence of their microenvironment. It is known that matrix chemistry controls stem cell differentiation. Single cell encapsulations of the Mesenchymal stem cells into a semi-permeable microgel, allows a greater control of the stem cell fate. In this study, a chip for single-cell encapsulation was designed and fabricated using microfluidic technology. By using microfluidic chip, human bone marrow mesenchymal stem cells (hBMSCs) are encapsulated inside alginate and alginate-poly-l lysine (PLL) microgels. The results of long-term viability of MSCs inside alginate-PLL microgels, shows a significant increase compared to alginate microgels. Mesenchymal stem cell proliferation in alginate-PLL microgels also increased significantly on days 14 and 21. It seems that PLL improves cell adhesion and function by creating a positively charged matrix. Microscopic studies indicate that the morphology of the cells inside the microgels is spherical. However, the average diameter and volume of cells in microgels containing PLL are smaller than others, which indicates more proliferation and space limitation inside the microgels. Therefore, single cell alginate-PLL microgels provide a suitable substrate in clinical studies for tissue engineering, organ transplantation and cell therapy.

Abstract Cell substrates play a crucial role in tissue engineering and biomaterial science. Various studies are performed to develop the appropriate cell substrates for using in vitro and in vivo. Therefore, a biocompatible substrate that mimics the native extracellular matrix properties with specified surface topography as a biomimicry factor is necessary under ''the novel cell substrates development'' approaches. Our aim in the current study was to design, synthesize, and characterize a substrate with aligned nanometric arrays on the surface. The rapid and easy capabilities of Polydimethylsiloxane to receive chemical and physical characteristics with simple modifications, make it a promised candidate for the cell substrate. The obtained results from the atomic force and scanning electron microscopy showed the formation of 305±19 and 571±141 nanometers wrinkled nanoarrays after regulating the substrate under lateral traction during the plasma treatment times of 100 and 200s. Then, the behavior of a human foreskin fibroblast cell, in terms of adhesion, growth, viability, and morphology on this substrate was investigated. Increasing the plasma treatment time increased both nanoarray size and surface hydrophilicity, resulting in improved 17 and 46% of cell attachment quality, respectively. Additionally, the presence of the designed nanowrinkles surprisingly improved the number of the attached cells. The nanowrinkles caused the cells to align perfectly through the substrate's surface due to the contact guidance phenomena. Consequently, the biocompatible Polydimethylsiloxane substrate of this study with suitable chemical, mechanical, and physical properties showed fit capacities as a novel aligned cell culture platform.

Abstract Abstract. FZD7 receptor is considered as an emerging target for the treatment of Wnt-βcatenin related cancers. This transmembrane receptor is overexpressed in many cancer types like breast cancer and ovarian carcinoma, and so selective targeting of this receptor has a great therapeutic capacity. On the other hand, one of the mechanisms proposed for the anticancer effect of Atrial natriuretic peptide (ANP) that known as a heart hormone at first, is Wnt-βcatenin inhibition through an FZD dependent manner but, the molecular mechanism of this inhibition is not clear. Here, using computational methods including molecular docking and molecular dynamics simulation, also designing a cellular system that enabled us to trace Wnt-βcatenin kinetics directly, we investigated the mechanism of the peptide inhibitory potential against the pathway. Our computational results show that ANP can directly interact with FZD7 and also, its binding site on FZD7 overlap to the binding region of the Wnt carboxyl-terminal domain (Wnt-CTD). The finding of the silencing experiments demonstrates the dependency of Wnt-βcatenin signaling of the cellular system to FZD7. The decrease of βcatenin in cells treated to ANP and Wnt is also significant to compare to the control experiments. Finally, our results show that ANP is a potential scaffold to design selective peptide against FZD7.

Abstract Chemotherapy is one of the most effective and common treatments for cancer. Multi-drug resistance and drug side effects are one of the major obstacles to successful chemotherapy. To address these limitations and achieve better drug efficacy, nanosystem-based combination therapy offers a promising approach. This study aimed to synthesize, characterize, and investigate the synergistic effect of nanoliposomes loaded with doxorubicin and epigallocatechin-3-gallate (EGCG) on MCF-7 breast cancer cell lines. In the present study, nanoliposomes were prepared by passive loading and thin-film hydration. The characterization of nanoliposomes such as size distribution, zeta potential, the loading rate, drug release profile, and toxicity were measured. The mean diameter of nanoliposomes was 82.5 nm, their surface charge was -24.2 mV and drug loading was about 80%. The interaction of doxorubicin and EGCG with nanoliposomes was mediated by electrostatic and van der Waals bonds and EGCG has a deceasing effect on the doxorubicin release profile but the observed differences are not significant. The toxicity and viability data indicate that the simultaneous use of these two drugs increased the toxicity of the cells. Nanoliposomes containing doxorubicin were not able to reduce viability to below 50% in monotherapy with 5×10^-3 μM. While, the amount of viability dramatically reduced to below 50%, in combination with EGCG, resulting as combination chemotherapy. Consequently, the concomitant administration of EGCG with doxorubicin may be a suitable candidate for chemotherapy.

Abstract Total-Internal-Reflection Fluorescence Microscopy (TIRFM) is a useful tool to visualize and record the phenomena that happens below 100 nm thickness of the sample surface. This unique property of TIRFM help to perform a "qualitative" study of cytoskeleton near the cell-substrate contact. Here, distribution of actin filaments at cell-substrate interface was imaged by a TIRFM set up. Then, staining the actins cytoskeleton of the human melanoma cell and implementing the prism-based total-internal-reflection fluorescence microscope. A method to "quantify" distribution of fluorophores at cell-substrate contact is proposed.

Abstract Graphene quantum dots (GQDs) have attracted increasing attention due to their unique properties such as high water solubility, photoluminescence activity, good biocompatibility, physical, chemical and electrical properties which makes them appropriate candidates for use in a variety of bio-applications, sensors and photocatalysts. The objective of this study is synthesis of GQDs and improving their surface properties via chemical modification.
Here, urea and citric acid as carbon precursor were used. Citric acid was self-assembled into graphene framework via hydrothermal method at 160 °C for 4 h. Then, the synthesized GQDs were carbonized and chemically activated by KOH treatment. The surface area and pore structures of GQDs were analyzed by nitrogen adsorption/desorption isotherms. The results showed that the specific surface area of carbonized-activated graphene quantum dots (CA-GQDs) have been increased from 0.06 to 1204.0 m²/g and pore structures have been enhanced significantly. The XRD pattern of GQDs confirmed the basic structure of graphite layer. The TEM images indicated the unique morphology of GQDs and the sizes of GQDs were less than 5 nm. Thus, our applied method is an effective approach in the formation of GQDs with large BET surface area and narrow pore structures which reveals their potential applicability in biomedical field.

Abstract In view of the constant increase of nanotechnology and nanomaterials applications in our daily life, to determine whether they are safe, “in vitro” and “in vivo” screening methods are needed. Obviously, application of models that are similar to the physiological tissues process of the human body could be a better candidate. The three-dimensional spheroid method, spheroid were generated using commercial microplates, has many benefits (in comparison with traditional methods or monolayer cell culture) such as the growth of the cells in 3D, similar to the body's physiological tissue, an alternative for animal models, cell-to-cell interactions, and better cell signaling. In this study, the toxicity of silver nanoparticles by using three factors such as metabolic activity, live/dead assay, and spheroid surface area was evaluated using two different methods (2D vs 3D) under treatment with various concentrations of silver nanoparticles at different times. The results showed that different cells types, cancer and/or normal lung cells, have significant differences. In addition, it was observed that distinct differences in terms of cytotoxicity of silver nanoparticles between 2D and 3D culture systems and also the rate of growth/non-growth of spheroids are highly depended on cell type and various concentrations have fundamental importance in such studies. The present study provides evidence that cellular dimensions (3D vs 2D) play a pivotal role in the results and outcomes of inflammation and cytotoxicity with nanoparticles due to the spatial-temporal structure.

Abstract Diatoms biosilica shell, frustule, is substitute biostructures to mesoporous silica particles, which possesses their wide surfaces, nano-diameter porosity, mechanical strength, and thermal stability, optical capabilities, and the ability to bind to biomolecules can be used in biosensing applications. In this study, diatom species called Chaetoceros muelleri, was used for the fabrication of the Fe₂O₃-Au-Biosilica magnetic package. After micro-algae cultivation, the synthesis of gold nanoparticles (AuNPs) on silica walls was carried out using the bio-synthesis method, which evaluations have demonstrated the continuous formation of spherical AuNPs on the walls and its surfaces. After this step, the magnetic iron oxide nanoparticles were attached to the silica surface of the diatom, this, in turn, leads to system guiding using a magnetic field. Surface modification of diatoms magnetic complex, by using the APTES, allowed the attachment of fluorescence Rhodamine and the Herceptin antibody (Trastuzumab) to the structure. As well as the attachment of the fabricated system to target cells (SKBR3) was confirmed by fluorescence microscopic analysis. The results of this study indicate the ability and specificity of the diatom silicone shell as a "multipurpose" package for diagnostic and therapeutic activities.