Excipients for the lyoprotection of MAPKAP kinase 2 inhibitory peptide nano-polyplexes

Abstract

Herein, excipients are investigated to ameliorate the deleterious effects of lyophilization on peptide-polymer nano-polyplex (NP) morphology, cellular uptake, and bioactivity. The NPs are a previously-described platform technology for intracellular peptide delivery and are formulated from a cationic therapeutic peptide and the anionic, pH-responsive, endosomolytic polymer poly(propylacrylic acid) (PPAA). These NPs are effective when formulated and immediately used for delivery into cells and tissue, but they are not amenable to reconstitution following storage as a lyophilized powder due to aggregation. To develop a lyophilized NP format that facilitates longer-term storage and ease of use, MAPKAP kinase 2 inhibitory peptide-based NPs (MK2i-NPs) were prepared in the presence of a range of concentrations of the excipients sucrose, trehalose, and lactosucrose prior to lyophilization and storage. All excipients improved particle morphology post-lyophilization and significantly improved MK2i-NP uptake in human coronary artery smooth muscle cells relative to lyophilized NPs without excipient. In particular, MK2i-NPs lyophilized with 300 mM lactosucrose as an excipient demonstrated a 5.23 fold increase in cellular uptake (p < 0.001), a 2.52 fold increase in endosomal disruption (p < 0.05), and a 2.39 fold increase in ex vivo bioactivity (p < 0.01) compared to MK2i-NPs lyophilized without excipients. In sum, these data suggest that addition of excipients, particularly lactosucrose, maintains and even improves the uptake and therapeutic efficacy of peptide-polymer NPs post-lyophilization relative to freshly-made formulations. Thus, the use of excipients as lyoprotectants is a promising approach for the long-term storage of biotherapeutic NPs and poises this NP platform for clinical translation.

Preparation and Characterization of PEG-PLA Genistein Micelles Using a Modified Emulsion-Evaporation Method

“…Trehalose is an efficient lyoprotectant and can be used in nanoparticle systems to increase the uptake and biological activity of drugs [45]. Trehalose inhibits the abnormal proliferation of human keratinocytes induced by UVB radiation and protects keratinocytes against virus infection by inducing autophagy [46], inhibits the proliferation of fibroblasts at a concentration of 5% in vivo and in vitro and the expression of vimentin and α-SMA at higher concentrations [47], and suppresses the BAX gene in cattle ovarian granulosa celn in cultured HSCs.…”

pH-Responsive Amphiphilic Carboxylate Polymers: Design and Potential for Endosomal Escape

“…(Wang, 2018;Evans et al, 2019). Albeit less renowned, recent studies show their emerging potentials for proteins, genes, and vaccine delivery (Mukalel et al, 2018;Qiu et al, 2018;Evans et al, 2019;Jacobson et al, 2019;Kopytynski et al, 2020).…”

“…Compared with polycationic polymeric carriers, anionic pH-responsive membrane permeabilizing polymers are less toxic, because of the repulsive charges against plasma membranes (Wang, 2018 ; Evans et al, 2019 ). Albeit less renowned, recent studies show their emerging potentials for proteins, genes, and vaccine delivery (Mukalel et al, 2018 ; Qiu et al, 2018 ; Evans et al, 2019 ; Jacobson et al, 2019 ; Kopytynski et al, 2020 ).…”

A proof of concept gene‐activated titanium surface for oral implantology applications

“…Rapid delivery of nanoplexes coding for therapeutic proteins to mucosal tissue could induce sustained expression and release of those proteins soon after implantation. The use of sucrose as a lyoprotectant has already been shown to enable the stabilization of particle formulations and PEI‐pDNA nanoplexes in other settings utilizing lyophilization; however, it has not been investigated in the context of surface coatings designed for gene therapy (Huang, Connell, Park, Mooney, & Rice, ; Kasper, Schaffert, Ogris, Wagner, & Friess, ; Mukalel et al, ). We demonstrate that this lyoprotectant‐stabilized surface coating can successfully transfect the robust HEK293T cell line with nanoplexes containing EGFP pDNA.…”