This study presents the chemical synthesis, purification, and characterization of a novel non-natural synthetic amino acid. The compound was synthesized in solution, purified, and characterized using NMR spectroscopy, polarimetry, and melting point determination. Dynamic Light Scattering (DLS) analysis demonstrated its ability to form aggregates with an average size of 391 nm, extending to the low micrometric size range. Furthermore, cellular biological assays revealed its ability to enhance fibroblast cell growth, highlighting its potential for tissue regenerative applications. Circular dichroism (CD) spectroscopy showed the ability of the synthetic amino acid to bind serum albumins (using bovine serum albumin (BSA) as a model), and CD deconvolution provided insights into the changes in the secondary structures of BSA upon interaction with the amino acid ligand. Additionally, molecular docking using HDOCK software elucidated the most likely binding mode of the ligand inside the BSA structure. We also performed in silico oligomerization of the synthetic compound in order to obtain a model of aggregate to investigate computationally. In more detail, the dimer formation achieved by molecular self-docking showed two distinct poses, corresponding to the lowest and comparable energies, with one pose exhibiting a quasi-coplanar arrangement characterized by a close alignment of two aromatic rings from the synthetic amino acids within the dimer, suggesting the presence of π-π stacking interactions. In contrast, the second pose displayed a non-coplanar configuration, with the aromatic rings oriented in a staggered arrangement, indicating distinct modes of interaction. Both poses were further utilized in the self-docking procedure. Notably, iterative molecular docking of amino acid structures resulted in the formation of higher-order aggregates, with a model of a 512-mer aggregate obtained through self-docking procedures. This model of aggregate presented a cavity capable of hosting therapeutic cargoes and biomolecules, rendering it a potential scaffold for cell adhesion and growth in tissue regenerative applications. Overall, our findings highlight the potential of this synthetic amino acid for tissue regenerative therapeutics and provide valuable insights into its molecular interactions and aggregation behavior.

In this study, a series of four surface-active compounds-N-alkyl betaine ethyl ester chlorides, CnBetC2Cl-were synthesized and characterized in aqueous solutions. As with other alkyl betaines, these amphiphiles can be practically used, for example, as co-surfactants and/or solubility enhancers acting according to hydrotropic or micellar mechanisms, depending on the alkyl chain length in the amine. We focused on the representatives of the medium alkyl chain length (C6-C12) to find the dependence between the alkyl chain length in N-alkyl betaine ethyl ester chlorides and the surface, volumetric, acoustic, and viscometric properties of their solutions. Ethyl esters, the derivatives of amino acids, were chosen to increase functionality and take advantage of possible hydrolysis in solutions at higher pH, which is also a key parameter in biodegradability. The micellization parameters were calculated based on the physicochemical characteristics. We focused our interest on the ester with a dodecyl substituent since we can compare and discuss its properties with some other C12 representatives that are available in literature. Surprisingly, its micellization characteristic is almost temperature-independent in the investigated temperature range, t = (15-45) °C. Particularly interesting are the results of dynamic light scattering (DLS), which show that the changes in physicochemical parameters of the C12 homolog around the CMC are caused by the two types of micelles of different sizes present in solutions.

OBJECTIVE: A comparative assessment was performed to evaluate the potential of particle sizing by an ensemble based conventional dynamic light scattering (DLS) technique and an emerging technology based on tunable resistive pulse sensing (TRPS) using particle by particle approach by evaluating three different types of vaccine formulations representing three case studies and showing the limitation of each technique, instrument variability, sensitivity, and the resolution in mixed population.
METHODS: Three types of in-house vaccine formulations- a protein antigen, an outer membrane vesicle and viral particles were simultaneously evaluated by TRPS based Exoid and two DLS instruments-Zetatrac and Zetasizer for particle size distribution, aggregates, and resolution of polydisperse species.
RESULTS: The data from first case study show the risk of possible size overestimation and size averaging in polydisperse samples in DLS measurements which can be addressed by the TRPS analysis. It also shows how TRPS may be utilized only to large size antigens due to its limited size range. The second case study highlights the difference in the sensitivities of two DLS instruments working on the same principle. The third case study show that how TRPS can better resolve the large aggregate species compare to DLS in polydisperse samples.
CONCLUSIONS: This analysis shows that TRPS can be used as an orthogonal technique in addition to conventional DLS based methods for more precise and in-depth characterization. Both techniques are efficient in size characterization and produce comparable results, however the choice will depend on the type of formulation and size range to be evaluated.

OBJECTIVE: Lipid nanoparticles containing a cationic lipid are increasingly used in drug and gene delivery as they can display improved cellular uptake, enhanced loading for anionic cargo such as siRNA and mRNA or exhibit additional functionality such as cytotoxicity against cancer cells. This research study tests the hypothesis that the molecular structure of the cationic lipid influences the structure of the lipid nanoparticle, the cellular uptake, and the resultant cytotoxicity.
METHODS: Three potentially cytotoxic cationic lipids, with systematic variations to the hydrophobic moiety, were designed and synthesised. All the three cationic lipids synthesised contain pharmacophores such as the bicyclic coumarin group (CCA12), the tricyclic etodolac moiety (ETD12), or the large pentacyclic triterpenoid \"ursolic\" group (U12) conjugated to a quaternary ammonium cationic lipid containing twin C12 chains. The cationic lipids were doped into monoolein cubosomes at a range of concentrations from 0.1 mol% to 5 mol% and the effect of the lipid molecular architecture on the cubosome phase behaviour was assessed using a combination of Small Angle X-Ray Scattering (SAXS), Dynamic Light Scattering (DLS), zeta-potential and cryo-Transmission Electron Microscopy (Cryo-TEM). The resulting cytotoxicity of these particles against a range of cancerous and non-cancerous cell-lines was assessed, along with their cellular uptake.
RESULTS: The molecular architecture of the cationic lipid was linked to the internal nanostructure of the resulting cationic cubosomes with a transition to more curved cubic and hexagonal phases generally observed. Cubosomes formed from the cationic lipid CCA12 were found to have improved cellular uptake and significantly higher cytotoxicity than the cationic lipids ETD12 and U12 against the gastric cancer cell-line (AGS) at lipid concentrations ≥ 75 µg/mL. CCA12 cationic cubosomes also displayed reasonable cytotoxicity against the prostate cancer PC-3 cell-line at lipid concentrations ≥ 100 µg/mL. In contrast, 2.5 mol% ETD12 and 2.5 mol% U12 cubosomes were generally non-toxic against both cancerous and non-cancerous cell lines over the entire concentration range tested. The molecular architecture of the cationic lipid was found to influence the cubosome phase behaviour, the cellular uptake and the toxicity although further studies are necessary to determine the exact relationship between structure and cellular uptake across a range of cell lines.

In this study, the complexation of potential chemo-therapeutic antibacterial drug, ciprofloxacin (CIP) with varying concentrations of surface active compounds (SACs) i.e., (N-(2-hydroxyethyl)-N,N-dimethyl-1-dodecanaminium bromide (12Cho.Br) and cetyltrimethylammonium bromide (CTAB) has been studied. Multispectroscopic techniques were exploited to carry out the study. The higher binding constant (Kb) value for CIP-CTAB than CIP-12Cho.Br obtained from fluorescence data revealed stronger binding of CTAB than 12Cho.Br, owing to the stronger hydrophobic-hydrophobic interaction betweeen CIP and CTAB compared to CIP and 12Cho.Br. The time resolve fluorescence decay shows changes in average lifetime (τavg) with the increasing concentration of 12Cho.Br and CTAB. The changes in τavg suggests that complex formation is taking place between CIP and 12Cho.Br / CTAB. Further, the formation of micelles by 12Cho.Br / CTAB and the effect of alkyl chain length was studied by dynamic light scattering (DLS) and zeta potential to confirm the drug complexation with 12Cho.Br and CTAB. The antibacterial activity has been performed for CIP and 12Cho.Br and CTAB. It was observed that in presence of lower concentrations of 12Cho.Br/ CTAB, the activity of the drug increased. The activity was also found cationic alkyl chain length dependent. Moreover, in-vitro cytotoxicity of CIP and its combinations with 12Cho.Br and CTAB was performed using MTT assay on HEK293 (Human embryonic kidney cells).

Cholesterol in a bio-membrane plays a significant role in many cellular event and is known to regulate the functional activity of protein and ion channel. In this study we report a significant effect of cholesterol on the ion-membrane interaction. We prepare large unilamellar vesicles, composed of zwitterionic lipid DOPC and anionic lipid DOPG with different cholesterol concentration. Electrostatics of anionic membranes containing cholesterol in the presence of NaCl has systematically been explored using dynamic light scattering and zeta potential. Negative zeta potential of the membrane decreases its negative value with increasing ion concentration for all cholesterol concentrations. However, zeta potential itself decreases with increasing cholesterol content even in the absence of monovalent ions. Electrostatic behaviour of the membrane is determined from well-known Gouy Chapmann model. Negative surface charge density of the membrane decreases with increasing cholesterol content. Binding constant, estimated from the electrostatic double layer theory, is found to increase significantly in the presence of cholesterol. Comparison of electrostatic parameters of the membrane in the presence and absence of cholesterol suggests that cholesterol significantly alter the electrostatic behaviour of the membrane.

The in vivo potency of polyphosphazene immunoadjuvants is inherently linked to the ability of these ionic macromolecules to assemble with antigenic proteins in aqueous solutions and form physiologically stable supramolecular complexes. Therefore, in-depth knowledge of interactions in this biologically relevant system is a prerequisite for a better understanding of mechanism of immunoadjuvant activity. Present study explores a self-assembly of polyphosphazene immunoadjuvant-PCPP and a model antigen-lysozyme in a physiologically relevant environment-saline solution and neutral pH. Three analytical techniques were employed to characterize reaction thermodynamics, water-solute structural organization, and supramolecular dimensions: isothermal titration calorimetry (ITC), water proton nuclear magnetic resonance (wNMR), and dynamic light scattering (DLS). The formation of lysozyme-PCPP complexes at near physiological conditions was detected by all methods and the avidity was modulated by a physical state and dimensions of the assemblies. Thermodynamic analysis revealed the dissociation constant in micromolar range and the dominance of enthalpy factor in interactions, which is in line with previously suggested model of protein charge anisotropy and small persistence length of the polymer favoring the formation of high affinity complexes. The paper reports advantageous use of wNMR method for studying protein-polymer interactions, especially for low protein-load complexes.

The properties of a lysozyme solution under laser-induced breakdown were studied. An optical breakdown under laser action in protein solutions proceeds with high efficiency: the formation of plasma and acoustic oscillations is observed. The concentration of protein molecules has very little effect on the physicochemical characteristics of optical breakdown. After exposure to optical breakdown, changes were observed in the enzymatic activity of lysozyme, absorption and fluorescence spectra, viscosity, and the sizes of molecules and aggregates of lysozyme measured by dynamic light scattering. However, the refractive index of the solution and the Raman spectrum did not change. The appearance of a new fluorescence peak was observed upon excitation at 350 nm and emission at 434 nm at exposure for 30 min. Previously, a peak in this range was associated with the fluorescence of amyloid fibrils. However, neither the ThT assay nor the circular dichroism dispersion confirmed the formation of amyloid fibrils. Probably, under the influence of optical breakdown, a small part of the protein degraded, and a part changed its native state and aggregated, forming functional dimers or \"native aggregates\".

Tau is an intrinsically disordered protein that binds and stabilizes axonal microtubules (MTs) in neurons of the central nervous system. The binding of Tau to MTs is mediated by its repeat domain and flanking proline-rich domains. The positively charged (basic) C-terminal half of Tau also mediates the assembly Tau into fibrillar aggregates in Alzheimer\'s disease (AD) and tauopathy brains. In recent years, another assembly form of Tau has been identified: Tau can undergo liquid-liquid phase separation (LLPS), which leads to its condensation into liquid-dense phases, either by complex coacervation with polyanions like heparin or RNA or through \"self-coacervation\" at high Tau concentrations. Condensation of Tau in the absence of polyanions can be enhanced by the presence of molecular crowding agents in a dilute Tau solution. In vitro experiments using recombinant purified Tau are helpful to study the physicochemical determinants of Tau LLPS, which can then be extrapolated into the cellular context. Tau condensation is a new aspect of Tau biology that may play a role for the initiation of Tau aggregation, but also for its physiological function(s), for example, the binding to microtubules. Here we describe how to study the condensation of Tau in vitro using light microscopy, including fluorescence recovery after photobleaching (FRAP), to assess the shape and molecular diffusion in the condensates, a proxy for the degree of condensate percolation. We also describe turbidity measurements of condensate-containing solutions to assess the overall amount of LLPS and time-resolved dynamic light scattering (trDLS) to study the formation and size of Tau condensates.

Green nanotechnology has emerged as a viable option for the production of nanoparticles. The purpose of the current investigation was to synthesize silver nanoparticles (AgNPs) using Eucalyptus camaldulensis and Terminalia arjuna extracts, as well as their combinations, as green reducing and capping agents. The parameters (concentration of silver nitrate solution and plant extract, time, pH, and temperature) were optimized for maximal yields, regulated size, and stability of silver nanoparticles. The ultraviolet-visible spectrophotometer (UV-Vis) and the surface plasmon resonance band (SPR) were used to validate the synthesis of AgNPs. The size, shape, and stability of nanoparticles were assessed using a zeta analyzer and a scanning electron microscope (SEM). The biomolecules responsible for the reduction of silver ion (Ag+) and the stability of silver nanoparticles generated with the plant extracts were identified using Fourier-transform infrared spectroscopy (FTIR). The agar-well diffusion method was used to test the antimicrobial activity of biosynthesized nanoparticles against Bacillus subtilis, Staphylococcus aureus, Pasteurella multocida, and Escherichia coli. When 1 mM of silver nitrate (AgNO3) was added to plant extracts and incubated for 60 min at 75°C in a neutral medium, maximum nanoparticles were produced. Biosynthesized silver nanoparticles were stable, spherical, and monodispersed according to zeta potential and scanning electron microscopy. Silver nanoparticles synthesized with combination 2 and T. arjuna showed the highest zone of inhibition (16 mm) against B. subtilis while combination 3 showed the largest zone of inhibition against S. aureus (17 ± 0.8). It was concluded that greenly produced silver nanoparticles showed good antibacterial activity while causing negligible cytotoxicity.