In this study, we developed SOD1-Pluronic conjugates and showed that Pluronic modification improves SOD1 intra-neuronal delivery and as a result decreases AngII-induced increase in intra-neuronal O2•−
levels. Two types of modifications strategies were explored for conjugation of SOD1 with amino-derivative of Pluronic. First is the modification of Lys and N-terminal amino groups of SOD1 using NHS-containing bifunctional cross-linkers, DSP and DSS. Second is the modification of Asp and Glu carboxyl groups of SOD1 using water-soluble carbodimide reagent, EDC. Both strategies produced catalytically active modified SOD1 with various amounts of Pluronic chains attached. Such conjugates at least in the case of DSP and DSS modifications retained up to 60% of the activity of the unmodified enzyme. However, the activity drastically decreased as the modification degree was increased above 5 Pluronic chains per the protein molecule. This may be due to a considerable change of global or local conformations of the enzyme, which affects the active center. In particular, native SOD1 exists in a homodimer form (M.W. 32 kDa), which, as suggested by Valentine et al., plays an important role in maintaining SOD1 function [36
]. One concern with Pluronic modification was a potential for SOD1 dimer dissociation, that may be accompanied by a loss of metals (Cu and Zn), crucial to maintaining the SOD1 activity. Indeed, we observed unmodified SOD1 monomers in SDS-PAGE () and mass spectra () in all SOD1-Pluronic conjugates. Furthermore, the mass spectra clearly suggested presence of various modified SOD1 monomers. However, these data may not truly reflect the dimer dissociation as a result of the modification. In particular, upon denaturing conditions of electrophoresis or sample ionization/dissociation processing in MALDI-TOF the monomeric species can be produced from both non-modified SOD1 dimer or/and modified SOD1 dimer (with Pluronic attached to either one or both SOD1 subunits). Purification of SOD1-Pluronic conjugates in non-denaturing conditions using SEC indicated no detectable monomeric species (Supplementary data Figure S1
). Surprisingly, the SOD1 in-gel activity assay, a non-denaturing electrophoresis, showed achromatic staining below the SOD1 dimer band. This indicated that the conjugation reaction might generate a small portion of Pluronic-modified monomeric SOD1, which cannot be determined using SEC but which is active. The catalytic activity displayed by such modified SOD1 may be due some stabilization effect of Pluronic chains that can shield the surface of the monomeric protein globule and stabilize the incorporated metals.
In addition to potential conformational changes and inactivation as a result of modification of the protein aminogroups by Pluronic the activity loss may be partially due to exposure of the enzyme to the organic solvents during modification. Such solvents include the aqueous-EtOH solution to increase the modification yield and cold acetone to purify the conjugates by acetone precipitation. As shown in our SEC purification experiment at least the latter step of acetone precipitation can be eliminated, because SEC can remove excess of unreacted Pluronic amine in non-denaturing conditions. This may be used in future to further increase the yield of active conjugate. However, the use of aqueous-organic solution during SOD1 and Pluronic conjugation appears to be necessary as it is likely to prevent association of polymer chains and, as such, increase the reaction yield. Yet, alternative aqueous-organic solutions can be also used for modifications that can further preserve the enzyme activity. In particular, we found that 20% 1,4-butanediol or 20% DMSO aqueous solutions resulted in less SOD1 activity loss and comparable reaction efficiency compared to aqueous-EtOH or aqueous-DMF solutions (data not shown).
The major result of this study is a clear demonstration that Pluronic-modified SOD1 can penetrate into neurons and display intra-neuronal enzymatic activity. Furthermore, we provide evidence that once inside the cell the SOD1-Pluronic is biologically functional. In particular, the SOD1-Pluronic conjugates attenuated the increase in 2-OH-E+
fluorescence in neurons loaded with DHE and stimulated with AngII. Although we assessed 2-OH-E+
fluorescence using an excitation of wavelength of 405 nm and confocal microscopy to specifically measure intracellular O2•−
, as previously described [29
], we understand the limitation of this method and thus performed similar experiments and measured 2-OH-E+
levels with HPLC. The use of HPLC to measure the oxidation products of DHE, both 2-OH-E+
and ethidium, in biological samples is now considered by some to be the “gold standard” [34
]. Importantly, our HPLC data corroborate our confocal microscopy data, and convincingly demonstrate that SOD1-Pluronic conjugates attenuate the AngII-induced increase in intra-neuronal O2•−
levels. As the SOD1 conjugates obtained and used in some of the cellular studies are a mixture of non-modified SOD1 and SOD1 conjugates, one potential concern was that non-modified SOD1 interfered with the interpretation of the results. To address this, we characterized the mixture obtained after SEC and showed that about 10% of non-modified SOD1 was present (based on area percentage in HPLC profile), and each fraction (non-modified SOD1, mono-Pluronic SOD1, and SOD1 modified by multiple Pluronic chains) showed similar activity as determined by pyrogallol kinetic assay (Supplementary data Figure S1
). Thus, we conclude that the majority (90%) of the mixture was modified SOD1, and the measured activity of conjugates and obtained cellular responses can be mainly attributed to the SOD1-Pluronic conjugates. Furthermore, taking advantage of the SEC purification method, the purified SOD1-Pluronic fractions were collected and used in our HPLC experiments (). These studies clearly show that even without the influence of non-modified SOD1, SOD1-Pluronic conjugates attenuate AngII-induced increase in intracellular O2•−
Numerous investigations have clearly shown that O2•−
in neurons is an important target for the improved treatment of AngII-dependent neuro-cardiovascular diseases. For example, injection of adenoviral vectors encoding SOD1 directly into the brain attenuates O2•−
levels in the brain and the elevated blood pressure in a mouse model of AngII-dependent hypertension, whereas gene transfer of extracellular SOD had no effect [6
]. Similarly in a heart failure model, which is associated with increased AngII signaling in the brain, overexpression of SOD1 in the brain attenuates sympathetic output and improves cardiac function [38
]. Although these previous studies clearly demonstrate a therapeutic benefit of overexpressing SOD1 in the brain in the pathogenesis of brain-related cardiovascular diseases, the potential toxicity associated with viral vectors and the inability of viral vectors to penetrate the BBB calls for the development of novel SOD1 delivery systems.
To address this concern, other investigators have developed PEG modified SOD1 and evaluated PEG-SOD1 antioxidant therapy in many disease models. For example, PEG modification has shown to stabilize the enzyme against degradation; however, little evidence indicates that PEG modification increases SOD1 penetration of neuronal cell membranes in vitro
or transport across the BBB in both normal and hypertension animals [20
]. In addition, the therapeutic effectiveness of intravenously injected PEG-SOD1 in hypertensive brain injury was suggested to be due to its action in the vascular wall or its extracellular activity [22
]. In the present study, PEG-SOD1 failed to significantly increase intra-neuronal SOD1 activity and did not inhibit that AngII-induced increase in intra-neuronal O2•−
levels. In comparison, SOD1-Pluronic does increase SOD1 activity in neurons and does scavenge elevated levels of O2•−
. Considering Pluronic modification of HRP enhances its BBB permeability in vitro
and in vivo
, as we previously reported [26
], it is tempting to speculate that Pluronic-modified SOD1 might also penetrate the BBB to exert its activity in the brain. Furthermore, similar to what we observed for Pluronic modified leptin [39
], Pluronic modification may increase SOD1 half-life and stability in circulation and provide an independent transportation route to cross the BBB.
The enhanced cellular uptake of the conjugates may be due to hydrophobic interactions of amphiphilic Pluronic chains with the neuronal cell membrane. Pluronics are neutral block copolymers consisting of hydrophobic PPO and hydrophilic PEO blocks. It was previously shown with HRP, a model protein, that the optimal Pluronic modifications for cellular uptake are Pluronic P85 and L81 (PPO40
) vs. Pluronic L121 and P123 (PPO70
]. In the current study, we further demonstrate that Pluronics can serve as synthetic transduction agents capable of facilitating neuronal cell penetration of a potential therapeutic protein. Modification of SOD1 by both Pluronic P85 (having intermediate hydrophobicity) and Pluronic L81 (which is more hydrophobic) resulted in enhanced cell membrane penetration without inducing cellular toxicity. A recent study has shown that Pluronic P85 employs a pathogen like mechanism for the cellular entry, which mirrors that of cholera toxin B [40
]. First, the copolymer chains bind with the cholesterol-rich domains in the cell membranes. Second, they enter cells through caveolae-mediated endocytosis or a caveolae- and clathrin-independent pathway. The efficient transport of Pluronic P85 in brain microvessel endothelial cells and primary neurons has also been demonstrated. Interestingly, in neurons the entry of Pluronic starts from accumulation in the cell body followed by anterograde trafficking towards axons/dendrites[41
]. Further studies are required to understand the mechanism of cellular entry and subsequent trafficking of SOD1-Pluronic conjugates.
In summary, the data presented herein demonstrate that Pluronic P85 and L81 modified SOD1 penetrate neuronal cell membranes resulting in an increase intracellular SOD1 activity. In addition, SOD1-Pluronic conjugates inhibit AngII-induced increase in intra-neuronal O2•− levels. These data suggest that Pluronic modification may be a new delivery system for SOD1 into neurons of the CNS, and may have therapeutic effects in cardiovascular diseases associated with increased AngII and O2•− levels signaling in the brain. Future studies, which are currently underway in our laboratory, are needed to investigate the BBB permeability of SOD1-Pluronics and to test their therapeutic effect in neuro-cardiovascular disease models.