Immunomodulatory Hydrogel Microspheres as a Sustained Release System of Angiogenic Growth Factors

Tawagi, Eric 1 ;  Santerre, Paul 1, 2 ;  Cheng, Hai-Ling Margaret 1, 3

1. Institute of Biomaterials and Biomedical Engineering, University of Toronto; 2. Faculty of Dentistry, University of Toronto; 3. Department of Electrical & Computer Engineering, University of Toronto

The growth of new blood vessels to address disorders related to compromised tissue perfusion has been identified as a promising strategy to treat the no-option patients with critical limb ischemia.  In the United States alone this disease state affects roughly on the order 3.5 million people. The revascularization approach involves delivering growth factors with short half-life (of minutes) in the body, to the ischemic limb for extended periods of time (weeks). Growth factors can be encapsulated into particles or taken up by particles to allow for a slow protein release, but pre-clinical and clinical success to date has been limited due in part to ineffective delivery systems and improper growth factors selection. Objective: This project looks to design a novel growth factor delivery platform with immunomodulatory microspheres composed of the degradable/polar/ hydrophobic/ionic polyurethane hydrogel, termed DPHI.2 

Methods:
The DPHI microspheres are synthesized by simultaneous emulsion and photopolymerization of a resin containing a lysine-based polycarbonate-urethane crosslinker, methacrylic acid and methyl methacrylate. The microsphere have a diameter of 20 µm (PDI = 0.4) and a 63 ± 7% recovery of the initial reagents. Proteins are encapsulated by adding the proteins to the pre-emulsion or they are taken up by microspheres, which are already polymerized and dry. 

Results and Discussion:
Release studies in phosphate buffered saline solution show that encapsulated bovine serum albumin (BSA) had a slower release (23 ± 5% at day 5) while the uptake of BSA by dry microspheres had a fast release (72 ± 7% at day 5). The incorporation of the basic fibroblast growth factor (bFGF) is currently being investigated. The effect of particle diameter on protein release is also being pursued. The microspheres are non-cytotoxic after 48 hr incubation with endothelial cells (HUVEC), and instead support an increased proliferation of HUVECs at 40 and 50 ng/cell microsphere concentrations. On-going work with a HUVEC sprouting assay in a fibrin gel is testing whether released bFGF maintains the capacity to induce HUVEC sprout formation. Future work is focused on defining the acute toxicity of the microspheres after injection to the hindlimb of rats. In conclusion, this report outlines the successful emulsification and polymerization of DPHI into microspheres and demonstrates two protein incorporation methods. 

Summary: The synthesis of DPHI microspheres loaded with growth factors is an important step towards the design of a protein delivery platform enabling the study of multiple growth factors and their administration into animal models and clinical settings of critical limb ischemia.

Acknowledgement:  Richard Lewar Heart and Stroke Centre of Excellence studentship, IBBME fellowship, NSERC Synergy grant # SYN 430828