In the present study a novel type of superparamagnetic nanoparticle formulation (MNP) applicable for targeted delivery of antioxidant enzymes, SOD and catalase, was developed and characterized. The underlying formulation approach is based on the entrapment of biologically active enzymes in non-polymeric MNP formed by controlled aggregation of oleate-stabilized nanocrystalline iron oxide in the presence of calcium, and enables high protein loading yields, controllable MNP size, and magnetic properties adequate for magnetically guided delivery and protection of ROS challenged endothelial cells.
In order to be effective, enzyme-based antioxidant therapy requires strategies for localizing the enzyme with therapeutically adequate functional activity to the site of ROS-mediated injury. Attempts have been made to address this goal by using polymeric nanocarriers. In prior studies with catalase biodegradable polymeric nanoparticles were shown to protect it from proteolysis [21
], modulate its release kinetics [22
], reduce the rate of clearance from circulation by the organs of reticuloendothelial system [24
], and enable the inclusion of targeting moieties to direct endothelial specific targeting [15
]. The use of organic solvents or high sheer emulsification as part of the formulation process and the poor stability of cargo proteins in the particle-forming polymer [7
] represent a challenging aspect of this approach, often associated with a substantial loss of enzymatic activity [23
Calcium oleate-based MNP designed and characterized in this study as antioxidant enzyme carriers have several important advantages, which are highly relevant for the efficient molecular therapy of oxidative stress. Antioxidant enzymes are encapsulated in a non-polymeric matrix of the particles formed under mild conditions. MNP are obtained by aggregation of iron oxide ferrofluid achieved simultaneously with precipitation of calcium oleate in the presence of a non-ionic colloidal stabilizer, Pluronic F-127. Oleate used as a non-polymeric structural component of MNP plays several important roles in this formulation:
- It has a distinct affinity for proteins mediated through hydrophobic and electrostatic interactions , enabling the high loading efficiency of the cargo enzymes demonstrated in the present study. Importantly, our results indicate that the association of catalase with calcium oleate-based MNP remains stable over a period of at least 48 hr upon dilution with plasma. This is in an obvious requirement for efficient protection of catalase, otherwise highly susceptible to proteolysis, from inactivation, and is in agreement with the high levels of enzymatic activity retained by MNP-encapsulated catalase after exposure to pronase observed in this study;
- Oleate anion strongly associates with the iron oxide surface and is essential for the high incorporation yield of small-sized, superparamagnetic nanocrystals in the nanoparticles, thereby providing the latter with a strong magnetic responsiveness in the absence of significant remanence. The resultant superparamagnetic MNP are thus unlikely to irreversibly aggregate upon exposure to a magnetic field, which is necessary for their safe clinical use, yet they can be effectively guided by a magnetic force, which makes them an attractive candidate for targeted delivery applications;
- The capacity of oleate, highly water-soluble as a sodium salt, to spontaneously precipitate as nanoparticles with controllable size upon addition of divalent calcium cations in the presence of a suitable colloidal stabilizer enables formation of the fatty acid-based nanoparticles under strictly aqueous conditions, without utilizing organic solvents typically employed for making polymeric carriers. This mechanism of MNP formation relying on the controlled aggregation/precipitation rather than emulsification appears to be responsible for the improved retention of biological activity by the nanoencapsulated enzymes observed in this study in comparison to previous publications ;
- Oleate is an endogenous substance that belongs to the class of monounsaturated fatty acids, and is an essential element in the composition of triglycerides and phospholipids in living cells and tissues. Its metabolism and in vivo elimination pathways have been extensively studied, and its biocompatibility, both as a nutrient and as a delivery system component, has been well-established [27, 28]. Furthermore, it has also been shown to have a vasculoprotective effect of its own , in part mediated by ROS scavenging [30, 31]. This property of oleate can potentially explain the small, yet detectable protective effect on cultured endothelial cells exhibited by blank calcium oleate-based MNP used as a control in the present study (). Interestingly, this is in contrast with the absence of any measurable levels of protection observed with catalase-loaded non-magnetic particles. Since the latter are not influenced by the high gradient field, and are thus not subject to the magnetically mediated uptake enhancement, their interaction with cells within the time frame of the cell protection experiment was likely inefficient, being rate-limited by their slower sedimentation kinetics  compared to the iron oxide-loaded MNP possessing a higher density.
As demonstrated by our results, the inclusion of iron oxide, while not essential for the formation of calcium oleate-based nanoparticles, provided the formulations with a capacity for enhanced cellular uptake driven by a high gradient magnetic field. Practically, the sufficiently rapid kinetics of endothelial uptake of catalase-loaded MNP and their cargo enzyme required for achieving the protective effect in ROS-challenged endothelial cells was observed within a clinically relevant exposure time only with magnetic guidance, but not in the absence of a magnetic field and/or with non-magnetic control nanoparticles. This observation confirming the essential dependence of the protective effect mediated by MNP-encapsulated catalase on the endothelial uptake of the carrier emphasizes the importance of implementing a cell-targeted delivery strategy for optimal antioxidant defense.
In the previous studies [33
] our group has explored the feasibility of magnetically targeted delivery as an experimental approach aimed at preventing in-stent restenosis, a life-threatening proliferative condition triggered by the trauma caused by stent deployment in atherosclerotic vessels. Our results suggested that the deep-penetrating magnetizing effect of a strong uniform field inducing high gradients on a stainless steel stent and generating a magnetic force acting on MNP, can provide the physical basis for targeted drug, gene or cell delivery to injured arteries [33
]. In combination with this magnetic guidance strategy, antioxidant enzyme-loaded MNP whose efficacy has been demonstrated in the present study may be an interesting candidate for a stent-targeted antirestenotic therapy. A rationale for exploring the potential of antioxidant enzymes, SOD and catalase, either alone or in combination, for preventing restenosis is based on the major contribution of ROS to the cascade of events eventually resulting in the vessel reobstruction [36
], with a proof-of-concept provided in previous studies using gene vectors encoding these proteins [38
Several additional aspects pertaining to the utility of calcium oleate-based MNP for cell targeting have remained beyond the scope of the present study. One example is the capacity of MNP decorated with Pluronic F-127 for surface modifications enabling their affinity binding to cell-specific ligands (E. Hood et al., manuscript in preparation). A combination of physical guidance with cell specific targeting has a potential to significantly extend the range of clinical applications and greatly improve the efficacy and safety of the experimental therapy. The utility of our formulation approach for encapsulation and targeted delivery of biologically active proteins other than SOD and catalase has also remained unexplored. However, based on our findings it is likely that it can be successfully applied with necessary modifications to a broad range of proteins contributing to the design of safer and more efficient clinical therapies.