We previously developed a platform nanotechnology comprised of electrostatically complexed, pH-responsive, endosomolytic nano-polyplexes for the enhanced delivery of intracellular-acting MAPKAP Kinase 2 inhibitory peptide (“MK2i-NPs”). The MK2i-NP system has been applied for peptide delivery to vascular tissue to inhibit vascular graft intimal hyperplasia and pathological vasoconstriction [1,2]. To further develop this approach commercially and provide a basis for clinical translation, we explored the effects of lyophilization of MK2i-NPs for long-term storage. Lyophilization of MK2i-NPs significantly affected particle morphology and decreased peptide uptake and bioactivity. Thus, we investigated addition of excipients to facilitate the lyoprotection of MK2i-NPs and to maintain MK2i-NP uptake and bioactivity. MK2i-NPs were prepared in the presence of a range of concentrations of the sugars sucrose, trehalose, and lactosucrose prior to lyophilization in order to elucidate their potential as lyoprotectants. The size, surface charge, cellular uptake, and pH-dependent membrane disruptive activity (as a measure for endosomal escape ability) of the resulting NP formulations were characterized following lyophilization and reconstitution with or without the addition of excipients. All excipients utilized were shown to maintain particle morphology relative to MK2i-NPs lyophilized without excipients. Furthermore, all excipients were found to partially or fully restore peptide uptake, with 300 mM lactosucrose outperforming all other excipients [enhanced uptake 2.38-fold (p = 0.0002) and 4.58-fold (p = 0.00002) compared to freshly prepared NPs and lyophilized NPs without excipient, respectively]. None of the excipients utilized were found to deleteriously effect the pH-responsive membrane disruptive activity of the NP formulations that facilitates peptide endosomal escape and cytosolic delivery. We are currently undertaking studies to investigate the effects of these excipients on NP cytocompatibility. Furthermore, the bioactivity of the reconstituted NP formulations is being investigated by assessing peptide-mediated effects on smooth muscle physiology in rat aortic tissue ex vivo. These results suggest that the addition of excipients to our previously established NP platform as lyoprotectants provide beneficial effects in terms of maintaining MK2i-NP morphology and restoring, if not even increasing, peptide uptake and bioactivity. Moreover, lactosucrose was found to be the most promising excipient and may serve as an effective lyoprotectant for the long-term storage of peptide containing NPs for the intracellular delivery of therapeutic peptides.
1. Evans BC et al. Nano-polyplex MK2 inhibitory peptide delivery Prevents Vascular Graft Intimal Hyperplasia. Science Translational Medicine. 2015; 7(291): 291ra95. DOI: 10.1126/scitranslmed.aa4549
2. Evans BC et al. An Endosomolyutic Nano-polyplex Platform Technology for Cytosolic Peptide Delivery to Inhibit Pathological Vasoconstriction. ACS Nano. 2015; DOI: 10.1021/acsnano.5b00491.