Developing oxidative stress-responsive neuroprotective nanotheranostic agents for the image-guided treatment of radiotherapy-induced brain injury.

Abstract

Rationale: Radiotherapy-induced brain injury (RIBI) is a chronic side effect, that affects up to ~90% of brain tumor survivors treated with radiotherapy. Chronic oxidative stress and neuroinflammation are key drivers of RIBI. Here, we developed oxidative stress-responsive polymeric nanotheranostic agents and evaluated their ability to reduce neuroinflammation in a preclinical mouse model of RIBI.

Method: Two oxidative stress-responsive amphiphilic block copolymers possessing varied numbers of phenylboronic acid pinacol ester (BAPE) moieties to scavenge reactive oxygen species (ROS) were designed, synthesized, and characterized by proton and carbon-13 nuclear magnetic resonance spectroscopy. Polymer P2b was designed to have twice as many BAPE moieties as polymer P2a, for a structure-activity relationship study. The polymers were then formulated into nanoparticles and characterized using fluorescence spectroscopy, dynamic light-scattering, transmission and scanning electron microscopy, and fluorescence imaging. The ability of the agents to prevent the degradation of fluorescent R-phycoerythrin (RPE) protein under oxidative stress was also evaluated. Then, the cellular uptake and toxicity of the agents were evaluated in human umbilical vein endothelial cells (HUVECs). Next, the in vivo therapeutic efficacies of the agents were evaluated in four groups of eight-week-old female BALB/c mice. Mice from one group (Group 1) were not irradiated and were used as controls, while the right brain hemispheres of mice from the other three groups were stereotactically irradiated at 80 Gy (1.7 Gy/minute). At two weeks post-irradiation, mice were intravenously treated with either a fluorescently labelled nanoparticle or phosphate buffered saline (PBS), as follows: Group 2 (PBS); Group 3 (P2a); Group 4 (P2b); and the delivery of the nanoparticles to the irradiated brain lesion was monitored with fluorescence imaging. Next, all the mice were monitored with multi-parametric magnetic resonance imaging (mp-MRI) and immunohistochemistry (CD68, IBA1, and GFAP) at 1.5 months after nanoparticle administration.

Results: Both polymers readily formed spherical nanoparticular micelles in aqueous milieu at low concentrations (~ 0.08 mg/mL), with comparable hydrodynamic diameters and zeta-potentials of 166 ± 51 nm and -2.52 ± 0.658 mV for P2a; and 183 ± 54 nm and -2.89 ± 1.58 mV for P2b. The activation kinetics of both nanoparticles was higher under oxidative stress than under normal physiological conditions. Additionally, both nanoparticles prevented the degradation of RPE protein under oxidative stress. Furthermore, both nanoparticles were taken up by HUVECs and did not cause any toxicity. In vivo, both nanoparticles were detected in the irradiated brain lesion at 4 h after intravenous administration and retained for at least seven days post-administration. Anatomical T₂-weighted MRI at 1.5 months after nanoparticle administration showed significantly less edema in both P2a-treated mice (1.46 ± 0.24, P = 0.0071, n = 3) and P2b-treated mice (1.48 ± 0.39, P = 0.0130, n = 3), than in PBS-treated mice (2.90 ± 0.43). However, contrast-enhanced T₁W MRI showed significantly less blood-brain barrier permeability in P2b-treated mice (10.72 ± 1.75) than in P2a-treated mice (15.74 ± 1.46, P = 0.0187, n = 3) and PBS-treated mice (14.56 ± 0.28, P = 0.0198, n = 3). Additionally, immunohistochemistry showed significantly less microglial activation (IBA1) in P2b-treated mice (1.98 ± 0.27) than in P2a-treated mice (3.88 ± 0.22, P = 0.0007, n = 3) and PBS-treated mice (6.25 ± 0.51, P = 0.0002, n = 3). Less infiltrative peripheral macrophages (CD68) were also detected in P2b-treated mice (4.36 ± 1.83) than in P2a-treated mice (20.79 ± 2.05, P = 0.00005, n = 3) and PBS-treated mice (22.95 ± 2.35, P = 0.00004, n = 3). Furthermore, significantly less astrogliosis (GFAP) was detected in P2b-treated mice (3.48 ± 0.36) than in P2a-treated mice (5.53 ± 0.92, P = 0.0019, n = 3) and PBS-treated mice (23.14 ± 4.66, P = 0.0030, n = 3).

Conclusion: Collectively, these results showed that although both nanotheranostic agent P2a and P2b significantly reduced radiation-induced neuroinflammation, nanotheranostic agent P2b (with twice as many ROS scavengers) was more effective at reducing radiation-induced neuroinflammation in the preclinical mouse model of RIBI.