Abstract
Superoxide dismutase 1 (SOD1) efficiently catalyzes dismutation of superoxide, but its poor delivery to the target sites in the body, such as brain, hinders its use as a therapeutic agent for superoxide-associated disorders. Here to enhance the delivery of SOD1 across the blood-brain barrier (BBB) and in neurons the enzyme was conjugated with poly(2-oxazoline) (POx) block copolymers, P(MeOx-b-BuOx) or P(EtOx-b-BuOx), composed of (1) hydrophilic 2-methyl-2-oxazoline (MeOx) or 2-ethyl-2-oxazoline (EtOx) and (2) hydrophobic 2-butyl-2-oxazoline (BuOx) repeating units. The conjugates contained from 2 to 3 POx chains joining the protein amino groups via cleavable -(ss)- or noncleavable -(cc)- linkers at the BuOx block terminus. They retained 30% to 50% of initial SOD1 activity, were conformationally and thermally stable, and assembled in 8 or 20 nm aggregates in aqueous solution. They had little if any toxicity to CATH.a neurons and displayed enhanced uptake in these neurons as compared to native or PEGylated SOD1. Of the two conjugates, SOD1-(cc)-P(MeOx-b-BuOx) and SOD1-(cc)-P(EtOx-b-BuOx), compared, the latter was entering cells 4 to 7 times faster and at 6 h colocalized predominantly with endoplasmic reticulum (41 ± 3%) and mitochondria (21 ± 2%). Colocalization with endocytosis markers and pathway inhibition assays suggested that it was internalized through lipid raft/caveolae, also employed by the P(EtOx-b-BuOx) copolymer. The SOD activity in cell lysates and ability to attenuate angiotensin II (Ang II)-induced superoxide in live cells were increased for this conjugate compared to SOD1 and PEG-SOD1. Studies in mice showed that SOD1-POx had ca. 1.75 times longer half-life in blood than native SOD1 (28.4 vs 15.9 min) and after iv administration penetrated the BBB significantly faster than albumin to accumulate in brain parenchyma. The conjugate maintained high stability both in serum and in brain (77% vs 84% at 1 h postinjection). Its amount taken up by the brain reached a maximum value of 0.08% ID/g (percent of the injected dose taken up per gram of brain) 4 h postinjection. The entry of SOD1-(cc)-P(EtOx-b-BuOx) to the brain was mediated by a nonsaturable mechanism. Altogether, SOD1-POx conjugates are promising candidates as macromolecular antioxidant therapies for superoxide-associated diseases such as Ang II-induced neurocardiovascular diseases.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 360-377 |
| Number of pages | 18 |
| Journal | Molecular Pharmaceutics |
| Volume | 10 |
| Issue number | 1 |
| DOIs | |
| State | Published - Dec 17 2012 |
| Externally published | Yes |
Bibliographical note
KAUST Repository Item: Exported on 2020-10-01Acknowledged KAUST grant number(s): KUK-F1-029-32
Acknowledgements: This study was supported by the United States National Institute of Health RO1 Grant NS051334, the United States Department of Defense (DoD) USAMRMC 06108004, and the Nanomaterials Core Facility of the Nebraska Center of Nanomedicine supported by NIH COBRE Grant RR021937 (all awarded to A.V.K.). R.L. is also thankful to the King Abdullah University of Science and Technology (KAUST Award No. KUK-F1-029-32, partial salary support for R.L.). Likewise J.T. has been in part supported by the Program of Excellence Graduate Assistantship from UNMC. We also gratefully acknowledge Professor Luis Marky (College of Pharmacy, UNMC) for kind assistance in CD and DSC experiments, Daria Filonova (College of Pharmacy, UNC-Chapel Hill) for her assistance in preparation of the Table of Contents Graphic, and the assistance of UNMC CLSM, Cell Analysis and MS core facilities.
This publication acknowledges KAUST support, but has no KAUST affiliated authors.