Assistant Professor Northwestern University, United States
Introduction: Mitochondria transplantation has emerged as a promising regenerative therapy for vascular disease due to the significant contribution of mitochondrial dysfunction in inflammation, ischemia-reperfusion injury, and oxidative stress. However, current transplantation strategies suffer from a lack of specificity, limited uptake, uncontrolled biodistribution, and overall subpar efficiency. To combat this, we have engineered targeted mitochondrial delivery systems (MDS) using modular polymer coatings that allow easy exchange and combination of tissue-specific and cell-penetrating peptides to improve the efficacy of mitochondrial transplantation. We previously showed that collagen binding peptide (CBP) preferentially binds with the basement membrane protein collagen IV, suggesting it can be exploited to target the vascular endothelium after injury. We pose that CBP can be combined with MDS coating to improve mitochondria transplantation to vascular endothelial cells after injury thereby enhancing healing.
Materials and
Methods: Mitotracker Red-labelled mitochondria (Mts) were isolated from mesenchymal stem cells (MSCs) derived from induced pluripotent stem cells (iPSCs). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)] (DSPE-PEG)-CBP conjugates were synthesized via Michael Addition at a 1:1 maleimide:thiol ratio and characterized via nuclear magnetic resonance spectroscopy. Isolated Mts were coated with DSPE-PEG-CBP at increasing polymer:mitochondria mass ratios. Successful coating was visualized using FITC-labelled CBP. Dynamic light scattering was used to monitor particle size. Mitochondria function after coating was determined using the Seahorse Mito Stress Test assay to measure the oxygen consumption rate. Uptake of uncoated and DSPE-PEG coated Mts was compared in vitro using confocal microscopy. The Seahorse Mito Stress Test and tube formation assays were utilized to measure the effects of isolated Mts on rescuing function of peroxide-treated endothelial cells. Intraluminal administration of uncoated and DSPE-PEG-CBP coated mitochondria was performed immediately after carotid balloon injury in a rat model. Biodistribution was determined using IVIS imaging at 3, 7, and 14d (n=6/timepoint). Cell-specific uptake was analyzed after euthanasia at 3, 7, and 14d. Vessel morphometry (i.e. neointimal hyperplasia, lumen area) and expression of inflammatory markers were determined histologically at 14d in vessels treated with uncoated Mts, coated Mts, deactivated Mts and vehicle controls (n=6/group).
Results, Conclusions, and Discussions:
Results: Successful DSPE-PEG-CBP characterization was confirmed using NMR. Isolated mitochondria (red) colocalized with polymer coating (green) under fluorescent microscopy. The polymer coating did not significantly change the average mitochondria particle diameter 399.6±149.3nm (uncoated), 426.4±76.2nm (1.5:1 mass ratio), and 411.7±152.9 nm (3:1 mass ratio). DSPE-PEG coating did not impact mitochondrial function, with no significant differences in the basal respiration, maximum respiration, and ATP production among groups. DSPE-PEG coating increased cell uptake in vitro compared to uncoated Mts and improved cell respiration and tube formation. In vivo, DSPE-PEG-CBP coated Mts accumulated at the injury site which was associated with improved morphometry and reduced expression of inflammatory markers.
Conclusion: In summary, DSPE-PEG peptide polymer conjugates are an effective method to coat mitochondria and preserve function for transplantation. Improved cell uptake and tissue targeting indicates MDS as a viable approach to improve mitochondria transplantation therapies.
