Jay C. Sy

Maleimide Cross‐Linked Bioactive PEG Hydrogel Exhibits Improved Reaction Kinetics and Cross‐Linking for Cell Encapsulation and In Situ Delivery

Engineered polyethylene glycol-maleimide matrices for regenerative medicine exhibit improved reaction efficiency and wider range of Young’s moduli by utilizing maleimide cross-linking chemistry. This hydrogel chemistry is advantageous for cell delivery due to the mild reaction that occurs rapidly enough for in situ delivery, while easily lending itself to “plug-and-play” design variations such as incorporation of enzyme-cleavable cross-links and cell-adhesion peptides.

Sustained release of a p38-inhibitor from non-inflammatory microspheres inhibits cardiac dysfunction

Cardiac dysfunction following acute myocardial infarction is a major cause of death in the world and there is a compelling need for new therapeutic strategies. In this report we demonstrate that a direct cardiac injection of drug-loaded microparticles, formulated from the polymer poly(cyclohexane-1,4-diylacetone dimethylene ketal) (PCADK), improves cardiac function following myocardial infarction. Drug-delivery vehicles have great potential to improve the treatment of cardiac dysfunction by sustaining high concentrations of therapeutics within the damaged myocardium. PCADK is unique among currently used polymers in drug delivery in that its hydrolysis generates neutral degradation products. We show here that PCADK causes minimal tissue inflammatory response, thus enabling PCADK for the treatment of inflammatory diseases, such as cardiac dysfunction. PCADK holds great promise for treating myocardial infarction and other inflammatory diseases given its neutral, biocompatible degradation products and its ability to deliver a wide range of therapeutics.

Microfluidic-Based Cell Sorting of Francisella tularensis Infected Macrophages Using Optical Forces

We have extended the principle of optical tweezers as a noninvasive technique to actively sort hydrodynamically focused cells based on their fluorescence signal in a microfluidic device. This micro fluorescence-activated cell sorter (microFACS) uses an infrared laser to laterally deflect cells into a collection channel. Green-labeled macrophages were sorted from a 40/60 ratio mixture at a throughput of 22 cells/s over 30 min achieving a 93% sorting purity and a 60% recovery yield. To rule out potential photoinduced cell damage during optical deflection, we investigated the response of mouse macrophage to brief exposures (<4 ms) of focused 1064-nm laser light (9.6 W at the sample). We found no significant difference in viability, cell proliferation, activation state, and functionality between infrared-exposed and unexposed cells. Activation state was measured by the phosphorylation of ERK and nuclear translocation of NF-kappaB, while functionality was assessed in a similar manner, but after a lipopolysaccharide challenge. To demonstrate the selective nature of optical sorting, we isolated a subpopulation of macrophages highly infected with the fluorescently labeled pathogen Francisella tularensis subsp. novicida. A total of 10,738 infected cells were sorted at a throughput of 11 cells/s with 93% purity and 39% recovery.

The delivery of superoxide dismutase encapsulated in polyketal microparticles to rat myocardium and protection from myocardial ischemia-reperfusion injury

Oxidative stress is increased in the myocardium following infarction and plays a significant role in death of cardiac myocytes, leading to cardiac dysfunction. Levels of the endogenous antioxidant Cu/Zn-superoxide dismutase (SOD1) decrease following myocardial infarction. While SOD1 gene therapy studies show promise, trials with SOD1 protein have had little success due to poor pharmacokinetics and thus new delivery vehicles are needed. In this work, polyketal particles, a recently developed delivery vehicle, were used to make SOD1-encapsulated-microparticles (PKSOD). Our studies with cultured macrophages demonstrated that PKSOD treatment scavenges both intracellular and extracellular superoxide, suggesting efficient delivery of SOD1 protein to the inside of cells. In a rat model of ischemia/reperfusion (IR) injury, injection of PKSOD, and not free SOD1 or empty particles was able to scavenge IR-induced excess superoxide 3 days following infarction. In addition, only PKSOD treatment significantly reduced myocyte apoptosis. Further, PKSOD treatment was able to improve cardiac function as measured by acute changes in fractional shortening from baseline echocardiography, suggesting that sustained delivery of SOD1 is critical during the early phase of cardiac repair. These data demonstrate that delivery of SOD1 with polyketals is superior to free SOD1 protein therapy and may have potential clinical implications.

Hoechst-IR: an imaging agent that detects necrotic tissue in vivo by binding extracellular DNA.

Cell necrosis is central to the progression of numerous diseases, and imaging agents that can detect necrotic tissue have great clinical potential. We demonstrate here that a small molecule, termed Hoechst-IR, composed of the DNA binding dye Hoechst and the near-infrared dye IR-786, can image necrotic tissue in vivo via fluorescence imaging. Hoechst-IR detects necrosis by binding extracellular DNA released from necrotic cells and was able to image necrosis generated from a myocardial infarction and lipopolysaccharide/d-galactosamine (LPS-GalN) induced sepsis.

Delivering Regenerative Cues to the Heart: Cardiac Drug Delivery by Microspheres and Peptide Nanofibers

Our understanding of signaling pathways and cues vital for cardiac regeneration is being refined by laboratories worldwide. As various mechanisms enabling cardiac regeneration are becoming elucidated, delivery vehicles suited for these potential therapeutics must also be developed. This review focuses on advances in two technologies, novel degradable microspheres for controlled release systems and self-assembling peptide nanofibers for cell and factor delivery. Polyketals, a new class of resorbable polymers, are well suited for treating inflammatory diseases due to biocompatible degradation products. Polyketals have been used to deliver small molecule inhibitors and antioxidant proteins to rat models of myocardial infarction with notable improvements in cardiac function. Self-assembling peptide nanofibers are a class of hydrogels that are well-defined scaffolds made up of 99% water and amenable to incorporation of a variety of bioactive cues. Work done by our laboratory and others have demonstrated functional improvements using these hydrogels as both a drug delivery vehicle for proteins as well as a defined microenvironment for transplanted cells. Combining non-inflammatory polymer microspheres for sustained release of drugs with self-assembling nanofibers yields multifunctional scaffolds that may soon drive the body’s healing response following myocardial infarction towards cardiac regeneration.

Surface functionalization of polyketal microparticles with nitrilotriacetic acid-nickel complexes for efficient protein capture and delivery.

Microparticle drug delivery systems have been used for over 20 years to deliver a variety of drugs and therapeutics. However, effective microencapsulation of proteins has been limited by low encapsulation efficiencies, large required amounts of protein, and risk of protein denaturation. In this work, we have adapted a widely used immobilized metal affinity protein purification strategy to non-covalently attach proteins to the surface of microparticles. Polyketal microparticles were surface modified with nitrilotriacetic acid-nickel complexes which have a high affinity for sequential histidine tags on proteins. We demonstrate that this high affinity interaction can efficiently capture proteins from dilute solutions with little risk of protein denaturation. Proteins that bound to the Ni-NTA complex retain activity and can diffuse away from the microparticles to activate cells from a distance. In addition, this surface modification can also be used for microparticle targeting by tethering cell-specific ligands to the surface of the particles, using VE-Cadherin and endothelial cells as a model. In summary, we show that immobilized metal affinity strategies have the potential to improve targeting and protein delivery via degradable polymer microparticles.

Targeting extracellular DNA to deliver IGF-1 to the injured heart

There is a great need for the development of therapeutic strategies that can target biomolecules to damaged myocardium. Necrosis of myocardium during a myocardial infarction (MI) is characterized by extracellular release of DNA, which can serve as a potential target for ischemic tissue. Hoechst, a histological stain that binds to double-stranded DNA can be conjugated to a variety of molecules. Insulin-like growth factor-1 (IGF-1), a small protein/polypeptide with a short circulating-half life is cardioprotective following MI but its clinical use is limited by poor delivery, as intra-myocardial injections have poor retention and chronic systemic presence has adverse side effects. Here, we present a novel delivery vehicle for IGF-1, via its conjugation to Hoechst for targeting infarcted tissue. Using a mouse model of ischemia-reperfusion, we demonstrate that intravenous delivery of Hoechst-IGF-1 results in activation of Akt, a downstream target of IGF-1 and protects from cardiac fibrosis and dysfunction following MI.