Novel multifunctional tacrine-donepezil hybrids against Alzheimer's disease: Design synthesis and bioactivity studies.

A series of tacrine-donepezil hybrids were synthesized as potential multifunctional anti-Alzheimer's disease (AD) compounds. For this purpose, tacrine and the benzylpiperidine moiety of donepezil were fused with a hydrazone group to achieve a small library of tacrine-donepezil hybrids. In agreement with the design, all compounds showed inhibitory activity toward both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with IC50 values in the low micromolar range. Kinetic studies on the most potent cholinesterase (ChE) inhibitors within the series showed a mixed-type inhibition mechanism on both enzymes. Also, the docking studies indicated that the compounds inhibit ChEs by dual binding site (DBS) interactions. Notably, tacrine-donepezil hybrids also exhibited significant neuroprotection against H2O2-induced cell death in a differentiated human neuroblastoma (SH-SY5Y) cell line at concentrations close to their IC50 values on ChEs and showed high to medium blood-brain barrier (BBB) permeability on human cerebral microvascular endothelial cells (HBEC-5i). Besides, the compounds do not cause remarkable toxicity in a human hepatocellular carcinoma cell line (HepG2) and SH-SY5Y cells. Additionally, the compounds were predicted to also have good bioavailability. Among the tested compounds, H4, H16, H17, and H24 stand out with their biological profile. Taken together, the proposed novel tacrine-donepezil scaffold represents a promising starting point for the development of novel anti-ChE multifunctional agents against AD.

Affinity-Based Magnetic Nanoparticle Development for Cancer Stem Cell Isolation.

Cancer is still the leading cause of death in the world despite the developing research and treatment opportunities. Failure of these treatments is generally associated with cancer stem cells (CSCs), which cause metastasis and are defined by their resistance to radio- and chemotherapy. Although known stem cell isolation methods are not sufficient for CSC isolation, they also bring a burden in terms of cost. The aim of this study is to develop a high-efficiency, low-cost, specific method for cancer stem cell isolation with magnetic functional nanoparticles. This study, unlike the stem cell isolation techniques (MACS, FACS) used today, was aimed to isolate cancer stem cells (separation of CD133+ cells) with nanoparticles with specific affinity and modification properties. For this purpose, affinity-based magnetic nanoparticles were synthesized and characterized by providing surface activity and chemical reactivity, as well as making surface modifications necessary for both lectin affinity and metal affinity interactions. In the other part of the study, synthesized and characterized functional polymeric magnetic nanoparticles were used for the isolation of CSC from the human osteosarcoma cancer cell line (SAOS-2) with a cancer stem cell subpopulation bearing the CD133 surface marker. The success and efficiency of separation after stem cell isolation were evaluated via the MACS and FACS methods. As a result, when the His-graft-mg-p(HEMA) nanoparticle was used at a concentration of 0.1 µg/mL for 106 and 108 cells, superior separation efficiency to commercial microbeads was obtained.

Surface Area of Graphene Governs Its Neurotoxicity.

Due to their unique physicochemical properties, graphene and its derivatives are widely exploited for biomedical applications. It has been shown that graphene may exert different degrees of toxicity in in vivo or in vitro models when administered via different routes and penetrated through physiological barriers, subsequently being distributed within tissues or located within cells. In this study, in vitro neurotoxicity of graphene with different surface areas (150 and 750 m2/g) was examined on dopaminergic neuron model cells. SH-SY5Y cells were treated with graphene possessing two different surface areas (150 and 750 m2/g) in different concentrations between 400 and 3.125 µg/mL, and the cytotoxic and genotoxic effects were investigated. Both sizes of graphene have shown increased cell viability in decreasing concentrations. Cell damage increased with higher surface area. Lactate dehydrogenase (LDH) results have concluded that the viability loss of the cells is not through membrane damage. Neither of the two graphene types showed damage through lipid peroxidation (MDA) oxidative stress pathway. Glutathione (GSH) values increased within the first 24 and 48 h for both types of graphene. This increase suggests that graphene has an antioxidant effect on the SH-SY5Y model neurons. Comet analysis shows that graphene does not show genotoxicity on either surface area. Although there are many studies on graphene and its derivatives on their use with different cells in the literature, there are conflicting results in these studies, and most of the literature is focused on graphene oxide. Among these studies, no study examining the effect of graphene surface areas on the cell was found. Our study contributes to the literature in terms of examining the cytotoxic and genotoxic behavior of graphene with different surface areas.

Role of Intermediate Filaments in Blood-Brain Barrier in Health and Disease.

The blood-brain barrier (BBB) is a highly selective cellular monolayer unique to the microvasculature of the central nervous system (CNS), and it mediates the communication of the CNS with the rest of the body by regulating the passage of molecules into the CNS microenvironment. Limitation of passage of substances through the BBB is mainly due to tight junctions (TJ) and adherens junctions (AJ) between brain microvascular endothelial cells. The importance of actin filaments and microtubules in establishing and maintaining TJs and AJs has been indicated; however, recent studies have shown that intermediate filaments are also important in the formation and function of cell-cell junctions. The most common intermediate filament protein in endothelial cells is vimentin. Vimentin plays a role in blood-brain barrier permeability in both cell-cell and cell-matrix interactions by affecting the actin and microtubule reorganization and by binding directly to VE-cadherin or integrin proteins. The BBB permeability increases due to the formation of stress fibers and the disruption of VE-cadherin interactions between two neighboring cells in various diseases, disrupting the fiber network of intermediate filament vimentin in different ways. Intermediate filaments may be long ignored key targets in regulation of BBB permeability in health and disease.

In Vitro Human Blood-Brain Barrier Model for Drug Permeability Testing.

Blood-brain barrier (BBB), although very important for protection of brain from major neurotoxins, negatively affects the treatment of central nervous system diseases by limiting the passage of neuropharmaceuticals from blood to the brain. Thus, researchers have to investigate the passage of the produced drug molecules through the BBB before they are introduced to the market. Although these experiments have been traditionally performed on experimental animals, drug permeability tests are now carried out mostly by in vitro BBB models due to ethical problems, differences between species, and expensive and troublesome in vivo test procedures. In this method, we explain how to model and characterize a realistic in vitro BBB model using human derived cells and perform a drug permeability test using this model.

Coculture model of blood-brain barrier on electrospun nanofibers.

The blood-brain barrier (BBB) is a control mechanism that limits the diffusion of many substances to the central nervous system (CNS). In this study, we designed an in-vitro 3-dimensional BBB system to obtain a fast and reliable model to mimic drug delivery characteristics of the CNS. A support membrane of polycaprolactone nanofiber surfaces was prepared using electrospinning. After confirming the fiber morphology and size, endothelial cells (HUVEC) and glial cells were cultured on either side of this membrane. The model's similarity to in vivo physiology was tested with a home-designed transmembrane resistance (TR) device, with positive and negative control molecules. Finally, 2 doses of methotrexate (MTX), a chemotherapy agent, were applied to the model, and its permeability through the model was determined indirectly by a vitality test on the MCF-7 cell line. Nicotine, the positive control, completed its penetration through the model almost instantly, while albumin, the negative control, was blocked significantly even after 2 days. MTX reached a deadly threshold 24 h after application. The TR value of the model was promising, being around 260 ohm.cm2. The provided model proposes a disposable and reliable tool for investigating drug permeability through the BBB and has the potential to reduce the number of animal experiments.

Wet-electrospun PHBV nanofiber reinforced carboxymethyl chitosan-silk hydrogel composite scaffolds for articular cartilage repair.

The objective of the study was to produce three-dimensional and porous nanofiber reinforced hydrogel scaffolds that can mimic the hydrated composite structure of the cartilage extracellular matrix. In this regard, wet-electrospun poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanofiber reinforced carboxymethyl chitosan-silk fibroin (PNFs/CMCht-SF) hydrogel composite scaffolds that were chemically cross-linked by poly(ethylene glycol) diglycidyl ether (PEGDE) were produced. To the best of our knowledge, this is the first study in cartilage regeneration where a three dimensional porous spongy composite scaffold was obtained by the dispersion of wet-electrospun nanofibers within a polymer matrix. All of the produced hydrogel composite scaffolds had an interconnected microporous structure with well-integrated PHBV nanofibers on the pore walls. The scaffold comprising an equal amount of PEGDE and polymer (PNFs/CMCht-SF1:PEGDE1) demonstrated comparable water content (91.4 ± 0.7%), tan δ (0.183 at 1 Hz) and compressive strength (457 ± 85 kPa) values to that of articular cartilage. Besides, based on the histological analysis, this hydrogel composite scaffold supported the chondrogenic differentiation of bone marrow mesenchymal stem cells. Consequently, this hydrogel composite scaffold presented a great promise for cartilage tissue regeneration.

Development and characterization of cancer stem cell-based tumoroids as an osteosarcoma model.

Three-dimensional (3D) cancer tumor models are becoming vital approaches for high-throughput drug screening, drug targeting, development of novel theranostic systems, and personalized medicine. Yet, it is becoming more evident that the tumor progression and metastasis is fueled by a subpopulation of stem-like cells within the tumor that are also called cancer stem cells (CSCs). This study aimed to develop a tumoroid model using CSCs. For this purpose CD133+ cells were isolated from SaOS-2 osteosarcoma cell line with magnetic-activated cell sorting. To evaluate tumoroid formation ability, the cells were incubated in different cell numbers in agar gels produced by 3D Petri Dish® method. Subsequently, CD133+ cells and CD133- cells were co-cultured to investigate CD133+ cell localization in tumoroids. The characterization of tumoroids was performed using Live&Dead staining, immunohistochemistry, and quantitative polymerase chain reaction analysis. The results showed that, CD133+ , CD133- and SaOS-2 cells were all able to form 3D tumoroids regardless of the initial cell number, but, while 72 hr were needed for CD133+ cells to self-assemble, 24 hr were enough for CD133- and SaOS-2 cells. CD133+ cells were located within tumoroids randomly with high cell viability. Finally, when compared to two-dimensional (2D) cultures, there were 5.88, 4.14, 6.95, and 1.68-fold higher messenger RNA expressions for Sox2, OCT3/4, Nanog, and Nestin, respectively, in CD133+ cells that were cultured within 3D tumoroids, showing longer maintenance of stem cell phenotype in 3D, that can allow more relevant screening and targeting efficiency in pharmaceutical testing. It was concluded that CSC-based tumoroids are propitious as 3D tumor models to fill the gap between conventional 2D in vitro culture and in vivo animal experiments for cancer research.

Mechanobiology of cells and cell systems, such as organoids.

Organoids are in vitro 3D self-organizing tissues that mimic embryogenesis. Organoid research is advancing at a tremendous pace, since it offers great opportunities for disease modeling, drug development and screening, personalized medicine, as well as understanding organogenesis. Mechanobiology of organoids is an unexplored area, which can shed light to several unexplained aspects of self-organization behavior in organogenesis. It is becoming evident that collective cell behavior is distinctly different from individual cells' conduct against certain stimulants. Inherently consisting of higher number of degrees of freedom for cell motility and more complex cell-to-cell and cell-to-extracellular matrix behavior, understanding mechanotransduction in organoids is even more challenging compared with cell communities in 2D culture conditions. Yet, deciphering mechanobiology of organoids can help us understand effects of mechanical cues in health and disease, and translate findings of basic research toward clinical diagnosis and therapy.

The use of bacterial cellulose as a basement membrane improves the plausibility of the static in vitro blood-brain barrier model.

There are several blood-brain barrier (BBB) models available for pharmaceutical research, but none of those are able to properly imitate the permeability of this special barrier. In this study, it is aimed to produce different BBB models with different cellular combinations and different basement membrane polymers, such as polyethylene terephthalate (PET) and bacterial cellulose (BC), which has not been used for BBB models before, to compare their barrier properties. Primary human brain microvascular endothelial cells were seeded on the luminal side and primary human astrocytes and/or primary human brain microvascular pericytes were seeded on the abluminal side of the membranes. Immunofluorescence (IF) staining results indicate that the expression of tight and adherence junction proteins increases on the 5th day of the cultivation. In accordance with Live-Dead staining results, IF images show that cells in the model lose their viability before the 10th day. Transendothelial electrical resistance (TEER) measurements indicate that BC membrane leads to statistically higher (p < 0.05) TEER values than the standard Transwell PET insert membrane. Sucrose and caffeine permeability values of all models are close to in vivo values. BC shows potential to be used as a more reliable basement membrane for BBB models for pharmaceutical research.

A bilayer scaffold prepared from collagen and carboxymethyl cellulose for skin tissue engineering applications.

Treatment of chronic skin wound such as diabetic ulcers, burns, pressure wounds are challenging problems in the medical area. The aim of this study was to design a bilayer skin equivalent mimicking the natural one to be used as a tissue engineered skin graft for use in the treatments of problematic wounds, and also as a model to be used in research related to skin, such as determination of the efficacy of transdermal bioactive agents on skin cells and treatment of acute skin damages that require immediate response. In this study, the top two layers of the skin were mimicked by producing a multilayer construct combining two different porous polymeric scaffolds: as the dermis layer a sodium carboxymethyl cellulose (NaCMC) hydrogel on which fibroblasts were added, and as the epidermis layer collagen (Coll) or chondroitin sulfate-incorporated collagen (CollCS) on which keratinocytes were added. The bilayer construct was designed to allow cross-talk between the two cell populations in the subsequent layers and achieves paracrine signalling. It had interconnected porosity, high water content, appropriate stability and elastic moduli. Expression of vascular endothelial growth factor (VEGF), basic-fibroblast growth factor (bFGF) and Interleukin 8 (IL-8), and the production of collagen I, collagen III, laminin and transglutaminase supported the attachment and proliferation of cells on both layers of the construct. Attachment and proliferation of fibroblasts on NaCMC were lower compared to performance of keratinocyte on collagen where keratinocytes created a dense and a stratified layer similar to epidermis. The resulting constructs succesfully mimicked in vitro the natural skin tissue. They are promising as grafts for use in the treatment of deep wounds and also as models for the study of the efficacy of bioactive agents on the skin.