Dual pH- and temperature-responsive microparticles for protein delivery to ischemic tissues

Injectable "smart" microspheres that are sensitive to both temperature and pH have been fabricated and tested for controlled delivery of therapeutic proteins to ischemic skeletal muscle. A library of copolymers composed of N-isopropyl acrylamide (NIPAAm), propyl acrylic acid (PAA), and butyl acrylate (BA) was used to fabricate microspheres using a double emulsion method, and an optimal formulation made from copolymers composed of 57 mol.% NIPAAm, 18 mol.% PAA and 25 mol.% BA copolymers was identified. At 37°C and pH representative of ischemic muscle (i.e. pH 5.2-7.2), these microspheres produced sustained, diffusion-controlled release, and at normal, physiological pH (i.e. pH 7.4), they underwent dissolution and rapid clearance. Delivery of fibroblast growth factor 2 was used to confirm that protein bioactivity was retained following microsphere encapsulation/release based on a dose-dependent increase in NIH3T3 fibroblast proliferation in vitro. Microsphere-loaded or free Cy5.5-labeled albumin was injected into ischemic and control gastrocnemii of mice following unilateral induction of hind limb ischemia to model peripheral arterial disease. In the ischemic limb at days 3.5 and 7, there was higher local retention of the protein delivered via microspheres relative to injected free protein (p<0.05). However, clearance of protein delivered via microspheres was equivalent to free protein at later time points that correspond to ischemic recovery in this model. Finally, histological analysis of the gastrocnemius revealed that the polymeric microspheres did not produce any microscopic signs of toxicity near the injection site. These combined results suggest that the pH- and temperature-responsive microspheres presented herein are a promising technological platform for controlled protein delivery to ischemic tissue.