Formulation and Efficacy of Catalase-Loaded Nanoparticles for the Treatment of Neonatal Hypoxic-Ischemic Encephalopathy

Abstract

Neonatal hypoxic-ischemic encephalopathy is the leading cause of permanent brain injury in term newborns and currently has no cure. Catalase, an antioxidant enzyme, is a promising therapeutic due to its ability to scavenge toxic reactive oxygen species and improve tissue oxygen status. However, upon in vivo administration, catalase is subject to a short half-life, rapid proteolytic degradation, immunogenicity, and an inability to penetrate the brain. Polymeric nanoparticles can improve pharmacokinetic properties of therapeutic cargo, although encapsulation of large proteins has been challenging. In this paper, we investigated hydrophobic ion pairing as a technique for increasing the hydrophobicity of catalase and driving its subsequent loading into a poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) nanoparticle. We found improved formation of catalase-hydrophobic ion complexes with dextran sulfate (DS) compared to sodium dodecyl sulfate (SDS) or taurocholic acid (TA). Molecular dynamics simulations in a model system demonstrated retention of native protein structure after complexation with DS, but not SDS or TA. Using DS-catalase complexes, we developed catalase-loaded PLGA-PEG nanoparticles and evaluated their efficacy in the Vannucci model of unilateral hypoxic-ischemic brain injury in postnatal day 10 rats. Catalase-loaded nanoparticles retained enzymatic activity for at least 24 h in serum-like conditions, distributed through injured brain tissue, and delivered a significant neuroprotective effect compared to saline and blank nanoparticle controls. These results encourage further investigation of catalase and PLGA-PEG nanoparticle-mediated drug delivery for the treatment of neonatal brain injury.

Keywords: catalase; hydrophobic-ion pairing; hypoxia-ischemia; molecular dynamics; nanomedicine; neonatal.

Figure 1

Characterization of DS-catalase complexation under various formulation conditions. (A) TA, (B) SDS, and (C) DS demonstrated an increasing trend of catalase binding efficiency with respect to increasing molar ratio. For DS-CAT complexes, more acidic pHs are associated with (D) increased binding efficiency but (E) increased loss of activity. (F) Binding efficiency in citrate buffer increases with increasing molar ratio. Values are represented as mean ± SD (n = 3).

Figure 2

Characterization of BSA structure and IP agent interactions with surface amino acids. (A) BSA backbone RMSD (in nanometers [nm]) vs. time (in nanoseconds [ns]), (B) Backbone RMSD of each BSA domain vs. time, (C) Fraction of surface residues with >95% occupancy vs. residue grouping.

Figure 3

Catalase loading and protection in PLGA-PEG nanoparticles by formulation method. (A) Nanoprecipitation and emulsion nanoparticles achieve non-significantly different catalase loading by activity, but (B) nanoprecipitation nanoparticles have significantly lower catalase loading by mass (p = 0.0003). (C) Both methods result in retention of catalase activity over 24 h in 0.2% pronase solution. Values are represented as mean ± SD (n = 4 for A and B; n = 3 for C).

Figure 4

Global brain injury is significantly reduced by treatment with catalase-loaded nanoparticles. (A) Rats were injured at P10, received treatment 30 min, 24 h, and 48 h after injury, and were sacrificed at 72 h for endpoint analysis. (B) Median (IQR) gross injury scores in the saline, blank nanoparticle, and catalase (CAT) nanoparticle groups are 0.5 (0–3), 2 (0–3.5), and 0 (0–2). (C) Median (IQR) area loss measurements in the saline, blank nanoparticle, and catalase nanoparticle groups are 13% (10–31%), 23% (16–41%), and 4.9% (0.61–27%). For both assessments, treatment with catalase is significantly neuroprotective compared to saline (p = 0.039 and p = 0.047, respectively) while blank nanoparticles have no significant effect.

Figure 5

Nanoparticle distribution and microglial response to treatment in the ipsilateral hemisphere. (A) PLGA-PEG nanoparticles (red) are observed in the cortex, dentate gyrus, and midbrain of the injured hemisphere, but appear trapped in the vasculature of the contralateral hemisphere. Neurons (green) and cell nuclei (blue) are also shown. Scale bars: 50 µm. (B) Microglia (green) have increased number and density in the ipsilateral compared to contralateral hemisphere in saline- and blank nanoparticle-treated pups. After catalase nanoparticle treatment, microglia number and density appear similar between hemispheres. Scale bars: 300 µm. All cell nuclei are shown in blue.