Endosomolytic Nano-Polyplex Platform Technology for Cytosolic Peptide Delivery To Inhibit Pathological Vasoconstriction

Abstract

A platform technology has been developed and tested for delivery of intracellular-acting peptides through electrostatically complexed nanoparticles, or nano-polyplexes, formulated from an anionic endosomolytic polymer and cationic therapeutic peptides. This delivery platform has been initially tested and optimized for delivery of two unique vasoactive peptides, a phosphomimetic of heat shock protein 20 and an inhibitor of MAPKAP kinase II, to prevent pathological vasoconstriction (i.e., vasospasm) in human vascular tissue. These peptides inhibit vasoconstriction and promote vasorelaxation by modulating actin dynamics in vascular smooth muscle cells. Formulating these peptides into nano-polyplexes significantly enhances peptide uptake and retention, facilitates cytosolic delivery through a pH-dependent endosomal escape mechanism, and enhances peptide bioactivity in vitro as measured by inhibition of F-actin stress fiber formation. In comparison to treatment with the free peptides, which were endowed with cell-penetrating sequences, the nano-polyplexes significantly increased vasorelaxation, inhibited vasoconstriction, and decreased F-actin formation in the human saphenous vein ex vivo. These results suggest that these formulations have significant potential for treatment of conditions such as cerebral vasospasm following subarachnoid hemorrhage. Furthermore, because many therapeutic peptides include cationic cell-penetrating segments, this simple and modular platform technology may have broad applicability as a cost-effective approach for enhancing the efficacy of cytosolically active peptides.

Mechanism of Enhanced Cellular Uptake and Cytosolic Retention of MK2 Inhibitory Peptide Nano-polyplexes

“…Anionic, pH-responsive carriers like the one described herein have been shown to reduce trafficking into acidifying compartments following CD22-dependent receptor mediated uptake 3 . While the pharmacodynamics of anionic, polymer-based nano-polyplexes (NPs) used for the delivery of both an intracellular acting MAPKAP kinase 2 inhibitory (MK2i) peptide and a peptide mimetic of phosphorylated heat shock protein 20 (p-HSP20 peptide) have been thoroughly investigated 18,19 , the mechanisms and kinetics of NP uptake and trafficking have not been rigorously studied. Because of the promise of MK2i-NPs as a prophylactic therapy to inhibit intimal hyperplasia and vasospasm of vascular grafts 18 , this specific formulation is the focus of the current studies designed to better elucidate endosomolytic NP intracellular pharmacokinetics.…”

“…Here, we sought to provide a comprehensive cellular pharmacokinetics analysis of the MK2i-NP formulation, which our group previously developed for the cytosolic delivery of MK2i to vascular smooth muscle cells to prevent intimal hyperplasia and vasospasm 18,19 . A previous cell trafficking study by Flynn et al found that the p-HSP20 peptide (therein known as AZX100) 22 , which shares the same CPP sequence with MK2i 18,19 , was internalized rapidly via a lipid raft dependent, caveolae mediated uptake process that was not significantly influenced by actin or dynamin inhibition.…”

Efficient Targeting of Adipose Tissue Macrophages in Obesity with Polysaccharide Nanocarriers

“…[18][19][20][21][22][23][24][25][26] To date, limitations of nanomedicines have been their low targeting efficiency of diseased tissue after systemic administration (>90% of dose is usually retained in liver), especially when targeting solid tumors, and frequent reliance on chemical agents that are not clinically approved, which slows clinical translation. [27][28][29][30][31][32] Here we describe the development of a nanomedicine strategy to ameliorate obesity-associated inflammation by targeting adipose tissue macrophages.…”

Heparin-functionalized polymer graft surface eluting MK2 inhibitory peptide to improve hemocompatibility and anti-neointimal activity

“…The changes in zeta-potential of HT/MK2i complex were analyzed by varying mixing ratio (w/w; [MK2i]/[HT] = 0.1 to 50) [2, 39]. Figure 3a shows zeta-potential values with increasing the MK2i concentration (0.1–50 mg/mL), while maintaining the consistent HT concentration (1 mg/mL).…”