Cross-linked antioxidant nanozymes for improved delivery to CNS

Abstract

Formulations of antioxidant enzymes, superoxide dismutase 1 (SOD1, also known as Cu/Zn SOD) and catalase were prepared by electrostatic coupling of enzymes with cationic block copolymers, polyethyleneimine-poly(ethylene glycol) or poly(L-lysine)-poly(ethylene glycol), followed by covalent cross-linking to stabilize nanoparticles. Different cross-linking strategies (using glutaraldehyde, bis-(sulfosuccinimidyl)suberate sodium salt or 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride with N-hydroxysulfosuccinimide) and reaction conditions (pH and polycation/protein charge ratio) were investigated that allowed immobilizing active enzymes in cross-linked nanoparticles, termed “nanozymes”. Bi-enzyme nanoparticles, containing both SOD1 and catalase were also formulated. Formation of complexes was confirmed using denaturing gel electrophoresis and western blotting and physicochemical characterization was conducted using dynamic light scattering and atomic force microscopy. In vivo studies of 125I-labeled SOD1-containing nanozymes in mice demonstrated its increased stability in both blood and brain and increased accumulation in brain tissues, compared to non-cross-linked complexes and native SOD1. Future studies will evaluate potential of these formulations for delivery of antioxidant enzymes to central nervous system to attenuate oxidative stress associated with neurological diseases.

Macrophages Offer A Paradigm Switch for CNS Delivery of Therapeutic Proteins

“…Such nanozymes displayed significant therapeutic efficacy in in vitro and in vivo models of neuroinflammation. These results are in accordance with our prior findings regarding the cross-linking of BICs with another antioxidant enzyme, superoxide dismutase 1, which resulted in improved enzyme stability in the blood and brain [21]. Our ultimate goal is to obtain injectable catalase nanoformulations that may be loaded into cell carriers directly into the bloodstream and delivered to disease sites by active targeted transport in macrophages.…”

Neuronal uptake of nanoformulated superoxide dismutase and attenuation of angiotensin II-dependent hypertension after central administration

“…Summary data (Figure 2B) clearly reveal a significant decrease in EPR spectra amplitude in all samples containing CuZnSOD as compared to vehicle. These data clearly validate the O 2 •− scavenging capacity of our PLL 50 -PEG CuZnSOD nanozyme formulations, and confirm that the nanozyme structure does not preclude access to the substrate nor does the protein need to be released for it to catalyze O 2 •− dismutation, as previously reported (35, 36). …”

“…We exploited the advantageous chemical properties of PLL 50 -PEG block copolymers and stabilized the complex by introducing reducible (disulfide bonds) or non-reducible (amide bonds) covalent bonds between the PLL 50 polymers using amine-reactive crosslinkers. Crosslinked nanozymes has been shown to significantly enhance delivery of nanoformulated complexes in vitro and in vivo (29–31). …”

Nanoparticles for Brain-Specific Drug and Genetic Material delivery, Imaging and Diagnosis

“…Several attempts have been made to use different cationic block copolymers as drug-delivery system to the CNS, such as PEI-poly(ethylene glycol) or PLL-PEG covalently cross-linked with superoxide dismutase 1 (SOD1). These complexes, when injected into mice, have shown to increase the enzyme-nanoparticle accumulation in brain tissues, although the potential to attenuate oxidative stress in neurodegenerative diseases has not been clearly established [39]. PLL-poly(ethylene glycol) administered by oropharyngeal aspiration mediate effective gene delivery to the brain, eyes and lung.…”