An orthogonal analytical platform for determining critical material attributes for in situ formation of thermosensitive hyaluronic acid-poly(N-isopropylacrylamide) copolymers

The developability assessment of the in situ formation of thermosensitive injectables requires the unequivocal determination of their lower critical solution temperatures (LCSTs). Herein, we established a standardized threshold for LCST detection by harmonizing and cross-validating the orthogonal methods using techniques such as nuclear magnetic resonance spectroscopy, light scattering, turbidimetry, backgrounded membrane imaging, and rheology. The selected techniques were in agreement and promptly disclosed unique features and instrumental biases of otherwise difficult interpretations in the characterization of thermosensitive polymers. Thus, various hyaluronic acid–poly ( N -isopropylacrylamide) copolymers were synthesized via strain-promoted azide–alkyne cycloaddition to understand their critical material attributes. Structure–function relationships and behaviors of the copolymers were tested under in vitro -simulated in vivo conditions. • Thermosensitive copolymers comprising of hyaluronic acid and poly ( N -isopropylacrylamide) were synthesized. • A comprehensive platform of orthogonal characterization methods was developed (NMR, SLS, DLS, Turbidity, BMI, and Rheology). • In vitro thermosensitive features were defined for future in vivo use.