Na podstawie uzyskanych wyników stwierdzono, że opracowane i scharakteryzowane pod kątem chemicznym i biologicznym matryce polimerowe na bazie chitozanu mają właściwości wiązania LPS, co daje możliwość ich zastosowanie do: oczyszczania rekombinowanych białek fagowych pozyskanych w systemie ekspresyjnym, opartym na bakteriach Gram-ujemnych, oraz jako nośniki dla bakteriofagów w komponowaniu opatrunków przeciwbakteryjnych o działania kontaktowym. Wynikiem przeprowadzonych w ramach realizacji pracy doktorskiej opisanych badań są dwa krajowe zgłoszenia patentowe na wynalazek, pt.: „Synteza i zastosowanie matrycy polimerowej do oczyszczania białek rekombinowanych z lipopolisacharydu bakteryjnego” (zgłoszenie do Urzędu Patentowego RP z dn. 31.03.2023, nr. P.443659) oraz: „Metoda syntezy i zastosowanie matrycy polimerowej do komponowania z preparatami bakteriofagów” (zgłoszenie do Urzędu Patentowego RP z dn. 31.03.2023, nr. P.443658).
The method of biosynthesis of therapeutic proteins using microbial expression systems by prokaryotic cells carries the risk of transferring gram-negative lipopolysaccharide (LPS). Therefore, it is important to have a concomitant, well-developed purification system for protein products that will be able to effectively bind harmful LPS and will not change the essential properties of the protein. Due to its high biological activity and the ability to bind micro and macro particles, chitosan can be proposed for the purification of therapeutic proteins from endotoxins. It is a biomaterial with high biocompatibility, non-toxic and biodegradable, due to its physicochemical properties and can be subjected to various chemical and physical modifications. In the first part of this work, two different biopolymer systems based on chitosan from shrimps of different microbiological purity were proposed - the form of granules and the form of a sponge. The templates were used to purify selected phage proteins (endolysins and depolymerases) obtained using bacterial expression systems. The matrices were characterized in terms of their physicochemical and mechanical properties, impact on living cells (cytotoxicity), activity of purified proteins, endotoxin binding capacity and the method of their binding to the chitosan chain (FT-IR). The results showed that both formulations: granulate and sponge are capable of binding LPS contaminating biotechnologically produced phage proteins. In addition, the microbiological purity of the starting material and the functional form of these polymers are important in the context of the ability to capture endotoxins in the tested protein preparations. The granulate turned out to be a good candidate for the initial purification of the product from LPS with a high recovery of purified material (protein). Chitosan sponge, on the other hand, allows to obtain the purest possible LPS-free product, with much lower recovery of the protein product. Difficult-to-heal wounds are one of the most common problems in clinical practice, especially in patients with various diseases (mainly diabetes), but also during post-operative convalescence or post-traumatic treatment at various stages. Wound healing is a dynamic and complex process where infection prevention is key. Many important changes occur during healing: from inflammation, to cell migration, angiogenesis and cell matrix synthesis, to collagen deposition and re-epithelialization. Chitosan, thanks to its bactericidal effect on Gram-positive and Gram-negative bacteria as well as anti-inflammatory and haemostatic properties, is an excellent candidate for the design of dressings for the treatment of hard-to-heal wounds. The great advantage of this biopolymer is its ability to be chemically modified, which allows the production of various utility forms depending on the needs and subsequent use.
In addition, chitosan may be an excellent polymer matrix for the immobilization of bacteriophages (phages) as a novel alternative/adjunctive approach to antibacterial therapy. The second part of this work concerns the preparation and characterization of a chitosan-based material in the form of a film with the addition of Pseudomonas lytic phages (KTN4, KT28 and LUZ19), which would show antibacterial activity as a potential dressing accelerating wound healing. A method for producing a polymer based on microcrystalline chitosan (MKCh) that serves as a template for phage deposition has been developed. Several important parameters were described, such as the average molar mass, swelling capacity, surface morphology, phage release profile and antibacterial activity tested in the Pseudomonas aeruginosa PAO1 bacterial model. It has been proven that chitosan polysaccharide interacts with phage particles, immobilizing them in the matrix structure. Nevertheless, due to the high hydrophilicity and swelling characteristics of the prepared material, the external bacterial culture solution was absorbed, and the phages came into direct contact with the bacteria, causing their lysis in the polymer matrix.
Jan 10, 2024
Jan 10, 2024
|Sikora, Monika, Synteza nowych materiałów na bazie chitozanu oraz ich wybrane zastosowania w biotechnologii i medycynie
|Jan 10, 2024