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Conjugates of Superoxide Dismutase 1 with Amphiphilic Poly(2-oxazoline) Block Copolymers for Enhanced Brain Delivery: Synthesis, Characterization and Evaluation in Vitro and in Vivo
journal contribution
posted on 2013-01-07, 00:00 authored by Jing Tong, Xiang Yi, Robert Luxenhofer, William A. Banks, Rainer Jordan, Matthew C. Zimmerman, Alexander V. KabanovSuperoxide 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.