Surface Engineering of FLT4-Targeted Nanocarriers Enhances Cell-Softening Glaucoma Therapy

Abstract

Primary open-angle glaucoma is associated with elevated intraocular pressure (IOP) that damages the optic nerve and leads to gradual vision loss. Several agents that reduce the stiffness of pressure-regulating Schlemm’s canal (SC) endothelial cells, in the conventional outflow pathway of the eye, lower IOP in glaucoma patients and are approved for clinical use. However, poor drug penetration and uncontrolled biodistribution limit their efficacy and produce local adverse effects. Compared to other ocular endothelia, FLT4/VEGFR3 is expressed at elevated levels by SC endothelial cells and can be exploited for targeted drug delivery. Here, we validate FLT4 receptors as clinically relevant targets on SC cells from glaucomatous human donors and engineer polymeric self-assembled nanocarriers displaying lipid-anchored targeting ligands that optimally engage this receptor. Targeting constructs were synthesized as lipid-PEG x -peptide, differing in the number of PEG spacer units (x), and were embedded in micelles. We present a novel proteolysis assay for quantifying ligand accessibility that we employ to design and optimize our FLT4-targeting strategy for glaucoma nanotherapy. Peptide accessibility to proteases correlated with receptor-mediated targeting enhancements. Increasing the accessibility of FLT4-binding peptides enhanced nanocarrier uptake by SC cells while simultaneously decreasing the uptake by off-target vascular endothelial cells. Using a paired longitudinal IOP study in vivo, we show that this enhanced targeting of SC cells translates to IOP reductions that are sustained for a significantly longer time as compared to controls. Confocal microscopy of murine anterior segment tissue confirmed nanocarrier localization to SC within 1 h after intracameral administration. This work demonstrates that steric effects between surface-displayed ligands and PEG coronas significantly impact the targeting performance of synthetic nanocarriers across multiple biological scales. Minimizing the obstruction of modular targeting ligands by PEG measurably improved the efficacy of glaucoma nanotherapy and is an important consideration for engineering PEGylated nanocarriers for targeted drug delivery.

Breaking Barriers in Eye Treatment: Polymeric Nano-Based Drug-Delivery System for Anterior Segment Diseases and Glaucoma

“…Cytotoxicity and systemic effects remain to be investigated for both delivery systems. Cell-softening agents, such as ligands targeting FLT-4/VEGFR3 receptors, were delivered through modified micelles, improving receptor targeting and lowering IOP [ 89 ]. Further improvement in corneal permeability and sustained release is required, given that effects remained up to only 48 h. Nanosuspensions were also reported to deliver acetazolamide by Donia et al (2020), where they demonstrated sustained drug release, improved acetazolamide solubility, and thereby improved bioavailability [ 90 ].…”

Harnessing the Therapeutic Potential of Biomacromolecules through Intracellular Delivery of Nucleic Acids, Peptides, and Proteins

“…For example, as recently reported, targeting ligands with a longer spacing PEG linker can better extend themselves from the micelle surface to avoid the crowded surface environment and allow for engagement with endocytic cell receptors. [ 222 ]…”

“…For example, as recently reported, targeting ligands with a longer spacing PEG linker can better extend themselves from the micelle surface to avoid the crowded surface environment and allow for engagement with endocytic cell receptors. [222] In addition to conjugating hydrophobic tails onto a single peptide domain, peptide amphiphiles also can be synthesized by polymerizing peptide-containing monomers into amphiphilic polymer brushes. This concept has been extensively explored by the Gianneschi group, and is named peptidepolymer amphiphile (PPA).…”

Dendritic peptide-conjugated polymeric nanovectors for non-toxic delivery of plasmid DNA and enhanced non-viral transfection of immune cells

“…Here, we develop a gene delivery system employing self-assembling block copolymer nanocarriers engineered for efficient cytosolic delivery of pDNA via incorporation of a cationic DP block ( Figure 1 ). We have previously demonstrated that poly (ethylene glycol)-block-poly (propylene sulfide) (PEG- b -PPS) nanocarriers effectively target macrophages and dendritic cells ( Vincent et al., 2021a ; Yi et al., 2016 , 2019 ), are capable of delivering diverse payloads intracellularly ( Allen et al., 2017 , 2018a bib_Allen_et_al_2017 ; Bobbala et al., 2020 ; Scott et al., 2012 ; Stack et al., 2020 , 2021 ; Vasdekis et al., 2012 ; Vincent et al., 2021c ), are non-immunogenic in human blood ( Vincent et al., 2021b ), and are both non-inflammatory and non-toxic in non-human primates ( Allen et al., 2018b ), humanized mice ( Dowling et al., 2017 ), and diverse mouse models of disease ( Yi et al., 2016 ). Furthermore, PEG- b -PPS copolymers can be formulated to permit the rapid self-assembly of nanostructures encapsulating biologic payloads upon the addition of aqueous medium ( Bobbala et al., 2021 ), which extends their practical usage to industrial or clinical settings.…”

“…In addition to the peptide aspects of our technology, the PEG- b -PPS polymers contribute multiple useful features for gene delivery. For example, PEG- b -PPS provides oxidation sensitivity via its hydrophobic PPS blocks that stabilize the nanostructure and enable disassembly within acidic endolysosomal compartments ( Bobbala et al., 2020 ; Scott et al., 2012 ; Stack et al., 2020 ; Vasdekis et al., 2012 ; Vincent et al., 2021c ). The hydrophilic methoxy-terminated PEG corona serves to improve biocompatibility and reduce toxicity ( Allen et al., 2018b ; Vincent et al., 2021b ).…”