20.500.12556/DKUM-83850 Preparation of amino acid and peptide polysaccharide derivatives and their application as biomaterials doctoral dissertation Priprava aminokislinskih in peptidnih polisaharidnih derivatov ter njihova uporaba kot biomateriali doktorska disertacija The derivatization of the polysaccharide dextran with N-protected amino acids (Boc-L-Phenylalanine, BocGlycine, Boc-L-Cysteine, and Boc-L-Cysteine-S-Trt) and peptides (Boc-L-DiPhenylalanine, Boc DiGlycine, and 2,5-diketopiperazine) as the basis for biomaterial preparation is presented in this Doctoral Dissertation. Such prepared dextran derivatives are intended to mimic the proteoglycan complex (PGs), one of the most important structural and functional biomacromolecules in the extracellular matrix (ECM) of tissue. Nowadays, developments in biomaterials are focusing increasingly on the preparation and use of biomimetic molecular structures to achieve positive results in tissue engineering (TE) and drug delivery. Designing and synthesizing these biomimetic materials, however, requires sophisticated chemical and material preparation methods, knowledge that is, currently, unexplored. In this work, we developed a suitable procedure for dextran derivatization, and investigated the most optimal reaction or deprotection conditions (temperature and time) and isolation/purification methods. The structures of the obtained BocPhe-Dex, BocGly-Dex, BocCys-Dex, and BocSTLC-Dex were analyzed with FTIR, NMR, SEC-MALS, and EA. The results showed that dextran derivatization was successful in all cases except in the case of dextran derivatization with BocCys. Investigation of the effect of the derivatization conditions and purification on the stability, purity, and other important chemical and physical properties of the obtained product, showed that the temperature and time of derivatization do not have a bigger effect on the products' properties, while the purification method, on the other hand, has. Its effect is visible in the product's purity and mass yields of products prepared under the same reaction conditions. Derivatization of dextran with peptides (Boc-L-DiPhenylalanine, BocDiGlycine, and 2,5-diketopiperazine) was performed using the CDI coupling agent or Amberlite-IR 120 as a catalyst. The products were analyzed with FTIR and 1H and 13C NMR. The results showed successful dextran derivatization in the case of BocDiPhenylalanine and BocDiGlycine, while, in the case of 2,5-diketopiperazine, a reaction covalent bond with the dextran was not confirmed. BocPhe-Dex and BocSTLC-Dex were selected as the most optimal amino acid-dextran derivatives for further preparation of 3D formulations in the shape of nanoparticles (NPs). Nanoparticles were prepared with the emulsion/solvent evaporation method from the obtained BocPhe-Dex and BocSTLC-Dex products (prepared in the first stage of this Doctoral Dissertation). SEM analysis showed that the prepared NPs were homogeneous and nicely spherical, with an average dry diameter of 325 ± 118 nm in the case of BocSTLC-Dex, and 1039 ± 382 nm in the case of NPs prepared from BocPhe-Dex. All the prepared NPs retained their proper spherical shape and stability during the acidic treatment, and so confirmed their potential for further functionalization and applications for drug delivery. The BocSTLC-Dex NPs were also evaluated with cell viability tests, which showed that the prepared NPs were not cytotoxic, one of the most important characteristics for the drug delivery applications of NPs. This work serves as a basis for further studies on the derivatization of polysaccharides with amino acids and peptides, and their application in tissue engineering or drug delivery. V tej doktorski disertaciji je predstavljena derivatizacija polisaharida dekstrana z N-zaščitenimi aminokislinami (Boc-L-Fenilalanin, BocGlicin, Boc-L-Cistein in Boc-L-Cistein-S-Trt) in peptidi (Boc-L-DiFenilalanin, Boc-DiGlicin, in 2,5-diketopiperazin) kot osnova za pripravo biomateriala. Tako pripravljeni derivati dekstrana so namenjeni posnemanju proteoglikanskega kompleksa (PG), ene najpomembnejših strukturnih in funkcionalnih biomakromolekul v zunajceličnem matriksu (ECM) tkiva. Dandanes se razvoj biomaterialov vse bolj osredotoča na pripravo in uporabo biomimetičnih molekularnih struktur za doseganje pozitivnih rezultatov v tkivnem inženirstvu (TE) in dostavi zdravil. Oblikovanje in sintetiziranje teh biomimetičnih materialov pa zahteva sofisticirane kemične in materialne metode priprave, znanje, ki je trenutno neraziskano. V tem delu smo razvili ustrezen postopek za derivatizacijo dekstrana in raziskali najbolj optimalne reakcijske pogoje (temperatura in čas) ter metode izolacije/čiščenja. Lastnosti dobljenih BocPhe-Dex, BocGly-Dex, BocCys-Dex in BocSTLC-Dex smo analizirali s FTIR, NMR, SEC-MALS in EA. Rezultati so pokazali, da je bila derivatizacija dekstrana uspešna v vseh primerih, razen v primeru derivatizacije dekstrana z BocCys. Raziskava vpliva pogojev derivatizacije in čiščenja na stabilnost, čistost in druge pomembne kemijske in fizikalne lastnosti dobljenega produkta je pokazala, da temperatura in čas derivatizacije nimata večjega vpliva na lastnosti produktov, medtem ko metoda čiščenja pa ima, saj je njen učinek viden v čistosti produkta in masnih izkoristkih produktov, pripravljenih pod enakimi reakcijskimi pogoji. Derivatizacija dekstrana s peptidi (Boc-L-DiFenilalanin, BocDiGlicin in 2,5-diketopiperazin) je bila izvedena z uporabo spojnega sredstva CDI ali Amberlite-IR 120 kot katalizatorja. Produkte smo analizirali s FTIR in NMR. Rezultati so pokazali uspešno derivatizacijo dekstrana v primeru BocDiFenilalanina in BocDiGlicina, medtem ko v primeru 2,5-diketopiperazina reakcijska kovalentna vez z dekstranom ni bila potrjena. BocPhe-Dex in BocSTLC-Dex sta bila izbrana kot najbolj optimalna aminokislinsko-dekstranska derivata za nadaljnjo pripravo 3D formulacij v obliki nanodelcev (NP). Iz dobljenih produktov BocPhe-Dex in BocSTLC-Dex (pripravljenih v prvi fazi te doktorske disertacije) smo pripravili nanodelce z metodo evaporacije emulzije/topila. Analiza SEM je pokazala, da so pripravljeni NP-ji homogeni in lepo sferični, s povprečnim suhim premerom 325 ± 118 nm v primeru BocSTLC-Dex in 1039 ± 382 nm v primeru NP-jev, pripravljenih iz BocPhe-Dex. Vsi pripravljeni NP-ji so ohranili pravilno sferično obliko in stabilnost med obdelavo s kislino in tako potrdili svoj potencial za nadaljnjo funkcionalizacijo in aplikacije za dostavo zdravil. NP-ji BocSTLC-Dex so bili ovrednoteni tudi s testi viabilnosti celic, ki so pokazali, da pripravljeni NP-ji niso citotoksični, kar je ena najpomembnejših značilnosti za aplikacije NP-jev za dostavo zdravil. To delo služi kot podlaga za nadaljnje študije o derivatizaciji polisaharidov z aminokislinami in peptidi ter njihovi uporabi v tkivnem inženiringu ali dostavi zdravil. Amino Acid-Dextran derivatives Peptide-Dextran derivatives Proteoglycan complex 3D formulation Nanoparticles Drug delivery Aminokislinski-Dekstranski derivati Peptidni-Dekstranski derivati Proteoglikanski kompleks 3D formulacija Nanodelci Ciljan vnos zdravilnih učnkovin true true false A. Bratuša Štern Angleški jezik Slovenski jezik Doktorsko delo/naloga 2023-02-23 13:20:24 2024-10-18 11:14:29 2026-07-15 09:15:53 0000-00-00 00:00:00 2024 0 Maribor 0 Maribor XX, 152 str. 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 [577.114.4:577.112]:615.015(043.3) 212555523 DOK_Bratusa_Stern_Ana_2024.pdf DOK_Bratusa_Stern_Ana_2024.pdf 1 7801AAAFC25390C53302CA64D026774E d6eb65a1ca5e4e987e852316284e4221ff2ec70cefa07dab22a443daaafc3018 0da3ce66-8d2a-11ef-a8b7-00155d000105 https://dk.um.si/Dokument.php?lang=slv&id=186649 Fakulteta za strojništvo 0 0 0