Abstract Body:

Introduction: Radiotherapy-induced brain injury (RIBI) is a chronic and debilitating side effect which affects up to 90% of brain tumor survivors treated with radiotherapy, especially pediatric brain tumor survivors.[1-3] Consequently, as the overall survival rates of pediatric brain tumor patients improve, there is a growing need for RIBI therapies.[1-3]

Hypothesis: Chronic oxidative stress and neuroinflammation are key contributors to RIBI. Thus, it has been postulated that RIBI can be mitigated by therapeutic agents that target oxidative stress and neuroinflammation.[2]

Objective: Here, we developed two pH/redox-responsive polymeric nanotheranostic agents and evaluated their ability to reduce neuroinflammation and improve outcomes in a RIBI mouse model.

Method: Two pH/redox-responsive amphiphilic block copolymers were synthesized and characterized by NMR spectroscopy.[4] The respective critical aggregation concentrations needed to form nanoparticles in aqueous milieu were determined with fluorescence spectroscopy. The hydrodynamic diameters, ζ-potentials and stabilities of the nanoparticles were then determined by dynamic light-scattering; and the shapes by transmission electron microscopy and scanning electron microscopy. Next, nanoparticle activation under different conditions of oxidative stress (varying hydrogen peroxide concentrations at pH 6.2) and under normal physiological conditions (pH 7.4) was determined by fluorescence spectroscopy and fluorescence imaging. The ability of the nanoparticles to prevent the degradation of fluorescent R-phycoerythrin (RPE) protein under oxidative stress was also evaluated. Cellular uptake and toxicity of the respective nanoparticles was then evaluated in human umbilical vein endothelial cells (HUVECs). Next, therapeutic efficacies of the nanoparticles were evaluated in a RIBI mouse model. Briefly, the right brain hemispheres of 8-weeks-old female balb/c mice were stereotactically irradiated at 80 Gy. [5, 6] The mice were next treated with either an 800 nm (red) fluorescently-labelled nanoparticle or phosphate buffered saline (PBS), two weeks post-irradiation as follows: Group 1 (PBS); Group 2 (P2a); Group 3 (P2b). The delivery of the respective nanoparticles to the brain was monitored after intravenous injection with in vivo fluorescence imaging. The mice were next monitored with multi-parametric magnetic resonance imaging (mp-MRI) and immunohistochemistry for neuroinflammation, 1.5 months after nanoparticle administration.  

Results: Two pH/REDOX-responsive amphiphilic block copolymers (P2a & P2b) possessing varied phenylboronic acid pinacol ester (BAPE) moieties to scavenge reactive oxygen species (ROS) were developed. P2b had twice as many BAPE moieties as P2a (A&B). Both polymers readily formed spherical nanoparticular micelles in aqueous milieu (C-F), and had hydrodynamic diameters and ζ-potentials of 164 ± 51 nm and – 2.52 ± 0.658 mV for P2a; and 164 ± 53 nm and – 2.89 ± 1.58 for P2b. The kinetics of activation of both nanoparticles was higher under oxidative stress compared to normal physiological conditions (G-O). Additionally, both nanoparticles prevented the degradation of RPE protein under oxidative stress (P-S). Both nanoparticles were also taken up by HUVECs (T) and did not cause toxicity (U). In vivo, both nanoparticles were detected in the brain 4 h post-administration and retained for up to 7 days post-administration (V-X). However, the uptake of P2b in the brain was significantly higher (1.3-fold) than that of P2a 4 h post-administration (X). Contrast-enhanced T1W MRI, 1.5 months after nanoparticle administration, showed significantly reduced disruption of the blood brain barrier in mice treated with P2b compared to PBS-treated control mice and P2a-treated mice (Y, Z). Additionally, T2W MRI showed significantly reduced edema/gliosis in P2b-treated and P2a-treated mice, compared to PBS-treated control mice (Z, Z1). Immunohistochemistry also showed significantly reduced microglial activation (IBA1) in P2b-treated and P2a-treated mice, compared to PBS-treated control mice (Z2, Z3). Reduced infiltrative macrophages (CD68) were also detected in P2b-treated mice compared to P2a-treated and PBS-treated control mice (Z2, Z3).

Conclusion: This shows that pH/REDOX-responsive nanoparticles with a high number of ROS scavengers can mitigate RIBI-associated neuroinflammation.

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Figure 1. Structure of copolymer P2a (A) and P2b (B). Transmission electron microscope images of polymeric micellar nanoparticle P2a (C) and P2b (D). Scanning electron microscope images of polymeric micellar nanoparticle P2a (E) and P2b (F). G) Schematic of pH/redox-activable drug release from nanoparticles. Release of an encapsulated fluorophore (Nile Red) under varying degrees of oxidative stress (varying concentrations of hydrogen peroxide (H₂O₂) at pH 6.2) compared to physiological conditions (pH 7.4 and no H₂O₂) from nanoparticle P2a (H) and P2b (I) (p ≤ 0.05). Fluorescence images of agarose gel phantoms under different conditions of oxidative stress (tubes 1 to 7), on which 700 nm (green) fluorescently-labelled polymeric nanoparticle P2a (J) and P2b (K) each encapsulating an 800 nm (red) fluorophore (drug surrogate) was placed (0 h). The release of the encapsulated red fluorophore (drug surrogate) from the green fluorescently-labelled polymeric nanoparticles was monitored over 360 h. Release and diffusion profile of an encapsulated red fluorophore (drug surrogate) from the green-fluorescently labelled P2a nanoparticle, under physiological conditions [0.0 mM H₂O₂ at pH 7.4 (Tube 1)] (L) and under oxidative stress [0.1 mM H₂O₂ at pH 6.2 (Tube 6)] (M). Release and diffusion profile of an encapsulated red fluorophore (drug surrogate) from the green fluorescently-labelled P2b nanoparticle, under physiological conditions [0.0 mM H₂O₂ at pH 7.4 (Tube 1)] (N) and under oxidative stress [0.1 mM H₂O₂ at pH 6.2 (Tube 6)] (O). P) Schematic of the degradation of fluorescent R-phycoerythrin (RPE) protein under oxidative stress and its protection by pH/redox-activable nanoparticle P2a or P2b. Q) Kinetics of fluorescent RPE protein (2.13 µg) degradation under oxidative stress (10 mM H₂O₂ at pH 7.0) and its protection in the presence of 1 µg of nanoparticles P2a and P2b, respectively (p ≤ 0.05). R) Change in the fluorescence signal intensity of fluorescent RPE protein 60 minutes after exposure to oxidative stress (10 mM H₂O₂ at pH 7.0) and its protection in the presence of 1 µg of nanoparticles P2a and P2b, respectively (p ≤ 0.05). S) Change in the fluorescence intensity signal of fluorescent RPE protein, 60 minutes after exposure to oxidative stress (10 mM H₂O₂ at pH 7.0) in the presence of varying amounts of nanoparticles P2a and P2b, respectively. RPE protection from degradation was obtained with as little as 1 µg of NPs. T) Nanoparticle uptake in human umbilical vein endothelial cells (HUVECs) shows that both nanoparticles were effectively taken up by the cells, with P2b being significantly more taken up than P2a (p ≤ 0.05). U) Cell viability of HUVECs after incubation with up to 20 µg (25 µg/µL) for 24 h, showed no toxicity from either nanoparticle. V) Schematic of in vivo experimental design used to evaluate the efficacy of nanoparticles P2a and P2b respectively. W) Fluorescence images of the delivery of 800 nm (red) fluorescently-labelled nanoparticles P2a and P2b respectively to the brain in a preclinical mouse model of radiotherapy-induced brain injury (RIBI). X) Quantification of the delivery of fluorescently-labelled nanoparticles P2a and P2b to the brain in a preclinical mouse model of RIBI (p ≤ 0.05). Y) In vivo contrast-enhanced T₁W MRI of representative mice from the respective groups 1.5 months after nanoparticle administration. The T₁W MRIs were acquired 15 mins after the intravenous administration of 200 µL of a 0.25 M contrast agent solution (Prohance®). This shows reduced disruption of the blood brain barrier (BBB) in mice treated with P2b. Z) Quantification of the MRI signals in the three groups 1.5 months after nanoparticle administration (p ≤ 0.05).  Z1) In vivo T₂W MRI of representative mice from each group. This shows more edema [hyperintensity (red arrow)] in the control group that was not treated with either of the nanoparticles. Z2) Neuroinflammation detection with CD68 (green) and IBA1 (red) immunohistochemistry of representative mice from the respective groups 1.5 months after nanoparticle administration. This shows reduced IBA1 detection in mice treated with P2a and P2b respectively and reduced CD68 detection in mice treated with P2b only. Z3) Quantification of neuroinflammation in representative mice from the respective groups 1.5 months after nanoparticle administration. This shows reduced IBA1 detection in mice treated with P2a and P2b respectively and reduced CD68 detection in mice treated with P2b only (p ≤ 0.05).

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