Design and Development of Renally Targeted Polymeric Nanoparticles for the Treatment of Mitochondrial Dysfunction

Abstract

Mitochondrial dysfunction plays a critical role in both acute and chronic kidney diseases throughout the world. Mitochondria are major intracellular organelles with a variety of critical roles including ATP, regulation of metabolism, maintaining reactive oxygen species levels and the regulation of apoptosis making them an attractive therapeutic target. Upon stress, mitochondrial dynamics are disrupted and membrane integrity is compromised resulting in mitophagy, cellular apoptosis, increased reactive oxygen species production and decreased ATP production which contributes to further injury and cell death. Research has also suggested that early damage to mitochondrial function is a critical determinant of tubular injury and progressive renal insufficiency in both acute and chronic renal disease. Despite this clear involvement in the pathogenesis of various diseases there are currently no effective and approved pharmacological therapies that target mitochondrial dysfunction. Compounds that restore mitochondrial dynamics, including those that induce mitochondrial biogenesis, have been shown to protect against acute and progressive renal injury in models of both AKI and DKD. One of these compounds is the FDA-approved β2-adrenergic receptor agonist formoterol fumarate dihydrate which has been shown to induce mitochondrial biogenesis in cells and rodents and enhance recovery in a mouse model of AKI. Formoterol however has demonstrated cardiotoxicity, resulting in immediate tachycardia and hypotension with sufficiently high systemic doses and cardiac hypertrophy and remodeling with continuous use. In this dissertation, formoterol has been incorporated into biodegradable and biocompatible polymeric nanoparticles. These nanoparticles are shown to accumulate preferentially to the tubules of the kidneys resulting in the localized sustained release of formoterol, resulting in mitochondrial biogenesis in the proximal tubule cells at a fraction of the required free drug dose. Daily administration of these nanoparticles in a model of acute kidney injury was shown to localize to the kidneys with 25-fold greater efficiency than the heart and other organs. Localized nanoparticle delivery of formoterol resulted in enhanced recovery from renal I/R induced AKI, even treatment was initiated 24-hours following injury. Formoterol nanoparticles showing greater recovery of serum biomarkers of loss of renal function KIM-1 and creatinine within 96-hours of injury and recovery of mitochondrial number and electron transport chain protein levels by 144-hours following injury. Formoterol nanoparticle treated groups showed marked improvement in renal histology by 144-hours including reduced tubular necrosis and fibrosis. This also indicates that formoterol nanoparticles reduced the likelihood of AKI to CKD transition in these mice, which is further substantiated by improved vascular and epithelial tight junction recovery as well as decreased serum NGAL which is correlated with CKD progression. Further, formoterol nanoparticle therapy was an improvement over formoterol free drug as it was able to achieve similar recovery with ~10-fold lower dose and with decreased dosing frequency, 2 days compared to 6 days with free formoterol. Additionally, nanoparticle drug delivery protects against acute cardiovascular toxicity and cardiac hypertrophy seen with formoterol free drug therapy. Weekly administration of formoterol containing nanoparticles in a progressive DKD model showed induction of mitochondrial biogenesis greater than that of 1 mg/kg formoterol free drug at ~10 fold lower weekly dose. Treatment of diabetic mice for 8 weeks showed decreased progression of key urinary markers of DKD including albumin:creatinine ratio, urinary KIM-1, urinary NGAL and glomerular hyperfiltration. Mice showed improved renal histology compared to vehicle treated diabetic mice. Formoterol nanoparticles resulted in decreased progression of glomerular hypertrophy and mesangial matrix expansion as well as reduced glomerular fibrosis. Tubular injury was also decreased in formoterol nanoparticle treated mice which showed decreased inflammation and fibrosis. Formoterol nanoparticles additionally protected against toxic cardiovascular effects of repeated formoterol free drug administration, preventing cardiac hypertrophy and severe fibrosis seen in formoterol treated mice as well as increasing the probability of survival compared to formoterol free drug groups. Taken together this research demonstrates the benefit of renally targeted drug delivery systems for acute and chronic kidney diseases as well as protecting against drug toxicity.