Examining Efficacy of “TAT-less” Delivery of a Peptide against the L‑Type Calcium Channel in Cardiac Ischemia–Reperfusion Injury

Increased calcium influx through the L-type Ca²⁺ channel or overexpression of the alpha subunit of the channel induces cardiac hypertrophy. Cardiac hypertrophy results from increased oxidative stress and alterations in cell calcium levels following ischemia–reperfusion injury and is an independent risk factor for increased morbidity and mortality. We find that decreasing the movement of the auxiliary beta subunit with a peptide derived against the alpha-interacting domain (AID) of the channel attenuates ischemia–reperfusion injury. We compared the efficacy of delivering the AID peptide using a trans-activator of transcription (TAT) sequence with that of the peptide complexed to multifunctional polymeric nanoparticles. The AID-tethered nanoparticles perfused through the myocardium more diffusely and associated with cardiac myocytes more rapidly than the TAT-labeled peptide but had similar effects on intracellular calcium levels. The AID-complexed nanoparticles resulted in a similar reduction in release of creatine kinase and lactate dehydrogenase after ischemia–reperfusion to the TAT-labeled peptide. Since nanoparticle delivery also holds the potential for dual drug delivery, we conclude that AID-complexed nanoparticles may provide an effective platform for peptide delivery in cardiac ischemia–reperfusion injuries

Nanoparticle-Mediated Dual Delivery of an Antioxidant and a Peptide against the L‑Type Ca²⁺ Channel Enables Simultaneous Reduction of Cardiac Ischemia-Reperfusion Injury

Increased reactive oxygen species (ROS) production and elevated intracellular Ca²⁺ following cardiac ischemia-reperfusion injury are key mediators of cell death and the development of cardiac hypertrophy. The L-type Ca²⁺ channel is the main route for calcium influx in cardiac myocytes. Activation of the L-type Ca²⁺ channel leads to a further increase in mitochondrial ROS production and metabolism. We have previously shown that the application of a peptide derived against the alpha-interacting domain of the L-type Ca²⁺ channel (AID) decreases myocardial injury post reperfusion. Herein, we examine the efficacy of simultaneous delivery of the AID peptide in combination with the potent antioxidants curcumin or resveratrol using multifunctional poly(glycidyl methacrylate) (PGMA) nanoparticles. We highlight that drug loading and dissolution are important parameters that have to be taken into account when designing novel combinatorial therapies following cardiac ischemia-reperfusion injury. In the case of resveratrol low loading capacity and fast release rates hinder its applicability as an effective candidate for simultaneous therapy. However, in the case of curcumin, high loading capacity and sustained release rates enable its effective simultaneous delivery in combination with the AID peptide. Simultaneous delivery of the AID peptide with curcumin allowed for effective attenuation of the L-type Ca²⁺ channel-activated increases in superoxide (assessed as changes in DHE fluorescence; Empty NP = 53.1 ± 7.6%; NP-C-AID = 7.32 ± 3.57%) and mitochondrial membrane potential (assessed as changes in JC-1 fluoresence; Empty NP = 19.8 ± 2.8%; NP-C-AID=13.05 ± 1.78%). We demonstrate in isolated rat hearts exposed to ischemia followed by reperfusion, that curcumin and the AID peptide in combination effectively reduce muscle damage, decrease oxidative stress and superoxide production in cardiac myocytes

Multimodal Analysis of PEI-Mediated Endocytosis of Nanoparticles in Neural Cells

Polymer nanoparticles are widely used as a highly generalizable tool to entrap a range of different drugs for controlled or site-specific release. However, despite numerous studies examining the kinetics of controlled release, the biological behavior of such nanoparticles remains poorly understood, particularly with respect to endocytosis and intracellular trafficking. We synthesized polyethylenimine-decorated polymer nanospheres (<i>ca.</i> 100–250 nm) of the type commonly used for drug release and used correlated electron microscopy, fluorescence spectroscopy and microscopy, and relaxometry to track endocytosis in neural cells. These capabilities provide insight into how polyethylenimine mediates the entry of nanoparticles into neural cells and show that polymer nanosphere uptake involves three distinct steps, namely, plasma membrane attachment, fluid-phase as well as clathrin- and caveolin-independent endocytosis, and progressive accumulation in membrane-bound intracellular vesicles. These findings provide detailed insight into how the intracellular delivery of nanoparticles is mediated by polyethylenimine, which is presently the most commonly used nonviral gene transfer agent. This fundamental knowledge may also assist in the preparation of next-generation nonviral vectors

Multimodal Analysis of PEI-Mediated Endocytosis of Nanoparticles in Neural Cells

Polymer nanoparticles are widely used as a highly generalizable tool to entrap a range of different drugs for controlled or site-specific release. However, despite numerous studies examining the kinetics of controlled release, the biological behavior of such nanoparticles remains poorly understood, particularly with respect to endocytosis and intracellular trafficking. We synthesized polyethylenimine-decorated polymer nanospheres (<i>ca.</i> 100–250 nm) of the type commonly used for drug release and used correlated electron microscopy, fluorescence spectroscopy and microscopy, and relaxometry to track endocytosis in neural cells. These capabilities provide insight into how polyethylenimine mediates the entry of nanoparticles into neural cells and show that polymer nanosphere uptake involves three distinct steps, namely, plasma membrane attachment, fluid-phase as well as clathrin- and caveolin-independent endocytosis, and progressive accumulation in membrane-bound intracellular vesicles. These findings provide detailed insight into how the intracellular delivery of nanoparticles is mediated by polyethylenimine, which is presently the most commonly used nonviral gene transfer agent. This fundamental knowledge may also assist in the preparation of next-generation nonviral vectors