Selection of collagen IV fragments forming the outer sphere of the native protein: Assessment of biological activity for regenerative medicine.

The aim of this research was to select the fragments that make up the outer layer of the collagen IV (COL4A6) protein and to assess their potential usefulness for regenerative medicine. It was expected that because protein-protein interactions take place via contact between external domains, the set of peptides forming the outer sphere of collagen IV will determine its interaction with other proteins. Cellulose-immobilized protein fragment libraries treated with polyclonal anti-collagen IV antibodies were used to select the peptides forming the outer sphere of collagen IV. In the first test, 33 peptides that strongly interacted with the polyclonal anti-collagen IV antibodies were selected from a library of non-overlapping fragments of collagen IV. The selected fragments of collagen IV (cleaved from the cellulose matrix) were tested for their cytotoxicity, their effects on cell viability and proliferation, and their impact on the formation of reactive oxygen species (ROS). The studies used RAW 264.7 mouse macrophage cells and Hs 680.Tr human fibroblasts. PrestoBlue, ToxiLight™, and ToxiLight 100% Lysis Control assays were conducted. The viability of fibroblasts cultured with the addition of increasing concentrations of the peptide mix did not show statistically significant differences from the control. Fragments 161-170, 221-230, 721-730, 1331-1340, 1521-1530, and 1661-1670 of COL4A6 were examined for cytotoxicity against BJ normal human foreskin fibroblasts. None of the collagen fragments were found to be cytotoxic. Further research is underway on the potential uses of collagen IV fragments in regenerative medicine.

Searching for EGF Fragments Recreating the Outer Sphere of the Growth Factor Involved in Receptor Interactions.

The aims of this study were to determine whether it is possible to use peptide microarrays obtained using the SPOT technique (immobilized on cellulose) and specific polyclonal antibodies to select fragments that reconstruct the outer sphere of proteins and to ascertain whether the selected peptide fragments can be useful in the study of their protein-protein and/or peptide-protein interactions. Using this approach, epidermal growth factor (EGF) fragments responsible for the interaction with the EGF receptor were searched. A library of EGF fragments immobilized on cellulose was obtained using triazine condensing reagents. Experiments on the interactions with EGFR confirmed the high affinity of the selected peptide fragments. Biological tests on cells showed the lack of cytotoxicity of the EGF fragments. Selected EGF fragments can be used in various areas of medicine.

Screening of Self-Assembling of Collagen IV Fragments into Stable Structures Potentially Useful in Regenerative Medicine.

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 ITGα1ß1. 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.

Modification of Alginates to Modulate Their Physic-Chemical Properties and Obtain Biomaterials with Different Functional Properties.

Modified alginates have a wide range of applications, including in the manufacture of dressings and scaffolds used for regenerative medicine, in systems for selective drug delivery, and as hydrogel materials. This literature review discusses the methods used to modify alginates and obtain materials with new or improved functional properties. It discusses the diverse biological and functional activity of alginates. It presents methods of modification that utilize both natural and synthetic peptides, and describes their influence on the biological properties of the alginates. The success of functionalization depends on the reaction conditions being sufficient to guarantee the desired transformations and provide modified alginates with new desirable properties, but mild enough to prevent degradation of the alginates. This review is a literature description of efficient methods of alginate functionalization using biologically active ligands. Particular attention was paid to methods of alginate functionalization with peptides, because the combination of the properties of alginates and peptides leads to the obtaining of conjugates with properties resulting from both components as well as a completely new, different functionality.

Antimicrobial and Antibiofilm N-acetyl-L-cysteine Grafted Siloxane Polymers with Potential for Use in Water Systems.

Antibiofilm strategies may be based on the prevention of initial bacterial adhesion, the inhibition of biofilm maturation or biofilm eradication. N-acetyl-L-cysteine (NAC), widely used in medical treatments, offers an interesting approach to biofilm destruction. However, many Eubacteria strains are able to enzymatically decompose the NAC molecule. This is the first report on the action of two hybrid materials, NAC-Si-1 and NAC-Si-2, against bacteria isolated from a water environment: Agrobacterium tumefaciens, Aeromonas hydrophila, Citrobacter freundii, Enterobacter soli, Janthinobacterium lividum and Stenotrophomonas maltophilia. The NAC was grafted onto functional siloxane polymers to reduce its availability to bacterial enzymes. The results confirm the bioactivity of NAC. However, the final effect of its action was environment- and strain-dependent. Moreover, all the tested bacterial strains showed the ability to degrade NAC by various metabolic routes. The NAC polymers were less effective bacterial inhibitors than NAC, but more effective at eradicating mature bacterial biofilms.

Interaction of ß(3) /ß(2) -peptides, consisting of Val-Ala-Leu segments, with POPC giant unilamellar vesicles (GUVs) and white blood cancer cells (U937)--a new type of cell-penetrating peptides, and a surprising chain-length dependence of their vesicle- and cell-lysing activity.

Many years ago, ß(2) /ß(3) -peptides, consisting of alternatively arranged ß(2) - and ß(3) h-amino-acid residues, have been found to undergo folding to a unique type of helix, the 10/12-helix, and to exhibit non-polar, lipophilic properties (Helv. Chim. Acta 1997, 80, 2033). We have now synthesized such 'mixed' hexa-, nona-, dodeca-, and octadecapeptides, consisting of Val-Ala-Leu triads, with N-terminal fluorescein (FAM) labels, i.e., 1-4, and studied their interactions with POPC (=1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) giant unilamellar vesicles (GUVs) and with human white blood cancer cells U937. The methods used were microfluidic technology, fluorescence correlation spectroscopy (FCS), a flow-cytometry assay, a membrane-toxicity assay with the dehydrogenase G6PDH as enzymatic reporter, and visual microscopy observations. All ß(3) /ß(2) -peptide derivatives penetrate the GUVs and/or the cells. As shown with the isomeric ß(3) /ß(2) -, ß(3) -, and ß(2) -nonamers, 2, 5, and 6, respectively, the derivatives 5 and 6 consisting exclusively of ß(3) - or ß(2) -amino-acid residues, respectively, interact neither with the vesicles nor with the cells. Depending on the method of investigation and on the pretreatment of the cells, the ß(3) /ß(2) -nonamer and/or the ß(3) /ß(2) -dodecamer derivative, 2 and/or 3, respectively, cause a surprising disintegration or lysis of the GUVs and cells, comparable with the action of tensides, viral fusion peptides, and host-defense antimicrobial peptides. Possible sources of the chain-length-dependent destructive potential of the ß(3) /ß(2) -nona- and ß(3) /ß(2) -dodecapeptide derivatives, and a possible relationship with the phosphate-to-phosphate and hydrocarbon thicknesses of GUVs, and eukaryotic cells are discussed. Further investigations with other types of GUVs and of eukaryotic or prokaryotic cells will be necessary to elucidate the mechanism(s) of interaction of 'mixed' ß(3) /ß(2) -peptides with membranes and to evaluate possible biomedical applications.

Chitosan and Its Derivatives - Biomaterials with Diverse Biological Activity for Manifold Applications.

Derived from chitin, chitosan is a natural polycationic linear polysaccharide being the second most abundant polymer next to cellulose. The main obstacle in the wide use of chitosan is its almost complete lack of solubility in water and alkaline solutions. To break this obstacle, the structure of chitosan is subjected to modification, improving its physic-chemical properties and facilitating application as components of composites or hydrogels. Derivatives of chitosan are biomaterials useful for different purposes because of their lack of toxicity, low allergenicity, biocompatibility and biodegradability. This review presents the methods of chemical modifications of chitosan which allow to obtain tailor- made properties required for a variety of biomedical applications. Selected pharmaceutical and biomedical applications of chitosan derivatives are also highlighted. Possibility to manage waste from arthropod and crab processing is also emphasized.

Analysis of structure and properties of antibacterial vascular patch used in abdominal aorta aneurysm surgeries.

BACKGROUND: Biocompatible materials are used for treatment of blood circulatory system diseases, especially abdominal aortic aneurysms. The most popular and often used are knitted and polymer vascular patches. The aim of this study was to optimize the manufacturing process of implantable materials, ensuring antibacterial activity useful for treating abdominal aorta aneurysms. METHODS: The vascular patch was manufactured from Trevira® yarn. The parameters of the intermediate product and vascular patch were tested according to standard procedures. RESULTS: The vascular patch, manufactured from microsilver-containing yarn, with crimps on the surface of the patch, has been found useful for treatment of abdominal aorta aneurysms. Introducing crimps on the surface of the patch resulted in reduction of water permeability and enabled cutting of the graft at various angles without fraying at the cut ends of the biomaterial. The final vascular patch was marked by a gradual release of silver within 48 hours. CONCLUSIONS: On the basis of the performed test, it has been demonstrated that an implantable material for the treatment of abdominal aorta aneurysms was obtained, and that it can be considered as an alternative for currently used vascular patches. The final vascular patch was marked by a gradual release of silver during the first period of incubation. The antibacterial properties of the final product were confirmed by observation of a significant reduction in the number of Staphylococcus aureus and Klebsiella pneumoniae bacterial colonies.

Examination of THPC as an oxygen scavenger impacting VIC dosimeter thermal stability and comparison of NVP-containing polymer gel dosimeters.

This work reports on the impact of tetrakis(hydroxymethyl)phosphonium chloride (THPC) on the properties of a VIC gel dosimeter (VIC is an abbreviated acronym of VIPARCT). THPC was used as a substitute oxygen scavenger in VIC (17% N-vinylpyrrolidone, 8% N,N'-methylenebisacrylamide, 12% tert-butyl alcohol, 7.5% gelatine, 0.02% hydroquinone and an oxygen scavenger of 0.007% ascorbic acid and 0.0008% CuSO4 × 5H2O). THPC reduced the gelation time of VIC from hours to minutes. The best composition (VIC-T) contained 14 mM THPC and a reduced gelatine concentration (5%) with respect to VIC, which allowed for gelation in about 3 min. VIC-T was characterised by the same dose sensitivity (0.176 ± 0.003 Gy-1 s-1 for VIC-T and 0.171 ± 0.002 Gy-1 s-1 for VIC), dose threshold (0.5 Gy) and dynamic dose range (0.5‒50 Gy) as VIC, and a lower linear dose range (20 Gy for VIC-T, 30 Gy for VIC) (0.47 T NMR measurements). VIC-T was stable for at least 10 days after irradiation, and 3D dose distribution was stable for over 4 months after irradiation. The dose response of VIC-T was independent of the radiation dose rate, type and energy of radiation for 6 and 15 MV photons and 12 MeV electrons. This is an improvement with respect to VIC which showed a different dose response for 6 MV photons than for 12 MeV electrons and 15 MV photons. Raman spectroscopy showed similarity in the rate of radiation-induced conversion of monomers in VIC and VIC-T, indicating interaction of THPC with gelatine in VIC-T, and showed ageing of gelatine in both dosimeters. Differential scanning calorimetry showed VIC-T stability at 0 °C-80 °C (VIC: 0 °C‒29.5 °C). The chemical polymerisation and crosslinking of gelatine with THPC is reported, the mechanism of which was analysed in detail. A comparison of N-vinylpyrrolidone-containing dosimeters is presented in this work.

Synthesis and serological interactions of H. pylori urease fragment 321-339 n-terminally immobilized on the cellulose.

Antigenic epitopes F8 (SIKEDVQF) and UB-33 (UreB fragment with residues 321-339: CHHLDKSIKEDVQFADSRI) in H. pylori urease that induces neutralizing antibody production were prepared on the cellulose plate from N- to C-terminus using CDMT as a coupling reagent. Reaction of both epitopes with sera of patients with medically confirmed atherosclerosis was studied. Strong, selective reactions of both peptides with some patients sera were observed.

N-lipidated oligopeptides immobilized on cellulose as new type of organocatalysts.

A library of supramolecular structures formed by self-organization of N-lipidated tripeptides, dipeptides and N-acylated amino acids attached to cellulose according to the TASP concept via aminophenylamino-1,3,5-triazine was synthesized and the catalytic activity of the structures was studied. Intensive catalytic activity in solvolysis of sterically hindered Z-Aib-Aib-ONp under ambient conditions was observed for structures bearing the catalytic triad as well as for structures with the peptide fragment shortened to a dipeptide or even a single Ser, Glu or His residue, but not for structures bearing alanine or phenylalanine residues. For all structures with a dipeptide or a single amino acid residue and for most of tripeptide structures the progress of solvolysis was stopped after the concentration of the nitrophenolate ion reached 0.5-0.7 x 10-4 M/L. Only in the case of catalysts with glutamic acid residues in the tripeptide fragment, solvolysis proceeded until all the substrate was consumed.