Abstract and Introduction
Abstract
Aims Active targeted transport of the nanoformulated redox enzyme, catalase, in macrophages attenuates oxidative stress and as such increases survival of dopaminergic neurons in animal models of Parkinson's disease. Optimization of the drug formulation is crucial for the successful delivery in living cells. We demonstrated earlier that packaging of catalase into a polyion complex micelle ('nanozyme') with a synthetic polyelectrolyte block copolymer protected the enzyme against degradation in macrophages and improved therapeutic outcomes. We now report the manufacture of nanozymes with superior structure and therapeutic indices.
Methods Synthesis, characterization and therapeutic efficacy of optimal cell-based nanoformulations are evaluated.
Results A formulation design for drug carriers typically works to avoid entrapment in monocytes and macrophages focusing on small-sized nanoparticles with a polyethylene glycol corona (to provide a stealth effect). By contrast, the best nanozymes for delivery in macrophages reported in this study have a relatively large size (~200 nm), which resulted in improved loading capacity and release from macrophages. Furthermore, the cross-linking of nanozymes with the excess of a nonbiodegradable linker ensured their low cytotoxicity, and efficient catalase protection in cell carriers. Finally, the 'alternatively activated' macrophage phenotype (M2) utilized in these studies did not promote further inflammation in the brain, resulting in a subtle but statistically significant effect on neuronal regeneration and repair in vivo.
Conclusion The optimized cross-linked nanozyme loaded into macrophages reduced neuroinflammatory responses and increased neuronal survival in mice. Importantly, the approach for nanoformulation design for cell-mediated delivery is different from the common requirements for injectable formulations.
Introduction
The restriction of macromolecules entry to the brain is a major impediment to the development of new treatment strategies for neurodegenerative and neuroinfectious diseases, which include Parkinson's (PD) and Alzheimer's diseases, meningitis, encephalitis, prion disease, HIV-associated neurocognitive disorders[1,2] and stroke.[3,4] The pathobiology of these disorders have an inflammatory component in common,[5] with production of reactive oxygen species (ROS) and proinflammatory factors, and consequential neurodegeneration.[6–9] The means to effectively treat such disorders by attenuating ROS production has met with limited success based, in part, on inefficient enzyme delivery to affected brain regions and across the blood–brain barrier (BBB).[10] To this end, our own works have sought to improve brain delivery of one of the most potent in nature redox enzymes, catalase.[11–14] This has been based on the concept that immunocytes readily home to the sites of inflammation and can be used as vehicles for CNS drug delivery of antioxidant enzymes.[15] Active targeted drug transport to disease sites by the cell carriers offers several advantages, which include: improved drug efficacy, prolonged half-lives, time-controlled drug release, and diminished immunogenicity and cytotoxicity. In addition to drug transport across the BBB, the efficient delivery of antioxidants to the brain endothelium, a substantive ROS source,[16] could also be beneficial for a range of neurologic disease-combating therapies.[17–19]
Our laboratory sought to optimize the structure of catalase nanoparticles for cell-mediated delivery and therapeutic gain. We proposed to incorporate catalase in the block ionomer complexes (BICs) with a cationic block copolymer, poly(ethyleneimine) (PEI)–polyethylene glycol (PEG; PEI–PEG), termed 'nanozyme', which protected the enzyme within macrophages.[13] Preservation of catalase enzymatic activity in macrophages by such complexes was, in part, due to the decreasing of acidification of the cell's endocytic compartments by the amino groups on the surface of the nanoparticles. This inhibited the protease activity and decreased the drug degradation in the host cells.[20] Here, we report further developments of drug nanoformulations for delivery in living cells. Contrary to a common approach for injectable drug formulations, the optimal particles were greater in size and stabilized by a nonbiodegradable cross-linker (Figure 1). This resulted in their higher loading capacity for macrophages, greater release and superior preservation of catalase enzymatic activity. 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.
Figure 1.
Nanozyme's structure stabilization. BICs spontaneously form in aqueous solution as a result of electrostatic coupling of catalase and a cationic block copolymer, poly(ethyleneimine)–polyethylene glycol. The obtained polyion complex, nanozyme, was further stabilized by cross-linking with an excess of biodegradable and nonbiodegradable linkers.
BIC: Block ionomer complex.
Nanomedicine. 2014;9(9):1403-1422. © 2014 Future Medicine Ltd.
