Controlling Drug Absorption, Release, and Erosion of Photopatterned Protein Engineered Hydrogels

Abstract

A protein-engineered triblock copolymer hydrogel composed of two self-assembling domains (SADs) has been fabricated by a photoactivatable diazirine group followed by ultraviolet (UV)-mediated crosslinking. The photocrosslinkable protein polymer CEC-D has been patterned into various features including different micrometer-scale stripes by using lithographic techniques. The patterned hydrogels are important for encapsulation of small molecules where a photopatterned fraction of 50% is optimal for maximum absorption. Stripe-patterned CEC-D100–100 exhibits slightly lower swelling ratios, an 8.9 times lower erosion profile, and a 2.6-fold higher drug release compared to the unpatterned hydrogel control, CEC-D0. Our studies demonstrate the potential of photocrosslinkable protein polymer hydrogels to be used as scaffolds for therapeutic delivery of small molecules. Through photolithographic techniques on the protein hydrogel, a variety of functionalities can be achieved by patterning different features enabling the mimicry of biological systems.

Effect of Divalent Metal Cations on the Conformation, Elastic Behavior, and Controlled Release of a Photocrosslinked Protein Engineered Hydrogel

“…As previously described, CEC was cloned into the PQE 30 vector. − , PQE 30 /CEC was transformed into AF-IQ Escherichia coli electrocompetent cells via electroporation at 2.5 kV. Expression and purification of CEC protein were carried out as discussed in previous papers. , Purity of fractions was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 12% polyacrylamide gels. Pure CEC protein fractions are collected and dialyzed in a stepwise fashion (with 4.0, 2.0, 1.0, and 0 M urea at 4 °C using SnakeSkin dialysis tubing with 10 kDa MWCO) into 50 mM PB buffer (pH 8.0).…”

“…The five lysine residues and the N-terminal amine on the CEC protein can react with the heterobifunctional photocrosslinker NHS-Diazirine (SDA) (succinimidyl 4,4-azipentanoate). The reaction procedure is as described in previous studies . Amino functional groups within the CEC protein can react with the ester of NHS-SDA and form stable amide bonds.…”

“…Amino functional groups within the CEC protein can react with the ester of NHS-SDA and form stable amide bonds. Exposure to UV light at 365 nm photoactivates the diazirine of the NHS-SDA crosslinker. , Modified CEC with the NHS-SDA crosslinker (CEC-D) of a 1:60 molar ratio can form freestanding protein hydrogels after drop casting three layers of CEC-D onto a silicon wafer of 1 cm 2 with 90.0 ± 3.1% of the lysine residues modified . The first and second layers of the CEC-D solution are exposed to UV light at 365 nm for 2 h at a light output of 8 W. The third layer of CEC-D is exposed using a mask aligner (Karl Suss MA-6) at 405 nm for 5 min under soft contact mode at a light output of 350 W with a clear chrome-quartz photomask.…”

“…Wavelength scans of the 10 μM polymerized CEC-D control and in the presence of different divalent metal solutions in 2.5 mM PB buffer at pH 8.0 were taken over at a range of 250–190 nm at 25 °C with a 1 nm step size using a quartz cuvette with a 1 mm path length. The observed ellipticity value (Θ) was converted into the mean residue ellipticity (Θ MRE ) using eq where l is the path length of the quartz cuvette in centimeters, C M is the molar concentration of the protein, and r is the number of residues in the protein. ,, Measurements were collected from three different sample preparations and subtracted from the background of 2.5 mM PB buffer at pH 8.0. The total structural content was investigated via the CDSSTR algorithm to determine the amount of helical, unordered, and strand structures and turns. , A low normalized root-mean-square deviation (NRMSD) was used to indicate the “goodness” of fit between the experimental spectrum and the calculated secondary structure …”

“…Previously, we engineered protein block copolymers that consist of two self-assembling domains (SADs): (1) a coiled-coil domain sequence from a cartilage oligomeric matrix protein (C) and (2) an elastin-like polypeptide motif (E) domain. The protein polymers have been rationally designed to combine functionalities from both SADs: small-molecule binding/delivery capacity of C and stimuli-responsive aggregation of E. − A photocrosslinkable triblock copolymer protein hydrogel, CEC-D, has been previously generated where the hydrogel is limited by its viscoelastic mechanical properties and moderately sustained release properties . Here, we investigate the impact of divalent metal ions such as Zn 2+ , Cu 2+ , and Ni 2+ on CEC-D hydrogels.…”