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Human retinal pigment epithelial (RPE) cells express the transmembrane Ca2+-dependent Cl− channel bestrophin-1 (hBest1) of the plasma membrane. Mutations in the hBest1 protein are associated with the development of distinct pathological conditions known as bestrophinopathies. The interactions between hBest1 and plasma membrane lipids (cholesterol (Chol), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and sphingomyelin (SM)) determine its lateral organization and surface dynamics, i.e., their miscibility or phase separation. Using the surface pressure/mean molecular area (π/A) isotherms, hysteresis and compressibility moduli (Cs−1) of hBest1/POPC/Chol and hBest1/SM/Chol composite Langmuir monolayers, we established that the films are in an LE (liquid-expanded) or LE-LC (liquid-condensed) state, the components are well-mixed and the Ca2+ ions have a condensing effect on the surface molecular organization. Cholesterol causes a decrease in the elasticity of both films and a decrease in the ΔGmixπ values (reduction of phase separation) of hBest1/POPC/Chol films. For the hBest1/SM/Chol monolayers, the negative values of ΔGmixπ are retained and equalized with the values of ΔGmixπ in the hBest1/POPC/Chol films. Shifts in phase separation/miscibility by cholesterol can lead to changes in the structure and localization of hBest1 in the lipid rafts and its channel functions.
Polyelectrolyte multilayer coatings (PEM) are prepared by alternative layer-by-layer deposition of cationic and anionic polyelectrolyte monolayers on charged surfaces. The thickness of the coatings ranges from nm to few μm. Their properties such as roughness, stiffness, surface charge and surface energy can be precisely tuned to fulfil different technical or biological requirements. The coating process is based on self-assembly of polyelectrolytes. Advantages of these coatings are their easy handling, no harsh chemistry and the possibility for coatings on complex geometries. The PEM coatings can be prepared from a variety of suitable polyelectrolytes. Their stability varies from very durable PEM coatings that are only soluble in strong solvents to quickly degradable, which may be applied as drug release system. One example of such a degradable PEM system is the one based on the polyelectrolyte pair Hyaluronan (HA) and Chitosan (CHI). These biopolymers originate from natural sources and show low toxicity towards human cells. However, HA/CHI multilayers show only weak adhesiveness for human umbilical vein endothelial cells (HUVEC). In this article, we summarize our approaches to enhance the HA/CHI multilayer by incorporation of a non-polymer substance –graphene oxide– to improve the cell adhesion and keep such properties as low cytotoxicity and biodegradability. Different approaches for incorporation of graphene oxide were performed and the cellular adhesion was tested by metabolic assay.
Human bestrophin-1 protein (hBest1) is a transmembrane channel associated with the calcium-dependent transport of chloride ions in the retinal pigment epithelium as well as with the transport of glutamate and GABA in nerve cells. Interactions between hBest1, sphingomyelins, phosphatidylcholines and cholesterol are crucial for hBest1 association with cell membrane domains and its biological functions. As cholesterol plays a key role in the formation of lipid rafts, motional ordering of lipids and modeling/remodeling of the lateral membrane structure, we examined the effect of different cholesterol concentrations on the surface tension of hBest1/POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and hBest1/SM Langmuir monolayers in the presence/absence of Ca2+ ions using surface pressure measurements and Brewster angle microscopy studies. Here, we report that cholesterol: (1) has negligible condensing effect on pure hBest1 monolayers detected mainly in the presence of Ca2+ ions, and; (2) induces a condensing effect on composite hBest1/POPC and hBest1/SM monolayers. These results offer evidence for the significance of intermolecular protein–lipid interactions for the conformational dynamics of hBest1 and its biological functions as multimeric ion channel.
Medical implants play a central role in modern medicine and both, naturally derived and synthetic materials have been explored as biomaterials for such devices. However, when implanted into living tissue, most materials initiate a host response. In addition, implants often cause bacterial infections leading to complications. Polyelectrolyte multilayer (PEM) coatings can be used for functionalization of medical implants improving the implant integration and reducing foreign body reactions. Some PEMs are also known to show antibacterial properties. We developed a PEM coating suggesting that it can decrease the risk of bacterial infections occurring after implantation while being highly biocompatible. We applied two different standard tests for evaluating the PEM’s antibacterial properties, the ISO norm (ISO 22196) and one ASTM norm (ASTM E2180) test. We found a reduction of bacterial growth on the PEM but to a different degree depending on the testing method. This result demonstrates the need for defining proper method to evaluate antibacterial properties of surface coatings.
High moisture permeability, excellent mechanical properties in a wet state, high water-holding capability, and high exudate absorption make bacterial nanocellulose (BNC) a favorable candidate for biomedical device production, especially wound dressings. The lack of antibacterial activity and healing-promoting ability are the main drawbacks that limit its wide application. Pullulan (Pul) is a nontoxic polymer that can promote wound healing. Zinc oxide nanoparticles (ZnO-NPs) are well-known as a safe antibacterial agent. In this study, aminoalkylsilane was chemically grafted on a BNC membrane (A-g-BNC) and used as a bridge to combine BNC with Pul-ZnO-NPs hybrid electrospun nanofibers. FTIR results confirmed the successful production of A-g-BNC/Pul-ZnO. The obtained dressing demonstrated blood clotting performance better than that of BNC. The dressing showed an ability to release ZnO, and its antibacterial activity was up to 5 log values higher than that of BNC. The cytotoxicity of the dressing toward L929 fibroblast cells clearly showed safety due to the proliferation of fibroblast cells. The animal test in a rat model indicated faster healing and re-epithelialization, small blood vessel formation, and collagen synthesis in the wounds covered by A-g-BNC/Pul-ZnO. The new functional dressing, fabricated with a cost-effective and easy method, not only showed excellent antibacterial activity but could also accelerate wound healing.
The effect of Hofmeister anions on the surface properties of polyelectrolyte multilayers built from hyaluronan and chitosan by layer-by-layer deposition is studied by ellipsometry and atomic force microscopy. The thickness, roughness and morphology of the resulting coatings were found to depend on the type of the anion. Relationship between the surface properties and the biological response of the polyelectrolyte multilayers is established by assessing the degree of protein (albumin) adsorption.
Human bestrophin-1 (hBest1) is a transmembrane Ca2+- dependent anion channel, associated with the transport of Cl−, HCO3- ions, γ-aminobutiric acid (GABA), glutamate (Glu), and regulation of retinal homeostasis. Its mutant forms cause retinal degenerative diseases, defined as Bestrophinopathies. Using both physicochemical - surface pressure/mean molecular area (π/A) isotherms, hysteresis, compressibility moduli of hBest1/sphingomyelin (SM) monolayers, Brewster angle microscopy (BAM) studies, and biological approaches - detergent membrane fractionation, Laurdan (6-dodecanoyl-N,N-dimethyl-2-naphthylamine) and immunofluorescence staining of stably transfected MDCK-hBest1 and MDCK II cells, we report:
1) Ca2+, Glu and GABA interact with binary hBest1/SM monolayers at 35 °C, resulting in changes in hBest1 surface conformation, structure, self-organization and surface dynamics. The process of mixing in hBest1/SM monolayers is spontaneous and the effect of protein on binary films was defined as “fluidizing”, hindering the phase-transition of monolayer from liquid-expanded to intermediate (LE-M) state;
2) in stably transfected MDCK-hBest1 cells, bestrophin-1 was distributed between detergent resistant (DRM) and detergent-soluble membranes (DSM) - up to 30 % and 70 %, respectively; in alive cells, hBest1 was visualized in both liquid-ordered (Lo) and liquid-disordered (Ld) fractions, quantifying protein association up to 35 % and 65 % with Lo and Ld. Our results indicate that the spontaneous miscibility of hBest1 and SM is a prerequisite to diverse protein interactions with membrane domains, different structural conformations and biological functions.
Controlling the surface properties and structure of thin nanosized coatings is of primary importance in diverse engineering and medical applications. Here we report on how the nanostructure, growth mechanism, thickness, roughness, and hydrophilicity of nanocomposites composed of weak natural or strong synthetic polyelectrolytes (PE) can be tailored by graphene oxide (GO) doping. GO reverses the build‐up mechanism affecting the internal structure and the hydrophilicity in a way depending on the type of the PE‐matrix. The extent of GO‐adsorption and its impact on the surface morphology was found to be independent on the type of the underlying PE‐matrix. The nanostructure of the hybrid films is not significantly altered when a single surface‐exposed GO‐layer is deposited, while increasing the number of embedded GO‐layers leads to pronounced surface heterogeneity. These results are expected to have valuable impact on the construction strategies of coatings with tunable surface properties.
Neurodegenerative disorders (NDDs) are complex, multifactorial disorders with significant social and economic impact in today’s society. NDDs are predicted to become the second-most common cause of death in the next few decades due to an increase in life expectancy but also to a lack of early diagnosis and mainly symptomatic treatment. Despite recent advances in diagnostic and therapeutic methods, there are yet no reliable biomarkers identifying the complex pathways contributing to these pathologies. The development of new approaches for early diagnosis and new therapies, together with the identification of non-invasive and more cost-effective diagnostic biomarkers, is one of the main trends in NDD biomedical research. Here we summarize data on peripheral biomarkers, biofluids (cerebrospinal fluid and blood plasma), and peripheral blood cells (platelets (PLTs) and red blood cells (RBCs)), reported so far for the three most common NDDs—Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS). PLTs and RBCs, beyond their primary physiological functions, are increasingly recognized as valuable sources of biomarkers for NDDs. Special attention is given to the morphological and nanomechanical signatures of PLTs and RBCs as biophysical markers for the three pathologies. Modifications of the surface nanostructure and morphometric and nanomechanical signatures of PLTs and RBCs from patients with AD, PD, and ALS have been revealed by atomic force microscopy (AFM). AFM is currently experiencing rapid and widespread adoption in biomedicine and clinical medicine, in particular for early diagnostics of various medical conditions. AFM is a unique instrument without an analog, allowing the generation of three-dimensional cell images with extremely high spatial resolution at near-atomic scale, which are complemented by insights into the mechanical properties of cells and subcellular structures. Data demonstrate that AFM can distinguish between the three pathologies and the normal, healthy state. The specific PLT and RBC signatures can serve as biomarkers in combination with the currently used diagnostic tools. We highlight the strong correlation of the morphological and nanomechanical signatures between RBCs and PLTs in PD, ALS, and AD.
Morphometry and stiffness of red blood cells - signatures of neurodegenerative diseases and aging
(2022)
Human red blood cells (RBCs) are unique cells with the remarkable ability to deform, which is crucial for their oxygen transport function, and which can be significantly altered under pathophysiological conditions. Here we performed ultrastructural analysis of RBCs as a peripheral cell model, looking for specific signatures of the neurodegenerative pathologies (NDDs) - Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS) and Alzheimer’s disease (AD), utilizing atomic force (AFM) and conventional optical (OM) microscopy. We found significant differences in the morphology and stiffness of RBCs isolated from patients with the selected NDDs and those from healthy individuals. Neurodegenerative pathologies’ RBCs are characterized by a reduced abundance of biconcave discoid shape, lower surface roughness and a higher Young’s modulus, compared to healthy cells. Although reduced, the biconcave is still the predominant shape in ALS and AD cells, while the morphology of PD is dominated by crenate cells. The features of RBCs underwent a marked aging-induced transformation, which followed different aging pathways for NDDs and normal healthy states. It was found that the diameter, height and volume of the different cell shape types have different values for NDDs and healthy cells. Common and specific morphological signatures of the NDDs were identified.