ABSTRACT
An inflammation-resolving polysialic acid-decorated PLGA nanoparticle (PolySia-NP) has been developed to treat geographic atrophy/age-related macular degeneration and other conditions caused by macrophage and complement over-activation. While PolySia-NPs have demonstrated pre-clinical efficacy, this study evaluated its systemic and intraocular safety. PolySia-NPs were evaluated in vitro for mutagenic activity using Salmonella strains and E. coli, with and without metabolic activation; cytotoxicity was evaluated based on its interference with normal mitosis. PolySia-NPs were administered intravenously in CD-1 mice and Sprague Dawley rats and assessed for survival and toxicity. Intravitreal (IVT) administration in Dutch Belted rabbits and non-human primates was assessed for ocular or systemic toxicity. In vitro results indicate that PolySia-NPs did not induce mutagenicity or cytotoxicity. Intravenous administration did not show clastogenic activity, effects on survival, or toxicity. A single intravitreal (IVT) injection and two elevated repeat IVT doses of PolySia-NPs separated by 7 days in rabbits showed no signs of systemic or ocular toxicity. A single IVT inoculation of PolySia-NPs in non-human primates demonstrated no adverse clinical or ophthalmological effects. The demonstration of systemic and ocular safety of PolySia-NPs supports its advancement into human clinical trials as a promising therapeutic approach for systemic and retinal degenerative diseases caused by chronic immune activation.
ABSTRACT
The alternative pathway of the complement system is implicated in the etiology of age-related macular degeneration (AMD). Complement depletion with pegcetacoplan and avacincaptad pegol are FDA-approved treatments for geographic atrophy in AMD that, while effective, have clinically observed risks of choroidal neovascular (CNV) conversion, optic neuritis, and retinal vasculitis, leaving room for other equally efficacious but safer therapeutics, including Poly Sialic acid (PSA) nanoparticle (PolySia-NP)-actuated complement factor H (CFH) alternative pathway inhibition. Our previous paper demonstrated that PolySia-NP inhibits pro-inflammatory polarization and cytokine release. Here, we extend these findings by investigating the therapeutic potential of PolySia-NP to attenuate the alternative complement pathway. First, we show that PolySia-NP binds CFH and enhances affinity to C3b. Next, we demonstrate that PolySia-NP treatment of human serum suppresses alternative pathway hemolytic activity and C3b deposition. Further, we show that treating human macrophages with PolySia-NP is non-toxic and reduces markers of complement activity. Finally, we describe PolySia-NP-treatment-induced decreases in neovascularization and inflammatory response in a laser-induced CNV mouse model of neovascular AMD. In conclusion, PolySia-NP suppresses alternative pathway complement activity in human serum, human macrophage, and mouse CNV without increasing neovascularization.
ABSTRACT
In addition to the intrinsic toxicity associated with the chemical composition of nanoparticles (NP) and their ligands, biofunctionalized NP can perturb specific cellular processes through NP-cell interactions and induce programmed cell death (apoptosis). In the case of the epidermal growth factor (EGF), nanoconjugation has been shown to enhance the apoptotic efficacy of the ligand, but the critical aspects of the underlying mechanism and its dependence on the NP morphology remain unclear. In this manuscript we characterize the apoptotic efficacy of nanoconjugated EGF as a function of NP size (with sphere diameters in the range 20-80 nm), aspect ratio (A.R., in the range of 4.5 to 8.6), and EGF surface loading in EGFR overexpressing MDA-MB-468 cells. We demonstrate a significant size and morphology dependence in this relatively narrow parameter space with spherical NP with a diameter of approx. 80 nm being much more efficient in inducing apoptosis than smaller spherical NP or rod-shaped NP with comparable EGF loading. The nanoconjugated EGF is found to trigger an EGFR-dependent increase in cytoplasmic reactive oxygen species (ROS) levels but no indications of increased mitochondrial ROS levels or mitochondrial membrane damage are detected at early time points of the apoptosis induction. The increase in cytoplasmic ROS is accompanied by a perturbation of the intracellular glutathione homeostasis, which represents an important check-point for NP-EGF mediated apoptosis. Abrogation of the oxidative stress through the inhibition of EGFR signaling by the EGFR inhibitor AG1478 or addition of antioxidants N-acetyl cysteine (NAC) or tempol, but not trolox, successfully suppressed the apoptotic effect of nanoconjugated EGF. A model to account for the observed morphology dependence of EGF nanoconjugation enhanced apoptosis and the underlying NP-cell interactions is discussed.
