The purpose of this study was to investigate whether Human Serum Albumin (HSA) can bind native human insulin and its A13–A19 and B12–B17 fragments, which are responsible for the aggregation of the whole hormone. To label the hormone and both hot spots, so that their binding positions within the HSA could be identified, 4-(1-pyrenyl)butyric acid was used as a fluorophore. Triazine coupling reagent was used to attach the 4-(1-pyrenyl)butyric acid to the N-terminus of the peptides. When attached to the peptides, the fluorophore showed extended fluorescence lifetimes in the excited state in the presence of HSA, compared to the samples in buffer solution. We also analyzed the interactions of unlabeled native insulin and its hot spots with HSA, using circular dichroism (CD), the microscale thermophoresis technique (MST), and three independent methods recommended for aggregating peptides. The CD spectra indicated increased amounts of the α-helical secondary structure in all analyzed samples after incubation. Moreover, for each of the two unlabeled hot spots, it was possible to determine the dissociation constant in the presence of HSA, as 14.4 µM (A13–A19) and 246 nM (B12–B17). Congo Red, Thioflavin T, and microscopy assays revealed significant differences between typical amyloids formed by the native hormone or its hot-spots and the secondary structures formed by the complexes of HSA with insulin and A13–A19 and B12–B17 fragments. All results show that the tested peptide-probe conjugates and their unlabeled analogues interact with HSA, which inhibits their aggregation.

This study investigates the propensity of short peptides to self-organize and the influence of aggregates on cell cultures. The dipeptides were derived from both enantiomers of identical aromatic amino acids and tripeptides were prepared from two identical aromatic amino acids with one cysteine or methionine residue in the C-terminal, N-terminal, or central position. The formation or absence of fibrous structures under physiological conditions was established using Congo Red and Thioflavine T assays as well as by microscopic examination using normal and polarized light. The in vitro stability of the aggregates in buffered saline solution was assessed over 30 days. Materials with potential for use in regenerative medicine were selected based on the cytotoxicity of the peptides to the endothelial cell line EA.hy 926 and the wettability of the surfaces of the films, as well as using scanning electron microscopy. The criteria were fulfilled by H-dPhedPhe-OH, H-dCysdPhedPhe-OH, H-CysTyrTyr-OH, H-dPhedPhedCys-OH, H-TyrTyrMet-OH, and H–TyrMetTyr–OH. Our preliminary results suggest that the morphology and cell viability of L919 fibroblast cells do not depend on the stereochemistry of the self-organizing peptides.

In this study, three independent methods were used to identify short fragment of both chains of human insulin which are prone for aggregation. In addition, circular dichroism (CD) research was conducted to understand the progress of aggregation over time. The insulin fragments (deca- and pepta-peptides) were obtained by solid-phase synthesis using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate (DMT/NMM/TosO-) as a coupling reagent. Systematic studies allowed identification of the new fragments, expected to be engaged in triggering aggregation of the entire structure of human insulin under physiological conditions. It was found that the aggregation process occurs through various structural conformers and may favor the formation of a fibrous structure of aggregate.

Current restrictions on the use of antibiotics, associated with increases in bacterial resistance, require new solutions, including materials with antibacterial properties. In this study, copper alginate fibers obtained using the classic wet method were used to make nonwovens which were modified with arginine-glycine-aspartic acid (RGD) derivatives. Stable polysaccharide-peptide conjugates formed by coupling with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate (DMT/NMM/TosO−), and materials with physically embedded RGD derivatives, were obtained. The materials were found to be characterized by very high antibacterial activity against S. aureus and K. pneumoniae. Cytotoxicity studies confirmed that the materials are not cytotoxic. Copper alginate conjugates with RGD peptides have strong potential for use in regenerative medicine, due to their biocompatibility and innate antibacterial activity.

In this study, N-methylated analogs of hot-spots of insulin were designed and synthesized, in the expectation that they would inhibit the aggregation of both insulin hot-spots and the entire hormone. Synthesis of insulin “amyloidogenic” analogs containing N-methylated amino acid residues was performed by microwave-assisted solid phase according to the Fmoc/tert-Bu strategy. As a coupling reagent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate (DMT/NMM/TosO-) was used. Three independent methods were applied in aggregation studies of the complexes of insulin with its N-methylated peptides. Additionally, circular dichroism (CD) measurements were used to confirm that aggregation processes did not occur in the presence of the N-methylated analogs of hot-spot insulin fragments, and that insulin retains its native conformation. Of the seven N-methylated analogs of the A- and B-chain hot-spots of insulin, six inhibited insulin aggregation (peptides 1 and 3–7). All tested peptides were found to have a lower ability to inhibit the aggregation of insulin hot-spots compared to the capability to inhibit native hormone aggregation.

The aim of the study was the assessment of the ability of short peptides to form aggregates under physiological conditions. The dipeptides studied were derived from different aromatic amino acids (heteroaromatic peptides). Tripeptides were obtained from two distinct aromatic amino acids and cysteine or methionine residue in the C‐terminal, N‐terminal, or central position. The ability of the peptides to form fibrous aggregates under physiological conditions was evaluated using three independent methods: the Congo Red assay, the Thioflavin T assay, and microscopic examinations using normal and polarized light. Materials potentially useful for regenerative medicine were selected based on their cytotoxicity to the endothelial cell line EA.hy 926 and physicochemical properties of films formed by peptides. The required parameters of biocompatibility were fulfilled by H−PheCysTrp−OH, H−PheCysTyr−OH, H−PheTyrMet−OH, and H−TrpTyr−OH.

The aims of this study were to identify the short aromatic peptides which are able to form highly ordered amyloid‐like structures in self‐assembling processes, to test the influence of length of hydrophobic peptides on tendency to aggregation, and to check if aggregated peptides fulfill requirements expected for materials useful for scaffolding. All tested hydrophobic peptides were prepared on solid phase by using DMT/NMM/TsO− as a coupling reagent. The progress of aggregation was studied by set of independent tests. All aggregated peptides were found stable under in vitro conditions. All fibrous material formed by self‐assembling of peptides does not show any cytotoxic effects on L929 fibroblast cells. Peptides containing tyrosine and tryptophan residues even effectively accelerated the proliferation and stimulated the activity of L929 fibroblasts.

The aim of the research was to check whether it is possible to use fragments of type IV collagen to obtain, as a result of self-assembling, stable spatial structures that could be used to prepare new materials useful in regenerative medicine. Collagen IV fragments were obtained by using DMT/NMM/TosO− as a coupling reagent. The ability to self-organize and form stable spatial structures was tested by the CD method and microscopic techniques. Biological studies covered: resazurin assay (cytotoxicity assessment) on BJ, BJ-5TA and C2C12 cell lines; an alkaline version of the comet assay (genotoxicity), Biolegend Legendplex human inflammation panel 1 assay (SC cell lines, assessment of the inflammation activity) and MTT test to determine the cytotoxicity of the porous materials based on collagen IV fragments. It was found that out of the pool of 37 fragments (peptides 1–33 and 2.1–2.4) reconstructing the outer sphere of collagen IV, nine fragments (peptides: 2, 4, 5, 6, 14, 15, 25, 26 and 30), as a result of self-assembling, form structures mimicking the structure of the triple helix of native collagens. The stability of spatial structures formed as a result of self-organization at temperatures of 4 °C, 20 °C, and 40 °C was found. The application of the MST method allowed us to determine the Kd of binding of selected fragments of collagen IV to ITGα1β1. The stability of the spatial structures of selected peptides made it possible to obtain porous materials based on their equimolar mixture. The formation of the porous materials was found for cross-linked structures and the material stabilized only by weak interactions. All tested peptides are non-cytotoxic against all tested cell lines. Selected peptides also showed no genotoxicity and no induction of immune system responses. Research on the use of porous materials based on fragments of type IV collagen, able to form stable spatial structures as scaffolds useful in regenerative medicine, will be continued.