10 research outputs found
Cathepsin-Mediated Cleavage of Peptides from Peptide Amphiphiles Leads to Enhanced Intracellular Peptide Accumulation
Peptides
synthesized in the likeness of their native interaction
domain(s) are natural choices to target protein–protein interactions
(PPIs) due to their fidelity of orthostatic contact points between
binding partners. Despite therapeutic promise, intracellular delivery
of biofunctional peptides at concentrations necessary for efficacy
remains a formidable challenge. Peptide amphiphiles (PAs) provide
a facile method of intracellular delivery and stabilization of bioactive
peptides. PAs consisting of biofunctional peptide headgroups linked
to hydrophobic alkyl lipid-like tails prevent peptide hydrolysis and
proteolysis in circulation, and PA monomers are internalized via endocytosis.
However, endocytotic sequestration and steric hindrance from the lipid
tail are two major mechanisms that limit PA efficacy to target intracellular
PPIs. To address these problems, we have constructed a PA platform
consisting of cathepsin-B cleavable PAs in which a selective p53-based
inhibitory peptide is cleaved from its lipid tail within endosomes,
allowing for intracellular peptide accumulation and extracellular
recycling of the lipid moiety. We monitor for cleavage and follow
individual PA components in real time using a Förster resonance
energy transfer (FRET)-based tracking system. Using this platform,
we provide a better understanding and quantification of cellular internalization,
trafficking, and endosomal cleavage of PAs and of the ultimate fates
of each component
Preclinical Evaluation of the Safety and Efficacy of Cryopreserved Bone Marrow Mesenchymal Stromal Cells for Corneal Repair
PurposeMesenchymal stromal cells (MSCs) have been shown to enhance tissue repair as a cell-based therapy. In preparation for a phase I clinical study, we evaluated the safety, dosing, and efficacy of bone marrow-derived MSCs after subconjunctival injection in preclinical animal models of mice, rats, and rabbits.MethodsHuman bone marrow-derived MSCs were expanded to passage 4 and cryopreserved. Viability of MSCs after thawing and injection through small-gauge needles was evaluated by vital dye staining. The in vivo safety of human and rabbit MSCs was studied by subconjunctivally injecting MSCs in rabbits with follow-up to 90 days. The potency of MSCs on accelerating wound healing was evaluated in vitro using a scratch assay and in vivo using 2-mm corneal epithelial debridement wounds in mice. Human MSCs were tracked after subconjunctival injection in rat and rabbit eyes.ResultsThe viability of MSCs after thawing and immediate injection through 27- and 30-gauge needles was 93.1% ± 2.1% and 94.9% ± 1.3%, respectively. Rabbit eyes demonstrated mild self-limiting conjunctival inflammation at the site of injection with human but not rabbit MSCs. In scratch assay, the mean wound healing area was 93.5% ± 12.1% in epithelial cells co-cultured with MSCs compared with 40.8% ± 23.1% in controls. At 24 hours after wounding, all MSC-injected murine eyes had 100% corneal wound closure compared with 79.9% ± 5.5% in controls. Human MSCs were detectable in the subconjunctival area and peripheral cornea at 14 days after injection.ConclusionsSubconjunctival administration of MSCs is safe and effective in promoting corneal epithelial wound healing in animal models.Translational relevanceThese results provide preclinical data to support a phase I clinical study