Binding & Release of Biomolecules from Hyaluronic Acid Macrogels with Cetylpyridinium Chloride as a Model Surfactant

Abstract: The purpose of this master thesis project was to investigate the penetration ofbiomolecules into hyaluronic acid (HA) macrogels. The investigations were performedboth in the presence and in the absence of the non-biodegradable surfactantcetylpyridinium chloride (CPC), which earlier has proved to form a micelle-rich shellat the gel surface. In this work investigations were performed to see if properties ofthe biomolecules used, such as size and charge, had any impact on the binding to theHA gels both with and without CPC. The biomolecules used were the proteinscytochrome c, lysozyme, hemoglobin and myoglobin and the polysaccharide dextranof different molecular weights and labeled with fluorescein isothiocyanate.Cetylpyridinium chloride was used as a non-biodegradable model surfactant for abiodegradable betaine ester surfactant. The cetylpyridinium chloride was thereforeserving well when performing control studies for the biodegradable betaine ester,because the two possesses similar properties. Investigations involving the betaineester surfactant was not included in this master thesis project. Also release studiesinvolving the labeled dextran and some of the proteins were performed in thepresence and absence of cetylpyridinium chloride.The binding of CPC to HA was investigated briefly as well as the microstructure ofHA gels saturated with CPC by means of small-angle X-ray scattering, SAXS, atdifferent salt concentrations. The microstructure-investigations indicated that at 10mM NaCl a cubic ordered phase with space group Pm3n was achieved. When the saltconcentration was increased to 40 mM the microstructure was altered to a clearface-centered cubic (FCC) structure. When increasing the NaCl concentrationfurther, to 150 mM, indication of an unordered micellar phase could be seen.Cytochrome c and lysozyme transport into HA gels, to which CPC had bound in anearlier step, could be registered using UV-VIS spectrophotometry. Indications showedthat CPC and cytochrome c was distributed to different parts of the gel frommicroscope pictures taken of the cross-section of gel samples at different time.From release experiments performed with fluorescein isothiocyanate-dextran noconclusions could be drawn on how the different molecular weights of dextranaffected the rate and extent of the amount substance released. Neither could theinfluence of CPC be elucidated. This since the extent of released amounts exceeded100 % for many samples and the duplicate samples investigated showed differentbehavior.The transport of cytochrome c, myoglobin and hemoglobin into HA gels with andwithout beforehand treatment with CPC was evaluated qualitatively. The transportinto gels treated with CPC was successful and complete with cytochrome c andmyoglobin at lower degree of binding but was limited for the bigger proteinhemoglobin. When investigating the release of cytochrome c, myoglobin andhemoglobin from HA gels the extent of released substance was lower with CPCpresent in the solution compared without CPC present. Also some indicationsshowed that the bigger size of hemoglobin affected the rate of release from the gel.