Piotr Bonarek

Despite the widespread use of cosolvents to modulate polypeptide conformation, their solvent-specific role in coupling polypeptide charge regulation in solution along with interfacial multilayer growth and mechanics remains poorly understood. Here, we systematically investigate how three common cosolvents, namely ethanol, urea, and dimethyl sulfoxide (DMSO), alter the solution behavior of poly-l-lysine (PLL) and poly-l-glutamic acid (PGA) across a broad pH range (3.0–11.0) at controlled ionic strength. We also examine how the cosolvent and the pH effects govern PLL and PGA layer-by-layer assembly on silica surfaces. By combining electrokinetic measurements, circular dichroism spectroscopy, molecular dynamics simulations, and in situ mechanical characterization, we identify distinct, solvent-dependent mechanisms of charge compensation and structural disruption. Ethanol and DMSO primarily promote counterion condensation and partial chain dehydration, whereas urea compensates charge through direct hydrogen bonding to the polypeptide backbone, leading to a more pronounced loss of secondary structure. These molecular-level differences directly translate into multilayer architecture and mechanics and affect multilayer build-up. Ethanol yields thin, compact, and mechanically stiff layers, while urea and DMSO produce thicker, softer, and more dissipative layers. Overall, this work establishes a direct, solvent-specific link between polypeptide charge regulation in solution and the growth and mechanical properties of polypeptide multilayers, providing a versatile strategy to tailor multilayer architectures through solvent control alone.

This study describes the interactions between two different pro-inflammatory factors produced by bacteria, lipopolysaccharide (LPS) from Gram-negative bacteria and the peptide BacSp222 produced by a Gram-positive zoonotic strain, Staphylococcus pseudintermedius 222. We demonstrate that the mentioned molecules interact, forming a complex, and this phenomenon selectively reduces their biological activities in vitro and in vivo. Specifically, the levels of tumor necrosis factor (TNF) and nitric oxide (NO) produced by monocyte-macrophage cells were lower in samples treated with both LPS and BacSp222 compared to those treated with LPS alone. This is most likely because BacSp222 limited the ability of LPS to stimulate the TLR4 receptor. In the Galleria mellonella larvae injected simultaneously with LPS and BacSp222, the activity of hemolymph phenoloxidase, a key component of the insect immune response, was lower than that observed in larvae injected with either LPS or BacSp222 alone. Moreover, LPS inhibited the antibacterial activity of the bacteriocin, while BacSp222 limited LPS's ability to activate a proenzyme in the Limulus amebocyte lysate test. The changes in the activities of BacSp222 and LPS were attributed to the electrostatic interactions between LPS micelles and bacteriocin molecules, resulting in a decrease in LPS aggregate size and the direct formation of a complex between them, as revealed by gel filtration and isothermal microcalorimetry.

A photoactive analogue of cisplatin was synthesized with two arylazopyrazole ligands, able to undergo trans-cis/cis-trans photoisomerizations. The cis photoisomer showed a dark half-life of 9 days. The cytotoxicities of both photoisomers of the complex were determined in several cancer and normal cell lines and compared to that of cisplatin. The trans photoisomer of the complex was much more cytotoxic than both the cis photoisomer and cisplatin, and was more toxic for cancer (4T1) than for normal (NMuMG) murine breast cells. 4T1 cell death occurred through necrosis. Photoisomerization of the trans and cis photoisomers internalized by the 4T1 cells increased and decreased their viability, respectively. The cellular uptake of the trans photoisomer was stronger than that of both the cis photoisomer and cisplatin. Both photoisomers interacted with DNA faster than cisplatin. The trans photoisomer was bound stronger by bovine serum albumin and induced a greater decrease in cellular glutathione levels than the cis photoisomer.

Zika virus (ZIKV), belonging to the family of flaviviruses, is an emerging mosquito‐borne pathogen causing microcephaly and other congenital abnormalities in newborns as well as various neurologic pathologies in adults. The climate change and the lifestyle of current societies, involving global mobility, result in a rapid broadening of ZIKV geographic distribution. There are no vaccines or drugs available to protect against or cure the diseases caused by that virus. Our in vitro studies have demonstrated that sodium polyanethole sulfonate (PASNa), a degradable polymer used in various biomedical applications, acts as an efficient ZIKV inhibitor, both in animal and human cells. The mechanistic studies indicated that PASNa bound to the ZIKV particles thus hindering their attachment to the host cells and also interfered in the late steps of viral replication. PASNa is characterized by very low cytotoxicity and its antiviral activity can be achieved at a concentration much lower than that at which its anticoagulant activity has to be considered. Isothermal titration calorimetry (ITC) studies have shown that the interaction of PASNa with human serum albumin (HSA), the most abundant blood protein, is the endothermic, entropy‐driven process resulting in the formation of stoichiometric (1:1) PASNa‐HSA complexes with a hydrodynamic diameter of 8.8 ± 0.8 nm and zeta potential (ζ) equal to −21.3 mV. The analysis of CD spectra indicated that PASNa did not significantly affect the HSA secondary structure on binding to that protein.

Curcumin, a natural product with recognized antiviral properties, is limited in its application largely due to its poor solubility. This study presents the synthesis of water-soluble curcumin-poly(sodium 4-styrenesulfonate) (Cur-PSSNa_n) covalent conjugates. The antiflaviviral activity of conjugates was validated in vitro by using the Zika virus as a model. In the development of these water-soluble curcumin-containing derivatives, we used the macromolecules reported by us to also hamper viral infections. Mechanistic investigations indicated that the conjugates exhibited excellent stability and bioavailability. The curcumin and macromolecules in concerted action interact directly with virus particles and block their attachment to host cells, hampering the infection process.

β‐Lactoglobulin (BLG) is a member of the lipocalin family. As other proteins from this group, BLG can be modified to bind specifically compounds of medical interests. The aim of this study was to evaluate the role of two mutations, L39Y and L58F, in the binding of topical anesthetic pramoxine (PRM) to β‐lactoglobulin. Circular dichroism spectroscopy, isothermal titration calorimetry (ITC), and X‐ray crystallography were used to understand the mechanisms of BLG–PRM interactions. Studies were performed for three new BLG mutants: L39Y, L58F, and L39Y/L58F. ITC measurements indicated a significant increase in the affinity to the PRM of variants L58F and L39Y. Measurements taken for the double mutant L39Y/L58F showed the additivity of two mutations leading to about 80‐fold increase in the affinity to PRM in comparison to natural protein BLG from bovine milk. The determined crystal structures revealed that pramoxine is accommodated in the β‐barrel interior of BLG mutants and stabilized by hydrophobic interactions. The observed additive effect of two mutations on drug binding opens the possibility for further designing of new BLG variants with high affinity to selected drugs.

Among potential macromolecule-based pharmaceuticals, polycations seem particularly interesting due to their proven antimicrobial properties and use as vectors in gene therapy. This makes an understanding of the mechanisms of these molecules’ interaction with living structures important, so the goal of this paper was to propose and carry out experiments that will allow us to characterize these phenomena. Of particular importance is the question of toxicity of such structures to mammalian cells and, in the work presented here, two lines, normal fibroblasts 3T3-L1 and A549 lung cancer, were used to determine this. In this work, three well-defined cationic derivatives of barley-derived betaglucans obtained in a reaction with glycidyltrimethylammonium chloride (BBGGTMAC) with different degrees of cationization (50, 70, and 100% per one glucose unit) and electrostatic charge were studied. The studies address interactions of these polymers with proteins (bovine serum proteins and BSA), nucleic acids (DNA), glycosaminoglycans (heparin), and biological membranes. The results described in this study make it possible to indicate that toxicity is most strongly influenced by interactions with biological membranes and is closely related to the electrostatic charge of the macromolecule. The presentation of this observation was the goal of this publication. This paper also shows, using fluorescently labeled variants of polymers, the penetration and impact on cell structure (only for the polymer with the highest substitution binding to cell membranes is observed) by using confocal and SEM (for the polymer with the highest degree of substitution, and the appearance of additional structures on the surface of the cell membrane is observed). The labeled polymers are also tools used together with dynamic light scattering and calorimetric titration to study their interaction with other biopolymers. As for the interactions with biological membranes, lipid Langmuir monolayers as model membrane systems were used.

Experiments and molecular dynamics simulations show that pH is a way to control the poly(l-lysine)/poly(l-glutamic acid) complexation. The associated changes in the charge and secondary structure can be used to control materials assembly.

A series of poly(ethylene glycol)-block-poly(sodium 4-styrenesulfonate) (PEG-b-PSSNa) copolymers were synthesized, and their antiviral activity against Zika virus (ZIKV) was determined. The polymers inhibit ZIKV replication in vitro in mammalian cells at nontoxic concentrations. The mechanistic analysis revealed that the PEG-b-PSSNa copolymers interact directly with viral particles in a zipper-like mechanism, hindering their interaction with the permissive cell. The antiviral activity of the copolymers is well-correlated with the length of the PSSNa block, indicating that the copolymers' ionic blocks are biologically active. The blocks of PEG present in copolymers studied do not hinder that interaction. Considering the practical application of PEG-b-PSSNa and the electrostatic nature of the inhibition, the interaction between the copolymers and human serum albumin (HSA) was evaluated. The formation of PEG-b-PSSNa-HSA complexes in the form of negatively charged nanoparticles well-dispersed in buffer solution was observed. That observation is promising, given the possible practical application of the copolymers. [GRAPHICS]