Soft micron-sized polypeptide microgels: preparation, crosslink density, topography and nanomechanics in swollen state

Abstract

We report an innovative preparation of soft micron-sized polypeptide microgels by horseradish peroxidase (HRP)-mediated crosslinking in inverse suspension. The prepared microgels were based on poly[N⁵-((2-hydroxypropyl)-L-glutamine)-ran-(N⁵-propargyl-L-glutamine)-ran-(N⁵-(6-aminohexyl)-L-glutamine)]-ran-(N⁵-[2-((4-hydroxyphenyl)ethyl)-L-glutamine)] (P2HPG-Tyr) polymer precursor. We tested effects of three different surfactants, namely, sorbitan monooleate (SPAN 80), polyoxyethylenesorbitan trioleate (TWEEN 85), and dioctyl sulfosuccinate sodium salt (AOT), on microgelation in inverse suspension without or with a pre-emulsification step. The prepared P2HPG-Tyr microgels were investigated using light and cryogenic microscopy. The HRP-mediated crosslinking in inverse suspension employing SPAN 80 and one hour pre-emulsification yielded high-quality, spherical, and colloidally stable ∼80 μm P2HPG-Tyr microgels. Innovatively, we immobilized these large swollen P2HPG-Tyr microgels on mica and glass substrates for subsequent topography and surface nanomechanical measurements of these hydrated and swollen microgels using atomic force microscopy (AFM) in peak force quantitative nanomechanical mapping (PF-QNM) mode. The topography analyses revealed surface irregularities of the developed P2HPG-Tyr microgels consisting of small holes with diameters ranging from 80 to 200 nm. The PF-QNM proved the viscoelasticity and softness of P2HPG-Tyr microgels documented with Young's modulus in the range of tens of kPa derived from force-separation curves. Finally, the crosslink density was evaluated using two methods of calculation, revealing comparable concentrations of elastically active network chains (EANCs) in the range from 0.489 × 10⁻³ to 0.812 × 10⁻³ mol cm⁻³.