Non-thermal plasma modulated l-tyrosine self-assemblies: a potential avenue for fabrication of supramolecular self-assembled biomaterials.

Aromatic amino acids (AAs) have garnered particular interest due to their pivotal roles in numerous biological processes and disorders. Variations in AA self-assembly not only affect protein structures and functions, but their non-covalent interactions such as hydrogen bonding, van der Waals forces, and π-π stacking, yield versatile assemblies vital in bio-inspired material fabrication. Tyrosine (Tyr), a non-essential aromatic amino acid, holds multifaceted significance in the body as a protein building block, neurotransmitter precursor, thyroid hormone contributor, and melanin synthesis regulator. The proficiency of Cold Atmospheric Plasma (CAP) in generating a spectrum of reactive oxygen and nitrogen species has spurred innovative research avenues in the studies of biomolecular components, including its potential for targeted cancer cell ablation and biomolecule modification. In this work, we have assessed the chemical as well as the structural changes in Tyrosine-derived self-assembled structures arising from the CAP-induced reactive species. For a comprehensive understanding of the mechanism, different treatment times, feed gases, and the role of solvent acidification are compared using various spectroscopic and microscopic techniques. LC-ESI-QQQ mass spectra unveiled the emergence of oxygenated and nitro derivatives of l-tyrosine following its interaction with CAP-derived ROS/RNS. SEM and TEM images demonstrated an enhanced surface size of self-assembled structures and the formation of novel nanomaterial-shaped assemblies following CAP treatment. Overall, this study aims to explore CAP's interaction with a single-amino acid, hypothesizing the creation of novel supramolecular structures and scrutinizing CAP-instigated transformations in l-tyrosine self-assembled structures, potentially advancing biomimetic-attributed nanomaterial fabrication which might present a novel frontier in the field of designing functional biomaterials.

Polyoxometalates Mediated Amyloid Fibrillation Dynamics and Restoration of Enzyme Activity of Hen Egg White Lysozyme Treated under Cold Atmospheric Pressure Plasma.

Neurodegenerative disorders are one of the most devastating disorders worldwide. Although a definite mechanistic pathway of neurodegenerative disorders is still not clear, it is almost clear that these diseases are initiated by protein misfolding. Hen Egg White Lysozyme (Lyz) can be converted to highly arranged amyloid fibrils and is therefore considered a good model protein for studying protein aggregation in connection to neurodegeneration. In this study, Lyz has been converted to fibrils using He-air gas fed single jet cold atmospheric plasma (CAP). The reactive oxygen species and the reactive nitrogen species produced by the plasma jet interact with the protein molecules and enhance the fibril formation. We monitored the fibrillation kinetics with the Thioflavin T (ThT) assay and observed that fibrils are formed when the samples are treated for 10 min with He-air gas fed CAP. Further, we studied the role of a special class of inorganic nanomaterials called polyoxometalates (POMs) in the process of the Lyz fibrillation using various biophysical techniques. The Keggin POMs used in this study are phosphomolybdic acid (PMA) and silico molybdic acid (SMA). Keggin POMs bring in structural self-assembly of the protein and disrupt the fibrils as evidenced in the ThT assay and TEM analysis. Molecular docking studies together with electrokinetic potential studies show the interactions between POMs and Lyz dominated via hydrogen bonding and electrostatic interactions. The enzyme activity of Lyz was assessed using the substrate Micrococcus lysodeikticus and after treatment with POMs results showed a significant increase in the activity. This study could pave way for looking into Keggin POMs for possible application in neurodegeneration.

Investigating the impact of inbuilt cold atmospheric pressure plasma on molecular assemblies of tryptophan enantiomers: in vitro fabrication of self-assembled supramolecular structures.

The advancements in understanding the phenomenon of plasma interactions with matter, coupled with the development of CAPP devices, have resulted in an interdisciplinary research topic of significant importance. This has led to the integration of various fields of science, including plasma physics, chemistry, biomedical sciences, and engineering. The reactive oxygen species and reactive nitrogen species generated from cold atmospheric plasma on interaction with biomolecules like proteins and peptides form various supramolecular structures. CAPP treatment of amino acids, which are the fundamental building blocks of proteins, holds potential in creating self-assembled supramolecular architectures. In this work, we demonstrate the process of self-assembly of aromatic amino acid tryptophan (Trp) enantiomers (l-tryptophan and d-tryptophan) into ordered supramolecular assemblies induced by the reactive species generated by a cold atmospheric pressure helium plasma jet. These enantiomers of tryptophan form organized structures as evidenced by FE-SEM. To assess the impact of CAPP treatment on the observed assemblies, we employed various analytical techniques such as zeta potential, dynamic light scattering and FTIR spectroscopy. Also, photoluminescence and time-resolved lifetime measurements revealed the transfiguration of individual Trp enantiomers. The LC-ESI-QTOF-MS analysis demonstrated that CAPP irradiation led to the incorporation of oxygenated ions into the pure Trp molecule. These studies of the self-assembly of Trp due to ROS and RNS interactions will help us to understand the assembly environment. This knowledge may be utilized to artificially design and synthesize highly ordered functional supramolecular structures using CAPP.

Self-cross-linked starch/chitosan hydrogel as a biocompatible vehicle for controlled release of drug.

A facile one-pot approach was adopted to prepare a polysaccharide-based hydrogel of oxidized starch (OS)-chitosan. The synthetic monomer-free, eco-friendly hydrogel was prepared in an aqueous solution and employed for controlled drug release application. The starch was first oxidized under mild conditions to prepare its bialdehydic derivative. Subsequently, the amino group-containing a modified polysaccharide, "chitosan" was introduced on the backbone of OS via a dynamic Schiff-base reaction. The bio-based hydrogel was obtained via a one-pot in-situ reaction, where functionalized starch acts as a macro-cross-linker that contributes structural stability and integrity to the hydrogel. The introduction of chitosan contributes to stimuli-responsive properties and thus pH-sensitive swelling behavior was obtained. The hydrogel showed its potential as a pH-dependent controlled drug release system and a maximum of 29 h sustained release period was observed for ampicillin sodium salt drug. In vitro studies confirmed that the prepared drug-loaded hydrogels showed excellent antibacterial ability. Most importantly, the hydrogel could find potential use in the biomedical field due to its facile reaction conditions, biocompatibility along with controlled releasing ability of the encapsulated drug.

Cold atmospheric plasma driven self-assembly in serum proteins: insights into the protein aggregation to biomaterials.

The self-assembly of proteins is crucial in many biomedical applications. This work deals with understanding the role of cold atmospheric plasma (CAP) on the self-assembly of two different proteins present in the serum - BSA and hemoglobin and to elucidate the process associated with the direct application of physical plasma on or in the human (or animal) body, which has implications in therapeutics. The work has been corroborated by several spectroscopic studies such as fluorescence spectroscopy, circular dichroism spectroscopy, and SEM analysis. Through steady-state fluorescence spectroscopy and by following the tryptophan fluorescence, we observed that the emission intensity was quenched for the protein when treated with plasma radiation. Circular dichroism spectroscopy revealed that the structure of the protein was altered both in the case of BSA and hemoglobin. N-Acetyl tryptophanamide (NATA), which resembles the tryptophan in the protein, was treated with CAP and we observed the similar quenching of fluorescence as in the proteins, indicating that the protein underwent self-assembly. Time-resolved fluorescence spectroscopy with a decrease in the lifetime revealed that the protein self-assembly was promoted with CAP treatment, which was also substantiated by SEM micrographs. The ROS/RNS produced in the CAP has been correlated with the protein self-assembly. This work will help to design protein self-assembled systems, and in the future, may bring possibilities of creating novel biomaterials with the help of plasma radiation.

Cold atmospheric pressure plasma for attenuation of SARS-CoV-2 spike protein binding to ACE2 protein and the RNA deactivation.

Cold atmospheric pressure (CAP) plasma has a profound effect on protein-protein interactions. In this work, we have highlighted the deactivation of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike protein by CAP plasma treatment. Complete deactivation of spike protein binding to the human ACE2 protein was observed within an exposure time of 5 minutes which is correlated to the higher concentration of hydrogen peroxide formation due to the interaction with the reactive oxygen species present in the plasma. On the other hand, we have established that CAP plasma is also capable of degrading RNA of SARS-CoV-2 virus which is also linked to hydrogen peroxide concentration. The reactive oxygen species is produced in the plasma by using noble gases such as helium, in the absence of any other chemicals. Therefore, it is a green process with no chemical waste generated and highly advantageous from the environmental safety prospects. Results of this work could be useful in designing plasma-based disinfection systems over those based on environmentally hazardous chemical-based disinfection and biomedical applications.

Insight into carbon quantum dot-vesicles interactions: role of functional groups.

Understanding carbon quantum dot-cell membrane interaction is essential for designing an effective nanoparticle-based drug delivery system. In this study, an attempt has been made to study the interaction involving phosphatidylcholine vesicles (PHOS VES, as model cell membrane) and four different carbon quantum dots bearing different functional groups (-COOH, -NH2, -OH, and protein bovine serum albumin coated) using various tools such as PL behavior, surface charge on vesicles, QCM, ITC, TEM, LSV, and FTIR. From the above studies, it was observed that the -NH2 terminating carbon dots were capable of binding strongly with the vesicles whereas other functional groups bearing carbon dots were not significantly interacting. This observation was also supported by direct visual evidence as shown by transmission electron microscopy, which shows that the polyethyleneimine carbon dot (PEICD) bearing -NH2 functionality has greater affinity towards PHOS VES. The mechanistic insight presented in the paper indicates greater possibility of higher H-bonding, signifying better interaction between -NH2 functionalized carbon dots and PHOS VES supported by FTIR, QCM, ITC and TEM. Moreover, the transport of neurotransmitters (which are generally amine compound) in neurons for cellular communication through synapse is only possible through vesicular platforms, showing that in our body, such interactions are already present. Such studies on the nano-bio interface will help biomedical researchers design efficient carbon-based nanomaterial as drug/gene delivery vehicles.

Functional characterisation of bioactive peptide derived from terrestrial snail Cryptozona bistrialis and its wound-healing property in normal and diabetic-induced Wistar albino rats.

A peptide might be an exciting biomaterial or template for the development of novel wound-healing agents. In this report, it was isolated from the terrestrial snail Cryptozona bistrialis by enzymatic digestion and was evaluated for its in vitro wound-healing activity in NIH/3T3 mouse fibroblasts cell line and in vivo wound-healing activity in normal and diabetic-induced Wistar albino rats. The C. bistrialis protein was digested by the papain enzyme, and 21.79 kDa peptide (Cb-peptide) was purified by reversed-phase high-performance liquid chromatography and identified by MALDI (matrix-assisted laser desorption/ionization)-TOF analysis. The isolated Cb-peptide was characterised by various analytical methods. The peptide demonstrated a capacity to prevent the development of pathogenic bacterial and fungal cultures and proved that it promotes significant wound-healing activity in the wound scratch assay method by rapid cell migration and closure of wound. Isolated Cb-peptide was lyophilised and formulated to ointment and analysed for in vivo wound-healing activity in normal and diabetic (alloxan monohydrate)-induced Wistar albino rats. Cb-peptide ointment-treated groups showed a greater degree of wound healing and early and complete period of epithelialisation in normal and diabetic-induced Wistar albino rats. Cb-peptide ointment-treated groups showed significant excision and incision wound-healing activity. A conclusion was reached that the peptide isolated from C. bistrialis showed greater wound-healing activity compared with vehicle control and standard control.

Advanced glycation end products induce differential structural modifications and fibrillation of albumin.

Glycation induced amyloid fibrillation is fundamental to the development of many neurodegenerative and cardiovascular complications. Excessive non-enzymatic glycation in conditions such as hyperglycaemia results in the increased accumulation of advanced glycation end products (AGEs). AGEs are highly reactive pro-oxidants, which can lead to the activation of inflammatory pathways and development of oxidative stress. Recently, the effect of non-enzymatic glycation on protein structure has been the major research area, but the role of specific AGEs in such structural alteration and induction of fibrillation remains undefined. In this study, we determined the specific AGEs mediated structural modifications in albumin mainly considering carboxymethyllysine (CML), carboxyethyllysine (CEL), and argpyrimidine (Arg-P) which are the major AGEs formed in the body. We studied the secondary structural changes based on circular dichroism (CD) and spectroscopic analysis. The AGEs induced fibrillation was determined by Congo red binding and examination of scanning and transmission electron micrographs. The amyloidogenic regions in the sequence of BSA were determined using FoldAmyloid. It was observed that CEL modification of BSA leads to the development of fibrillar structures, which was evident from both secondary structure changes and TEM analysis.

Role of Interfacial Viscosity and pH in L-Phenylalanine, L-Tryptophan Molecular Rotors.

Protein folding involves the aminoacid sequence to come forth and form an energy minimized structure. Recently molecular crowding leading to increase in viscosity is said to be one of the major concerns affecting protein folding. Many external fluorescent probes are used to detect such increases in viscosity. Since most of the protein sequences contain L-Phe and L-Trp, in this study we have used these aminoacids as probes to detect changes in viscosity. This study will help to advance the knowledge on molecular crowding effects in protein folding.

Does L to D-amino acid substitution trigger helix→sheet conformations in collagen like peptides adsorbed to surfaces?

The present work reports on the structural order, self assembling behaviour and the role in adsorption to hydrophilic or hydrophobic solid surfaces of modified sequence from the triple helical peptide model of the collagenase cleavage site in type I collagen (Uniprot accession number P02452 residues from 935 to 970) using (D)Ala and (D)Ile substitutions as given in the models below: Model-1: GSOGADGPAGAOGTOGPQGIAGQRGVV GLOGQRGER. Model-2: GSOGADGP(D)AGAOGTOGPQGIAGQRGVVGLOGQRGER. Model-3: GSOGADGPAGAOGTOGPQG(D)IAGQRGVVGLOGQRGER. Collagenase is an important enzyme that plays an important role in degrading collagen in wound healing, cancer metastasis and even in embryonic development. However, the mechanism by which this degradation occurs is not completely understood. Our results show that adsorption of the peptides to the solid surfaces, specifically hydrophobic triggers a helix to beta transition with order increasing in peptide models 2 and 3. This restricts the collagenolytic behaviour of collagenase and may find application in design of peptides and peptidomimetics for enzyme-substrate interaction, specifically with reference to collagen and other extra cellular matrix proteins.

Role of viscogens on the macromolecular assemblies of fibrinogen at liquid/air and solid/air interfaces.

In this study, an attempt has been made to understand the organization and association of fibrinogen (Fg) in solvent environment induced by viscogens such as 1-ethyl 3-methyl imidazolium ethyl sulfate (IL-emes), Ficoll, and Trehalose. The author observed that Fg in IL-emes adsorbed on solid surface shows higher ß-sheet conformation. Shear viscosity measured using quartz crystal microbalance, for Fg in IL-emes was highest with a corresponding higher adsorbed mass 3.26 µg/cm(2). Associated assemblies of the protein at the liquid/air interface were monitored with changes in surface tension and were used to calculate work of adhesion. Changes in work of adhesion were used as a tool to measure the adsorption of Fg to solid surfaces in presence of viscogens and highest adsorption was observed for hydrophilic surfaces. Scanning electron microscopy images show Fg in trehalose forms elongated bead like structures implying organization of the protein at the interface. Crowding in the solvent environment induced by viscogens can slow down organization of Fg, leading to macromolecular assemblies near the interface.

Microviscosity-induced conformational transition in ß-lactoglobulin in the presence of an ionic liquid.

This study reports on the helix-beta conformation transition of bovine ß-lactoglobulin (ßLG) prepared at two different pH conditions (pH 4 and 7.5) and in the presence of the ionic liquid 1-ethyl-3-methylimidazolium ethyl sulfate (IL-emes). The investigation was carried out by combining a range of techniques such as circular dichroic (CD) spectroscopy, steady-state fluorescence spectroscopy, isothermal titration calorimetry (ITC), and transmission electron microscopy. The influence of microviscosity induced by IL-emes on the secondary structure of ßLG was studied using a quartz crystal microbalance and correlated with the steady-state fluorescence emission. The effect of heat on the helix-beta transition in ßLG was directly measured by ITC by titrating ßLG with IL-emes. The net effect of heat after subtraction of the heat of dilution was negative in both cases, suggesting that the protein moves to a stable conformation. The changes in the overall aggregated structures were confirmed by transmission electron microscopy, where a shift in the size and morphology of aggregates was found, from large clusters (size of 70 nm) at pH 4 to smaller aggregates (size of 20 nm) at pH 7.5, which reduced to 7 nm in the presence of the IL. The transformation of helical to beta structure at pH 4 show that the folding pathway in the presence of the ionic liquid is hierarchical, whereas at neutral pH, it appeared to be nonhierarchical and the final native structure was acquired by nonlocal interactions through typical forces involved in the stabilization of the tertiary structure.

Reversible and irreversible conformational transitions in myoglobin: role of hydrated amino acid ionic liquid.

Hydrated phenylalanine ionic liquid (Phe-IL) has been used to solubilize myoglobin (Mb). Structural stability of Mb in Phe-IL analyzed using fluorescence and circular dichroism spectroscopy shows that for low levels of hydration of Phe-IL there is a large red shift in the fluorescence emission wavelength and the protein transforms to complete ß sheet from its native helical conformation. Rehydration or dilution reverses the ß sheet to an α helix which on aging organizes to micrometer-sized fibrils. At concentrations higher than 200 µM, the protein changes from ß to a more random coiled structure. Organization of the protein in Phe-IL in a Langmuir film at the air/water interface has been investigated using the surface pressure-molecular area isotherm and shows nearly the same surface tension for both pure Mb and Mb in Phe-IL. Scanning electron microscopy of the films of Mb in Phe-IL transferred using the Langmuir-Blodgett film technique show layered morphology. This study shows that the conformation of Mb is completely reversible going from ß â†’ helix → ß sheet up to 200 µM of Phe-IL. Similar surface tension values for Mb in water and in Phe-IL suggests that direct ion binding interactions with the protein coupled with the change in local viscosity from the IL seems to not only alter the secondary structure of individual proteins but also drives the self-assembly of the protein molecules leading finally to fibril formation.

Evaluation of hydropathy of amino acids from a comparison of their viscosities inside vesicles and on supported lipid bilayers.

The viscosity of amino acids enclosed in giant lipid vesicles (η(out)) subjected to a shear flow near a solid surface has been studied using quartz crystal microbalance (QCM). This viscosity has been compared with shear viscosity for the different amino acids adsorbed on supported bilayers (SLBs) (η(in)) of the lipids on quartz. Using a first approximation of vesicles as model rigid spheres, the measured viscosities and the extent of deformation of vesicles observed using optical microscopy, two non-dimensional parameters: the reduced volume and the ratio of (η(in))/(η(out)) have been analyzed as a function of physical parameters: vesicle substrate distance (vesicle vs. supported lipid bilayers), vesicle size and their variation as a function of the viscosity. The kinematics of the vesicles with the amino acids compared with the shear at supported lipid bilayers seems to describe a reasonable hydropathy scale for the amino acids. The results show that there is a direct correlation between the above parameters and the polarity variations in amino acids suggesting that the viscous force may be an important parameter and should be taken into account in studies on membrane proteins interacting with cells and cell adhesion in flow chambers where cell membrane and the adhesive substrate are in relative motion.

Interfacial viscoelasticity of myoglobin at air/water and air/solution interfaces: role of folding and clustering.

This study describes the folding and organization of myoglobin (Mb) at the solution/air interface at different pH values of 2.5, 3.5, 5.5, 7.5, and 8.5. Dynamic surface tension and the associated dilational and shear viscoelasticity for Mb at these pH's have been studied using a sinusoidal surface compression and expansion for frequencies ranging from 0.01 to 0.4 Hz. The changes in dilational viscosity, elasticity, and fluorescence lifetime measurements have been related to the conformational changes of the protein films at the interface. It is observed that while acid-induced denaturation of the protein does not lead to large changes in dilational properties, the shear properties on the other hand are strongly influenced by it, and the protein behaves like a shear-thickening fluid. At higher pH, particularly at the isoelectric point, Mb is pseudoplastic indicating an increase in the shear viscosity. These results are strongly suggestive of formation of hydrophobic clusters at the protein-buffer interface because of the change in the overall charge distributions.

Interaction of chromium(III) complexes with model lipid bilayers: implications on cellular uptake.

To understand molecular cytotoxicity of chromium(III) and how it affects the stability of biological membranes, studies on the interaction of chromium(III) complexes aquapentaminechromium complex (complex I) and trans- [Cr(5-methoxysalcyclohex) (H(2)O) (2)] ClO(4) (complex II) with model biomembranes have been carried out. Langmuir films of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidic acid (DPPA), dioctadecyldimethylammoniumbromide (DOMA) at air/water interface interacting with the chromium(III) complexes have been characterized using the surface pressure-molecular area (π-A) isotherms. Initial surface pressures changes for the two complexes show that the chromium(III) complexes inserted in the Langmuir films and complex I interacted strongly compared to complex II. Supported bilayers (SB) of the lipids on solid substrates formed by hydrating their Langmuir-Blodgett films (LB films) have been characterized using linear dichroic spectra, low angle X-ray diffraction and steady state fluorescence anisotropy. Depending on the geometry of the ligands and concentration, the complexes either insert in the alkyl or in the head group region of the SB and sometimes in both regions. The Supported lipid bilayers are well-layered and at low concentration, the metal complexes are incorporated near the head group region. Order and increase in lamellar spacing show stronger interaction of complex I with the lipids compared with complex II. This study provides some insights into the mechanism of chromium(III) toxicity and uptake of chromium(III) by the cells.

Assembling fibrinogen at air/water and solid/liquid interfaces using Langmuir and Langmuir-Blodgett films.

Langmuir films of pure fibrinogen (Fg) and Fg spread at the air/buffer interface and subphase containing electrolytes, NaCl, KCl, CaCl(2), and ZnCl(2), have been analyzed to understand the role of the surface/interface in mediating the organization of the protein eventually to fibrils. These films have been characterized by the surface pressure and surface potential-molecular area ((pi-A) and (DeltaV-A)) isotherms and Brewster angle microscopy (BAM). The Langmuir-Blodgett (LB) films of the protein transferred to the solid substrates have been characterized by scanning electron microscopy (SEM) and circular dichroism (CD). Our results suggest that fibrils are formed during organization at air/solution interface and also in LB films. The rate of formation of the fibril is the maximum for Fg with ZnCl(2). Adsorption of Fg to surfaces coated with a neutral lipid, dimyristoylphosphatidylcholine (DMPC), and a cationic lipid, dioctadecyldimethylammonium bromide (DOMA), from a range of solution concentrations has been studied using a quartz crystal microbalance (QCM). The work of adhesion of the protein on the solid surface shows fibril formation and positive adhesion for Fg in the presence of electrolytes. SEM results show that the adherent protein exhibits the widely reported nodulelike structure in the presence of CaCl(2) and ZnCl(2). These results provide definite evidence that specifically designed surfaces can promote adhesion of Fg and also activate fibril formation even in the absence of thrombin.