Vliv kationtu Li+ na vnitřní pnutí slitinových povlaků na bázi niklu

Prezenční výpůjčkaVŠB - Technická univerzita Ostrava. Fakulta strojní. Katedra (345) mechanické technologi

The characterization of hyaluronic acid by gel permeation chromatography coupled to multi-angle light scattering

Cieľom tejto dezertačnej práce bolo pripraviť vzorky hyaluronanu s rozličnou mólovou hmotnosťou, experimentálne zistiť navhodnejšie podmienky pre GPC-MALS analýzu kyseliny hyalurónovej a porovnať túto metódu s ostatnými často používanými metódami stanovenia mólovej hmotnosti. Ďalším čiastkovým cieľom bolo objasnenie molekulárnej štruktúry tohto zaujímavého biopolyméru. V úvode sa práca zaoberá teóriou stanovenia mólovej hmotnosti, vysvetlením separačného mechanizmu a inštrumentácie. Ďalej sú predstavené alternatívne metódy stanovenia mólovej hmotnosti a molekulárnej distribúcie hyaluronanu. Experimentálna časť zahŕňa zoznam použitých chemikálií a spotrebného materiálu, spôsoby prípravy vzoriek a medodológiu používanú pre meranie. Posledná časť práce je rozdelená do niekoľkých kapitol, ktoré sa zaoberajú prípravou hyaluronanu s rozličnou mólovou hmotnosťou, optimalizáciou separačných podmienok GPC-MALS analýzy a porovnaním metódy s ostatnými často používanými technikami stanovenia mólovej hmotnosti, a to s frakcionáciou tokom v poli a analytickou ultracentrifugáciou. Posledná časť je venovaná štúdiu roztokových vlastností hyaluronanu v širokom rozsahu mólových hmotností pomocou viskozimetrie s použitím klasického kapilárneho Ubbelohdeho viskozimetru a kombinácie gélovej permeačnej chromatografie s viac uhlovým detektorom rozptylu svetla a on-line viskozimetrom.The aim of this thesis was prepare the hyaluronan samples with various molar masses, determine the most suitable conditions for GPC-MALS analysis of hyaluronic acid, and compare the method with other commonly used techniques. The last partial aim was to study the molecular structure of this interesting and important biopolymer in solution. At the beginning work deals with theoretical basics of method, explanation of separation mechanism, instrumentation and with introducing other techniques that can be used for evaluation of molar mass and molar mass distribution of hyaluronan. Experimental part covers up the list of used materials, preparation of samples and methodology of accomplished measurements. Final part of this work is divided into several chapters. The first chapter deals with the preparation of sodium hyaluronate with various molar masses, the second one demonstrates optimization of the GPC separation conditions and in the third part the GPC-MALS is compared to other commonly used techniques, such as field-flow fractionation and analytical ultracentrifugation. The final section demonstrates the study of solution properties of sodium hyaluronate covering broad molar mass range carried auto by classical viscosimetry using an Ubbelohde capillary viscometer and by a combination of gel permeation chromatography coupled to multi-angle light scattering detector and an no-line capillary viscometer.Ústav chemie a technologie makromolekulárních látekPředseda komise prof. Šňupárek představil disertantku Mgr. Martinu Hermannovou. Konstatoval, že přítomno je šest z osmi členů komise a všichni tři jmenovaní oponenti. Předseda komise přečetl životopis uchazečky, stanovisko školícího pracoviště a doporučení školitelky. Poté uchazečka seznámila komisi s obsahem své disertační práce. Oponenti přečetli své posudky, uchazečka zaujala stanovisko k připomínkám oponentů, své odpovědi předala v písemné formě a jsou přílohou tohoto zápisu. Poté předseda vyzval k diskusi i ostatní členy komise. Mgr. Martina Hermannová reagovala na dotazy přítomných, které uspokojivě zodpověděla. Diskuse se zúčastnili všichni členové komise. Diskuse se týkala problematiky podmínek analýzy, mechanismu dělení na GPC kolonách, otázky větvení a tzv. síťování makromolekul, standardizace analytických metod pro farmacii a kosmetický průmysl, oblasti použití hyaluronové kyseliny v souvislosti se stupněm čistoty a obsahem proteinů. V neveřejném zasedání komise zhodnotila vystoupení Mgr. Martiny Hermannové a tajným hlasováním všemi hlasy (6/0) rozhodla pro udělení akademického titulu doktor - Ph.D. Výsledek hlasování byl oznámen uchazečce.Dokončená práce s úspěšnou obhajobo

The rate and evenness of the substitutions on hyaluronan grafted by dodecanoic acid influenced by the mixed-solvent composition

In this work, low molecular weight (17 kDa) hyaluronan was modified by dodecanoyl substituents. The activation of dodecanoic acid was mediated by benzoyl chloride towards the preparation of a mixed anhydride, which reacts in a second step with HA in water mixed with an organic solvent. The effect of the cosolvent was studied and showed an even distribution of substituents and higher reaction rate in water: 1,4-dioxane compared to water:tert-butanol where substituents occupy adjacent positions. The chemical characterization of the prepared derivatives was elucidated by NMR, FTIR spectroscopy, thermal analyses, and gas chromatography, while the distribution of substituents was evaluated by enzymatic degradation. Molecular-dynamics simulations reveal opposite solvent separations around HA and dodecanoyl chains, that is stronger in water:tert-butanol solution. The resulting incompatibility of solvation-shells of the two entities repels the reaction intermediates from the HA chain and drives them towards the already bound substituents, explaining the observed differences in the distribution evenness. Thus, the influence of the solvent on the reaction selectivity is observed by shielding reactive sites around HA. Therefore, a control of the distribution of the substituents was obtained by defining the concentration of HA and used cosolvent. © 2021 Elsevier B.V.National Grid Infrastructure MetaCentrum provided under the program "Projects of Large Research, Development, and Innovations Infrastructures" [CESNET LM2015042]; Ministry of Education, Youth and Sports under the project "IT4 Innovations National Supercomputing Center" [LM2015070]; Tomas Bata University in Zlin [IGA/FT/2016/011, IGA/FT/2017/009, IGA/FT/2018/010]; Technology Agency of the Czech Republic: Program of Industrial Research and Experimental Development 2020-2027 (TREND) [FW01010060]CESNET LM2015042; Technology Agency of the Czech Republic, TACR: FW01010060; Ministerstvo Školství, Mládeže a Tělovýchovy, MŠMT: LM2015070; Univerzita Tomáše Bati ve Zlíně: IGA/FT/2016/011, IGA/FT/2017/009, IGA/FT/2018/01

In vivo monitoring of tumor distribution of hyaluronan polymeric micelles labeled or loaded with near-infrared fluorescence dye

Development of delivery systems which allow real-time visual inspection of tumors is critical for effective therapy. Near-infrared (NIR) fluorophores have a great potential for such an application. To overcome NIR dyes short blood circulation time and increase tumor accumulation, a NIR dye, cypate, was associated with oleyl hyaluronan, which can self-assemble into polymeric aggregates. The cypate association with oleyl hyaluronan was performed either by a covalent linkage, or physical entrapment. The two systems were compared for tumor targeting and contrast enhancement using BALB/c mice bearing 4T1 breast cancer tumors. Independently on the way of cypate association, it took more than 24 h from intravenous administration to detect NIR signal in tumors and the tumors were clearly visualized for 2 following weeks without substrate reinjection. Covalently linked cypate generated 2–3 fold stronger fluorescence signal than physically loaded cypate. This study demonstrates the potential of HA matrix to be used as carrier of contrast agents for non-invasive long-term tumor visualization. © 2018 Elsevier LtdMEYS, Ministry of Education, Youth and ScienceInternal Founding Agency of Tomas Bata University in Zlin [IGA/FT/2018/001]; National Program of Sustainability II (MEYS CR) [LQ1605]; program Institute Contipr

The Degradation of Hyaluronan in the Skin

Hyaluronan (HA) comprises a fundamental component of the extracellular matrix and participates in a variety of biological processes. Half of the total amount of HA in the human body is present in the skin. HA exhibits a dynamic turnover; its half-life in the skin is less than one day. Nevertheless, the specific participants in the catabolism of HA in the skin have not yet been described in detail, despite the essential role of HA in cutaneous biology. A deeper knowledge of the processes involved will act to support the development of HA-based topical and implantable materials and enhance the understanding of the various related pathological cutaneous conditions. This study aimed to characterize the distribution and activity of hyaluronidases and the other proteins involved in the degradation of HA in healthy human full-thickness skin, the epidermis and the dermis. Hyaluronidase activity was detected for the first time in healthy human skin. The degradation of HA occurred in lysates at an acidic pH. HA gel zymography revealed a single band corresponding to approximately 50 kDa. This study provided the first comprehensive view of the distribution of canonic HA-degrading proteins (HYAL1 and HYAL2) in human skin employing IHF and IHC. Furthermore, contrary to previous assumptions TMEM2, a novel hyaluronidase, as well as CEMIP, a protein involved in HA degradation, were localized in the human epidermis, as well as in the dermis

Structure and dynamics of the hyaluronan oligosaccharides and their solvation shell in water: organic mixed solvents

Hyaluronan (HA) is a natural polysaccharide occurring ubiquitously in the connective tissues of vertebrates widely used in the cosmetic and pharmaceutic industries. In numerous applications HA oligosaccharides are being chemically modified using reactions incompatible with aqueous solutions, often carried out in water:organic mixed solvents. We carry out molecular-dynamics (MD) simulations of HA oligosaccharides in water:1,4-dioxane and water:tert-butanol mixtures of different compositions. HA molecule causes a separation of the solvent components in its surroundings, especially in tert-butanol containing solutions, constituting thus a solvation shell enriched by water. Furthermore, interactions with ions are stronger than in pure water and depend on the solvent composition. Consequently, the dynamics of the HA chain varies with the solvent composition and causes observable conformational changes of the HA oligosaccharide. Composition of mixed solvents thus enables us to modify the interaction of HA with other molecules as well as its reactivity. © 2022 Elsevier LtdMinisterstvo Školství, Mládeže a Tělovýchovy, MŠMT: 90140; Univerzita Tomáše Bati ve Zlíně: IGA/FT/2016/011, IGA/FT/2017/009, IGA/FT/2018/010, IGA/FT/2021/010, IGA/FT/2022/00

Nanostructure of hyaluronan acyl-derivatives in the solid state

Acylované deriváty hyaluronanu (acyl-HA) jsou perspektivní materiály pro biomedicínské aplikace. V závislosti na délce acylové skupiny a stupni substituce mohou mít tyto deriváty formu polymerů rozpustných ve vodě se schopností samouspořádání až po materiály ve vodném prostředí nerozpustné. Již bylo studováno chování acyl-HA v roztocích, avšak málo pozornosti jim bylo zatím věnováno v pevném stavu, přestože jsou významné pro takové aplikace, jako je například výroba zdravotnických prostředků. Proto jsme s pomocí rentgenového rozptylu a elektronové mikroskopie zkoumali nanostrukturu acyl-HA v pevném stavu. Sada vzorků zahrnovala různé substituenty, stupně substituce a molekulové hmotnosti. Získaná data ukázala, že všechny studované materiály acyl-HA obsahují struktury o rozměrech v řádu nanometrů, které nejsou přítomné v nemodifikovaném hyaluronanu. Rozměr nanostruktur se zvyšoval s délkou acylové skupiny, zatímco vliv stupně substituce a molekulové hmotnosti byl zanedbatelný. Zjištěná nanostrukura pravděpodobně odpovídá distribuci hydrofobních domén v hydrofilní matrici nemodifikovaného hayluronanuAcyl derivatives of hyaluronan (acyl-HA) are promising materials for biomedical applications. Depending on the acyl length and the degree of substitution, these derivatives range from self-assembling watersoluble polymers to materials insoluble in aqueous environments. The behaviour of acyl-HA was studied in solution, but little attention was paid to the solid state, despite its importance for applications such as medical device fabrication. We thus used X-ray scattering and electron microscopy to explore the solidstate nano-structure of acyl-HA. The set of samples included various substituents, substitution degrees and molecular weights. The obtained data showed that all studied acyl-HA materials contained structures with dimensions on the order of nanometers that were not present in unmodified HA. The size of the nanostructures increased with the acyl length, while the degree of substitution and molecular weight had negligible effects. We suggest that the observed nanostructure corresponds to a distribution of hydrophobic domains in a hydrophilic matrix of unmodified HA segment

Vhled do problematiky mazání a adhezních vlastností hyaluronanu pro oční léčiva

Hyaluronan (HA) is widely used for eye drops as lubricant to counteract dry eye disease. High and low molecular weight HA are currently used in ophthalmology. However, a large portion of the current literature on friction and lubrication addresses articular (joint) cartilage. Therefore, eye drops compositions based on HA and its derivatized forms are extensively characterized providing data on the tribological and mucoadhesive properties. The physiochemical properties are investigated in buffers used commonly in eye drops formulations. The tribological investigation reveals that amphiphilic HA-C12 decreases the friction coefficient. At the same time, the combination of trehalose/HA or HAC12 enhances up to eighty-fold the mucoadhesiveness. Thus, it is predicted a prolonged residence time on the surface of the eye. The incorporation of trehalose enhances the protection of human keratinocytes (HaCaT) cells, as demonstrated in an in-vitro cell-desiccation model. The presence of trehalose increases the friction coefficient. Medium molecular weight HA shows significantly lower friction coefficient than high molecular weight HA. This research represents a first, wide array of features of diverse HA forms for eye drops contributing to increase the knowledge of these preparations. The results here presented also provide valuable information for the design of highly performing HA-formulations addressing specific needs before preclinic.Hyaluronan (HA) je široce používaná surovina do očních kapek pro léčbu syndromu suchého oka. Vysokomolekulární a nízkomolekulární HA se v současnosti používá v oftalmologii. Složení očních kapek na bázi HA a jejích derivátů široce charakterizováno a poskytuje údaje o tribologických a mukoadhezivních vlastnostech. Tribologický výzkum ukazuje, že amfifilní HA-C12 snižuje součinitel tření. Kombinace trehalóza/HA nebo HA-C12 zároveň zvyšuje až osmdesátinásobně mukoadhezivitu. Předpokládá se tedy prodloužená doba setrvání účinné látky na povrchu oka. Začlenění trehalózy zvyšuje ochranu buněk lidských keratinocytů (HaCaT), jak bylo prokázáno v modelu vysoušení buněk in vitro. Přítomnost trehalózy zvyšuje součinitel tření. HA se střední molekulovou hmotností vykazuje výrazně nižší součinitel tření než HA s vysokou molekulovou hmotností. Tento výzkum představuje první rozsáhlou škálu vlastností různých forem HA pro oční kapky, které přispívají ke zvýšení znalostí o těchto léčivech. Zde prezentované výsledky také poskytují cenné informace pro návrh vysoce účinných HA-přípravků, které řeší specifické potřeby před preklinickým vyšetřením

Nanofibrous material from hyaluronan derivatives preserving fibrous structure in aqueous environment

Nanofibrous materials produced from natural polymers have wide range of potential uses in regenerative medicine. This paper focuses on preparation of nanofibrous layers produced from intentionally hydrophobized derivatives of hyaluronan, which is known for its ability to promote wound healing. This structural modification of hyaluronan expands the range of potential uses of this promising material, which is otherwise limited due to the hydrophilic nature of hyaluronic acid. The aim of this research was preparation of nanofibrous material that would retain its fibrous structure and dimensional stability even after getting into contact with an aqueous medium, which is impossible to achieve with layers composed solely of native hyaluronan. As a result, such material would be able to retain its breathability and good mechanical properties when both dry and wet. Furthermore, all prepared materials were proved non-toxic for cells. This self-supporting nanofibrous matrix can be used as a scaffold, or porous wound dressing. © 2021 Elsevier Lt

Hyaluronan polymeric micelles for topical drug delivery

Nanosized materials offer promising strategy for topical drug delivery due to their enhancing effect on drug percutaneous transport across the stratum corneum barrier. In this work, polymeric micelles made from hydrophobized hyaluronic acid (HA) were probed for skin delivery. Compared to non-polymeric micelle solutions containing similar drug amount, in vitro skin penetration analysis indicated 3 times larger deposition of drug in the epidermis and 6 times larger drug deposition in the dermis after 5 h of topical treatment in Franz diffusion cells. The drug deposition was further increased with prolonged time of topical treatment. Laser confocal microscopy revealed the accumulation of both, the HA forming the vehicle and the payload, in the epidermis and dermis. Although fluorescent labeling of the HA would suggest co-transport of the HA and the drug, loading FRET pair dyes in the micellar core clearly demonstrated gradual micelle disruption with increasing skin depth. Transcellular penetration was the predominant pathway for the loaded drug. The HA polymeric micelles also demonstrated increased bioactivity of loaded compound in vitro and in vivo. In addition, the loaded micelles were found to be stable in cream formulations and thus they have great potential for topical applications for cosmetic and pharmaceutical purposes. © 2016Internal Funding Agency of the Tomas Bata University in Zlin [IGA/FT/2016/001