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Sruthi Santhanam, Rinku Baid, Nathan Ravi, ; Optimization of Thiolation for Biomimetic Vitreous Substitute. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5278.
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The overall objective of our research is to develop a biomimetic in situ forming hydrogel. Past research has shown that water soluble polymers containing pendant thiol groups cross-link spontaneously in the presence of oxygen under physiological conditions of pH, temperature, and ionic strength. However, derivatizing these polymers to a predetermined extent has been an Achilles tendon. Consequently, we have studied the reaction of introducing thiol amides groups using hyaluronic acid (HA) as a surrogate. Typically, amidation of biological molecules is conducted by activation of a carboxyl group using carbodiimide (EDC) with n-hydroxyl succinimide (NHS) present as a stabilizing intermediate. Final amidation is subsequently formed by reacting with a thiolated primary amine, in this case cystamine (Cys). The extent of the reaction also is a function of pH, temperature and duration. The process was optimized using a fractional factorial design of the above five factors.
A 2-level fractional factorial design of experiments (DoE) of mole ratio of (1) HA:Cys, (2) HA:EDC, (3) HA:NHS, and (4) pH and (5) duration was investigated. The experiments were carried out in a random order at 37oC and degree of amidation was determined by modified Ellman’s Test. Using Design Expert software, main parameters affecting the reaction were determined by analyzing the normal and half normal probability plots. A model of significant parameters were constructed and validated by analysis of variance (ANOVA), and residual analysis.
The degree of amidation response varied from a minimum of 15 % to a maximum of 91 % with a mean of 45 % and standard deviation of 24 %. A 10 molar Cys, 15 molar EDC, and 0.1 molar NHS at pH 4.5 for 6 hours resulted in near quantitative yield. The main parameters affecting the reaction in order of importance were HA: Cys mole ratio, HA: EDC mole ratio, and reaction duration. Two factor interaction of Cys- EDC, EDAC - time, EDAC - pH, pH - time, and pH - NHS were also significant.
The degree of amidation has an effect on other properties of the gel such as pore size, mechanical strength, and diffusion of molecules. In this study we were able to control the degree of amidation by designing the experiment using 2- level fraction factorial DoE. Application of these findings will facilitate the future planning of our vitreous substitutes.
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