Polymeric nanoparticles for diagnosis and treatment of radiotherapy-induced brain injury

Abstract

The present disclosure provides polymeric nanoparticles including block copolymers which possess reactive oxygen species (ROS) quenching units to reduce oxidative stress to prevent neurodegeneration and which can also be used to encapsulate an active pharmaceutical ingredient, a diagnostic marker, or a combination thereof in a pharmaceutical composition. The polymeric nanoparticles of the present disclosure are designed to release ROS quenchers, the active pharmaceutical ingredient, the diagnostic marker, or the combination thereof upon exposure to a pH below about 6.5, exposure to a reactive oxygen species, or a combination thereof. Methods of diagnosing or treating radiotherapy-induced brain injuries in a patient are also described.

Description

PATENTATTORNEY DOCKET NO. JHU4770-1WOPOLYMERIC NANOPARTICLES FOR DIAGNOSIS AND TREATMENT OF RADIOTHERAPY-INDUCED BRAIN INJURYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 688,694, filed on August 29, 2024, which is hereby incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] This invention was made with government support under Award Numbers R01CA262887 and R21HD097357 awarded by the National Cancer Institute (NCI) and the Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD), respectively, of the National Institutes of Health. The government has certain rights in the invention.FIELD OF THE INVENTION

[0003] The present disclosure relates to compositions and methods for diagnosing and treating radiotherapy -induced brain injuries in a patient and more specifically, to polymeric nanoparticles, possessing reactive oxygen species (ROS) quenching units for reducing oxidative stress to prevent neurodegeneration and also for encapsulating active pharmaceutical ingredients, diagnostic markers, or combinations thereof.BACKGROUND INFORMATION

[0004] Radiotherapy-induced brain injury (RIBI) and cognitive decline are chronic side effects that affect up to 90% of brain tumor survivors treated with radiotherapy. Given the greater vulnerability of the developing brain to ionizing radiation, survivors of childhood brain tumors are at a greater risk of developing RIBI. With more patients surviving childhood cancers, there is a growing need for noninvasive imaging biomarkers capable of detecting RIBI early on, for its effective management. Current methods to detect RIBI and its associated cognitive decline rely on neuropsychological evaluations. However, RIBI management could be greatly improved with noninvasive imaging biomarkers. These biomarkers could aid in patient stratification, the longitudinal monitoring of RIBI progression, and responses to RIBI therapies.11623347761.1PATENT ATTORNEY DOCKET NO. JHU4770-1WO

[0005] Although the pathogenesis of RIBI has not yet been fully elucidated, the vascular hypothesis of RIBI pathogenesis postulates that it is driven by oxidative stress and neuroinflammation, which lead to ischemia, cerebrovascular damage, neurodegeneration, and cognitive decline. Preclinical rodent models of RIBI could play a critical role in elucidating the pathogenesis of RIBI and also in evaluating the efficacy of potential RIBI prophylactic and therapeutic agents. Additionally, preclinical RIBI models could play a critical role in enabling the better identification and characterization of noninvasive and clinically translatable longitudinal imaging biomarkers of RIBI.

[0005] There remains a need for diagnostic tools and effective drug delivery methods for the treatment of RIBI in patients.BRIEF DESCRIPTION

[0006] In some aspects, the techniques described herein relate to a polymeric nanoparticle including a block copolymer which includes a structure of Formula I:Formula I wherein o is an integer between 60 and 100; wherein p is an integer between 20 and 60; wherein m is an integer between 10 and 20; and wherein n is an integer between 5 and 15.

[0007] In some aspects, the techniques described herein relate to a polymeric nanoparticle, wherein the block copolymer includes a structure of Formula II:21623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WOFormula II

[0008] In some aspects, the techniques described herein relate to a polymeric nanoparticle, wherein the block copolymer includes a structure of Formula III:Formula III

[0009] In some aspects, the techniques described herein relate to a polymeric nanoparticle, wherein the block copolymer has a critical aggregation concentration of about 0.08 mg / mL to about 0.1 mg / mL.

[0010] In some aspects, the techniques described herein relate to a polymeric nanoparticle, having a diameter of about 165 nm at a pH of 7.4.31623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO

[0011] In some aspects, the techniques described herein relate to a polymeric nanoparticle, having a diameter of about 15 nm to about 25 nm at a pH of 6.2, in the presence of a reactive oxygen species, or a combination thereof.

[0012] In some aspects, the techniques described herein relate to a pharmaceutical composition, including: an active pharmaceutical ingredient, a diagnostic marker, or a combination thereof encapsulated in a pH and redox-responsive polymeric nanoparticle including a block copolymer which includes a structure of Formula I:Formula 1 wherein o is an integer between 60 and 100; wherein p is an integer between 20 and 60; wherein m is an integer between 10 and 20; and wherein n is an integer between 5 and 15.

[0013] In some aspects, the techniques described herein relate to a pharmaceutical composition, wherein the active pharmaceutical ingredient, the diagnostic marker, or the combination thereof is released from the polymeric nanoparticle upon exposure to a pH below about 6.5, exposure to reactive oxygen species, or a combination thereof.

[0014] In some aspects, the techniques described herein relate to a pharmaceutical composition, wherein the pharmaceutical composition includes an active pharmaceutical ingredient including a neuroprotective agent.

[0015] In some aspects, the techniques described herein relate to a pharmaceutical composition, wherein the pharmaceutical composition includes a diagnostic marker for detecting oxidative stress, reducing oxidative stress or a combination thereof.41623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO

[0016] In some aspects, the techniques described herein relate to a pharmaceutical composition, wherein the block copolymer includes a structure of Formula II:Formula II

[0017] In some aspects, the techniques described herein relate to a pharmaceutical composition, wherein the block copolymer includes a structure of Formula III:Formula III

[0018] In some aspects, the techniques described herein relate to a method of diagnosing or treating a radiotherapy-induced brain injury in a patient in need thereof, including: administering to the patient a therapeutically effective amount of a pharmaceutical composition, including: an active pharmaceutical ingredient, a diagnostic marker, or a combination thereof encapsulated in a polymeric nanoparticle including a block copolymer which includes a structure of Formula I:51623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WOFormula I wherein o is an integer between 60 and 100; wherein p is an integer between 20 and 60; wherein m is an integer between 10 and 20; and wherein n is an integer between 5 and 15; thereby diagnosing or treating the radiotherapy-induced brain injury in the patient.

[0019] In some aspects, the techniques described herein relate to a method, wherein the pharmaceutical composition includes an active pharmaceutical ingredient including a neuroprotective agent.

[0020] In some aspects, the techniques described herein relate to a method, wherein the pharmaceutical composition includes an active pharmaceutical ingredient which is released from the polymeric nanoparticle upon exposure to a pH below about 6.5, exposure to a reactive oxygen species, or a combination thereof, thereby treating the radiotherapy-induced brain injury in the patient.

[0021] In some aspects, the techniques described herein relate to a method, wherein the pharmaceutical composition includes a diagnostic marker for detecting oxidative stress in the patient.

[0022] In some aspects, the techniques described herein relate to a method, wherein the pharmaceutical composition includes a diagnostic marker which is released from the polymeric nanoparticle upon exposure to a pH below about 6.5, exposure to a reactive oxygen species, or a combination thereof, and further including detecting the diagnostic marker in the patient, thereby diagnosing the radiotherapy-induced brain injury in the patient.61623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO

[0023] In some aspects, the techniques described herein relate to a method, wherein the pharmaceutical composition includes a diagnostic marker including an MRI-detectable biomarker, and further including evaluating the patient by MRI.

[0024] In some aspects, the techniques described herein relate to a method, wherein the block copolymer includes a structure of Formula II:Formula 11

[0025] In some aspects, the techniques described herein relate to a method, wherein the block copolymer includes a structure of Formula III:Formula III71623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WOBRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIGS. 1A-1D. FIG. 1A shows a PEG homopolymer starting material and two exemplary block copolymers synthesized via RAFT polymerization. FIG. IB is 1H NMR spectrum of PEGMEA 480 homopolymer. FIG. 1C and FIG. ID are *HNMR spectra of block copolymers P2a and P2b, respectively.

[0027] FIGS. 2A-2B. FIG. 2A shows the mechanism of activation of the polymeric nanoparticles under oxidative stress conditions (presence of hydrogen peroxide and at pH of 6.2). FIG. 2B shows an activation mechanism wherein the ROS scavengers are first released from the block co-polymer at pH values below 6.5, before being oxidized by reactive oxygen species (ROS). Thus, the kinetics of release and the kinetics of tissue retention of the ROS scavengers greatly impact the long-term efficacy.

[0028] FIGS. 3A-3D. FIG. 3A is a schematic of the experimental design. FIG. 3B shows quantification of the fluorescence signals of DAPI and y-H2AX in the irradiated brain hemisphere normalized to the non-irradiated brain hemisphere 1 h and 3 days after stereotactic irradiation. A statistically significant difference was detected in the DNA double strand breakage (y-H2AX) signal 1 h post-irradiation compared to 3 days post-irradiation (P = 0.01, n = 3). No statistically significant difference was detected in the DAPI signal, 1 h postirradiation compared to 3 days post-irradiation. This suggested the rapid clearance of the DNA damaged cells shortly after irradiation.. FIG. 3C shows quantification of the respective MRI signals in the irradiated brain hemisphere normalized to the non-irradiated brain hemisphere of the same mouse. FIG. 3D shows quantification of the contrast-enhanced T1W MRI signal in mice irradiated at 5-weeks-old compared to 8-weeks-old.

[0029] FIG. 4A-4C shows T2W MRI detection of RIBI in mice stereotactically irradiated with 80 Gy of an X-ray beam at age 8-weeks-old compared to age 5-weeks-old. FIG. 4A) Coronal view and axial view. No differences were detected visually between the irradiated brain hemispheres and non-irradiated brain hemispheres in either group. FIG. 4B) Quantification of the T2W MRI hyperintensity signal in mice irradiated at 8-weeks-old compared to 5-weeks-old. No statistically significant difference was detected between both groups (n =3). FIG. 4C) Quantification of the T2W MRI hypointensity signal in mice irradiated at 8-weeks-old compared to 5-weeks-old. A statistically significant difference (P = 0.005, n81623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO=3) was calculated 0.5 months post-irradiation between both groups (even though no difference could be visually detected on the images)..

[0030] FIGS. 5A-5B. FIG. 5A shows contrast-enhanced T1W MRIs (grayscale Z- proj ections of six 0.5 mm coronal slices and six 0.5 mm axial slices respectively). The images show the kinetics of contrast enhancement one-month and two months post-irradiation in mice irradiated with 80 Gy of an X-ray beam at age 8-weeks-old. Contrast enhancement could be detected 2 to 60 minutes after the administration of the contrast agent. FIG. 5B shows quantification of the kinetics of T1W MRI contrast enhancement one month and two months post-irradiation, in mice irradiated with 80 Gy of an X-ray beam at age 8-weeks-old.

[0031] FIGS. 6A-6F. FIG. 6A-6D shows contrast-enhanced T1W MRIs (grayscale) of longitudinal blood-brain barrier (BBB) permeability changes over a 12-month period after irradiation. FIG. 6A) Coronal view of Z-proj ections of 6 slices. FIG. 6B) Axial view of Z- proj ections of 6 slices. FIG. 6C) Coronal view of a single slice. FIG. 6D) Axial view of a single slice. FIG. 6E) Quantification of the contrast-enhanced T1W MRI signal in the irradiated brain hemisphere normalized to the non-irradiated brain hemisphere 15- and 30 minutes post-contrast administration respectively, in mice irradiated at 8-weeks-old and monitored at different time points post-irradiation (P < 0.05, n >3). FIG. 6F) Quantification of the changes in the contrast-enhanced T1W MRI signal in the irradiated brain hemisphere normalized to the non-irradiated brain hemisphere over a period of 12-months post-irradiation (P < 0.05, n >3).

[0032] FIGS. 7A-7G shows MRI of longitudinal blood-brain barrier (BBB) permeability changes and anatomical changes after irradiation. FIG. 7A) Longitudinal in vivo T2W MRIs of a representative irradiated mouse brain (coronal and axial views), over a 12-month postirradiation period. FIG. 7B) Quantification of the changes in the T2W MRI hyperintensity and hypointensity signals respectively, in the irradiated brain hemisphere normalized to the nonirradiated brain hemisphere in mice irradiated at 8-weeks-old, and monitored at different time points post-irradiation (P < 0.05, n >3). FIG. 7C) Longitudinal in vivo T2*W MRIs of a representative irradiated mouse brain (coronal and axial views), over a 12-month postirradiation period. FIG. 7D) Quantification of the changes in the T2*W MRI hypointensity signal, in the irradiated brain hemisphere normalized to the non-irradiated brain hemisphere in mice irradiated at 8-weeks-old, and monitored at different time points post-irradiation (P <91623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO0.05, n >3). FIG. 7E) In vivo longitudinal detection of atrophy on T2W MRIs of a representative irradiated mouse brain (coronal and axial views), over a 12-month postirradiation period. FIG. 7F) Quantification of atrophy of the irradiated brain hemispheres normalized to the non-irradiated brain hemisphere in mice irradiated at 8-weeks-old and monitored at different time points post-irradiation, compared to the non-irradiated mice (P < 0.05; n > 3). FIG. 7G Quantification of the changes in the T2W MRI hyperintensity and hypointensity signals respectively, in the irradiated brain hemisphere normalized to the nonirradiated brain hemisphere in mice irradiated at 8-weeks-old and monitored at different time points post-irradiation (P < 0.05, n >3).

[0033] FIGS. 8A-8D shows Correlating longitudinal MRI biomarkers of RIBI to immunohistochemical biomarkers of RIBI. FIG. 8A) Quantification of the respective fluorescence signals and the contrast-enhanced T1W MRI signals in the irradiated brain hemispheres normalized to the non-irradiated brain hemispheres at different time points postirradiation (P < 0.05, n >3). FIG. 8B) Quantification of the respective fluorescence signals and the T2W MRI hyperintensity signals in the irradiated brain hemispheres normalized to the nonirradiated brain hemispheres at different time points post-irradiation (P < 0.05, n >3).. FIG. 8C) Quantification of the H&E signal and the T2W MRI hyperintensity signals in the irradiated brain hemispheres normalized to the non-irradiated brain hemispheres at different time points post-irradiation (P < 0.05, n >3). FIG. 8D) Quantification of the Peris’ Prussian blue staining signals and the T2*W MRI hypointensity signals in the irradiated brain hemispheres normalized to the non-irradiated brain hemispheres at different time points post-irradiation (P < 0.05, n >3).

[0034] FIGS. 9A-9E shows correlation of longitudinal MRI and IHC biomarkers of RIBI to behavioral and survival outcomes. The freezing behavior of irradiated mice (Group 1) compared to non-irradiated mice (Group 2), in the fear-conditioning test at different time points post-irradiation (FIGS. 9A-9E).

[0035] FIGS. 10A-10C. FIG. 10A) Quantification of the changes in the T2W MRI hypointensity signals and T2*W MRI hypointensity signals, respectively, in the irradiated brain hemispheres normalized to the nonirradiated brain hemispheres of mice irradiated at 8- weeks-old and monitored over a period of 12-month post irradiation (P < 0.05, n = 3). FIG. 10B) Weight changes in irradiated mice (Group 1) versus non-irradiated mice (Group 2), at101623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO different time points post-irradiation. FIG. IOC) Survival curves of irradiated mice (Group 1) versus non-irradiated mice (Group 2) at different time points post-irradiation.

[0036] FIGS. 11A-11F. FIG. 11A CACs of the polymers determined by fluorescence measurement of Nile red against concentration of the polymers. FIG. 11B Size of the polymeric nanoparticles under normal conditions (pH 7.4) and under oxidative stress (pH 6.2 in presence of H2O2). FIG. 11C Sizes and zeta potentials of the nanoparticles before activation (pH 7.4) and after activation (pH 6.2 in presence of H2O2). FIG. 11D TEM images of the respective nanoparticles. FIG. HE SEM images of the respective nanoparticles. FIG. HF Stability experiment of the nanoparticles.

[0037] FIGS. 12A-12G. FIG. 12A Schematic of cargo release. FIG. 12B, 12C Release of encapsulated Nile red from nanoparticles at physiological conditions (pH 7.4) compared to under varying degrees of oxidative stress (pH 6.4 and varying concentration of hydrogen peroxide). FIG 12D-12G show further cargo release studies of encapsulated Nile red from nanoparticles.

[0038] FIG. 13 Schematic representation of the encapsulation of 800 nm NIR fluorophores in 700 nm fluorescently labeled polymeric nanoparticles.

[0039] FIGS. 14A-14B. FIG. 14A shows degradation of the nanoparticles. FIG. 14B shows release of encapsulated dye.

[0040] FIGS. 15A-15E. FIG. 15A) Schematic of the degradation of fluorescent R- phycoerythrin (RPE) protein under oxidative stress and its protection by pH / redox-activable nanoparticle P2a or P2b. 15B) Changes in the fluorescence spectra of fluorescent RPE protein over 60 minutes, at physiological pH 7.4 and under oxidative stress (10 mM H202 at pH 7.0). 15C) Kinetics of fluorescent RPE protein (2.13 pg) degradation under oxidative stress (10 mM H2O2 at pH 7.4) and its protection in the presence of 1 pg of nanoparticles P2a and P2b, respectively (p < 0.05). 15D) Change in the fluorescence signal intensity of fluorescent RPE protein 60 minutes after exposure to oxidative stress (10 mM H2O2 at pH 7.4) and its protection in the presence of 1 pg of nanoparticles P2a and P2b, respectively (p < 0.05). 15E) Change in the fluorescence intensity signal of fluorescent RPE protein, 60 minutes after exposure to oxidative stress (10 mM H2O2 at pH 7.4) in the presence of varying amounts of nanoparticles111623347761.1PATENT ATTORNEY DOCKET NO. JHU4770-1WOP2a and P2b, respectively. RPE protection from degradation was obtained with as little as 1 pg ofNPs.

[0041] FIGS. 16A-16E. FIGS. 16A-16C show SSC-A versus Comp-FL3-A. FIG. 16D) 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). 16E) Cell viability of HUVECs after incubation with up to 20 pg (25 pg / pL) for 24 h, showed no toxicity from either nanoparticle.

[0042] FIG. 17) Schematic of in vivo experimental design used to evaluate the efficacy of nanoparticles P2a and P2b respectively.

[0043] FIGS. 18A-18C. FIG. 18A) Structure of fluorescent R-phycoerythrin (RPE) protein. FIG. 18B) Changes in the fluorescence spectra of fluorescent RPE protein over 60 minutes, at physiological pH 7.4 and under oxidative stress (10 mM H202 at pH 7.0). FIG. 18C) Kinetics of fluorescent RPE protein degradation under oxidative stress (10 mM H2O2 at pH 7.0) and its protection in the presence of 1 - 4 pg of nanoparticle P2a.

[0044] FIG. 19 Quantification of the delivery of fluorescently -labelled nanoparticles P2a and P2b to the brain in a preclinical mouse model of RIB I (p < 0.05).

[0045] FIG. 20 Complete blood counts in mice that received the 10 pg / pL of the nanoparticles P2a or P2b compared to mice that received phosphate buffered serum (PBS).

[0046] FIG. 21A shows total fluorescence vs. time after administration and mean fluorescence vs. time after administration.

[0047] FIG. 21B: Comprehensive serum chemistry in mice that received the 10 pg / pL of either nanoparticles P2a or P2b compared to mice that received phosphate buffered serum (PBS).

[0048] FIGS. 22A-22D: 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 pL of a 0.25 M contrast agent121623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO solution (Prohance®). Grayscale Z-projection T1 W MRIs of four 0.5 mm coronal (A) and axial (B) slices. Grayscale T1W MRIs of a single 0.5 mm coronal (C) and axial (D) slices.

[0049] FIGS. 23A-23C. FIG. 23A) In vivo contrast-enhanced T iW MRI of representative mice from the respective groups 1.5 months after nanoparticle administration. The TiW MRIs were acquired 15 mins after the intravenous administration of 200 pL of a 0.25 M contrast agent solution (Prohance®). 23B) In vivo T2W MRI of representative mice from each group. 23C) Quantification of the MRI signals in the three groups 1.5 months after nanoparticle administration (p < 0.05).

[0050] FIG. 24) Quantification of neuroinflammation in representative mice from the respective groups 1.5 months after nanoparticle administration.

[0051] FIGS. 25A-25B. FIG. 25A shows weight changes of mice used in the study over a period of 7 months post-irradiation. FIG. 25B shows survival curves of mice used in the study over a period of 6 months post-irradiation.

[0052] FIGS. 26A-26J: pH-dependent and power-dependent CEST MRI detection of nanotheranostic agent P2a at concentrations of 10 pg / pL. CEST MRI Z-spectra of 10 pg / pL of nanoparticle P2a solutions acquired at different powers (FIG. 26A to FIG. E); CEST MRI magnetization transfer asymmetry (MTRASYM) ratios of 10 pg / pL of nanoparticle P2a solutions acquired at different powers (FIG. 26F to FIG. 26J).

[0053] FIGS. 27A-27J: pH-dependent and power-dependent CEST MRI detection of nanotheranostic agent P2b at concentrations of 10 pg / pL. CEST MRI Z-spectra of 10 pg / pL of nanoparticle P2b solutions acquired at different powers (FIG. 27A to 27E); CEST MRT magnetization transfer asymmetry (MTRASYM) ratios of 10 pg / pL of nanoparticle P2a solutions acquired at different powers (FIG. 27F to FIG. 27J).

[0054] FIGS. 28A-28D: Summary of the pH-dependent and power-dependent CEST MRI detection of nanotheranostic agent P2a and P2b at concentrations of 10 pg / pL. FIG. 28A) Power-dependent CEST MRIs of nanoparticle p2a. at different pH values. FIG. 28B) Powerdependent CEST MRIs of nanoparticle p2b at different pH values. FIG. 28C) pH-dependent CEST MRIs of nanoparticle p2a, at different powers. FIG. 28D) pH-dependent CEST MRIs of nanoparticle p2b, at di fferent powers.131623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO

[0055] FIGS. 29A / 29B: pH-dependent and power-dependent CEST MRI detection of nanotheranostic agent P2a and P2b. Magnetization transfer asymmetry (MTRASYM) plots of 10 pg / pL nanoparticle solutions acquired at different powers: FIG, 29 A) Nanoparticle p2a and FIG. 29B) Nanoparticle p2b.

[0056] FIG. 29A-29J: Concentration-dependent and power-dependent CEST MRIs detection of nanotheranostic agent P2a. CEST MRI Z-spectra of different concentrations ofP2a solutions acquired at different powers (FIG. 29A to FIG. 29E); CEST MRI magnetization transfer asymmetry (MTRASYM) ratios of different concentrations of P2a solutions acquired at different powers (FIG. 29F to FIG. 29 J).

[0057] FIGS. 30A-30B: Summary of the concentration-dependent CEST MRI detection of nanotheranostic agent P2a. FIG. 30A) Power-dependence of the P2a CEST MRI signal at different concentrations. FIG. 30B) Concentration-dependence of the P2a CEST MRI signal of 10 nig / niL P2a phantoms at different MRI acquisitioning powers.

[0058] FIG. 31: Complete blood counts in mice that received the 10 pg / pL of the nanoparticles P2a or P2b compared to mice that received phosphate buffered serum (PBS).

[0059] FIG. 32: Comprehensive serum chemistry in mice that received the 10 pg / pL of either nanoparticles P2a or P2b compared to mice that received phosphate buffered serum (PBS).

[0060] FIG. 33 is a schematic of an experimental design.

[0061] FIGS. 34A-34E: Detection of neuroinflammalion m RIBI usingnC-DPA PET-MRI PET-MRIs of the radioactivity distribution of thenC-DPA tracer in the brain before, 2, 5, 10, 15 and 20 minutes after the bolus intravenous administration off’C-DPA: FIG. 34A) Before irradiation (non-irradiated control mice); FIG. 34B) one-month post-irradiation versus before irradiation (F = 0.0001, n = 3); FIG. 34C) two-months post-irradiation versus before irradiation (P = 0.0260, n = 3); FIG. 34D) six-montli post-irradiation versus before irradiation: and FIG. 34E) nme-months post-irradiation versus before irradiation.

[0062] FIGS. 35A-35B: Summary' of the detection of neuroinflammation in RIBI usingnC- DPA PET-MRI over a 9 months post-irradiation period. Representative PET-MRIs of the radioactivity' distribution of theUC-DPA tracer in mouse brains over the 9 months post-141623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO irradiation study period. The PET-MRIs were acquired before, 10, 15 and 20 minutes after the bolus intravenous administration ofnC-DPA: FIG. 35A) Quantification graphs of the ratios of the time-radioactivity' kinetic curves of the irradiated (right) brain hemispheres normalized to the corresponding contralateral (left) brain hemispheres of the respective mice, over 30 minutes after the bolus intravenous administration ofUC-DP A, at different time points before and after irradiation (P < 0.05). FIG. 35B) Radioactivity ratios of the irradiated (right) brain hemispheres normalized to the corresponding contralateral (left) brain hemispheres of the respective mice, 0, 10, 13, 15, 18, and 20 minutes after the bolus intravenous administration ofnC-DPA, at different time points before and after irradiation (P < 0.05).

[0063] FIGS. 36A-36C:nC-DPA PET-MRI blocking studies. PET-MRIs of the radioactivity distribution of the ' '(’-DPA tracer in the brain before. 2, 5, 10, 15 and 20 minutes after the bolus intravenous administration of "C-DPA: FIG. 36A) Before irradiation (nonirradiated control mice), FIG. 36B) two-month post-irradiation versus before irradiation (P::::0.0001, n:::3); FIG. 36C) two-months post-irradiation without the blocker versus two-months post-irradiation with the blocker versus before irradiation (P = 0.0001, n = 3).10064] FIGS. 37A-37C. FIG. 37A) Quantification of the ratio of the respective fluorescence signals in the irradiated brain hemispheres normalized to the corresponding contralateral nonirradiated brain hemispheres of the mice at different time point in the study, shown in comparison tonC-DPA PET, and FIG. 37B) contrast-enhance TiW MRI. FIG. 37C) Comparison of thenC-DPA PET and contrast-enhance TiW MRI signals in the study. A high correlation was observed between the neuroinflammation signals detected with immunohistochemistry,nC-DPA PET,nC-CPPC PET, and contrast-enhance TiW MRI. This suggested the possibility of using contrast-enhance TiW MRI (which doesn't involve ionizing radiation) as a proxy / surrogate biomarker of neuroinflammation in radiotherapy -induced brain injury.

[0065] FIG. 38: Schematic of experimental design of long-term nanoparticle efficacy study.

[0066] FIG. 39A-C: MRI signal changes in the different mouse groups over a 12-rnonth postirradiation period.151623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO

[0067] FIG. 40: Weight changes in the different mouse groups over a 12-month postirradiation period.

[0068] FIG. 41: Kaplan Meier survival curves of a long-term nanoparticle study.DETAILED DESCRIPTION

[0069] The present disclosure provides a technology related to polymeric nanoparticles. These nanoparticles are composed of a block copolymer structure, which may include a compound with a specific formula. The block copolymer structure may be designed to have certain properties, such as specific molecular weights or integer values, which can be adjusted to achieve desired characteristics of the nanoparticles.

[0070] In some aspects, the polymeric nanoparticles may be used in pharmaceutical compositions. These compositions may encapsulate an active pharmaceutical ingredient, a diagnostic marker, or a combination thereof. The encapsulated substances may be released from the nanoparticles under certain conditions, such as exposure to specific pH levels or reactive oxygen species. This release mechanism may allow for targeted delivery of the encapsulated substances, potentially improving the effectiveness of the pharmaceutical compositions.

[0071] In other aspects, the polymeric nanoparticles and the pharmaceutical compositions contained in them may be used in methods for diagnosing or treating certain medical conditions. For example, they may be used in diagnosing or treating radiotherapy-induced brain injury and neurodegenerative disorders that are driven by oxidative stress. The active pharmaceutical ingredient or the diagnostic marker encapsulated in the nanoparticles may be specifically chosen for their potential effectiveness in diagnosing or treating such conditions.

[0072] In yet other aspects, the block copolymer structure of the nanoparticles may have different structures, potentially providing flexibility in designing the nanoparticles for specific applications. The block copolymer structure may also contribute to the properties of the nanoparticles, such as their size or their response to certain environmental conditions.161623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO

[0073] Overall, the polymeric nanoparticle technology disclosed herein may provide a versatile platform for developing pharmaceutical compositions and methods for diagnosing or treating various medical conditions. The specific characteristics of the nanoparticles, such as their block copolymer structure and their encapsulation and release mechanisms, may be tailored to meet the needs of specific applications, potentially providing advantages in terms of effectiveness, specificity, and versatility.

[0074] In some embodiments, the polymeric nanoparticles may comprise a block copolymer which includes a compound of a specific formula, referred to here as Formula I.Formula I

[0075] . In some embodiments, the block copolymer may be structured such that it includes integer values for certain variables in Formula I. The block copolymer may include a fixed or variable number of pH-responsive CEST-MRI sensitive quaternary amine monomer units. For instance, the variable ‘o’ in Formula I refers to the number of repeating units, and ‘o’ may be an integer between 60 and 100, such as 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100.

[0076] Similarly, the variable ‘p’ in Formula I refers to the number of repeating phenylboronic acid pinacol ester (BAPE) units to scavenge reactive oxygen species (ROS) at injured or diseased sites and reduce oxidative stress. In some embodiments, ‘p’ may be an integer between 20 and 60, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60.171623347761.1PATENT ATTORNEY DOCKET NO. JHU4770-1WO

[0077] In some embodiments, the block copolymer may also include the variable ‘m’ in Formula I. The variable ‘m’ refers to the number of repeating units, and may be an integer between 10 and 20, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0078] In some embodiments, the variable ‘n’ in Formula I refers to the number of repeating polyethylene glycol units for improved nanoparticle circulation in the blood stream, and may be an integer between 5 and 15, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0079] In some embodiments, the specific integer values for ‘o’, ‘p’, ‘m’, and ‘n’ may be selected based on the desired properties of the polymeric nanoparticles. For example, different integer values may result in nanoparticles with different sizes, shapes, or other physical or chemical properties. This flexibility in the design of the block copolymer may allow for the customization of the polymeric nanoparticles for specific applications.

[0080] In some embodiments, the block copolymer may be synthesized using techniques known in the art, and the specific integer values for ‘o’, ‘p’, ‘m’, and ‘n’ may be controlled during the synthesis process. In some embodiments, block copolymer is synthesized via reversible addition fragmentation chain-transfer (RAFT) polymerization. FIG. 1A shows a PEG homopolymer starting material and two exemplary block copolymers synthesized via RAFT polymerization.

[0081] The resulting polymeric nanoparticles may then be characterized using techniques such as nuclear magnetic resonance spectroscopy, gel permeation chromatography, or other suitable methods to confirm the structure and properties of the block copolymer.

[0082] In some aspects, the block copolymer that forms the polymeric nanoparticles may comprise a structure represented by Formula II.181623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WOFormula II

[0083] . The structure of Formula I may be a variation of the block copolymer structure represented by Formula I, with specific modifications or substitutions that result in the structure of Formula II. The structure of Formula II may be designed to provide certain properties to the polymeric nanoparticles, such as specific responses to environmental conditions, encapsulation and release characteristics, or other desired properties.

[0084] In some aspects, the block copolymer that forms the polymeric nanoparticles may comprise a structure represented by Formula III.Formula III

[0085] . The structure of Formula III may be another variation of the block copolymer structure represented by Formula I, with specific modifications or substitutions that result in the structure of Formula III. The structure of Formula III may be designed to provide certain191623347761.1PATENT ATTORNEY DOCKET NO. JHU4770-1WO properties to the polymeric nanoparticles, such as specific responses to environmental conditions, encapsulation and release characteristics, or other desired properties.

[0086] In some embodiments, the block copolymer has a critical aggregation concentration of about 0.08 mg / mL to about 0.1 mg / mL, such as about 0.08 mg / mL, about 0.085 mg / mL, about 0.09 mg / mL, about 0.095 mg / mL, about 0.1 mg / mL, or any value contained with a range formed by any two of the preceding values. The critical aggregation concentration refers to the concentration above which the block copolymer starts to aggregate and form nanoparticles. This property may be important for the stability of the nanoparticles in solution and their ability to encapsulate and release substances. For instance, a lower critical aggregation concentration may indicate a higher stability of the nanoparticles in solution, which may be beneficial for certain applications, such as drug delivery.

[0087] In some instances, the critical aggregation concentration of the polymeric nanoparticles may be determined using techniques known in the art, such as fluorescence spectrometry, transmission electron microscopy, scanning electron microscopy, dynamic light scattering or nanoparticle tracking analysis. The critical aggregation concentration may be used as a parameter to modify the properties of the polymeric nanoparticles for specific applications. For example, the critical aggregation concentration may be adjusted to achieve a desired stability or encapsulation capacity of the nanoparticles.

[0088] In some aspects, the block copolymer forming the polymeric nanoparticles may have a molecular weight between about 10,000 and about 20,000 Da. The molecular weight of the block copolymer may influence various properties of the polymeric nanoparticles, such as their size, stability, and encapsulation capacity. For instance, block copolymers with higher molecular weights may form larger nanoparticles, while those with lower molecular weights may form smaller nanoparticles. The molecular weight of the block copolymer may also affect the stability of the nanoparticles, with higher molecular weights potentially leading to more stable nanoparticles.

[0089] In some cases, the molecular weight of the block copolymer may be controlled during the synthesis process. For example, the reaction conditions, such as the reaction time, temperature, and the concentration of the reactants, may be adjusted to achieve a desired201623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO molecular weight. The resulting block copolymer may then be used to form polymeric nanoparticles with specific properties, based on the molecular weight of the block copolymer.

[0090] In some embodiments, the block copolymer disclosed herein forms polymeric nanoparticles in aqueous media. The size, shape, and stability of the nanoparticles may be evaluated by methods including dynamic light-scattering (DLS) spectroscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM), the like, and combinations thereof.

[0091] In some aspects, the polymeric nanoparticles may exhibit a specific diameter at certain pH levels. For instance, the polymeric nanoparticles may have a diameter of about 165 nm at a pH of 7.4. The diameter of the nanoparticles at this pH level, equivalent to physiological condition, may be influenced by the properties of the block copolymer, such as its structure and molecular weight, as well as the specific integer values for ‘o’, ‘p’, ‘m’, and ‘n’ in Formula I.

[0092] The diameter of the polymeric nanoparticles at a pH of 7.4 may have implications for their use in certain applications. For example, nanoparticles with a diameter of about 165 nm may be suitable for encapsulating certain active pharmaceutical ingredients or diagnostic markers. The size of the nanoparticles may also influence their stability in solution, their ability to penetrate certain biological barriers, and their interactions with cells or tissues.

[0093] In some cases, the diameter of the polymeric nanoparticles at a pH of 7.4 may be determined using techniques known in the art, such as dynamic light scattering or nanoparticle tracking analysis. These techniques may provide accurate measurements of the nanoparticle size, which may be used to confirm the properties of the nanoparticles and their suitability for specific applications.

[0094] In other aspects, the diameter of the polymeric nanoparticles at a pH of 7.4 may be different from the aforementioned value. For instance, the diameter may be larger or smaller than 165 nm, depending on the specific properties of the block copolymer and the conditions under which the nanoparticles are formed. This flexibility in the size of the nanoparticles may allow for the customization of the nanoparticles for specific applications.211623347761.1PATENT ATTORNEY DOCKET NO. JHU4770-1WO

[0095] In some aspects, the polymeric nanoparticles may exhibit a change in diameter under certain conditions. For instance, the diameter of the nanoparticles may be about 15 nm to about 25 nm at a pH of 6.2, in the presence of a reactive oxygen species, or a combination thereof. This change in diameter may be due to the properties of the block copolymer, such as its structure and molecular weight, as well as the specific integer values for ‘o’, ‘p’, ‘m’, and ‘n’ in Formula I.

[0096] The change in diameter of the polymeric nanoparticles at a pH of 6.2 or in the presence of reactive oxygen species may have implications for their use in certain applications. For example, the reduced size of the nanoparticles under these conditions may facilitate their penetration into certain biological barriers, such as the blood-brain barrier. This may be particularly beneficial for applications involving the delivery of active pharmaceutical ingredients or diagnostic markers to the brain.

[0097] In some cases, the change in diameter of the polymeric nanoparticles at a pH of 6.2 or in the presence of reactive oxygen species may be determined using techniques known in the art, such as dynamic light scattering or nanoparticle tracking analysis. These techniques may provide accurate measurements of the nanoparticle size under different conditions, which may be used to confirm the properties of the nanoparticles and their suitability for specific applications.

[0098] In other aspects, the change in diameter of the polymeric nanoparticles at a pH of 6.2 or in the presence of reactive oxygen species may be different from the aforementioned values. For instance, the diameter may be larger or smaller than 15 nm to 25 nm, depending on the specific properties of the block copolymer and the conditions under which the nanoparticles are formed. This flexibility in the size of the nanoparticles may allow for the customization of the nanoparticles for specific applications.

[0099] In some embodiments, the polymeric nanoparticles maintain their stability in aqueous pH at pH 7.4 including when diluted 100-fold.

[0100] In some embodiments, there is provided a pharmaceutical composition which includes an active pharmaceutical ingredient, a diagnostic marker, or a combination thereof encapsulated in the polymeric nanoparticles described herein. The pharmaceutical221623347761.1PATENT ATTORNEY DOCKET NO. JHU4770-1WO composition may include an active pharmaceutical ingredient, a diagnostic marker, or a combination thereof encapsulated in polymeric nanoparticles which include a block copolymer including the structure of Formula I, the structure of Formula II, the structure of Formula III, or any combination thereof.

[0101] The active pharmaceutical ingredient may be any substance or combination of substances that may have a therapeutic, prophylactic, or diagnostic effect. In some cases, the active pharmaceutical ingredient may be a neuroprotective agent, which may be beneficial for treating conditions such as radiotherapy-induced brain injury and other neurodegenerative disorders. The specific neuroprotective agent may be selected based on its potential effectiveness in treating the condition.

[0102] In other aspects, the diagnostic marker encapsulated in the polymeric nanoparticles may be any substance or combination of substances that may be used to detect or monitor a specific condition or disease. For instance, the diagnostic marker may be a marker for detecting oxidative stress, which may be associated with various conditions, including radiotherapy- induced brain injury and other neurodegenerative disorders. In some embodiments, the diagnostic marker may be a dye, an MRI-detectable biomarker, or a combination thereof, such that when the pharmaceutical composition is administered to a patient, the diagnostic marker is released if the patient is experiencing oxidative stress, such as oxidative stress associated with RIBI. The specific diagnostic marker may be selected based on its potential effectiveness in diagnosing or monitoring the condition.

[0103] In some cases, the pharmaceutical composition may comprise a combination of an active pharmaceutical ingredient and a diagnostic marker encapsulated in the polymeric nanoparticles. This combination may provide a dual-functionality to the pharmaceutical composition, potentially allowing for the simultaneous treatment and monitoring of a specific condition. The specific active pharmaceutical ingredient and diagnostic marker may be selected based on their potential effectiveness in treating and diagnosing the condition, respectively.

[0104] In some aspects, the encapsulation of the active pharmaceutical ingredient, the diagnostic marker, or the combination thereof in the polymeric nanoparticles may be achieved using techniques known in the art. For instance, the encapsulation may be achieved through a231623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO process of nanoprecipitation, emulsion, or other suitable methods. The encapsulation may provide certain advantages, such as protecting the encapsulated substances from degradation, enhancing their stability, and allowing for their controlled release.

[0105] In some aspects, the release of the encapsulated substances from the polymeric nanoparticles may occur under certain conditions, such as exposure to specific pH levels or reactive oxygen species. FIG. 2 shows the mechanism of activation of the polymeric nanoparticles under oxidative stress conditions (presence of hydrogen peroxide and a pH of 6.2). This release mechanism may allow for the targeted delivery of the encapsulated substances, potentially improving the effectiveness of the pharmaceutical composition. The specific conditions for the release of the encapsulated substances may be tailored based on the needs of the specific application.

[0106] In some embodiments, the release of the encapsulated substances from the polymeric nanoparticles may be triggered by exposure to a pH below about 6.5, exposure to a reactive oxygen species, or a combination thereof. This pH-responsive and reactive oxygen species- responsive release mechanism may be due to the properties of the block copolymer, such as its structure and molecular weight, as well as the specific integer values for ‘o’, ‘p’, ‘m’, and ‘n’ in Formula I.

[0107] In some cases, the active pharmaceutical ingredient, the diagnostic marker, or the combination thereof may be encapsulated in the polymeric nanoparticles in such a way that they are released upon exposure to these specific conditions. This release may allow for the targeted delivery of the encapsulated substances to specific sites in the body, such as areas of inflammation or oxidative stress, where the pH may be lower than normal or where reactive oxygen species may be present.

[0108] In other aspects, the release of the encapsulated substances from the polymeric nanoparticles may be controlled to achieve a desired release profile. For instance, the release may be slow and sustained over a period of time, or it may be rapid and immediate upon exposure to the specific conditions. The specific release profile may be tailored based on the needs of the specific application, such as the type of active pharmaceutical ingredient or diagnostic marker encapsulated in the nanoparticles, the condition being treated or diagnosed, and the desired therapeutic or diagnostic effect.241623347761.1PATENT ATTORNEY DOCKET NO. JHU4770-1WO

[0109] In some cases, the release of the encapsulated substances from the polymeric nanoparticles upon exposure to a pH below about 6.5, exposure to a reactive oxygen species, or a combination thereof may be confirmed using techniques known in the art. For instance, the release may be monitored using techniques such as fluorescence spectroscopy, ultraviolet- visible spectroscopy, fluorescence imaging, magnetic resonance imaging (MRI), or other suitable methods. These techniques may provide accurate measurements of the release of the encapsulated substances, which may be used to confirm the properties of the nanoparticles and their suitability for specific applications.

[0110] In other cases, the release of the encapsulated substances from the polymeric nanoparticles upon exposure to a pH below about 6.5, exposure to a reactive oxygen species, or a combination thereof may be different from the aforementioned conditions. For instance, the release may be triggered by a different pH level, a different type of reactive species, or other environmental conditions, depending on the specific properties of the block copolymer and the encapsulated substances. This flexibility in the release mechanism may allow for the customization of the polymeric nanoparticles for specific applications.

[0111] In some embodiments, the active pharmaceutical ingredient encapsulated in the polymeric nanoparticles may be a neuroprotective agent. Neuroprotective agents are substances that have the potential to preserve neuronal structure and / or function. These agents may be beneficial in conditions where neuronal damage is present, such as in radiotherapy- induced brain injury and other neurodegenerative disorders. The specific neuroprotective agent may be selected based on its potential effectiveness in treating the condition.

[0112] In some cases, the neuroprotective agent may be a small molecule drug, a peptide, a protein, genetic material such as mRNAs or other suitable substances. The specific type of neuroprotective agent may be selected based on factors such as its mechanism of action, its pharmacokinetic properties, its potential side effects, and its compatibility with the polymeric nanoparticles.

[0113] In some cases, the release of the neuroprotective agent from the polymeric nanoparticles may occur under certain conditions, such as exposure to specific pH levels or reactive oxygen species. This release mechanism may allow for the targeted delivery of the251623347761.1PATENT ATTORNEY DOCKET NO. JHU4770-1WO neuroprotective agent, potentially improving its effectiveness in treating conditions such as radiotherapy -induced brain injury and other neurodegenerative disorders.

[0114] In some embodiments, the pharmaceutical composition may comprise a diagnostic marker for detecting oxidative stress. Oxidative stress refers to an imbalance between the production of free radicals and the ability of the body to counteract or detoxify their harmful effects through neutralization by antioxidants. This condition may be associated with various diseases and conditions, including radiotherapy -induced brain injury and other neurodegenerative disorders. The specific diagnostic marker for detecting oxidative stress may be selected based on its potential effectiveness in diagnosing or monitoring the condition.

[0115] In some cases, the diagnostic marker for detecting oxidative stress may be a small molecule, a peptide, a protein, an antibody or other suitable substances. The specific type of diagnostic marker may be selected based on factors such as its sensitivity and specificity for detecting oxidative stress, its stability, and its compatibility with the polymeric nanoparticles.

[0116] In some cases, the release of the diagnostic marker from the polymeric nanoparticles may occur under certain conditions, such as exposure to specific pH levels or reactive oxygen species. This release mechanism may allow for the targeted delivery of the diagnostic marker, potentially improving its effectiveness in diagnosing conditions associated with oxidative stress.

[0117] In some embodiments, there is provided a method for diagnosing or treating a radiotherapy -induced brain injury in a patient in need thereof, including administering to the patient a therapeutically effective amount of a pharmaceutical composition as described herein. In some embodiments, the pharmaceutical composition may include an active pharmaceutical ingredient, a diagnostic marker, or a combination thereof encapsulated in a polymeric nanoparticle as described herein, such as a polymeric nanoparticle including a block copolymer which includes the structure of Formula I, the structure of Formula II, the structure of Formula III, or any combination thereof.

[0118] In some embodiments, the patient is a human. In some embodiments, the patient is an animal, such as a mammal including canine, feline, murine, ovine, porcine, equine, bovine, and the like.261623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO

[0119] In some embodiments, the active pharmaceutical ingredient, the diagnostic marker, or the combination thereof encapsulated in the polymeric nanoparticles may be released upon exposure to a pH below about 6.5, exposure to a reactive oxygen species, or a combination thereof.

[0120] In some cases, the active pharmaceutical ingredient may be a neuroprotective agent, a chemotherapeutic agent, or a combination thereof, which may be beneficial for treating radiotherapy -induced brain injury. The active pharmaceutical ingredient may be released from the polymeric nanoparticles upon exposure to a pH below about 6.5, exposure to a reactive oxygen species, or a combination thereof. This release mechanism may allow for the targeted delivery of the neuroprotective agent to the site of the brain injury, potentially improving the effectiveness of the treatment.

[0121] In other aspects, the diagnostic marker may be a marker for detecting oxidative stress, which may be associated with radiotherapy-induced brain injury. The diagnostic marker may be released from the polymeric nanoparticles upon exposure to a pH below about 6.5, exposure to a reactive oxygen species, or a combination thereof. This release mechanism may allow for the targeted delivery of the diagnostic marker to the site of the brain injury, potentially improving the effectiveness of the diagnosis.

[0122] In some cases, the diagnostic marker may be an MRI-detectable biomarker. The release of the MRI-detectable biomarker from the polymeric nanoparticles may allow for the detection of the biomarker in the patient using MRI, potentially providing a non-invasive method for diagnosing radiotherapy-induced brain injury.

[0123] In some embodiments, the process of administering a therapeutically effective amount of the pharmaceutical composition to a patient may involve various methods known in the art. For instance, the pharmaceutical composition may be administered orally, intravenously, intramuscularly, subcutaneously, intranasally, or by other suitable routes. The specific route of administration may be selected based on factors such as the nature of the active pharmaceutical ingredient or diagnostic marker, the condition being treated or diagnosed, and the desired therapeutic or diagnostic effect.271623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO

[0124] In some cases, the therapeutically effective amount of the pharmaceutical composition may be determined based on factors such as the potency of the active pharmaceutical ingredient or diagnostic marker, the severity of the condition being treated or diagnosed, the patient's weight, age, and overall health status, and other relevant factors. The therapeutically effective amount may be adjusted as needed to achieve the desired therapeutic or diagnostic effect, while minimizing potential side effects.

[0125] In other aspects, the pharmaceutical composition may be administered in a single dose or in multiple doses over a period of time. The specific dosing regimen may be selected based on factors such as the pharmacokinetic properties of the active pharmaceutical ingredient or diagnostic marker, the nature of the condition being treated or diagnosed, and the desired therapeutic or diagnostic effect.

[0126] In some cases, the pharmaceutical composition may be administered in combination with other treatments or therapies. For instance, the pharmaceutical composition may be administered in combination with stem cell therapies, extracellular vesicles, chemotherapeutic agents, or other treatments for radiotherapy-induced brain injury. The combination of the pharmaceutical composition with other treatments or therapies may provide a synergistic effect, potentially improving the effectiveness of the diagnosis or treatment. In other embodiments, the method may include administering the pharmaceutical composition alone to the patient, such that the patient is not also undergoing other treatments or receiving other therapies.

[0127] In other aspects, the process of administering the pharmaceutical composition may be monitored using techniques known in the art. For instance, the release of the active pharmaceutical ingredient or diagnostic marker from the polymeric nanoparticles may be monitored using techniques such as MRI, fluorescence imaging, fluorescence spectroscopy, ultraviolet-visible spectroscopy, or other suitable methods. The monitoring of the administration process may provide valuable information about the effectiveness of the pharmaceutical composition and may allow for adjustments to the dosing regimen or the administration method as needed.

[0128] In some embodiments, the method including diagnosing RIBI in the patient and includes administering to the patient the pharmaceutical composition as described herein,281623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO wherein the pharmaceutical composition includes a diagnostic marker which is released from the polymeric nanoparticle upon exposure to a pH below about 6.5, exposure to a reactive oxygen species, or a combination thereof, and further comprising detecting the diagnostic marker in the patient, thereby diagnosing the radiotherapy -induced brain injury in the patient. The diagnostic marker may be released in a patient experiencing oxidative stress, and the release of the diagnostic marker may be monitored and evaluated by methods including but not limited to MRI, PET, or combinations thereof, thereby diagnosing RIBI in the patient.

[0129] In some embodiments, the diagnostic marker encapsulated in the polymeric nanoparticles may be an MRI-detectable biomarker. MRI-detectable biomarkers are substances that can be detected using magnetic resonance imaging (MRI), a non-invasive imaging technique that uses a powerful magnetic field and radio waves to create detailed images of the inside of the body. The use of MRI-detectable biomarkers may provide a non- invasive method for diagnosing or monitoring certain conditions, such as radiotherapy -induced brain injury.

[0130] The specific MRI-detectable biomarker may be selected based on factors such as its sensitivity and specificity for detecting the condition, its stability, and its compatibility with the polymeric nanoparticles. In some cases, the MRI-detectable biomarker may be a contrast agent, a radiolabeled substance, or other suitable substances that can be detected using MRI.

[0131] In some aspects, the method for diagnosing or treating a radiotherapy-induced brain injury in a patient may further include evaluating the patient by MRI. This evaluation may involve the detection of the MRI-detectable biomarker released from the polymeric nanoparticles. The MRI evaluation may provide detailed images of the brain, potentially allowing for the detection of changes associated with radiotherapy-induced brain injury. The specific MRI techniques used for the evaluation may be selected based on factors such as the type of MRI-detectable biomarker, the condition being diagnosed or treated, and the desired diagnostic or therapeutic effect.

[0132] In some embodiments, the diagnostic marker encapsulated in the polymeric nanoparticles may be a PET-detectable biomarker. PET-detectable biomarkers are substances that can be detected using positron emission tomography (PET) which may provide a non- invasive method for diagnosing or monitoring certain conditions, such as radiotherapy -induced291623347761.1PATENT ATTORNEY DOCKET NO. JHU4770-1WO brain injury. The specific PET-detectable biomarker may be selected based on factors such as its sensitivity and specificity for detecting the condition, its stability, and its compatibility with the polymeric nanoparticles. In some cases, the PET-detectable biomarker may be a contrast agent, a radiolabeled substance, or other suitable substances that can be detected using PET.

[0133] In some aspects, the method for diagnosing or treating a radiotherapy-induced brain injury in a patient may further include evaluating the patient by PET. This evaluation may involve the detection of the PET-detectable biomarker released from the polymeric nanoparticles. The PET evaluation may provide detailed images of the brain, potentially allowing for the detection of changes associated with radiotherapy-induced brain injury. The specific PET techniques used for the evaluation may be selected based on factors such as the type of PET-detectable biomarker, the condition being diagnosed or treated, and the desired diagnostic or therapeutic effect.

[0134] In some embodiments, the method includes treating RIBI in the patient and includes administering to the patient the pharmaceutical composition as described herein, wherein the pharmaceutical composition includes an active pharmaceutical ingredient which is released from the polymeric nanoparticle upon exposure to a pH below about 6.5, exposure to a reactive oxygen species, or a combination thereof, and further comprising detecting the diagnostic marker in the patient, thereby diagnosing the radiotherapy -induced brain injury in the patient. The active pharmaceutical ingredient may include neuroprotective agents.

[0135] In some embodiments, the method includes both diagnosing and treating RIBI in the patient and includes administering to the patient the pharmaceutical composition as described herein, wherein the pharmaceutical composition includes both an active pharmaceutical ingredient and a diagnostic marker encapsuled in the polymeric nanoparticles disclosed herein.EXAMPLES

[0136] The following examples are provided to further illustrate the embodiments of the present invention, but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.EXAMPLE 1301623347761.1PATENT ATTORNEY DOCKET NO. JHU4770-1WODeveloping magnetic resonance imaging biomarkers of radiotherapy-induced brain injury in a preclinical mouse model

[0137] Introduction: Radiotherapy-induced brain injury (RIBI) and cognitive decline are chronic side effects that affect up to 90% of brain tumor survivors, treated with radiotherapy. Current methods to detect RIBI rely on neuropsychological evaluations. However, RIBI management could be greatly improved with noninvasive imaging biomarkers. These biomarkers could aid in patient stratification, the longitudinal monitoring of RIBI progression and responses to RIBI therapies.

[0138] Objective: Here, we elucidated translatable magnetic resonance imaging (MRI) biomarkers of RIBI in a preclinical mouse model.

[0139] Method: 8-week-old female, immune competent BALB / c mice, were stereotactically irradiated with a single dose of 80 Gy (dose rate of 1.7 Gy / minute). The irradiated mice were then monitored longitudinally with MRI, behavioral tests and immunohistochemistry, in comparison to non-irradiated mice.

[0140] Summary of Results: Blood-brain barrier (BBB) permeability was detected with contrast-enhanced T1 -weighted MRI, 2 weeks post-irradiation. This permeability peaked one- month post-irradiation, then decreased over time. The onset of transient cognitive impairment and neuroinflammation were also detected around these early time points, with the fearconditioning behavioral test and immunohistochemistry respectively. Edema, necrosis, hemorrhage, and atrophy, were detected at later time points, with T2-weighted MRI. Whereas edema was detected as a hyperintense signal, necrosis and hemorrhage were detected as hypointense signals, and validated with T2*-weighted MRI. These MRI biomarkers of late injury preceded significant weight loss, severe cognitive impairment and decreased survival in the irradiated mice, compared to the non-irradiated mice.

[0141] Conclusion: These translational MRI biomarkers of RIBI could play a pivotal role in the management of RIBI in brain tumor survivors.

[0142] Radiotherapy-induced brain injury (RIBI) and cognitive impairment are progressive and chronic side effects that affect up to 90% of brain tumor survivors, treated with radiotherapy. Given the greater vulnerability of the developing brain to ionizing radiation,311623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO survivors of childhood brain tumors are at a greater risk of developing RIBI. Recent diagnostic and therapeutic advances have greatly improved the overall survival of pediatric brain tumor patients. Consequently, with more pediatric brain tumor patients surviving childhood cancers, there is a growing need for surveillance strategies capable of detecting RIBI early on, for its effective management. Current methods to detect RIBI and its associated cognitive decline rely on neuropsychological evaluations. However, neuropsychological evaluations could be greatly supplemented with robust noninvasive imaging biomarkers, that could be used to detect subtle molecular changes indicative of the early onset of RIBI. These imaging biomarkers could also aid in: 1) Patient stratification; 2) The longitudinal monitoring of RIBI progression; and 3) Monitoring patient responses to potential RIBI therapies.

[0143] Although the pathogenesis of RIBI has not yet been fully elucidated, the vascular hypothesis of RIBI pathogenesis postulates that it is driven by oxidative stress and neuroinflammation, which lead to ischemia, cerebrovascular damage, neurodegeneration, and cognitive decline. Preclinical rodent models of RIBI could play a critical role in elucidating the pathogenesis of RIBI and also in evaluating the efficacy of potential RIBI prophylactic and therapeutic agents. Additionally, preclinical RIBI models could play a critical role in enabling the better identification and characterization of noninvasive and clinically translatable longitudinal imaging biomarkers of RIBI.

[0144] Magnetic resonance imaging (MRI) offers several advantages for imaging brain tumor patients and brain tumor survivors, especially pediatric patients and survivors. These advantages include: The possibility of longitudinally monitoring patients noninvasively in the absence of ionizing radiation, and at great spatial resolution. Previously, we elucidated early MRI biomarkers of RIBI and correlated these early MRI biomarkers with early cognitive changes in a preclinical RIBI mouse model, using behavioral tests of memory and learning. Here, we expand on our previous findings and evaluate the longitudinal evolution of these MRI biomarkers in relation to neuroinflammation and cognitive changes in a preclinical RIBI mouse model, over a 12-month post-irradiation period (FIG. 3A). Ultimately, these translatable MRI biomarkers could enable the longitudinal evaluation of potential RIBI prophylactic and therapeutic agents that are currently being developed; and also enable the better characterization, detection and management of RIBI in brain tumor survivors.321623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO

[0145] FIG. 3A is a schematic of the experimental design. FIG. 3B shows fluorescence microscopy images of immunohistochemical detection of DNA double strand breakage (y- H2AX) and cellular nuclei (DAPI) in a 4 pm mouse brain section, harvested 1 h after stereotactic irradiation with 80 Gy of an X-ray beam (2.5 mm scalebar). FIG. 3C shows quantification of the fluorescence signals from y-H2AX and DAPI respectively, in the irradiated brain hemisphere normalized to the non-irradiated brain hemisphere of the same mouse brain section. A statistically significant difference was detected in the y-H2AX signal ratio compared to the DAPI signal ratio, 1 h post-irradiation (P = 0.006, n = 3). Multiparametric MRI (contrast-enhanced T1W MRI, T2W MRI, and T2*W MRI) detection of RIBI two weeks post-irradiation in mice stereotactically irradiated with 80 Gy of an X-ray beam at 8-weeks of age: FIG. 3D shows a coronal view of multi -parametric MRI detection of RIBI two weeks post-irradiation in mice; FIG. 3E shows an axial view of multi-parametric MRI detection of RIBI two weeks post-irradiation in mice. RIBI could be detected with contrast- enhanced T1W MRI, as early as two weeks post-irradiation, in mice irradiated at 8-weeks of age. This shows that contrast-enhanced T1W MRI was most sensitive at detecting RIBI at this early time point. RIBI could not be detected with either T2W MRI or T2*W MRI at this early time point. FIG. 3F shows quantification of the respective MRI signals in the irradiated brain hemisphere normalized to the non-irradiated brain hemisphere of the same mouse. A statistically significant difference was detected in the contrast-enhanced T1W MRI signal ratio two weeks post-irradiation (P = 0.0006, n = 3). However, no statistically significant difference was detected with either T2W MRI or T2*W MRI at this early time point. This suggested that contrast-enhanced T1W MRI was most sensitive at detecting RIBI early-on. FIG. 3G shows contrast-enhanced T1W MRI detection of RIBI in mice stereotactically irradiated with 80 Gy of an X-ray beam at age 5-weeks-old compared to age 8-weeks-old. RIBI was detected as early as one-week post-irradiation in mice irradiated at 5-weeks-old; compared to detection at two weeks post-irradiation in mice irradiated at age 8-weeks-old. This suggested that RIBI occurs earlier in mice irradiated at a younger age compared to mice irradiated at an older age. FIG. 3H shows quantification of the contrast-enhanced T1W MRI signal in mice irradiated at 5- weeks-old compared to 8-weeks-old. A statistically significant difference in the contrast- enhanced T1W MRI signal was detected one-week post-irradiation in the irradiated brain hemispheres of the 5-weeks-old mice compared to the 8-weeks-old mice (P = 0.012, n = 3). However, no statistically significant difference was detected between both groups, two weeks,331623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO one month and two months post-irradiation (n >3). FIG. 31 shows contrast-enhanced T1W MRIs showing the kinetics of contrast enhancement one month and two months postirradiation, in mice irradiated with 80 Gy of an X-ray beam at age 8-weeks-old. Contrast enhancement could be detected 2 to 60 minutes after the administration of the contrast agent. FIG. 5B shows quantification of the kinetics of T1W MRI contrast enhancement one month and two months post-irradiation, in mice irradiated with 80 Gy of an X-ray beam at age 8- weeks-old. This showed a similar kinetic trend one month and two months post-irradiation.

[0146] Results:

[0147] Early detection of radiotherapy-induced brain injury (RIBI). Fluorescence microscopy images of brain tissue samples harvested 1 h after the irradiation of 8-week-old mice (Group 3) and stained for DNA double strand breakage using y-H2AX immunohistochemistry, showed specific DNA damage only in the irradiated brain hemispheres as was expected (FIG. 3B, 3C and FIG. 4). Quantitative pixel analysis of the y-H2AX fluorescence signal ratios of the irradiated brain hemispheres compared to the non-irradiated brain hemispheres revealed a 12.14 ± 3.58-fold increase in y-H2AX staining in the irradiated brain hemispheres, 1 h post-irradiation (P = 0.006, n =3). This confirmed the specific irradiation of a single brain hemisphere in the irradiated mice.

[0148] Two weeks after the stereotactic irradiation of 8-week-old mice (Group 1), contrast- enhanced T1W MRI showed the specific disruption of the blood-brain barrier (BBB) in the irradiated brain hemispheres of the mice. Quantitative pixel analysis of the contrast-enhanced T1 W MRI signal ratios of the irradiated brain hemispheres compared to the non-irradiated brain hemispheres revealed a 12.10 ± 2.01-fold increase (P = 0.0001, n =3) in the irradiated (Group 1) mice compared to the control non-irradiated (Group 2) mice. This detection of BBB permeability indicated the onset of RIBI. Interestingly, no differences between the irradiated brain hemispheres and the non-irradiated brain hemispheres were detected at this time point with either T2 -weighted (T2W) MRI or T2*-weighted (T2*W) MRI in these mice. These results suggested that contrast-enhanced T1W MRI was the most sensitive of the MRI pulse sequences evaluated, for detecting the onset of RIBI, via the detection of BBB permeability.

[0149] Additionally, contrast-enhanced T1 W MRI detected BBB disruption at an earlier time point in mice irradiated at a younger age. Briefly, BBB disruption was detected one-week post-341623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO irradiation in mice irradiated at 5-weeks-old, compared to detection at two weeks postirradiation in mice irradiated at 8-weeks-old. A statistically significant difference (P = 0.012, n =3) between the contrast-enhanced T1W MRI signals from the respective age groups was detected one-week post-irradiation. Briefly, quantitative pixel analysis of the contrast- enhanced T1W MRI signal ratios in the irradiated brain hemispheres compared to the nonirradiated brain hemispheres of the respective age group mice revealed a 7.15 ± 2.64-fold higher ratio, in the mice irradiated at 5-weeks-old, one-week post-irradiation. No contrast- enhanced T1W MRI signal was detected one-week post-irradiation, in mice irradiated at 8- weeks of age. Additionally, no statistically significant differences between the contrast- enhanced T1W MRI signals from both age groups, were detected at any other time points postirradiation.

[0150] Interestingly, no visual differences between the irradiated brain hemispheres and the non-irradiated brain hemispheres were visible on the T2W MRIs or T2*W MRIs of mice irradiated at age 5-weeks-old compared to 8-weeks-old, either one week or two weeks postirradiation (FIG. 4A). However, quantitative pixel analysis of the irradiated brain hemispheres compared to the non-irradiated brain hemispheres using the T2W MRI hypointensity signal, showed a statistically significant difference between both age groups (P = 0.004, n = 3) two weeks post-irradiation (FIG. 4B). This suggested that RIBI in younger mice could be detected quantitatively, but not visually with the T2W MRI hypointense signal, as early as two weeks post-irradiation.

[0151] Imaging longitudinal changes in the blood-brain barrier (BBB) permeability after irradiation. In order to use contrast-enhanced T1W MRI to evaluate how the permeability of the BBB changed after irradiation, the kinetics of T1W MRI contrast enhancement was determined in mice irradiated at 8-weeks of age. Briefly, contrast-enhanced T1W MRIs were acquired sequentially before and up to 60 minutes post-contrast administration, one month and two months post-irradiation. The contrast enhancement kinetic study was done at these time points (one month and two months post-irradiation), since at these time points, no statistically significant differences were detected between the contrast-enhanced T1 W MRI signals of mice irradiated at 5-weeks-old compared to mice irradiated at 8-weeks-old.

[0152] Our results showed that, although the contrast-enhanced T1W MRI signal intensity ratio of the irradiated brain hemispheres compared to the nonirradiated brain hemispheres was351623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO higher (P = 0.005, n = 3) at one-month post-irradiation (18.49 ± 1.10) compared to two months post-irradiation (13.80 ± 0.37), similar contrast enhancement kinetic patterns were detected at both time points (FIG. 31, 3 J and FIG. 5A). Briefly, at both time points, contrast enhancement was detected in the irradiated brain hemispheres compared to the nonirradiated brain hemispheres as early as 2 minutes post-contrast administration (11.78 ± 1.56, one-month postirradiation (IR) versus 6.34 ± 2.35, two months post-IR) and increased from 2 to 15 minutes post-contrast administration (16.90 ± 1.37, one-month post-IR versus 12.03 ± 0.30, two months post-IR ), then plateaued between 15 to 60 minutes (18.49 ± 1.10, one-month post-IR versus 13.80 ± 0.37, two months post-IR). These plateaued contrast-enhanced T1W MRI signal intensity ratios 15 to 60 minutes post-contrast administration suggested that contrast- enhanced T1W MRIs acquired between 15 to 30 minutes post-contrast administration could be used to reliably evaluate changes in the permeability of the BBB at different time points after irradiation.

[0153] FIG. 9 shows MRI of longitudinal blood-brain barrier (BBB) permeability changes and anatomical changes after irradiation. In vivo contrast-enhanced T1W MRI detection of BBB permeability in mice stereotactically irradiated with 80 Gy of an X-ray beam at age 8- weeks-old. Contrast-enhanced T1W MRI showed that BBB permeability was detected as early as two weeks post-irradiation and remained permeable 12 months post-irradiation. However, the degree of BBB permeability changed over the 12-month post-irradiation period. FIG. 6E) Quantification of the contrast-enhanced T1W MRI signal in the irradiated brain hemisphere normalized to the non-irradiated brain hemisphere 15- and 30 minutes post-contrast administration respectively, in mice irradiated at 8-weeks-old and monitored at different time points post-irradiation (P < 0.05, n >3). This showed comparable T1W MRI contrastenhancement of the irradiated brain hemispheres 15- and 30 minutes post-contrast administration, at the respective time points post-irradiation. FIG. 6F) Quantification of the changes in the contrast-enhanced T1W MRI signal in the irradiated brain hemisphere normalized to the non-irradiated brain hemisphere over a period of 12-months post-irradiation (P < 0.05, n >3). This showed that the degree of BBB permeability changed statistically significantly (P < 0.05, n >3) over the 12-months post-irradiation period. FIG. 7A) Longitudinal in vivo T2W MRIs of a representative irradiated mouse brain (coronal and axial views), over a 12-month post-irradiation period. T2W MRI hyperintensity (red arrow) indicative of edema was detected one to two months post-irradiation; while T2W MRI361623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO hypointensity (blue arrow) indicative of hemorrhage was detected four months to 12 months post-irradiation. FIG. 7B) Quantification of the changes in the T2W MRI hyperintensity and hypointensity signals respectively, in the irradiated brain hemisphere normalized to the nonirradiated brain hemisphere in mice irradiated at 8-weeks-old, and monitored at different time points post-irradiation (P < 0.05, n >3). FIG. 7C) Longitudinal in vivo T2*W MRIs of a representative irradiated mouse brain (coronal and axial views), over a 12-month postirradiation period. T2*W MRI hypointensity (blue arrow) was detected as early as one-month post-irradiation and continued up until 12 months post-irradiation. FIG. 7D) Quantification of the changes in the T2*W MRI hypointensity signal, in the irradiated brain hemisphere normalized to the non-irradiated brain hemisphere in mice irradiated at 8-weeks-old, and monitored at different time points post-irradiation (P < 0.05, n >3). FIG. 7E) In vivo longitudinal detection of atrophy on T2W MRIs of a representative irradiated mouse brain (coronal and axial views), over a 12-month post-irradiation period. Atrophy (red arrow) was visible on T2W MRIs two months to 12 months post-irradiation. FIG. 7F) Quantification of atrophy of the irradiated brain hemispheres normalized to the non-irradiated brain hemisphere in mice irradiated at 8-weeks-old and monitored at different time points post-irradiation, compared to the non-irradiated mice (P < 0.05; n > 3).

[0154] Using contrast-enhanced T1W MRI, the BBB permeability was evaluated over a 12- month post-irradiation period in mice irradiated at 8-weeks of age. As previously mentioned, contrast-enhanced T1W MRI detected BBB permeability in these mice as early as 0.5 months post-irradiation, and this indicated the onset of injury (FIGS.6A-F). Quantitative pixel analysis of the contrast-enhanced T1W MRI signal ratio of the irradiated brain hemispheres compared to the non-irradiated brain hemispheres revealed a value of 12.10 ± 2.01 (P = 0.0001, n =3) at this time point. This contrast-enhanced T1W MRI signal intensity ratio peaked at one-month post-irradiation (18.03 ± 2.28), and this indicated a peak in the BBB permeability (FIGS. 6A- 6F). The contrast-enhanced T1W MRI signal ratio then gradually decreased (P = 0.001, n = 3), over the following eleven months post-irradiation to a value of 5.69 ± 2.23 at 12 months postirradiation (FIGS. 6A-6F). This indicated a decrease in the BBB permeability over time. Overall, a contrast-enhanced T1 W MRI signal was detected in the irradiated brain hemispheres throughout the 12-month post-irradiation period. This indicated BBB permeability throughout the 12-months post-irradiation study period.371623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO

[0155] Imaging longitudinal anatomical changes in the brain after irradiation. T2W MRI was used to monitor anatomical changes in the brain after irradiation. A hyperintense T2W MRI signal was detected in the irradiated brain hemisphere of mice irradiated at age 8-weeks-old, one month and two months post-irradiation (FIG. 9E, 9F, 9K and 9M). Quantitative pixel analysis of the hyperintense T2W MRI signal ratio of the irradiated brain hemispheres compared to the non-irradiated brain hemispheres revealed statistically significant differences (P < 0.05, n =3) one month (1.82 ±1.03) and two months (1.86 ±1.42) post-irradiation (FIG. 9E, 9F, 9K and 9M). However, this hyperintense signal was not statistically significantly at four months post-irradiation and beyond (FIG. 9E, 9F, 9K and 9M). This hyperintense signal suggested the presence of edema at this phase of injury, one month and two months postirradiation.

[0156] Additionally, a hypointense T2W MRI signal was detected in the irradiated brain hemispheres of mice irradiated at age 8-weeks-old, at four months post-irradiation, and this signal increased over time (FIG. 9E, 9F, 9K and 9M). Quantitative pixel analysis of the hypointense T2W MRI signal ratio of the irradiated brain hemispheres compared to the nonirradiated brain hemispheres revealed statistically significant ratios (P < 0.05, n =3) at four months post-irradiation (1.96 ±1.11) and beyond (FIG. 9E, 9F, 9K and 9M). This hypointense T2W MRI signal suggested the presence of hemorrhage and necrosis at the later phase of injury, four months post-irradiation and beyond.

[0157] Collectively, two main T2W MRI biomarkers were identified (FIG. 9E, 9F, and FIG. 12): A hyperintense T2W MRI biomarker that occurred at the mid-phase of injury, one month and two months post-irradiation, which was indicative of edema; and a hypointense T2W MRI signal that occurred at the later phase of injury, four months post-irradiation and beyond, which was indicative of hemorrhage and necrosis.

[0158] Given the higher sensitivity of T2* -weighted (T2*W) MRI to detect hemorrhage and necrosis, it was further used to validate the hypointense signal detected on T2W MRI. Briefly, a hypointense T2*W MRI signal was detected in the irradiated brain hemispheres of mice irradiated at age 8-weeks-old, as early as two months post-irradiation and this signal increased over time (FIG. 9G, 9H, 9K and 9N). Quantitative pixel analysis of the hypointense T2*W MRI signal ratio of the irradiated brain hemispheres compared to the non-irradiated brain381623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO hemispheres revealed statistically significant differences (P < 0.05, n =3) at two months postirradiation (1.13 ±0.91) and beyond (FIG. 9G, 9H, 9K and 9N).

[0159] Furthermore, atrophy was detected with T2W MRI, in the irradiated brain hemispheres of mice irradiated at age 8-weeks-old, as early as two months post-irradiation and this phenomenon increased over time (FIG. 91, 9J and 9M). Quantitative size analysis of the irradiated brain hemispheres compared to the non-irradiated brain hemispheres on T2W MRIs revealed statistically significant decreases (P < 0.05, n =3) in the sizes of the irradiated brain hemispheres at two months post-irradiation and beyond (FIG. 91, 9J and 9M).

[0160] Correlating multi-parametric MRI biomarkers of RIBI to immunohistochemical (IHC) biomarkers of RIBI. Using Group 3 mice that were irradiated at 8-weeks of age and transcardially perfused at different time points over a 12-month post-irradiation period, we correlated the longitudinal MRI biomarkers of RIBI to immunohistochemical biomarkers of RIBI.

[0161] FIG. 8 shows Correlating longitudinal MRI biomarkers of RIBI to immunohistochemical biomarkers of RIBI. FIG. 8A) Quantification of the respective fluorescence signals and the contrast-enhanced T1W MRI signals in the irradiated brain hemispheres normalized to the non-irradiated brain hemispheres at different time points postirradiation (P < 0.05, n >3). Maximum neuroinflammation was detected one-month postirradiation and this corresponded with maximum BBB permeability detected one-month postirradiation with in vivo contrast-enhanced T1W MRI. FIG. 8B) Quantification of the respective fluorescence signals and the T2W MRI hyperintensity signals in the irradiated brain hemispheres normalized to the non-irradiated brain hemispheres at different time points postirradiation (P < 0.05, n >3). Maximum astrogliosis was detected two months post-irradiation and corresponded with the maximum in vivo T2W MRI hyperintensity signal also detected two-months post-irradiation. No statistically significant neuronal loss was detected within the study’s 12-month post-irradiation time frame. FIG. 8C) Quantification of the H&E signal and the T2W MRI hyperintensity signals in the irradiated brain hemispheres normalized to the nonirradiated brain hemispheres at different time points post-irradiation (P < 0.05, n >3). A tissue porosity (cell loss) trend similar to the in vivo T2W MRI hyperintensity signal trend was detected. FIG. 8D) Quantification of the Peris’ Prussian blue staining signals and the T2*W MRI hypointensity signals in the irradiated brain hemispheres normalized to the non-irradiated391623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO brain hemispheres at different time points post-irradiation (P < 0.05, n >3). An increase in Peris’ Prussian blue staining (iron deposition) was detected over time in the irradiated brain hemispheres and this correlated with an increase in the in vivo T2*W MRI hypointensity signals detected in the irradiated brain hemispheres over time.

[0162] Neuroinflammation was detected in these mouse brain samples using cluster of differentiation 68 (CD68) and ionized calcium binding adaptor molecule 1 (IBA1) immunohistochemistry. CD68 was used to detect infiltrating macrophages, while IBA1 was used to detect microglial activation. Fluorescence microscopy images of brain tissue samples showed maximum CD68 (green) and IBA1 (red) fluorescence signals one-month postirradiation (FIG. 8). Quantitative pixel analysis of the respective CD68 (green) and IBA1 (red) fluorescence signal ratios of the irradiated brain hemispheres compared to the non-irradiated brain hemispheres revealed 23.60 ± 4.40 and 23.18 ± 6.72-fold increases respectively in the irradiated brain hemispheres, one-month post-irradiation (P = 0.0008, n =3 and P = 0.004, n =3, respectively). This maximum neuroinflammation signal detected one-month postirradiation corresponded with the maximum BBB permeability detected one-month postirradiation, with in vivo contrast-enhanced T1W MRI. The neuroinflammation signals then gradually decreased (P = 0.008, n = 3 and P = 0.03, n =3, respectively), over the following 12 months post-irradiation to ratios of 7.39 ± 3.55 and 7.68 ± 4.04, respectively (FIG. 8). Overall, this neuroinflammation trend detected with IHC and fluorescence microscopy correlated with the BBB permeability trend detected in vivo with contrast-enhanced T1 W MRI. This suggested that the contrast-enhanced T1W MRI signal could be used as a surrogate (or proxy) biomarker of neuroinflammation.

[0163] Next, astrogliosis and neuronal loss were evaluated with glial fibrillary acidic protein (GFAP) and neuronal nuclear antigen (NeuN) immunohistochemistry respectively, at different time points post-irradiation. Fluorescence microscopy images of brain tissue samples showed maximum GFAP (red) fluorescence signals in the irradiated brain hemispheres, two months post-irradiation (FIG. 8). Quantitative pixel analysis of the GFAP (red) fluorescence signal ratios of the irradiated brain hemispheres compared to the non-irradiated brain hemispheres revealed a 9.13 ± 4.21-fold increase in the irradiated brain hemispheres, two months postirradiation (P = 0.02, n =3). This maximum GFAP signal detected two months post-irradiation corresponded with the maximum in vivo T2W MRI hyperintensity signal detected two months401623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO post-irradiation. The GFAP signal then decreased over the following 12 months postirradiation to 2.68 ± 1.27 (FIG. 8). Overall, this GFAP (astrogliosis) trend detected with fluorescence microscopy correlated with the in vivo T2W MRI hyperintensity signal trend. This suggested that the T2W MRI hyperintensity signal could be used as a surrogate (or proxy) biomarker of astrogliosis.

[0164] No statistically significant change in the NeuN (green) fluorescence signal was detected in the irradiated brain hemispheres compare to the non-irradiated brain hemispheres, over the 12-month post-irradiation study period (FIG. 8).

[0165] Next, using hematoxylin and eosin (H&E) staining we evaluated general cell loss (tissue porosity) at different time points post-irradiation, in mice irradiated at 8-weeks-old and monitored for 12 months post-irradiation (FIG. 8). Statistically significant differences in the tissue porosities of the irradiated brain hemispheres compared to the non-irradiated brain hemispheres were detected from two weeks to 12 months post-irradiation (P < 0.05, n >3), using a one tailed t-test. Overall, a tissue porosity trend similar to the in vivo T2W MRI hyperintensity signal trend was detected (FIG. 8). This suggested that in addition to astrogliosis, tissue porosity could enable the accumulation of fluids in tissue (edema) and also contribute to the in vivo T2W MRI hyperintensity signal.

[0166] Finally, using Peris ’ Prussian blue staining we evaluated hemorrhage (iron deposition) at different time points post-irradiation, in mice irradiated at 8-weeks-old and monitored for 12 months post-irradiation (FIG. 8). Statistically significant differences in Peris’ Prussian blue staining of the irradiated brain hemispheres compared to the non-irradiated brain hemispheres were detected from one month to 12 months post-irradiation (P < 0.05, n >3), using a one tailed t-test. Overall, an increase in Peris’ Prussian blue staining (iron deposition) was detected over time in the irradiated brain hemispheres and this correlated with the increase in the hypointensity signal detected over time in the irradiated brain hemispheres, with in vivo T2*W MRI (FIG. 8). This suggested that hemorrhage (iron deposition) was responsible for the in vivo T2*W MRI hypointensity signal.

[0167] FIG. 9: Correlating longitudinal MRI and IHC biomarkers of RIBI to behavioral and survival outcomes. The freezing behavior of irradiated mice (Group 1) compared to nonirradiated mice (Group 2), in the fear-conditioning test at different time points post-irradiation411623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO(9A-E). This suggested the onset of mild and transient cognitive impairment in irradiated mice compared to non-irradiated mice, almost one month post-irradiation (P = 0.001; n =10). At 12- months post-irradiation, the direct statistically significant difference (P = 0.018, n =5) detected in the freezing behavior of the irradiated mice compared to the non-irradiated mice in the shortterm memory (STM) context FC test, indicated more severe cognitive impairment at this time point.

[0168] FIG. 10B) Weight changes in irradiated mice (Group 1) versus non-irradiated mice (Group 2), at different time points post-irradiation. Statistically significant weight loss was detected in the irradiated mice compared to the non-irradiated mice, from the 7-months postirradiation time point, onwards (P < 0.05; n= 10). FIG. 10C) Survival curves of irradiated mice (Group 1) versus non-irradiated mice (Group 2) at different time points post-irradiation. The first death of an irradiated mouse was observed eight months post-irradiation and this increased until the end of the study, 12 months post-irradiation. A statistically significant difference was detected in the survival of the irradiated mice compared to the non-irradiated mice 12 months post-irradiation (P = 0.002, n = 10).

[0169] Correlating multi-parametric MRI biomarkers and immunohistochemical biomarkers of RIBI to behavioral outcomes. To evaluate the correlation between the degree of BBB permeability and neuroinflammation on cognitive impairment, fear-conditioning tests were performed at different time points post-irradiation (FIG. 9). Our results showed a statistically significant difference (P = 0.002, n = 10) in the freezing behavior of the irradiated mice (Group 1) compared to the non-irradiated mice (Group 2) during the habituation phase of the fearconditioning test, three weeks (almost one-month) post-irradiation. By comparing the freezing behaviors of the respective mouse groups during the habituation phase versus the short-term memory (STM) context phase of the fear-conditioning test, a statistically significant difference (P = 0.001, n = 10) was detected in the non-irradiated (Group 2) mice (as was expected), but not in the irradiated (Group 1) mice. This indirect detection suggested the onset of mild cognitive impairment in the irradiated mice compared to the non-irradiated mice, almost one- month post-irradiation, when maximum BBB permeability and maximum neuroinflammation were also detected.

[0170] Interestingly, no statistically significant differences in the freezing behaviors of the irradiated mice compared to the non-irradiated mice were detected in the fear-conditioning test,421623347761.1PATENT ATTORNEY DOCKET NO. JHU4770-1WO two months to 10 months post-irradiation, as the BBB permeability and neuroinflammation decreased. This suggested that the early cognitive impairment detected almost one-month postirradiation, which coincided with the maximum BBB permeability, was transient in nature. This also suggested the possibility of using the in vivo contrast-enhanced T1W MRI biomarker of RIB I to noninvasively predict the onset of transient cognitive impairment in RIBI.

[0171] Finally, a statistically significant difference (P = 0.028, n = 5) in the freezing behavior of the irradiated mice compared to the non-irradiated mice was detected directly in the shortterm memory (STM) context phase of the fear-conditioning test, 12 months post-irradiation (FIG. 4L) This direct detection suggested the onset of severe cognitive impairment. At this later time point, despite a decrease in the permeability of the BBB, an increase in infiltrative CD68-positive macrophages was detected. This infiltration of CD68-positive macrophages in the irradiated brain hemisphere could be due to the increase in hemorrhage and iron deposition in the irradiated brain hemisphere detected in vivo, with T2W MRI and T2*W MRI at later time points post-irradiation. Taken together, this severe cognitive impairment could result from a combination of factors including: iron deposition, neuroinflammation, and atrophy of the irradiated brain hemisphere.

[0172] Correlating multi-parametric MRI biomarkers and immunohistochemical biomarkers of RIBI to overall RIBI outcomes. The in vivo detection of hemorrhage (hypointensity) in the irradiated brain hemispheres of Group 1 mice, 6-months post-irradiation with T2W MRI and T2*W MRI, preceded the onset of severe weight loss, severe cognitive impairment and ultimately mouse deaths, after irradiation. Statistically significant weight loss was detected in the irradiated mice (Group 1) compared to the non-irradiated mice (Group 2), from the seven months post-irradiation time point to the end of the study at 12 months post-irradiation (P < 0.05; n= 10). The first death of an irradiated mouse was observed eight months post-irradiation and this number increased until the end of the study, 12 months post-irradiation. A statistically significant difference was detected in the survival of the non-irradiated mice compared to the irradiated mice, 12 months post-irradiation (P = 0.002). Collectively, this suggested the possibility of using the in vivo hypointense T2W and T2*W MRI biomarkers of late injury as noninvasive predictive biomarkers of poor overall RIBI outcomes.

[0173] Discussion431623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO

[0174] In this study, we used conventional multi-parametric MRI to identify potential noninvasive MRI biomarkers of RIBI, that could be used to noninvasively detect the early onset of RIBI and also to longitudinally monitor RIBI progression.

[0175] Overall, our results showed that contrast-enhanced T1W MRI was the most sensitive of the MRI pulse sequences evaluated, for detecting the onset of RIBI via the detection of BBB disruption at the early stages of injury, in mice irradiated at any age. Contrast-enhanced T1W MRI also detected BBB disruption at an earlier time point (one week earlier) in mice that were irradiated at a younger age (5-weeks-old) compared to mice that were irradiated at an older age (8-weeks-old). This is approximately the human equivalent of contrast-enhanced T1W MRI detection of RIBI, approximately 5.5 years earlier in humans irradiated as early adolescents (11-14-years-old) compared to humans irradiated as middle to late adolescents (15-21-years- old). This finding is in agreement with previous reports which show that RIBI is detected earlier in patients treated with radiotherapy, at an earlier age.

[0176] Additionally, although contrast-enhanced T1W MRI detected BBB permeability throughout the 12-months post-irradiation study period, it was also able to longitudinally detect subtle changes in the degree of BBB permeability over the 12-months post-irradiation study period. These changes in the BBB permeability, coincided with changes in neuroinflammation (that were detected with fluorescence microscopy), and also with changes in transient cognitive impairment, (that were detected with the fear-conditioning test). This correlation between BBB permeability and cognitive impairment has also been suggested in several neurodegenerative disorders. Ultimately, this finding suggested the possibility of using the contrast-enhanced T1W MRI biomarker, as a surrogate (or proxy) biomarker of neuroinflammation and transient cognitive impairment in RIBI.

[0177] Our results also identified two types of T2W MRI biomarkers: 1) A hyperintense T2W MRI biomarker, that occurred at the middle phase of injury, one month and two months postirradiation and; 2) A hypointense T2W MRI biomarker, that occurred at the later phase of injury, four months post-irradiation and beyond. The in vivo hyperintense T2W MRI biomarker trend coincided with both the astrogliosis (GFAP) trend (that was detected with fluorescence microscopy), and the edema (tissue porosity) trend (that was detected with H&E staining and optical microscopy). This suggested that the T2W MRI hyperintense biomarker could be used as a surrogate (or proxy) biomarker of astrogliosis and edema.441623347761.1PATENT ATTORNEY DOCKET NO. JHU4770-1WO

[0178] The hypointense T2W MRI biomarker detected at the later phases of injury, was validated with the more hemorrhage-sensitive T2*W MRI, and its trend coincided with the hemorrhage (iron deposition) trend detected with Peris’ Prussian blue staining and optical microscopy. This suggested that the T2W MRI and T2*W MRI hypointense biomarkers could be used as a surrogate (or proxy) biomarker of hemorrhage. These in vivo hypointense T2W MRI and T2*W MRI biomarkers preceded the onset of severe weight loss, severe cognitive impairment and ultimately mouse deaths, after irradiation. This finding is in agreement with other reports which show a correlation between iron accumulation in the brain and severe cognitive impairment. Thus, this suggested the possibility of using the in vivo hypointense T2W and T2*W MRI biomarkers of late injury as noninvasive predictive biomarkers of poor overall RIBI outcomes.

[0179] In summary, these multi-parametric MRI biomarkers can be grouped into three main categories: 1) An early MRI biomarker of RIBI (the contrast-enhanced T1W MRI biomarker); 2) A middle MRI biomarker of RIBI (the T2W MRI hyperintense biomarker); and 3) Late MRI biomarkers of RIBI (the T2W and T2*W MRI hypointense biomarkers). The early MRI biomarker of RIBI (the contrast-enhanced T1W MRI biomarker) was best suited to detect the onset of RIBI, and also to detect subtle longitudinal changes in BBB permeability, which could be predictive of changes in neuroinflammation and transient cognitive impairment. The middle MRI biomarker of RIBI (the T2W MRI hyperintense biomarker), on the other hand, could be used to predict the status of astrogliosis and edema. Finally, the late MRI biomarkers of RIBI were best suited to detect hemorrhage in late injury and precedes weight loss, severe cognitive impairment, and poor survival. Consequently, these late biomarkers could be used as prognostic biomarker of poor overall RIBI outcomes.

[0180] Conclusion: In this study, we used conventional multi-parametric MRI to identify three categories of noninvasive MRI prognostic biomarkers of RIBI, in a preclinical mouse model of RIBI. These translatable MRI prognostic biomarkers could enable better detection; characterization, and management of RIBI in brain tumor survivors. These MRI prognostic biomarkers could also enable the longitudinal evaluation of potential RIBI prophylactic and therapeutic agents that are currently being developed. Ultimately, these multi-parametric MRI prognostic biomarkers could aid in the personalization of RIBI therapeutic regimens.

[0181] Materials and Methods451623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO

[0182] Animals. Female three-to-four-week-old immune competent BALB / cAnNTac (BALB / c) mice were obtained from Taconic Biosciences. All animal procedures were approved by the Johns Hopkins University Animal Care and Use Committee (JHU ACUC).

[0183] Experimental Design. Four groups of 8-week-old female BALB / c mice were used in this study to identify and characterize biomarkers of RIBI (FIG.1A) .Briefly, the right brain hemispheres of Group 1 mice were stereotactically irradiated with a single X-ray beam at a dose of 80 Gy and a dose rate of 1.7Gy / minute using a 3 mm x 3 mm collimator, and a small animal radiation research platform (SARRP), as previously described. [32-35] The mice were then monitored longitudinally over a 12-month post-irradiation period, with multi -parametric MRI and the fear-conditioning behavioral test of memory and learning. The weight changes and survival of the mice were also monitored. Group 1 mice were studied in comparison to non-irradiated control Group 2 mice. Group 3 mice were irradiated similarly to Group 1 mice, and sacrificed at different time points post-irradiation (0.03, 0.1, 0.5, 1, 2, 4, 6, 9 and 12 months, post-irradiation) and evaluated with immunohistochemistry (IHC) and histology, for IHC biomarkers of RIBI. Group 3 mice were studied in comparison to non-irradiated control Group 4 mice. All mice were sacrificed for humane reasons when a weight loss greater than 20% of the body weight was detected, in accordance with the JHU ACUC protocol.

[0184] To evaluate the effect of mouse age at the time of irradiation on the induction of RIBI, a younger cohort of mice (5-week-old mice) was added to Group 1 and also evaluated.

[0185] Irradiation. Mice were anesthetized with an isoflurane (2%) and oxygen mixture, and irradiated using a small animal radiation research platform (SARRP) as previously described. [32-35] Briefly, computed tomography (CT) images were acquired and a target location in the right brain hemisphere, with iso-center at the hippocampus, was chosen on the CT images using the following coordinates: Anterior posterior (AP) = +3 mm relative to the lambda; medial lateral (ML) = +0.5 mm relative to the mi dime; and dorsal ventral (DV) = -2 mm relative to the skull base. The mice, were then irradiated with a single X-ray beam at a dose of 80 Gy and a dose rate of 1.7Gy / min, using a 3 mm x 3 mm collimator.

[0186] Magnetic Resonance Imaging (MRI). Mice were anesthetized with isoflurane (2% in air), and all MRIs were acquired using an 11.7T Bruker Biospec horizontal bore scanner,461623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO equipped with a 23 mm Bruker mouse head volume radiofrequency coil. The Paravision 6.1.0 software was used for all image acquisitions.

[0187] All in vivo contrast-enhanced Tl-weighted images were acquired following the bolus intravenous administration of 200 pL of a 0.25 M of Prohance® solution, diluted in 0.01M phosphate buffered saline (PBS). Tl-weighted images were acquired using a spin echo pulse sequence with the following acquisition parameters. Sequence = rapid acquisition with refocused echoes (RARE); echo time = 5.5 ms; effective echo time = 5.5 ms; RARE factor = 4; repetition time = 557 ms; number of averages = 3; field of view = 18 x 18 mm; matrix size = 256 x 256 pixels; and slice thickness = 0.5 mm. Final image analyses were performed with the NIH ImageJ software, using bright pixel analyses, previously reported. [22, 23]

[0188] All in vivo anatomical T2-weighted images were acquired using a spin echo pulse sequence with the following acquisitioning parameters: Sequence = rapid acquisition with refocused echoes (RARE); echo time = 6.1 ms; effective echo time = 18.3 ms; RARE factor = 8; repetition time = 1500 ms; number of averages = 2; field of view = 18 x 18 mm; matrix size = 256 x 256 pixels; and slice thickness = 0.5 mm. Final image analyses were performed with the NIH ImageJ software. T2W MRI hyperintensity signal quantification was done using bright pixel analyses, as previously reported. [22, 23] T2W MRI hypointensity signal quantification was done on the other hand, using dark pixel analyses, as previously reported. [36-38]

[0189] All in vivo T2*-weighted images were acquired using a gradient echo pulse sequence. Sequence: multiple gradient echo; echo time = 3 ms; echo spacing = 3.5 ms; number of echoes = 7; effective echo times (EET) =3, 6.5, 10, 13.5, 17, 20.5, 24 ms; repetition times = 800 ms; number of averages = 4; number of repetitions = 1 ; field of view (FOV) = 18 x 18 mm; matrix size (MS) = 128 x 128 pixels; and slice thickness = 0.5 mm. Final image analyses were performed on the images with EETs of 24 ms, using the NIH ImageJ software and black pixel analyses, as previously reported. [36-38]

[0190] Fear-conditioning Behavioral Tests of Memory and Learning. Fear conditioning tests were conducted at the following time points: three weeks post-irradiation, two months postirradiation, four months post-irradiation, six months post-irradiation, nine months postirradiation and 12 months post-irradiation respectively, as previously reported, and matched to the closest time point images. [22, 23] Fear conditioning tests were conducted in Med471623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WOAssociates fear conditioning chambers (Med Associates, Inc., Fairfax, VT, USA) and consisted of five sessions over two consecutive days. Habituation, training, and one-hour or short-term memory (STM) context test sessions were conducted on day one, while 24-hour or long-term memory (LTM) context and 24-hour or LTM cue test sessions were conducted on day two. Briefly, mice were initially placed in the conditioning chambers (habituation) for five minutes and then returned to their cages. One hour following habituation, the mice were returned to the conditioning chambers where a tone (2000 Hz) was played three minutes post placement for 30s. This was immediately followed by a foot-shock using scrambled alternating currents delivered through the grid floor by a constant current shock source (5 mA) for two seconds. The mice were returned to their cages after a total of five minutes. One hour following the training session, the short-term contextual fear memory of the mice was assessed. During this session, the mice were returned to the conditioning chambers for five minutes during which neither tones nor shocks were administered. This was repeated, 24 hours following the training session, to assess the long-term contextual fear memory of the mice. One hour following this, the cued fear memory was assessed by re-exposing the mice to the tone (2000 Hz, for 30 seconds), three minutes post-placement in a new context and in the absence of a foot shock. The freezing behaviors in each session was automatically scored using Med Associates, Inc. Video Freeze Software (Med Associates, Inc., Fairfax, VT, USA). Data from the first minute of each session was compared as previously reported. [22, 23]

[0191] Histology and Immunohistochemistry. Mice were transcardially perfused with 30 mL of heparinized PBS (1% heparin in PBS), followed by 30 mL of a 4% paraformaldehyde (PFA) solution (4% PFA in PBS), at a flow rate of lO mL / min. The mouse skulls were stored overnight at 4 °C in 4% PFA, then at 4 °C in PBS for 48 hours. The brains were next extracted from the skulls, paraffin-embedded, sectioned into 4 pm slices, and stained for immunohistochemistry. For DNA double strand breakage, a rabbit anti-gamma H2AX primary antibody (1: 200, ab243906, Abeam) was used followed by an Alexa Flour® 488 goat anti-rabbit secondary antibody (1:200, abl 50077, Abeam). For neuroinflammation detection, a rabbit anti-CD68 (1:100, ab283654, Abeam) and a rat anti-IBAl (1:100, ab283346, Abeam) primary antibody respectively were used followed by an Alexa Flour® 488 goat anti-rabbit (1:200, abl50077, Abeam) and an Alexa Flour® 647 goat anti-rat (1:1000, abl 50077, Abeam) secondary antibody respectively. For astrogliosis and neuron loss, a rat anti-GFAP (1:100, ab279291, Abeam) and rabbit Anti NeuN (1:100, abl77487, Abeam) primary antibody respectively were481623347761.1PATENT ATTORNEY DOCKET NO. JHU4770-1WO used, followed by an Alexa Flour® 647 goat anti -rat (1:1000, ab!50077, Abeam) and an Alexa Flour® 488 goat anti-rabbit (1:200, ab!50077, Abeam) secondary antibody respectively. All slides were counter stained with DAPI in fluoroshield mounting media (abl04139, Abeam). Image acquisitioning was performed using a Zeiss Axio Scan.Z.l Slide Scanner. Adjacent tissue sections were also stained with Peris’ Prussian blue (PPB) and hematoxylin and eosin (H&E) for histology as previously described. [39, 40] Image acquisitioning was performed using a Zeiss upright brightfield microscope.

[0192] Statistical Analyses. All data points were presented as the mean ± standard deviation of at least three independent experiments. Statistical comparisons were made using paired two- tailed student t-tests, except when otherwise stated. The results were considered statistically significant at P < 0.05.EXAMPLE 2Developing magnetic resonance (MRI) imaging biomarkers of radiotherapy-induced brain injury (RIB I) in a preclinical mouse model of RIBI

[0193] Introduction: Radiotherapy-induced brain injury (RIBI) and neurocognitive decline are chronic side effects that occurs in up to 90% of brain tumor survivors, treated with radiotherapy. Current methods to detect RIBI and neurocognitive decline rely on neuropsychological evaluations. However, neuropsychological evaluations could greatly benefit from the development of robust noninvasive imaging biomarkers, that could be used to detect subtle changes indicative of the early onset of RIBI. These imaging biomarkers could also aid in patient stratification; the longitudinal monitoring of RIBI progression; and responses to RIBI therapies.

[0194] Objective: Here, we evaluated the feasibility of using multi-parametric magnetic resonance imaging (mp-MRI) to identify potential imaging biomarker of RIBI, that could be used to noninvasively detect the early onset of RIBI and also to longitudinally monitor RIBI progression in a preclinical mouse model of RIBI.

[0195] Hypothesis: Although the pathogenesis of RIBI has not yet been fully elucidated, the vascular hypothesis of RIBI postulates that it is characterized by cerebrovascular damage and neuroinflammation. These lead to ischemia, neurodegeneration, and neurocognitive decline.491623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO

[0196] Method: Four groups of mice were used in this study (A). Group 1 mice were stereotactically irradiated and used to identify noninvasive mp-MRI and behavioral biomarkers of RIBI; while non- irradiated Group 2 mice were used as mp-MRI and behavioral controls. Group 3 mice were also stereotactically irradiated and were used to identify immunohistochemical and histological biomarkers of RIBI; while non-irradiated Group 4 mice were used as histological controls.

[0197] Briefly, in Group 1, the right brain hemispheres of ten 8-week old female balb / c mice were stereotactically irradiated at a dose of 80 Gy and a dose rate of 1.7Gy / min, using a 3 mm x 3 mm collimator. The mice were then monitored with mp-MRI and behavioral tests at 0.5, 1, 2, 4, 6, 9 and 12-months post-irradiation. Mp-MRI included: Dynamic contrast enhanced Tl- weighted MRI, following the bolus administration of 200 pL of a 0.25 M contrast agent (Prohance®) solution, to evaluate the permeability of the blood brain barrier (BBB); T2- weighted MRI was used to monitor anatomical changes such as edema, necrosis, hemorrhage and atrophy; and T2*-weighted MRI was used to validate the detection of necrosis and hemorrhage, given its higher sensitivity. Behavioral tests included: fear-conditioning and novel object recognition tests. The weights and survival of the mice were also monitored. Immunohistochemical biomarkers of neuroinflammation (CD68 and IBA1) were next studied in Group 3 mice.

[0198] Results: BBB permeability was detected with contrast-enhanced Tl-weighted MRI in Group 1 mice at 0.5 months post-IR, and this indicated the onset of injury. This BBB permeability peaked at 1 -month post-IR, then decreased over time (B & C). The onset of neurocognitive decline in Group 1 was also detected at 0.5 months post-IR, with the fearconditioning behavioral test (D). These findings also coincided with significant neuroinflammation (CD68 and IBA1 staining) detected around this early time point, that then decreased over time (E). Edema, necrosis, hemorrhage, and atrophy, were also detected in Group 1 with T2-weighted MRI, at later time points. Whereas edema, was detected as a hyperintense signal (C & F); necrosis and hemorrhage were detected as a hypointense signal (C & F); and validated with the more sensitive T2* MRI, (C & G). Significant atrophy was also detected over time (H). These MRI biomarkers of late injury preceded significant weight loss (I) and decreased survival (J) in the Group 1 mice, compared to Group 2 controls.501623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO

[0199] Conclusion: mp-MRI more robustly detects changes associated with RIBI compared to behavioral tests. Noninvasive MRI biomarkers of RIBI could play a pivotal role in the management of RIBI in brain tumor survivors.EXAMPLE 3Responsive drug nanocarriers for image-guided treatment of radiotherapy-induced brain injuries

[0200] Description of Technology: We have developed two oxidative stress-responsive nanotheranostic agents, that can be used to reduce oxidative stress and neuroinflammation which is associated with radiotherapy-induced brain injury and cognitive decline in brain tumor survivors.

[0201] We have successfully synthesized two amphiphilic block copolymers (P2a & P2b) with fixed a fixed number of CEST-MRI sensitive quaternary amine units and a variation in the pH-responsive monomeric units via the reversible addition fragmentation chain-transfer (RAFT) polymerization technique (FIG. 1A).

[0202] JH and12C NMR spectroscopy were used to characterize the polymers and to determine the number of monomer unit for each polymer segment (FIG. 1B-1D).

[0203] These polymers readily form nanoparticular micelles in aqueous medium as demonstrated by their critical aggregation concentrations (CACs) of 0.094 and 0.085 mg / mL for P2a and P2b, respectively (FIG. 11A), determined with fluorescence spectroscopy. The size, shape, and stability of the nanoparticles was determined by dynamic light-scattering spectroscopy (DLS), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). These spherical polymeric nanoparticles were approximately 160 nm in diameter (FIG. 11B-11E), and are very stable even after a 100-fold dilution (FIG. 11F).

[0204] The activation of the nanoparticles under oxidative stress (pH 6.2 and in the presence of hydrogen peroxide) compared to physiological condition (pH 7.4) and was determined by studying the release of an encapsulated fluorescent dye (Nile red) using fluorescence spectroscopy (FIG. 12B, 12C).511623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO

[0205] To evaluate the feasibility of detecting these nanoparticles in vivo, a 700 nm nearinfrared (NIR) fluorescence dye was conjugation to the respective polymers and confirmed via UV-Visible spectroscopy (FIG. 13). A subsequent fluorescence-based nanoparticle activation study using an encapsulated 800 nm near-infrared fluorescence dye was then performed in agarose gel for the further confirmation about the responsive release profile of the payload from the polymer NPs.

[0206] Conclusion: Our newly synthesized amphiphilic polymers readily form nanoparticles in aqueous medium and can be used to encapsulate a variety of therapeutic agents. These nanoparticles are responsive to pH changes and changes in reactive oxygen species (ROS). Thus, these nanoparticles can be used to control oxidative stress and neuroinflammation associated with RIBI, especially when used in combination with encapsulated neuroprotective agent for an enhanced therapeutic effect.

[0207] See also FIG. 22A-22D: 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 pL of a 0.25 M contrast agent solution (Prohance®). Grayscale Z-projection T1 W MRIs of four 0.5 mm coronal (A) and axial (B) slices. Grayscale T1W MRIs of a single 0.5 mm coronal (C) and axial (D) slices. This shows reduced disruption of the blood brain barrier (BBB) in mice treated with P2b (red arrow).EXAMPLE 4Developing neuroprotective pH / redox- activable polymeric nanotheranostic agents for the multimodal image-guided mitigation of radiotherapy-induced brain injury in a preclinical mouse model

[0208] Keywords: Stimuli-responsive polymer nanoparticles, theranostic nanoparticles, NIR imaging, multimodal probes, neuroinflammation, radiation injury, neuroimaging.

[0209] 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. Consequently, as the overall survival rates of pediatric brain tumor patients improve, there is a growing need for RIBI therapies.521623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO

[0210] 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.

[0211] 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.

[0212] Method: Two pH / redox-responsive amphiphilic block copolymers were synthesized and characterized by NMR spectroscopy. The respective critical aggregation concentrations needed to form nanoparticles in aqueous milieu were determined with fluorescence spectroscopy. The hydrodynamic diameters, ^-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).

[0213] 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. The mice were next treated with either an 800 nm (red) fluorescently-labelled nanoparticle or phosphate buffered saline (PBS), two weeks postirradiation 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.

[0214] 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).531623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WOBoth polymers readily formed spherical nanoparticular micelles in aqueous milieu (C-F), and had hydrodynamic diameters and ^-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).

[0215] 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, Zl). Immunohistochemistry also showed significantly reduced microglial activation (IBA-1) 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).

[0216] Conclusion: This shows that pH / REDOX-responsive nanoparticles with a high number of ROS scavengers can mitigate RIBI-associated neuroinflammation and improve RIBI outcomes.

[0217] FIG. 15A) Schematic of the degradation of fluorescent R-phycoerythrin (RPE) protein under oxidative stress and its protection by pH / redox-activable nanoparticle P2a or P2b. FIG. 15C) Kinetics of fluorescent RPE protein (2.13 pg) degradation under oxidative stress (10 mM H2O2 at pH 7.4) and its protection in the presence of 1 pg of nanoparticles P2a and P2b, respectively (p < 0.05). FIG. 15D) Change in the fluorescence signal intensity of fluorescent RPE protein 60 minutes after exposure to oxidative stress (10 mM H2O2 at pH 7.4) and its protection in the presence of 1 pg of nanoparticles P2a and P2b, respectively (p < 0.05). FIG. 15E) Change in the fluorescence intensity signal of fluorescent RPE protein, 60 minutes after exposure to oxidative stress (10 mM H2O2 at pH 7.4) in the presence of varying amounts541623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO of nanoparticles P2a and P2b, respectively. RPE protection from degradation was obtained with as little as 1 pg of NPs.

[0218] FIG. 16D) 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). FIG. 16E) Cell viability of HUVECs after incubation with up to 20 pg (25 pg / pL) for 24 h, showed no toxicity from either nanoparticle. FIG. 17) Schematic of in vivo experimental design used to evaluate the efficacy of nanoparticles P2a and P2b respectively. FIG. 19) Quantification of the delivery of fluorescently-labelled nanoparticles P2a and P2b to the brain in a preclinical mouse model of RIBI (p < 0.05).

[0219] FIG. 23A) In vivo contrast-enhanced TiW MRI of representative mice from the respective groups 1.5 months after nanoparticle administration. The TiW MRIs were acquired 15 mins after the intravenous administration of 200 pL of a 0.25 M contrast agent solution (Prohance®). This shows reduced disruption of the blood brain barrier (BBB) in mice treated with P2b. FIG. 23C) Quantification of the MRI signals in the three groups 1.5 months after nanoparticle administration (p < 0.05). FIG. 23B) 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. FIG. 24) 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).EXAMPLE 5Improving transplanted stem cell survival and neurorepair using the nanoparticle P2b

[0220] In vivo evaluation of prolong transplanted stem cell survival and improved neurorepair. We have also started evaluating the feasibility of improving neurorepair by transplanted stem cells, using the P2b nanotheranostic agent developed, which has a high number of phenyl-boronic acid pinacol ester (BAPE) moieties to scavenge reactive oxygen species (ROS), and sustainably reduce oxidative stress and neuroinflammation in our RIBI model in immunodeficient mice.551623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO

[0221] Method: Briefly, the right brain hemispheres of 8-weeks-old female severe combined immuno-deficient (SCID) mice were stereotactically irradiated at 80 Gy (dose rate of 1.7 Gy / min). Mes-enchymal stem cells (one million cells) where then stereotactically administered to the irradiated hemispheres of the mice, one-week post-irradiation. Starting at two weeks post-irradiation, Ipg of the pH / REDOX-responsive nanoparticle P2b (with a high number of ROS scavenging moie-ties), was intravenously administered bi-weekly to the mice for six months. The weights of the mice and their survival was also monitored. The mice were then monitored with multi -parametric MRI. This group of mice (Group 1) that were stereotactically irradiated (80 Gy), and received both stem cell transplantation (one million) and P2b nanoparticles (biweekly) was studied in comparison to six other groups of mice:

[0222] Group 2: Mice stereotactically irradiated (80 Gy) + stem cells (one million)

[0223] Group 3: Mice stereotactically irradiated (80 Gy) + P2b nanoparticle (Ipg biweekly, for six months)

[0224] Group 4: Mice stereotactically irradiated (80 Gy) + received sham surgery

[0225] Group 5: Mice stereotactically irradiated (80 Gy) only

[0226] Group 6: Mice that received sham surgery only

[0227] Group 7: Control naive mice (with no irradiation or surgery)

[0228] Results: Weight measurements showed no significant differences between the groups up to seven months post irradiation. This suggested that the biweekly administration of the P2b nano-particle was not toxic to the mice. Additionally, Group 1 mice that received the mesenchymal stem cell transplant and P2b nanoparticles biweekly after irradiation, showed the best survival trend of the irradiated mouse groups (FIG. 25A: Weight changes of mice used in the study over a period of 7 months post-irradiation. FIG. 25B) survival curves of mice used in the study over a period of 6 months post-irradiation.).

[0229] Conclusion: This data shows that pH / REDOX-responsive nanoparticles with a high number of phenylboronic acid pinacol ester (BAPE) moieties to scavenge reactive oxygen species (ROS), can also improve neurorepair and RIBI outcomes when used in combination with transplanted stem cells.561623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WOEXAMPLE 6Developing Positron Emission Tomography (PET) imaging biomarkers of radiotherapy-induced brain injury (RIB I) in a preclinical mouse model of RIBI

[0230] We have successfully used the radiotracer [(11)C]DPA-713 to noninvasively image neuroinflammation associated with RIBI. [(11)C] DPA-713 detection of the upregulation of activated microglia was validated with immunohistochemistry.

[0231] Future Direction: This longitudinal study with the [(11)C] DPA-713 PET tracer is still going-on. We are also concurrently evaluating the efficacy of another PET tracer [(11)C] CPPC to noninvasively detect neuroinflammation. We intend to use these PET biomarkers of RIBI together with the multi-parametric MRI biomarkers of RIBI previously described, to longitudinally monitor responses to RIBI therapies (including as polymeric nanoparticles) in the next part of this project.EXAMPLE 7Evaluating noninvasive imaging biomarkers of neuroinflammation in a preclinical mouse model of radiotherapy-induced brain injury: A comparison of 11C-DPA-713 PET, 11C-CPPC PET and contrast-enhanced Tl-weighted magnetic resonance imaging

[0232] Key words: Radiation injury; brain injury ; neuromflammation; predictive biomarkers; imaging biomarkers; MRI biomarkers; and PET biomarkers.

[0233] Introduction: Radiotherapy-induced brain injury (RIBI) is a chronic side effect that affects up to 90% of pediatric brain tumor survivors who were treated with radiotherapy.! -4 Neuromflammation has been identified as is a key contributor to RIB1.4 Thus, there is a need for imaging strategies capable of noninvasively monitoring the status of neuroinflammation in pediatric brain tumor survivors.

[0234] Objective: Here, we evaluated the feasibility of using contrast-enhanced T'l -weighted magnetic resonance imaging (T1W MRI) as a proxy imaging biomarker of neuroinflammation. This could provide a means to noninvasively monitor RIBI progression in pediatric brain tumor survivors without the use of ionizing radiation, which is involved in positron emission tomography (PET), and which could be detrimental to pediatric patients.571623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO

[0235] Hypothesis: We hypothesized that the direct detection of neuroinflammation during RTBI using the 11 C-DPA and 11C-CPPC radiotracers respectively for PET will be comparable to the indirect detection of neuroinflammation via the detection of blood-brain barrier (BBB) permeability, using contrast-enhanced T1W MRI.

[0236] Method: Four groups of 8-week-old female BALB / c mice were stereotactically irradiated with a single X-ray beam at a dose of 80 Gy and a dose rate of 1.7 Gy / 'min (A), as previously described.5 -7 The mice were then monitored longitudinally for neuroinflammation over a period of 9 months post-irradiation (IR), using 11C-DPA PET (Group 1); 11C-CPPC PET (Group 2); contrast-enhanced T1W MRI (Group 3) and TSPO, IBA1, and CD68 immunohistochemistry (Group 4). Mice from each group were imaged at the following time points: 1) Pre-IR; 2) one-month post-IR; 3) two months post-IR; 4) six months post-IR; and 5) nine months post-IR.

[0237] The 11C-DPA and 11C-CPPC radiotracers were synthesized and obtained with radioactive purity greater than 95%, as previously reported.7-9 A preclinical 7T Bruker Biospec 70 / 30 horizontal bore PET-MRT scanner was used to acquire all dynamic PET scans. At each time point, Group 1 and Group 2 anesthetized mice were intravenously administered 150 pCi (5.55 MBq) of the respective radiotracers, via catheters, one minute after the start of the dynamic PET scans. Blocking studies were also conducted at two months post-IR, using unlabeled DPA and CPPC to evaluate the specificity of the respective tracers. In Group 3 mice, contrast-enhanced T1W MRIs were acquired after the bolus intravenous administration of 200 pl. of a 0.25 M (2 mmol / Kg) gadoteridol (ProHance®) solution, prepared in 0.01M phosphate buffered saline (PBS), as previously reported.? In Group 4, the mice were transcardially perfused at the different time points, paraffin embedded, sectioned and stained for neuroinflammation using TSPO, IBA1, and CD68 respectively .

[0238] Results: Maximum neuroinflammation was detected using both 11C-DPA PET and T1C-CPPC PET at one-month post-IR (P < 0.0001, n - 3). The 11C-DPA and 1IC-CPPC PET signals then respectively decreased at two-months post-IR (P < 0.0001, n = 3). These PET signals continued to decrease through six months post-IR, and remained low through nine- months post-IR (B-D). Blocking studies conducted at two months post-IR, using unlabeled DPA and CPPC confirmed the specificity of the respective tracers. These neuroinflammation trends detected using 11 C-DPA PET and 11 C-CPPC PET respectively correlated with the BBB581623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO permeability trend that was detected using contrast-enhanced T1W MRI (E-H). Furthermore, the neuroinflammation trend detected using PET and contrast-enhanced T1W MRI respectively, correlated with the neuroinflammation trend that was detected using TSPO, 1BA1 , and CD68 immunohistochemistry (I-K).

[0239] Conclusion: These findings suggest that contrast-enhanced T1W MRI could be used as a proxy imaging biomarker of neuroinflammation to noninvasive monitor RIBI progression in pediatric brain tumor survivors, without the use of ionizing radiation.EXAMPLE 8Imaging the delivery and efficacy of neuroprotective nanotheranostic agents by chemical exchange saturation transfer (CEST) MRI

[0240] FIG. 26: pH-dependent and power-dependent CEST MRI detection of nanotheranostic agent P2a at concentrations of 10 pg / pL. CEST MRI Z-spectra of 10 pg / pL of nanoparticle P2a solutions acquired al different powers (FIGg. 26A to E); CEST MRI magnetization transfer asymmetry (MTRASYM) ratios of 10 pg / pL of nanoparticle P2a solutions acquired at different powers (FIG. 26F to J).

[0241] FIG. 27: pH-dependent and power-dependent CEST MRI detection of nanotheranostic agent P2b at concentrations of 10 pg / pL. CEST MRI Z-spectra of 10 pg / pL of nanoparticle P2b solutions acquired at different powers (FIG. 27 A to E); CEST MRI magnetization transfer asymmetry (MTRASYM) ratios of 10 pg / pL of nanoparticle P2a solutions acquired at different powers (FIG. 27F to J).

[0242] FIG. 28: Summary of the pH-dependent and power-dependent CEST MRI detection of nanotheranostic agent P2a and P2b at concentrations of 10 pg / pL. FIG. 28A) Powerdependent CEST MRIs of nanoparticle p2a, at different pH values. FIG. 28B) Powerdependent CEST MRIs of nanoparticle p2b at different pH values. FIG. 28C) pH-dependent CEST MRIs of nanoparticle p2a, at different powers. FIG. 2SD) pH-dependem CEST MRIs of nanoparticle p2b, at different powers.

[0243] FIG. 29: Concentration -depen dent and power-dependent CEST MRIs detection of nanotheranostic agent P2a. CEST MRI Z-spectra of different concentrations of P2a solutions591623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO acquired at different powers (FIG. 29 A to E); CEST MRI magnetization transfer asymmetry (MTRASYM) ratios of different concentrations of P2a solutions acquired at different powers (FIG. 29F to J).

[0244] FIG. 30: Summary of the concentration-dependent CEST MRI detection of nanotheranostic agent P2a. FIG. 308) Power-dependence of the P2a CEST MRI signal at different concentrations. FIG. 30C) Concentration-dependence of the P2a CEST MRI signal of 10 mg / niL P2a phantoms at different MRI acquisitioning powers.

[0245] IGg. 31: Complete blood counts in mice that received the 10 pg / pL of the nanoparticies P2a or P2b compared to mice that received phosphate buffered serum (PBS). No statistically significant differences were detected between the mouse group. This suggests that it is safe to use these higher nanoparticle doses in vivo.

[0246] FIG. 32. Comprehensive serum chemistry in mice that received the 10 ug / uL of either nanoparticies P2a or P2b compared to mice that received phosphate buffered serum (PBS). No statistically significant differences were detected between the mouse group. This suggests that it is safe to use these higher nanoparti cie doses in vivo.

[0247] Objective: Here, we evaluated the feasibility of noninvasively detecting our previously developed nanotheranosties agents P2a and P2b using CEST MRI in phantoms: 1) Before redox-activation at pH 7.4 and; 2) After redox-activation at and below pH 6.2.

[0248] Hypothesis: We hypothesized that whereas the detection of the nanotheranostic agents before redox-activation (at pH 7.4) could provide a means of noninvasively monitoring the delivery of the nanotheranostic agents in vivo, the detection of the nanotheranostic agents after redox-activation (at and below' pH 6.2) could provide a means of noninvasively monitoring the efficacy of the nanotheranostic agents in vivo. Ultimately, this could provide a means to noninvasively and longitudinally monitor the delivery and therapeutic efficacy of the nanotheranostic agents in the clinical setting for the image-guided treatment of RIBI.

[0249] Results: Effects of pH and CEST MRI acquisitioning power on CEST MRI signal. Both nanotheranostic agents P2a and P2b showed pH-dependent CEST MRI signals before and after redox-activation. In both nanoparticies, high CEST MRI signals were detected at acid pH values close to pH 4.5 (typically encountered in tissues under very high oxidative stress), and601623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO low CEST MRI signals were detected at pH values close to the physiological pH 7,4 (typically encountered in normal tissues). These findings support our premise of using these theranostic nanoparticies for the image-guided treatment of RIBI.

[0250] Effects of Nanoparticie Concentration on CEST MRI Detection. At the nanoparticie concentrations of 0.1 pg / pL that was used in the in vivo preclinical experiments, CEST MRI signals could not be detected from the nanotheranostic agents at any of the CEST MRI acquisitioning powers tested (1 pT to 5 pT). However, CEST MRI signals could be detected from the nanotheranostic agents at higher concentrations ranging from 1.0 rig / pL to 10.0 pg / pL (FIG. 29). The CEST MRI signals of the nanotheranostic agents were both concentrationdependent and power-dependent. This supports our premise of using these theranostic nanoparticies quantitatively, tn the image-guided treatment of RIBI.

[0251] Ultimately, this CEST MRI method could provide a means to noninvasivdy and longitudinally monitor the delivery' and therapeutic efficacy of the nanotheranostic agents in the clinical setting for the image-guided treatment of RIBI.

[0252] Nanoparticle Toxicology Study at 10 mg / mL. We also carried out a toxicology study (complete blood counts and comprehensive serum chemistry') in non-irradiated mice and showed that no systemic toxicity was detected in mice that received either 10.0 tig / iiL of nanoparticie P2a or P2b at two weeks post-administration, w-hen compared to the control mice that did not receive any of the nanoparticies but only phosphate buffered sahne (FIG. 31 and FIG, 32). 'Illis further validated the feasibility of using these nanotheranostic agents at high nanoparticie concentrations that can be detected by CEST MRI.

[0253] On-going studies: We are currently evaluating the feasibility of noninvasively imaging the delivery of nanoparticies P2a & P2b and tissue responses to the agents in our previously developed preclmical mouse model of RIBI,EXAMPLE 9Establishing a RIBI mouse model and developing imaging biomarkers of radiotherapy- induced brain injury (RIBI)611623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO

[0254] Multi -parametric magnetic resonance imaging (mp-MRI) biomarkers of RIBI. Previously, we identified multi-parametric magnetic resonance imaging (mp-MRI) biomarkers of RIBI.(l) We showed that contrast-enhanced Tl-weighted (T1W) MRI, using gadoteridol (PROHANCE®) as a contrast agent, was the most sensitive of the conventional mp-MRI pulse sequences evaluated in the study, for detecting the onset of RIBI via the detection of the bloodbrain barrier (BBB) permeability. We also showed that contrast-enhanced T1W MRI could be used to longitudinally detect subtle changes in the BBB permeability over a 12-month postirradiation period. Furthermore, we showed that the maximum BBB permeability detected using contrast-enhanced T1W MRI coincided with the maximum neuroinflammation detected using cluster of differentiation 68 (CD68) and ionized calcium binding adaptor molecule 1 (IBA1) immunohistochemistry'. Finally, we showed that the detection of this simultaneous maximum BBB permeability and maximum neuroinflammation detection also coincided with the detection of the onset of cognitive impairment in our preclinical model of RIBI. These findings suggested the possibility of using contrast-enhanced T1W MRI as a surrogate (or proxy) biomarker of neuroinflammation in RIBI. These findings were published in the journal Investigative Radiology.Developing positron emission tomography (PET) biomarkers of RJBI

[0255] Background: There are several positron emission tomography (PET)-based radioligands for imaging neuroinflammation, such as the 1 1 C-DPA radiotracer for imaging the 18-kDa translocator protein (TSPO), and the 11C-CPPC radiotracer for imaging the colonystimulating factor 1 receptor (CSF1R). Both of these receptors are upregulated during neuroinfl animation. While PET provides a means to detect subtle changes in vivo with high sensitivity, PET involves the use of ionizing radiation, which could be particularly' detrimental to pediatric patients, who face the risk of RIBI.

[0256] Hypothesis: Thus, based on our MRI biomarker findings above, we hy pothesized that the indirect detection of neuroinflammation via the detection of BBB permeability, using contrast-enhanced T1W MRI, will be as effective as the direct detection of neuroinflammation using 11 C-DPA PET and 11C-CPPC PET respectively, in our preclinical model of RIBI.

[0257] Ultimately, this method could provide a simple yet effective means of noninvasively probing the status of neuroinflammation in RIBI progression using conventional MRI, which621623347761.1PATENT ATTORNEY DOCKET NO. JHU4770-1WO is already used routinely in the clinical setting to monitor and manage brain tumor patients and survivors. Additionally, this method could provide a clinically translatable means of longitudinally monitoring the therapeutic efficacy of potential R1BI prophylactic and therapeutic agents, without exposure to ionizing radiation that is involved in PET.

[0258] Objective: Thus, to test our hypothesis, we expanded on our previous MRI findings, and earned out a comparative study to see if the indirect detection of neuroinflammation via the detection of BBB permeability’, using contrast- enhanced T1W MRI will be as effective as the detection of iieuroiiiflannnation using llC-DPA-713 PET and 11C-CPPC PET respectively, in our previously developed preclinical model of RIB I (FIG. 33).

[0259] Method: Four groups of 8-week-old female BALB / c mice were stereotactically irradiated with a single X-ray beam at a dose of 80 Gy and a dose rate of 1.7 Gy / min (A), as previously described. 'The mice were then monitored longitudinally for neuroinflammation over a period of 9 months post-irradiation (IR), using 11C-DPA PET (Group 1); I IC-CPPC PET (Group 2); contrast-enhanced T1W MRI (Group 3) and TSPO, IB Al, and CD68 immunohistochemistry’ (Group 4). Mice from each group were imaged at the following time points: 1) Pre-IR; 2) one-month post-IR: 3) two months post-IR: 4) six months post-IR: and 5) nine months post-IR.

[0260] The ”C-DPA and ”C-CPPC radiotracers were synthesized and obtained with radioactive purity greater than 95%. A preclinical 7T Broker Biospec 70 / 30 horizontal bore PET-MRI scanner was used to acquire ah dynamic PET scans. At each time point. Group 1 and Group 2 anesthetized mice were intravenously’ administered 150 pd (5.55 MBq) of the respective radiotracers, via catheters, one minute after the start of the dynamic PET scans. Blocking studies were also conducted at two months post-IR, using unlabeled DPA and CPPC to evaluate the specificity of the respective tracers. In Group 3 mice, contrast-enhanced T1W MRIs were acquired after the bolus intravenous administration of 200 ■if of a 0.25 M (2 mmol / Kg) gadoteridol (ProHance®) solution, prepared in 0.01M phosphate buffered saline (PBS), as previously reported. (1) In Group 4, the mice were transcardially perfused at the different time points, paraffin embedded, sectioned and stained for iieuroiiiflannnation using TSPO, IB Al, and CD68 respectively.631623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO

[0261] Results: Detection of neuroinflammation in RIBI using “C-DPA and “C-CPPC PET- MRI, Maximum neuroinfl animation was detected using both “C-DPA PET and “C-CPPC at one-month post-IR (P <• 0.0001, n = 3). The PET signals then decreased at two-months post- IR (P < 0.0001 , n::::3) and continued to decrease through six months post-IR, and then remained low through nine-months post-IR, respectively. FIG. 34 and FIG. 35 show data from the 1 1 C- DPA PET radiotracer.

[0262] FIG. 34: Detection of neuroinflammation in RIBI using “C-DPA PET-MRI. PET- MRIs of the radioactivity distribution of the “C-DPA tracer in the brain before, 2. 5, 10, 15 and 20 minutes after the bolus intravenous administration ofnC-DPA; This “C-DPA accumulation in the irradiated brain hemispheres of the mice at these early time points postirradiation was indicative of the presence of neuroinflammation. Quantification graphs of the ratios of the time-radioactivity kinetic curves of the irradiated (right) brain hemispheres normalized to the corresponding contralateral (left) brain hemispheres of the mice, after the bolus intravenous administration of “C-DPA: FIG. 34A) Before irradiation (non-irradiated control mice); FIG. 34B) one-month post-irradiation versus before irradiation (P = 0.0001, n ” 3); FIG. 34C) two-months post-irradiation versus before irradiation (P ------ 0.0260, n === 3); FIG. 34D) six-month post-irradiation versus before irradiation; and FIG. 34E) nine-months post-irradiation versus before irradiation. Statistically significant accumulation and retention of the “C-DPA radiotracer was detected in the irradiated brain hemispheres compared to the corresponding contralateral non-irradiated brain hemispheres of the mice one-month ( / ’::::0.0001, n:::3) and two-months (P::::0.026, n:::3) post-irradiation. This high “C-DPA accumulation and retention at these early time points post-irradiation were indicative of the presence of neuioinflammation in the irradiated brain hemispheres.

[0263] FIG. 35: Summary of the detection of neuroinflammation in RIBI using “C-DPA PET-MRI over a 9 months post-irradiation period. Representative PET-MRIs of the radioactivity distribution of the11C-DPA tracer in mouse brains over the 9 months postirradiation study period. Ihe PET-MRIs were acquired before, 10, 15 and 20 minutes after the bolus intravenous administration of1 fC-DPA: FIG. 35A) Quantification graphs of the ratios of the time-radioactivity kinetic curves of the irradiated (right) brain hemispheres normalized to the corresponding contralateral (left) brain hemispheres of the respective mice, over 30 minutes after the bolus intravenous administration of “C-DPA, at different time points before641623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO and after irradiation (P < 0.05). FIG. 35B) Radioactivity ratios of the irradiated (right) brain hemispheres normalized to the corresponding contralateral (left) brain hemispheres of the respective mice, 0, 10, 13, 15, 18, and 20 minutes after the bolus intravenous administration of ”C-DPA, at different time points before and after irradiation (P < 0.05).

[0264] nC-DPA andnC-CPPC PET-MRI blocking studies: Blocking studies conducted at two months post-IR, using unlabeled DPA confirmed the specificity of the l lC-DPA radiotracer (FIG. 36). A similar neuroinflammatioii trend was detected using ”C-CPPC PET (data not shown)

[0265] FIG. 36:1 fC-DPA PET-MRI blocking studies. Quantification graphs of the ratios of the time-radioactivity' kinetic curves of the irradiated (right) brain hemispheres normalized to the corresponding contralateral (left) brain hemispheres of the mice, after the bolus intravenous administration ofnC-DPA: FIG. 36A) Before irradiation (non-irradiated control mice); FIG. 36B) two-month post-irradiation versus before irradiation (P ------ 0.0001, n === 3); FIG. 36C) two- months post-irradiation without the blocker versus two-rnonths post-irradiation with the blocker versus before irradiation (P = 0.0001 , n = 3). A decrease was detected in thenC-DPA ratio of the mice that received the blocking agent compared to the mice that did not receive the blocking agent at two-month post irradiation. This, JC-DPA ratio in the mice that received the blocking agent at two-month post irradiation was similar to that detected in the non-irradiated control mice. This confirmed the specificity' of the;iC-DPA to detected TSPO in the preclinical mouse model of RIBI.

[0266] A decrease was detected in the! !C-DPA ratio of the mice that received the blocking agent compared to the mice that did not receive the blocking agent at two-month post irradiation. This confirmed the specificity of thellC-DPA to detected TSPO in the preclinical mouse model of RIBI.

[0267] Correlation ofllC-DPA PET,nC-CPPC PET, contrast-enhanced T1W MR1 and TSPO, IBA1, and CD68 immunohistochemistry'. The neuroinflammation trends detected using l lC-DPA PET andnC-CPPC PET respectively correlated with the BBB permeability trend that was detected using contrast-enhanced T1W MRT, and previously reported. Furthermore, the neuroinflamination trend detected using PET and contrast-enhanced T1W MRI651623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO respectively, correlated with the neuroinflammation trend that was detected using TSPO, IB Al , and CD68 immunohistochemistry-.

[0268] FIG. 37 A) Quantification of the ratio of the respective fluorescence signals in the irradiated brain hemispheres normalized to the corresponding contralateral non-irradiated brain hemispheres of the mice at different time point in the study, shown in comparison to1SC-DPA PET, and FIG.378) contrast-enhance TiW MRI. FIG.37C) Comparison of the “C-DPA PET and contrast-enhance TiW MRI signals in the study. A high correlation was observed between the nemoinflammation signals detected with immunohistochemistry,T1C-DPA PEI', ’ ’ C-CPPC PET, and contrast-enhance TjW MRI. This suggested the possibility of using contrast-enhance TiW MRI (which doesn’t involve ionizing radiation) as a proxy / s arrogate biomarker of neuroinflammation in radiotherapy-induced brain injury'.

[0269] Conclusion: These findings suggest that contrast-enhanced TIW MRI could be used as a proxy imaging biomarker of neuroinflammation to noninvasive monitor RIBI progression in pediatric brain tumor survivors, without the use of ionizing radiation. Ultimately, this method could provide a simple yet effective means of noninvasively probing the status of neuroinflamination in RIBI progression using conventional MRI, which is already used routinely in the clinical setting to monitor and manage brain tumor patients and survivors. Additionally, this method could provide a clinically translatable means of longitudinally monitoring the therapeutic efficacy of potential RIBI prophylactic and therapeutic agents, without exposure to ionizing radiation that is involved in PET.EXAMPLE 10Improving transplanted stem cell survival and neurorepair using the nanoparticle P2b

[0270] In vivo evaluation of prolong transplanted stem cell survival and unproved neurorepair. We have also started evaluating the feasibility of improving neurorepair by transplanted stem cells, using the P2b nanotheranostic agent developed, which has a high number of phenyl-boroiiic acid pinacol ester (BAPE) moieties to scavenge reactive oxygen species (ROS), and sustainably reduce oxidative stress and nemoinflammation in our RIBI model in immunodeficient mice.661623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO

[0271] Method: Briefly, the right brain hemispheres of 8-weeks-old female severe combined immuno-deficient (SCID) mice were stereotacticaHy irradiated al 80 Gy (dose rate of 1.7 Gy / min). Mes-enchymal stem cells (one million cells) where then stereotacticaHy administered to the irradiated hemispheres of the mice, one- week post-irradiation. Starting at two weeks post-irradiation, fpg of the pH / REDOX-responsive nanopartide P2b (with a high number of ROS scavenging moie-ties). was intravenously administered bi-weekly to the mice for six months. The weights of the mice and their survival was also monitored. The mice were then monitored with multi -parametric MRI. This group of mice (Group 1) that were stereotacticaHy irradiated (80 Gy), and received both stem cell transplantation (one million) and P2b nanoparticles (biweekly) was studied in comparison to six other groups of mice:

[0272] Group 2: Mice stereotacticaHy irradiated (80 Gy) + stem cells (one million)

[0273] Group 3: Mice stereotacticaHy irradiated (80 Gy) + P2b nanoparticle (igg biweekly, for six months)

[0274] Group 4: Mice stereotacticaHy irradiated (80 Gy) + received sham surgery

[0275] Group 5: Mice stereotacticaHy irradiated (80 Gy) only

[0276] Group 6: Mice that received sham surgery only

[0277] Group 7: Control naive mice (with no irradiation or surgery)

[0278] Results: Weight measurements showed no significant differences between the groups up to seven months post irradiation. This suggested that the biweekly administration of the P2b nano-particle was not toxic to the mice. Additionally, Group 1 mice that received the mesenchymal stem cell transplant and P2b nanoparticles biweekly after irradiation, showed the best survival trend of the irradiated mouse groups.

[0279] Conclusion: This data shows that pH / REDOX-responsive nanoparticles with a high number of phenylboronic acid pinacol ester (RAPE) moieties to scavenge reactive oxygen species (ROS), can also improve neurorepair and RIBI outcomes when used in combination with transplanted stem cells.EXAMPLE 11671623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WOEvaluation of the long-term therapeutic efficacy of nanotheranostic agents in a RIBI model.

[0280] Here, we expanded on our previous short-term nanoparticle efficacy study findings, and investigated the long-term feasibility of using our nanoparticles to mitigate neuroinflammation and improve outcomes in the long-term (over a period of 12 months post- irradiation), in apreclinical model of RIBI FIG. 38: Schematic of experimental design of long- term nanoparticle efficacy study).

[0281] Hypothesis: We hypothesized that the significant reduction of neuroinflammation (microglial activation and the recruitment of infiltrative peripheral macrophages); blood-brain barrier disruption; and edema7astrogtiosis detected at 1.5 months after nanoparticle administration, in nanoparticle-treated irradiated mice compared to PBS-treated irradiated control mice, would lead to significantly improved outcomes in our preclinical model of RIBI.

[0282] Objective: To test this hypothesis, we expanded on our previous short-term (two months post-IR) nanotheranostic study, and carried out a long-term (12 months post-IR) nanotheranostic study to evaluate if the reduction of neuroinfl animation at the early stages of RIBI, improved outcomes in the long-term,

[0283] Method: Four groups of 8-week-old female BALB / c mice were used in this long-term study. N on-irradiated mice treated with phosphate buffered saline (PBS) were used as controls (Group 1 ) in the study. Three groups of mice were stereo tactically irradiated with a single X- ray beam at a dose of 80 Gy and a dose rate of 1.7 Gy / min (FIG. 1), as previously described. The mice were next treated either nanoparticles or phosphate buffered saline (PBS), biweekly from two weeks post-irradiation to 4 months post-irradiation as follows: irradiated control mice treated with PBS (Group 2); irradiated mice treated with 20 ug of nanoparticle P2a (Group 3); irradiated mice PBS treated with 20 pg of nanoparticle P2b (Group 4), The mice were then monitored longitudinally using multi-parametric MR1 and behavioral tests of memory' and learning at the following intervals: 1) one-month post-IR; 2) three / four months post-IR; 3) Six months post-IR; and 5) twelve months post-IR. At 12 months post-IR, blood samples were collected from the mice, and the mice were then transcardially perfused, the organs harvested, paraffin embedded, sectioned and stained for histology / toxicology.681623347761.1PATENT ATTORNEY DOCKET NO. JHU4770-1WO

[0284] Results.

[0285] FIG. 39: MRI signal changes in the different mouse groups over a 12-month postirradiation period.

[0286] Multi-Parametric MRI results: Contrast-enhanced TIW MRI results showed that nanoparticie P2a was more effective than P2b in sustainably reducing blood-brain barrier (BBB) permeability over the 12-month post-irradiation period (FIG. 39A). Additionally, anatomical T2W weighted MRI showed that P2a was also more effective than P2b in sustainably reducing hemorrhage, detected as a hypointense T2W weighted MRI signal over the 12-month post-irradiation period (FIG. 39C). No statistically significant differences in edema were detected between the mouse groups at the time points evaluated in the long-term study (FIG. 39B) detected as a hypointense T2W weighted MRT signal over the 12-month postirradiation period.

[0287] Mouse weight changes: Statistically significant differences were detected between the non-irradiated control group and all the irradiated mice groups over the 12-month postirradiation period (FIG. 40). Interestingly whereas a statistically significant difference was detected between the P2b- treated irradiated mice and the PBS-treated irradiated mice, no statistically significant difference was detected between the P2a-treated irradiated mice and the PBS-treated irradiated mice. Additionally, a statistically significant difference was detected between the P2b-treated irradiated mice and the P2a-treated irradiated mice (FIG. 40). This suggested that P2b was better suited to control weight loss changes in the irradiated mice compared to P2a.

[0288] FIG. 40: Weight changes in the different mouse groups over a 12-month postirradiation period.

[0289] Improved survival outcomes in nanoparticie- treated mice. Our results showed that the irradiated mice treated with nanoparticie P2ahad belter survival outcomes than irradiated mice treated with nanoparticie P2b. No statistically significant differences were detected between the mice that received nanoparticie P2a, and the non-irradiated control mice (P =0.0679). However, a statistically significant difference was detected between mice that received nanoparticie P2a and the irradiated control mice that received PBS (P =0.0407). This improved691623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO survival outcome detected with P2a nanoparticle-treated mice was attributed to the slower P2a nanoparticle activation and clearance kinetics compared to nanoparticle P2b. This slower P2a nanoparticle activation and clearance kinetics enabled the reactive oxygen scavenging (ROS) moieties [phenyl boronic acid pinacol ester (BAPE) moieties] released from the activated nanoparticle P2a to be retained in the irradiated regions of the brain for longer periods of time, and subsequently improved the long-term performance of nanoparticle P2a compared to nanoparticle P2b.

[0290] Although nanoparticle P2b was designed to possess twice as many phenyl boronic acid pinacol ester (BAPE) moieties to scavenge reactive oxygen species (ROS) than P2a. and even though it showed a greater ami -inflammatory' effect at the early time point (1.5 months post- nanoparticle administration) in the short-term study (2 months post-irradiation study), it showed poorer long-term survival outcomes than nanoparticle P2a in the 12 months postirradiation study. This poorer long-term RIBI control detected with nanoparticle P2b compared to P2a was attributed to its faster nanoparticle activation kinetics. Additionally, the reactive oxygen scavenging (ROS) moieties (BATE) released from the activated nanoparticle P2b was rapidly cleared from the irradiated region of the brain compared to nanoparticle P2a. Subsequently this rapid BAPE clearance kinetics in the brain could have contributed to the poorer long-term RIBI control by the agent.

[0291] There was a statistically significant difference between the mice that received nanoparticle P2b and the non-irradiated control mice (P =0.0115). Additionally, no statistically significant difference was detected between the mice that received nanoparticle P2b and the irradiated control mice that received PBS (P =0.6118). This finding emphasizes the importance of nanoparticle activation kinetics; BAPE retention and clearance kinetics in improving the long-term performance of the neuroprotective nanotheranostic agents, and ultimately in improving the overall survival outcomes in radiotherapy induced-brain injury.

[0292] FIG. 41: Kaplan Meier survival curves of a long-term nanoparticle study. The irradiated mice treated with nanoparticle P2a had better overall survival outcomes than irradiated mice treated with nanoparticle P2b. This improved long-term performance of nanoparticle P2a compared to nanoparticle P2b (despite P2b having twice as many ROS scavengers than P2a), was attributed to the slower activation kinetics of nanoparticle P2a compared to P2b, and also the slower clearance kinetics of the BAPE moiety released from P2a701623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO compared to that released from P2b. This slower activation kinetics of nanoparticle P2a and the subsequent slower clearance kinetics of the BAPE moiety released from P2a, enabled BAPE to be retained in the irradiated regions of the brain for longer periods of time, and subsequently improved the long-term performance of nanoparticle P2a, compared to nanoparticle P2b.EXAMPLE 12Developing Magnetic Resonance Imaging Biomarkers of Neuroinflammation, Cognitive Impairment, and Survival Outcomes for Radiotherapy-Induced Brain Injury in aPreclinical Mouse Model

[0293] Objective: Radiotherapy-induced brain injury (RIBI) is a chronic side effect that affects up to 90% of brain tumor survivors treated with radiotherapy. Here, we used multiparametric magnetic resonance imaging (MRI) to identify nonin vasive and clinically translatable biomarkers of RIBI.

[0294] Method: 8-week-old female, immune competent BALB / c mice were stereotactically irradiated with a single dose of 80 Gy, at a dose rate of 1.7 Gy / minute. The irradiated mice were then monitored longitudinally with MRI, behavioral tests of learning and memory, and immunohistochemistry, in comparison to nonirradiated mice.

[0295] Results: Three types of MRI biomarkers of RIBI were identified. A contrast enhanced T1 -weighted MRI biomarker was identified as being best suited to detect the onset of injury, by detecting changes in the blood-brain barrier (BBB) permeability. Maximum BBB permeability' (18.95 ± 1.75) was detected with contrast-enhanced T1 -weighted MRI at 1 -month post irradiation in irradiated mice (P < 0.0001, n = 3). Interestingly, maximum neuroinflamniation (24.14 ± 6.72) was also detected using IBA1 and CD68 immunohistochemistry at 1 -month post irradiation in irradiated mice (P::::0.0041, n::::3). This simultaneous maximum BBB permeability and neuroinflammation detection also coincided with the detection of the onset of transient cognitive impairment, detected using the fear conditioning behavioral test at 1-month post irradiation in irradiated mice compared to nonirradiated mice (P:::0.0017, n:::10). AT2-weighted MRI hyperintensity biomarker was also identified, and determined to be best suited to detect intermediate injury. Maximum T2-711623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO weighted MRI hyperintensity (3.97 ± 2,07) was detected at 2-month post irradiation in the irradiated mice compared to nonirradiated mice (P = 0.0368, n = 3). This T2-weighted MRT hyperintensity also correlated with maximum astrogliosis (9.92 ± 4.21), which was also detected at 2-month post irradiation using GFAP immunohistochemistry in the irradiated mice compared to nonirradiated mice (P = 0.0215, n = 3). Finally, T2-weighted and T2*-weighted MRI hypointensity biomarkers were identified as being best suited to detect late injury', from 4-month post irradiation. These biomarkers correlated with increased iron deposition from late vascular damage, which was validated with Peris' Prussian bine histology’ (P < 0.05, n::::3). These hypointense MRI biomarkers of late injury' also preceded significant weight loss, severe cognitive impairment, and decreased survival in the irradiated mice compared to die nonirradiated mice.

[0296] Conclusions: Here, we identified 3 types of translational MRI biomarkers of RIBI that could enable the noninvasive longitudinal evaluation of potential RIBI prophylactic and therapeutic agents. These translational MRI biomarkers could also play a pivotal role in the management of RIBI in brain tumor survivors.

[0297] Key Words: radiation injury’, brain injury, MRI biomarkers, neuroinflanimation biomarkers, cognitive impairment biomarkers, predictive biomarkers, and brain injury mouse model

[0298] Radiotherapy -induced brain injury (RIBI) and its associated cognitive impairment are debilitating, progressive, and chronic side effects that affect up to 90% of brain tumor survivors treated with radiotherapy. Given the greater vulnerability of developing brains to ionizing radiation, survivors of childhood brain tumors are at a greater risk of developing RIBI. Recent advances in pediatric brain tumor diagnostic and therapeutic strategies have greatly improved the overall survival rates of pediatric brain tumor patients.

[0299] However, with more pediatric patients surviving childhood brain tumors, and growing into adulthood, there has been an increase in the incidence of RIBI. This is because RIBI usually becomes symptomatic and is typically detected several years after treatment with radiotherapy. Consequently, there is a growing need for noninvasive diagnostic methods capable of detecting RIBI early on, for its effective management.721623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO

[0300] Current methods to detect RIBI and its associated cognitive impairment rely on neuropsychological evaluations. However, neuropsychological evaluations detect RIBI at the later stages, when it is already symptomatic. Neuropsychological evaluations could be greatly supplemented with robust nonin vasive imaging biomarkers, which could be used to detect subtle molecular changes in the brain, which are indicative of the early onset of RIBI and cogniti ve impairment.

[0301] These imaging biomarkers could also aid in 1) patient stratification; 2) the longitudinal monitoring of RIBI progression; and 3) monitoring patient responses to potential RIBI therapies. Although the pathogenesis of RIBI has not yet been fully elucidated, the vascular hypothesis of RIBI pathogenesis postulates that it is driven by oxidative stress and neuroinflammation, which lead to cerebrovascular damage, ischemia, neurodegeneration, and cognitive impairment. Several reports have postulated that irradiation generates reactive oxygen species, which cause oxidative stress and destroy vulnerable endothelial cells. The damaged endothelial cells release cytokines, which then stimulate the immune system via the activation of resident microglial cells. Microglial cells play' an important role in the phagocytosis of damaged cells, and also secrete cytokines which further exacerbate neuroinflammation, by recruiting infiltrative peripheral macrophages.

[0302] The damaged endothelial cells also cause the disruption of the blood-brain barrier (BBB), which further enhances neuroinflammation by facilitating the infiltration of peripheral macrophages. Furthermore, the damaged endothelial cells and the activated microglial cells release cytokines, which activate astrocytes and cause astrogliosis. Collectively, these activated microglial cells and astrocytes cause further cerebrovascular damage, which leads to ischemia, glial and neuronal cell death, neurodegeneration, and cognitive impairment.

[0303] Preclinical rodent models of RIBI could play a critical role in enabling the better identification and characterization of noninvasive and clinically translatable longitudinal imaging biomarkers of RIBI. Furthermore, these noninvasive imaging biomarkers could aid in the evaluation of potential RIBI prophylactic and therapeutic agents, which are currently being developed.

[0304] Magnetic resonance imaging (MRI) is the primary' imaging modality used for the diagnosis of brain tumors, and it offers several advantages for imaging brain tumor survivors.731623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO especially pediatric survivors. These advantages include the possibility of longitudinally monitoring survivors noninvasively in the absence of ionizing radiation, and at high spatial resolution. Thus, developing translational MRI biomarkers of RIBI could significantly improve the management of RIBI in brain tumor survivors. Additionally, given the significant role of neuroinfl amination in the progression of RIBI, developing MRI biomarkers capable of reporting on the status of neuroinflammation could help in the management of RIBI.

[0305] Previously, we carried out a short-term study that lasted for a period of 3 months, and we showed that we could use contrast-enhanced T1 -weighted MRI to detect the onset of RIBI in a preclinical mouse model of RIBI, by detecting changes in the BBB permeability. We also showed that the detection of the onset of RIBI using contrast enhanced Tl -weighted MRI coincided with early transient changes in the short-term memory (STM) of the mice, detected using behavioral tests of memory and learning.

[0306] In this study, given the significant role of neuroinflammation in the progression of RIBI, we hypothesized that the degree of BBB permeability at different time points after irradiation would affect the degree of neuroinfl amination and that the degree of neuroinflanimation would subsequently affect the degree of cognitive impairment in RIBI. We also hypothesized that it might be possible to predict the degree of neuroinflammation and cognitive impairment during RIBI progression, using conventional rnultiparametric MRI. Thus, we expanded on our previous findings, and carried out a longer-term study that lasted for a period of 12.5 months, to see how multiparametnc MRI biomarkers of RIBI changed over time in relation to neuroinflammation and cognitive impairment changes in a preclinical RIBI mouse model.

[0307] Ultimately, these translatable MRI biomarkers and the information obtained from this study could enable the better detection, characterization, and management of RIBI in brain tumor survivors, who were treated with radiotherapy. These MRI biomarkers could also enable the longitudinal evaluation of potential RIBI prophylactic and therapeutic agents that are currently in development.

[0308] MzlTERlALS AND METHODS

[0309] Animals741623347761.1PATENT ATTORNEY DOCKET NO. JHU4770-1WO

[0310] Female 3- to 4-week-old immune competent BALB / cAnNTac (BALB / c) mice were obtained from laconic Biosciences. One cohort of mice in group 1 and all group 3 mice were irradiated at age 8-weeks-old. The other cohort of mice in group 1 was irradiated at age 5- weeks-old to evaluate the age-dependent effects of radiation. The mice irradiated at age 8- weeks-old are roughly the equivalent of humans irradiated as middle to late adolescents (15- lo 21-years-old), while the mice irradiated at age 5-weeks-old are roughly the equivalent of humans irradiated as early adolescents (11- to 14-years-old).

[0311] All animal procedures were approved by the Johns Hopkins University' Animal Care and Use Committee. Because male mice were used in our previous studies, female mice were used in our current study to evaluate any differences in the early detection lime frame, previously reported.

[0312] Study Design

[0313] Four groups of 8-week-old female BALB / c mice were used in this study FIG. 58). The right brain hemispheres of group 1 mice were stereotactically irradiated as previously described. The mi ce were then monitored longitudinally over a 12-month postirradiation period using multiparametric MRI and the fear-conditioning behavioral lest of learning and memory'. The weight changes and survival of the mice were also monitored. Group 1 mice were studied in comparison to nonirradiated control group 2 mice. Group 3 mice were irradiated similarly to group 1 mice and sacrificed at different time points after irradiation (0.03, 0.1, 0.25, 0.5, 1 , 2. 4, 6, 9, and 12 months after irradiation) and evaluated using immunohistochemistry (IHC) and histology for IHC biomarkers of R1B1. Group 3 mice were studied in comparison to nonirradiated control group 4 mice. All mice were transcardially perfused and their brains harvested at the end of the study (12.5 months after the study's initiation). However, mice were also sacrificed for humane reasons (after transcardial perfusion), when a weight loss greater than 20% of the body weight was detected or when any other signs of severe distress such as hunched postures, and reluctance to move were detected, in accordance with the Johns Hopkins University Animal Care and Use Committee protocol. To evaluate the effect of mouse age at the time of irradiation on the induction of RIBL a cohort of 5 -week-old mice was added to group 1, and also evaluated.

[0314] Irradiation751623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO

[0315] Mice were anesthetized with an isofl urane (2%) and oxygen mixture and irradiated using a small animal radiation research platform (SARRP, Xstrahl, Suwanee, GA) as previously described. Briefly, computed tomography (CT) images were acquired and a target location in the right brain hemisphere, with iso-center at the hippocampus, was chosen on the CT images using the following coordinates: anterior posterior = +3 mm relative to the lambda: medial lateral = +0.5 mm relative to the midline: and dorsal ventral = -2 mm relative to the skull base. The mice w-ere then irradiated with a single x-ray beam at a dose of 80 Gy and a dose rate of 1.7 Gy per minute, using a 3 mm x 3 mm collimator.

[0316] Magnetic Resonance Imaging

[0317] Mice were anesthetized with isoflurane (2% in air), and all MRIs were acquired using an 1 1.7 T Broker Biospec horizontal bore scanner (Billerica, MA), equipped with a 23 mm Broker mouse head volume radiofrequency coil. The Paravision 6.1.0 software was used for all image acquisitions. All in vivo contrast-enhanced T1 -weighted images were acquired follo wing the bolus intravenous administration of 200 pL of a 0,25 M (2 mmol / Kg) gadoteridol (PROHANCE) solution (Bracco Diagnostics Inc,, Singers, Germany). The gadoteridol solution was prepared in 0.01 M phosphate buffered saline (PBS). T1 -weighted images were acquired using a spin echo pulse sequence with the following acquisition parameters. Sequence::::rapid acquisition with refocused echoes; echo time:::5.5 milliseconds (ms), effective echo time:::5.5 ms: rapid acquisition with refocused echo factor = 4; repetition time = 557 ms; number of averages = 3; field of view = 18 x 18 mm; matrix size = 256 x 256 pixels; and slice thickness:::0.5 mm. Final contrast-enhanced T1 -weighted MRI analyses were performed using the NIH Image! software.

[0318] Two regions of interest (ROIs) were drawn on each axial image to cover the entire brain for the analyses. One ROI was drawn around the whole right hemisphere (irradiated), while the second ROI was drawn around the whole left hemisphere (control). Whole brain hemisphere ROIs were used to keep the sizes of the ROIs constant throughout the study, especially since the size of the region of contrast enhancement changed over the study period. Bright pixel analysis of the defined ROIs was then performed, as previously reported. A sample size of 3 was used for all contrast enhanced T1 -weighted MRI analyses.1623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO

[0319] Illustrations of Region of Interests (ROIs) used in the study. Illustrations of the two region ROIs used in the MRI studies. The white lines around the different brain hemispheres indicate the two ROIs used in the MRI studies. Illustrations of the two ROIs used in the immunohistochemistry studies. The white lines around the different brain hemispheres indicate the two ROIs used in the MRI studies. Illustrations of the two ROIs used in the histology studies. The white squares around each image indicate the ROIs used tn the H&E study. The black squares around each image indicate the ROIs used in the Perls’Prussian Blue study.

[0320] All in vivo anatomical T2-weighted MRIs were acquired using a spin echo pulse sequence with the following acquisitioning parameters: sequence = rapid acquisition with refocused echoes; echo time = 6.1 ms; effective echo time = 18.3 ms; rapid acquisition with refocused echo factor = 8; repetition time = 1500 ms; number of averages = 2; field of view = 18 x 18 mm; matrix size = 256 x 256 pixels; and slice thickness = 0.5 mm. Final T2-weighted MRI analyses were performed with the NIH Image.! software. Two ROIs were drawn on each axial image to cover the entire brain for the analyses, as described above. T2-weighted MRI hyperintensity signal quantification was then done using bright pixel analyses, as previously reported. T2-weighted MRI hypointensity signal quantification was done using dark pixel analyses, as previously reported. A sample size of 3 was used for all T2- weighted MRI analyses.

[0321] All in vivo T2* -weighted images were acquired using a gradient echo pulse sequence. Sequence: multiple gradient echoes; echo tune = 3 ms; echo spacing = 3.5 ms; number of echoes:::7; effective echo times =3, 6.5, 10, 13.5, 17, 20.5, 24 ms, repetition times = 800 ms; number of averages = 4; number of repetitions = 1 ; field of view:::18 18 mm, matrix size::::128 128 pixels; and slice thickness = 0.5 mm. Final 12* -weighted MRI analyses were performed on the images with an effective echo time of 24 ms, using the NIH Imaged software. Two ROIs were drawn on each axial image to cover the entire brain for the analyses, as described above, and dark pixel analysis was carried out. as previously reported. A sample size of 3 was used for all T2*-weighted MRI analyses.

[0322] Fear Conditioning Behavioral Tests of Learning and Memory

[0323] The fear conditioning behavioral test was used to evaluate learning and memory' in the mice, since the hippocampi of the mice (which are involved in learning and memory) were771623347761.1PATENT ATTORNEY DOCKET NO. JHU4770-1WO irradiated in this study. Fear conditioning tests were conducted at the following time points: 3 vveeks after irradiation, 2 months after irradiation, 4 month after irradiation, 6 months after irradiation, 9 months after irradiation, and 12 months after irradiation, and matched to the closest time point images.

[0324] Fear conditioning tests were conducted in Med Associates fear conditioning chambers (Med Associates, Inc., Fairfax, VT). and consisted of 5 sessions over 2 consecutive days. Habituation, training, and a 1-hour STM context test sessions were conducted on day 1, while 24-hour long-term memory' (LTM) context and 24-hour LTM cue test sessions were conducted on day 2, as previously reported.

[0325] Mice were initially placed in the conditioning chambers (habituation) for 5 minutes and then returned to their cages. One hour following habituation, the mice were returned to the conditioning chambers where a tone (2000 Hz) was played 3 minutes after placement for 30s.This was immediately followed by a foot-shock using scrambled alternating currents delivered through the grid floor by a constant current shock source (5 mA) for 2 seconds. The mice were returned to their cages after a total of 5 minutes. One hour following the training session, the short-term contextual fear memory of the mice was assessed. During this session, the mice were returned to the conditioning chambers for 5 minutes during which neither tones nor shocks were administered. This was repeated 24 hours following the training session to assess the long-term contextual fear memory of the mice. One hour following this, the cued fear memory' was assessed by re-exposing the mice to the tone (2000 Hz, for 30 seconds), 3 minutes after placement in the same chamber and in the absence of a foot shock. The freezing behavior in each session was automatically scored using Med Associates, Inc. Video Freeze Software (Med Associates, Inc.. Fairfax, VT). Data from the first minute of each session was compared as previously reported. Less freezing behavior was used to indicate memory impairment, as previ ously reported.

[0326] A sample size of 10 was used for the behavioral tests (from 3 weeks after irradiation to 9 months after irradiation). However, a sample size of 5 was used at 12-montb postirradiation, due to the lower survival of the irradiated mice at this time point. The large sample size was used to compensate for large variabilities that are generally observed in animal behavior. Furthermore, to minimize potential confounding factors that could affect the behavioral outcomes the following measures were taken: 1) All the mice were housed at the781623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO same density of 5 mice per cage, and the cages were kept at the same rack positions in the same colony room, which was kept on the standard light cycle throughout the study. 2) All behavioral testing at each time point was completed prior to anesthesia administration during the imaging of the mice. This was a deliberate decision to avoid potential effects of anesthesia on behavior. 3) All the mice were habituated to the testing room for at least 30 minutes prior to testing. Testing was performed at the same location, under the same lighting conditions (lowest setting on the lightbox in the Med Associates, Inc., fear conditioning chamber), and at the same time of day (all tests were completed in the morning for each time point), throughout the study. 4) All the experiments were performed by a single female handler blinded to the study design.

[0327] Histology and Immunohistochemistry

[0328] Mice were transcardially perfused with 30 mL of heparinized phosphate buffered saline (1% heparin in 0.01 M PBS), followed by 30 mL of a 4% paraformaldehyde (PFA) solution (4% PFA in 0.01 M PBS), at a flow rate of 10 mL / min. The mouse skulls were stored overnight at 4°C in 4% PFA, then at 4°C in PBS for 48 hours. The brains were next extracted from the skulls, paraffin-embedded, sectioned into 4-pm slices, and stained for IHC. For DNA double-strand breakage, a rabbit anti-gamma H2AX primary antibody (1: 200, ab243906, Abeam) was used followed by an Alexa Fluor 488 goat anti-rabbit secondary antibody (1:200, abl50077, Abeam). For neuroinflammation detection, a rabbit anti-CD68 (1 :100, ab283654, Abeam) and a rat anti-IBAl (1 : 100, ab283346, Abeam) primary antibody respectively were used followed by an Alexa Fluor 488 goat anti-rabbit (1 :200, abl50077, Abeam) and an Alexa Fluor 647 goat anti-rat (1 : 1000, abl50077, Abeam) secondary antibody respectively. For astrogliosis and neuron loss, a rat anti-GFAP (1 TOO, ab279291, Abeam) and rabbit Anti-NeuN (1: 100, abl77487, Abeam) primary antibody were used, followed by an Alexa Fluor 647 goat anti-rat (1 : 1000, abl50077, Abeam) and an Alexa Fluor 488 goat anti-rabbit (1 :200, abl50077, Abeam) secondary antibody, respectively. All slides were counter stained with DAPI in fluoroshield mounting media (abl04139, Abeam). Image acquisitioning was performed using a Zeiss Axio Scan.Z.l Slide Scanner. Fluorescence image analyses were performed using the NIH ImageJ software. Two ROIs were drawn on each axial image to cover the entire brain for the analyses, as described above. Pixel analysis of the defined ROIs were then performed, as described above.791623347761.1PATENT ATTORNEY DOCKET NO. JHU4770-1WO

[0329] Adjacent tissue sections were also stained with Peris' Prussian blue and hematoxylin and eosin (H&E) for histology as previously described. Image acquisitioning was performed using a Nano Zoomer S210 Hamamatsu. Optical image analyses were performed using the NIH ImageJ software. Pixel analysis of the images were then performed, as previously reported.801623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO

[0330] Statistical Analyses

[0331] All MRI, histology, and IHC data points were presented as the mean ± standard deviation of 3 independent experiments. The behavioral data points were presented as the mean ± standard deviation of 10 independent experiments, except for the 12-month post irradiation time point when 5 independent experiments were used, due to the lower survival of the irradiated mice at this time point. All statistical analyses were done using the GraphPad Prism 10.4.1 software. Multiple comparisons were made using the ordinary 2-way analysis of variance test. Two dataset comparisons were made using unpaired 2-tailed Student's t-tests, except when otherwise stated. The results were considered statistically significant at P < 0.05.

[0332] RESULTS

[0333] Detection of DNA Double Strand Breakage After Irradiation

[0334] Fluorescence microscopy images of brain tissue samples harvested 1 hour after the irradiation of 8-week-old mice (group 3), and stained to show DNA double-strand breakage using y-H2AX IHC indicated specific DNA damage only in the irradiated brain hemispheres, as was expected. Quantitative pixel analysis of the y-H2AX fluorescence signal ratios of the irradiated brain hemispheres compared to the nonirradiated brain hemispheres revealed a 12. 14 ± 3.58-fold increase in y-H2AX staining in the irradiated brain hemispheres 1 hour after irradiation (P = 0.006, n = 3). This confirmed the specific irradiation of a single brain hemisphere in the irradiated mice.

[0335] Contrast-Enhanced T1 -Weighted MRI Detection of Radiotherapy-Induced Brain Injury

[0336] Two weeks after the stereotactic irradiation of 8-week-old mice (Group 1), contrast- enhanced T1 -weighted (T1W) MRI showed the specific disruption of the BBB in the irradiated brain hemispheres of the mice. Quantitative pixel analysis of the contrast-enhanced T1 W MRI signal ratios of the irradiated brain hemispheres compared to the nonirradiated brain hemispheres revealed a 13.42 ± 2.01-fold increase (P = 0.0005, n = 3) in the irradiated (Group 1) mice compared to the control nonirradiated (Group 2) mice. This detection of BBB permeability indicated the onset of injury. Interestingly, no differences between the irradiated brain hemispheres and the nonirradiated brain hemispheres were detected at this time point811623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO with either T2 -weighted (T2W) MRI or T2*-weighted (T2*W) MRI in these mice. These results suggest that contrast-enhanced T1W MRI was the most sensitive of the MRI pulse sequences evaluated for detecting the onset of RIBI, via the detection of BBB permeability.

[0337] The Effect of Age at the Time of Irradiation on Injury Induction

[0338] Contrast-enhanced T1 W MRI detected BBB disruption at an earlier time point in mice irradiated at a younger age. BBB disruption was detected 1-week post irradiation in mice irradiated at 5 -weeks-old, compared to detection at 2-week post irradiation in mice irradiated at 8-weeks-old. Statistically significant differences between the contrast-enhanced T1W MRI signals from the respective age groups were detected 1 week to 1 month after irradiation. Interestingly, no visual differences between the irradiated brain hemispheres and the nonirradiated brain hemispheres were visible on the T2W MRIs or T2*W MRIs of mice irradiated at age 5-weeks-old compared to 8-weeks-old at either 1 week or 2 weeks after irradiation. However, quantitative pixel analysis of the irradiated brain hemispheres compared to the nonirradiated brain hemispheres using the T2W MRI hypointensity signal showed a statistically significant difference between both age groups (P = 0.0046, n = 3) 2 weeks after irradiation. This finding suggests that RIBI in younger mice can be detected quantitatively, but not visually, using the T2W MRI hypointense signal as early as 2 weeks after irradiation.

[0339] Determining the Kinetics of T1 W MRI Contrast Enhancement in RIBI

[0340] In order to use contrast-enhanced T1W MRI to evaluate how the permeability of the BBB changed after irradiation, the kinetics of T1W MRI contrast enhancement was determined in mice irradiated at 8 weeks of age. Contrast-enhanced T1W MRIs were acquired sequentially before and up to 60 minutes after contrast administration, 1 month, and 2 months after irradiation. Our results showed that although the contrast-enhanced T1W MRI signal intensity ratio of the irradiated brain hemispheres compared to the nonirradiated brain hemispheres was higher (P = 0.005, n = 3) at 1-month post irradiation (18.97 ± 1.23) compared to at 2-month post irradiation (14.32 ± 0.28), similar contrast enhancement kinetic patterns were detected at both time points. The plateaued contrast-enhanced T1W MRI signal intensity ratios 15-60 minutes after contrast administration suggested that contrast-enhanced T1W MRIs acquired between 15-30 minutes after contrast administration could be used to reliably evaluate changes in the permeability of the BBB at different time points after irradiation.821623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO

[0341] Imaging Longitudinal Changes in the BBB Permeability After Irradiation

[0342] Using contrast-enhanced T1W MRI, the BBB permeability was evaluated over a 12- month post irradiation period in mice irradiated at 8 weeks of age. As mentioned above, contrast-enhanced T1W MRI detected BBB permeability in these mice as early as 0.5 months after irradiation. This contrast-enhanced T1W MRI signal intensity ratio peaked at 1 -month post irradiation (18.95 ± 1.75), and this indicated a peak in the BBB permeability. The contrast- enhanced T1W MRI signal ratio then gradually decreased over the following 11 months after irradiation to a value of 5.69 ± 2.23, at 12-month post irradiation. This indicated a decrease in the BBB permeability over time. Overall, a contrast-enhanced T1W MRI signal was detected in the irradiated brain hemispheres throughout the 12-month post irradiation period, indicating BBB permeability throughout the 12-month post irradiation study period.

[0343] Imaging Longitudinal Anatomical Changes in the Brain After Irradiation

[0344] T2W MRI was used to monitor anatomical changes in the brain after irradiation. A statistically significant hyperintense T2W MRI signal was detected in the irradiated brain hemisphere of mice irradiated at age 8-weeks-old, 1 month (3.52 ± 0.97) and 2 months (3.97 ± 2.07) after irradiation,. This hyperintense signal suggested the presence of edema at this phase of injury, 1 month and 2 months after irradiation.

[0345] Additionally, a statistically significant hypointense T2W MRI signal was detected in the irradiated brain hemispheres of mice irradiated at age 8-weeks-old at 4-month postirradiation (3.34 ± 0.81), and this signal increased over time. This hypointense T2W MRI signal suggested the presence of hemorrhage and necrosis at the later phase of injury, 4 months after irradiation and beyond.

[0346] Collectively, 2 main types T2W MRI biomarkers were identified: A hyperintense T2W MRI biomarker that occurred at the intermediate phase of injury, 1 month and 2 months after irradiation, which was indicative of edema; and a hypointense T2W MRI signal that occurred at the later phase of injury, 4 months after irradiation and beyond, which was indicative of hemorrhage and necrosis. Given the higher sensitivity of T2* -weighted (T2*W) MRI to detect hemorrhage and necrosis, it was used to further validate the hypointense signal detected on T2W MRI. A statistically significant hypointense T2*W MRI signal was detected831623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO in the irradiated brain hemispheres of mice irradiated at age 8-weeks-old as early as 1 month after irradiation (1.75 ± 0.27), and this signal increased over time.

[0347] Furthermore, statistically significant atrophy was detected using T2W MRI in the irradiated brain hemispheres of mice irradiated at age 8-weeks-old as early as 2 month after irradiation, compared to nonirradiated control mice. This phenomenon increased over time.

[0348] Correlating Multiparametric MRI Biomarkers of RIBI to IHC and Histological Biomarkers of RIBI

[0349] We next correlated the longitudinal MRI biomarkers of RIBI to immunohistochemical biomarkers of RIBI, using group 3 and 4 mice.

[0350] Neuroinflammation Detection

[0351] Neuroinflammation was detected in the mouse brain samples using cluster of differentiation 68 (CD68) and ionized calcium binding adaptor molecule 1 (IBA1) IHC. CD68 was used to detect infiltrating macrophages, while IBA1 was used to detect microglial activation. Fluorescence microscopy images of brain tissue samples showed maximum CD68 (24.48 ± 4.40) and IBA1 (24.14 ± 6.72) fluorescence signals 1 month after irradiation. This maximum neuroinflammation signal detected 1 month after irradiation corresponded with the maximum BBB permeability also detected 1 month after irradiation using in vivo contrast- enhanced T1W MRI. The neuroinflammation signals then gradually decreased over the following 12 months after irradiation to ratios of 7.39 ± 3.55 and 7.68 ± 4.04, respectively. Overall, this neuroinflammation trend detected using IHC and fluorescence microscopy correlated with the BBB permeability trend detected in vivo using contrast enhanced T1W MRI. These findings suggest that the contrast-enhanced T1W MRI signal could be used as a surrogate (or proxy) biomarker of neuroinflammation.

[0352] Astrogliosis Detection

[0353] Astrogliosis was evaluated using glial fibrillary acidic protein (GFAP) IHC at different time points after irradiation. Fluorescence microscopy images of brain tissue samples showed maximum GFAP (9.92 ± 4.21) fluorescence signals in the irradiated brain hemispheres at 2 months after irradiation. This maximum GFAP signal detected 2 months after irradiation841623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO corresponded with the maximum in vivo T2W MRI hyperintensity signal detected 2 months after irradiation. The GFAP signal then decreased over the following 12 months after irradiation to 3.46 ± 1.27. Overall, this GFAP (astrogliosis) trend detected using fluorescence microscopy correlated with the in vivo T2W MRI hyperintensity signal trend. These findings suggest that the T2W MRI hyperintensity signal could be used as a surrogate (or proxy) biomarker of astrogliosis.

[0354] Cell Loss Detection

[0355] Neuronal loss was evaluated using neuronal nuclear antigen (NeuN) IHC at different time points after irradiation. No statistically significant change in the NeuN (green) fluorescence signal was detected in the irradiated brain hemispheres compared to the nonirradiated brain hemispheres over the 12-month post irradiation study period. Next, using H&E staining, we evaluated general cell loss (tissue porosity) at different time points after irradiation in mice irradiated at 8-weeks-old and monitored for 12 months post irradiation. Maximum tissue porosity (11.83 ± 5.78) was detected in the irradiated brain hemispheres compared to the nonirradiated brain hemispheres at 2 months post irradiation. Overall, a tissue porosity trend similar to the in vivo T2W MRI hyperintensity signal trend was detected. This finding suggests that in addition to astrogliosis, tissue porosity may enable the accumulation of fluids in tissue (edema) and contribute to the in vivo T2W MRI hyperintensity signal.

[0356] Hemorrhage Detection

[0357] Using Peris' Prussian blue staining, we evaluated hemorrhage (iron deposition) at different time points after irradiation in mice irradiated at 8-weeks-old and monitored for 12 months post irradiation. Statistically significant differences in Peris' Prussian blue staining of the irradiated brain hemispheres compared to the nonirradiated brain hemispheres were detected from 1 month to 12 month post irradiation using an unpaired 1-tail t-test. Overall, an increase in Peris' Prussian blue staining (iron deposition) was detected over time in the irradiated brain hemispheres and this correlated with the increase in the hypointensity signal detected over time in the irradiated brain hemispheres, using in vivo T2*W MRI. This finding suggests that hemorrhage (iron deposition) is responsible for the in vivo T2*W MRI hypointensity signal.851623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO

[0358] Correlating Multiparametric MRI Biomarkers and Immunohistochemical Biomarkers of RIB I to Cognitive Outcomes

[0359] To evaluate the correlation between the degree of BBB permeability and neuroinflammation on cognitive impairment, fear- conditioning tests were performed at different time points after irradiation. Our results showed a statistically significant difference (P = 0.0017, n = 10) in the freezing behavior of the irradiated mice (Group 1) compared to the nonirradiated mice (Group 2) during the habituation phase of the fear conditioning test at 3 weeks (almost 1 month) after irradiation. By comparing the freezing behaviors of the respective mouse groups during the habituation phase versus the STM context phase, a statistically significant difference (P = 0.0012, n = 10) was detected in the nonirradiated (Group 2) mice as was expected, but not in the irradiated (Group 1) mice. This indirect detection of differences in STM suggested the onset of mild cognitive impairment in the irradiated mice compared to the nonirradiated mice at nearly 1 month after irradiation, when maximum BBB permeability and maximum neuroinflammation were also detected.

[0360] Interestingly, no statistically significant differences in the freezing behaviors of the irradiated mice compared to the nonirradiated mice were detected using the fear conditioning test 2 months to 9 months after irradiation, as the BBB permeability and neuroinflammation decreased. This suggested that the early cognitive impairment detected almost 1 month after irradiation, which correlated with the maximum BBB permeability, was transient in nature. This finding also suggested the possibility of using the in vivo contrast-enhanced T1W MRI biomarker of RIBI to noninvasively predict the onset of transient cognitive impairment in RIBI.

[0361] Interestingly, statistically significant differences in the freezing behaviors of the irradiated mice compared to the nonirradiated mice were detected in the STM context phase (P = 0.0049, n = 5) and in the LTM cued phase (P = 0.0069, n = 5) of the fear conditioning test at 12 months post irradiation (FIG. 12L). This direct detection of differences in memory between the 2 groups at 12 months post irradiation (compared to the indirect detection at almost 1- month post irradiation) suggested the onset of severe cognitive impairment. At this later time point, despite a decrease in the permeability of the BBB, an increase in infiltrative CD68- positive macrophages was detected. This increase in the infiltration of phagocytic CD68- positive macrophages in the irradiated brain hemisphere was attributed to the increase in iron deposition in the irradiated brain hemisphere detected in vivo with T2W MRI and T2*W MRI861623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO at the later time points after irradiation. This is plausible, because phagocytic macrophages are responsible for clearing up systemic debris, including deposited iron. Taken together, this severe cognitive impairment detected at 12 months post irradiation could result from a combination of factors, including iron deposition, neuroinflammation, and atrophy of the irradiated brain hemisphere.

[0362] Correlating Multiparametric MRI Biomarkers and Histological Biomarkers of RIBI to Overall RIBI Outcomes

[0363] The in vivo detection of hemorrhage (hypointensity) in the irradiated brain hemispheres of group 1 mice at 6 months post irradiation with T2W MRI and T2*W MRI, preceded the onset of severe weight loss, severe cognitive impairment, and ultimately mouse deaths after irradiation. Statistically significant weight loss was detected in the irradiated mice (Group 1) compared to the nonirradiated mice (Group 2), from the 7 months after irradiation time point to the end of the study at 12 months post irradiation. The first death of an irradiated mouse was observed 8 months after irradiation, and this number increased until the end of the study at 12 months post irradiation. A statistically significant difference was detected in the survival of the nonirradiated mice compared to the irradiated mice at 12 months post irradiation (P = 0.0016). Collectively, this suggests the possibility of using the in vivo hypointense T2W and T2*W MRI biomarkers of late injury as noninvasive predictive biomarkers of poor overall RIBI outcomes.

[0364] DISCUSSION

[0365] In this study, we used conventional multiparametric MRI to identify potential noninvasive MRI biomarkers of RIBI, which could be used to noninvasively detect the early onset of RIBI and longitudinally monitor RIBI progression. Overall, our results showed that contrast-enhanced T1 W MRI was the most sensitive of the MRI pulse sequences evaluated for detecting the onset of RIBI via the detection of BBB disruption at the early stages of injury in mice irradiated at any age. Contrast-enhanced T1W MRI also detected BBB disruption at an earlier time point (1 week earlier) in mice that were irradiated at a younger age (5-weeks-old) compared to mice that were irradiated at an older age (8-weeks-old). This is roughly the human equivalent of contrast-enhanced T1W MRI detection of RIBI approximately 5.5 years earlier in humans irradiated as early adolescents (11- to 14-years-old) compared to humans irradiated871623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO as middle to late adolescents (15- to 21 -years-old). This finding is in agreement with previous reports, which show that RIBI is detected earlier in patients treated with radiotherapy, at a younger age.

[0366] Additionally, although contrast-enhanced T1W MRI detected BBB permeability throughout the 12-month post irradiation study period, it was also able to longitudinally detect subtle changes in the degree of BBB permeability over the course of the study. These changes in the BBB permeability correlated with changes in neuroinflammation that were detected with IHC and fluorescence microscopy and also with changes in transient cognitive impairment that were detected with the fear conditioning behavioral test. This correlation between BBB permeability and cognitive impairment has also been suggested in several neurodegenerative disorders. Ultimately, this finding suggests the possibility of using the contrast-enhanced T1 W MRI biomarker, as a surrogate (or proxy) biomarker of neuroinflammation and transient cognitive impairment in RIBI.

[0367] Our results also identified 2 types of T2W MRI biomarkers: 1) a hyperintense T2W MRI biomarker that occurred at the intermediate phase of injury at 1 month and 2 months after irradiation and 2) a hypointense T2W MRI biomarker that occurred at the later phase of injury, 4-months after irradiation and beyond. The in vivo hyperintense T2W MRI biomarker trend correlated with both the astrogliosis (GFAP) trend that was detected with IHC and fluorescence microscopy, and the edema (tissue porosity) trend that was detected with H&E histology and optical microscopy. This suggests that the T2W MRI hyperintense biomarker could be used as a surrogate (or proxy) biomarker of astrogliosis and edema.

[0368] The hypointense T2W MRI biomarker detected at the later phases of injury was validated with the more hemorrhage-sensitive T2*W MRI, and it correlated with the hemorrhage (iron deposition) trend detected with Peris' Prussian blue staining and optical microscopy. This finding suggests that the T2W MRI and T2*W MRI hypointense biomarkers could be used as a surrogate (or proxy) biomarker of hemorrhage. These in vivo hypointense T2W MRI and T2*W MRI biomarkers preceded the onset of severe weight loss, severe cognitive impairment, and ultimately mouse deaths after irradiation. This finding is in agreement with other reports which show a correlation between iron accumulation in the brain and severe cognitive impairment. Thus, this further suggests the possibility of using the in vivo881623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO hypointense T2W and T2*W MRI biomarkers of late injury as noninvasive predictive biomarkers of poor overall RIBI outcomes.

[0369] In summary, these multiparametric MRI biomarkers can be grouped into 3 main categories: 1) an early MRI biomarker of RIBI (the contrast-enhanced T1W MRI biomarker); 2) an intermediate MRI biomarker of RIBI (the T2W MRI hyperintense biomarker); and 3) late MRI biomarkers of RIBI (the T2W and T2*W MRI hypointense biomarkers). The early MRI biomarker of RIBI (the contrast-enhanced T1W MRI biomarker) was best suited to detect the onset of RIBI and also to detect subtle longitudinal changes in BBB permeability, which could be predictive of changes in neuroinflammation and transient cognitive impairment. The intermediate MRI biomarker of RIBI (the T2W MRI hyperintense biomarker), on the other hand, could be used to predict the status of astrogliosis and edema. Finally, the late MRI biomarkers of RIBI were best suited to detect hemorrhage in late injury and preceded weight loss, severe cognitive impairment, and poor survival. Consequently, these late biomarkers could be used as prognostic biomarkers of poor overall RIBI outcomes.

[0370] However, several limitations are associated with this study including the following: 1) Since RIBI is a side effect that is detected several years after treatment in humans, to compensate for the relatively shorter lifespans of mice compared to humans, a radiation dose higher than that typically used in the clinical setting was used in this study to accelerate the occurrence of RIBI. This enabled the detection of RIBI within the lifespan of the mice. 2) Given the relatively small size of the mouse brain, a single radiation dose regiment was used to minimize variable irradiation of the target region, as opposed to the fractionated radiation regimens typically used in clinical settings. 3) A nontumor bearing mouse model was used in this study to minimize the potential confounding complexities of variable brain tumor responses to radiotherapy, and also to improve the long-term survival (12 month) of the mice and to enable the feasibility of the study. 4) A relatively small cohort size of adolescent mice of the same sex were used in this study. 5) Only the fear conditioning behavioral test (which involves less human interaction with mice) was used to assess learning and memory in the mice, in order to minimize potentially confounding mouse handling effects on the behavioral results. However, the fear conditioning behavioral test itself can induce stress in animals through the electric shock and this can cause anxiety in mice. Because stress is also known to impact neuroinflammation, this could compound the radiation impact. 6) Finally, a high891623347761.1

Claims

PATENTATTORNEY DOCKET NO. JHU4770-1WO standard deviation was observed for some variables in the study (such as the T2W MRI hyperintense biomarker) at different time points, especially when a small effect size was associated with the variable. Thus, these limitations could potentially restrict the generalizability of our findings. Our future studies include validating these translatable MRI biomarkers in radiation-responsive brain tumor mouse models, prior to validating these MRI biomarkers in the clinical setting. We will also evaluate the feasibility of using these MRI biomarkers to distinguish RIBI from tumor responses to radiotherapy and other brain pathologies.[0371] CONCLUSIONS[0372] In this study, we used multiparametric MRI to identify 3 categories of noninvasive MRI biomarkers of RIBI in a preclinical mouse model of RIBI. These translatable MRI biomarkers could enable the better detection, characterization, and management of RIBI in brain tumor survivors, especially since MRI is the primary imaging modality used in diagnosing brain tumors and also in monitoring brain tumor survivors. These MRI biomarkers could also enable the longitudinal evaluation of potential RIBI prophylactic and therapeutic agents that are currently being developed. Ultimately, these multiparametric MRI biomarkers could aid in the early detection of RIBI and the personalization of RIBI therapeutic regimens, especially in pediatric brain tumor survivors.[0373] Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.901623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WOWhat Is Claimed Is:

1. A polymeric nanoparticle comprising a block copolymer which comprises a structure of Formula I:Formula 1 wherein o is an integer between 60 and 100; wherein p is an integer between 20 and 60; wherein m is an integer between 10 and 20; and wherein n is an integer between 5 and 15.

2. The polymeric nanoparticle of claim 1, wherein the block copolymer comprises a structure of Formula II:Formula II911623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO3. The polymeric nanoparticle of claim 1, wherein the block copolymer comprises a structure of Formula III:Formula III4. The polymeric nanoparticle of claim 1, wherein the block copolymer has a critical aggregation concentration of about 0.08 mg / mL to about 0.1 mg / mL.

5. The polymeric nanoparticle of claim 1, having a diameter of about 165 nm at a pH of 7.4.

6. The polymeric nanoparticle of claim 1, having a diameter of about 15 nm to about 25 nm at a pH of 6.2, in the presence of a reactive oxygen species, or a combination thereof.921623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO7. A pharmaceutical composition, comprising: an active pharmaceutical ingredient, a diagnostic marker, or a combination thereof encapsulated in a polymeric nanoparticle comprising a block copolymer which comprises a structure of Formula I:Formula I wherein o is an integer between 60 and 100; wherein p is an integer between 20 and 60; wherein m is an integer between 10 and 20; and wherein n is an integer between 5 and 15.

8. The pharmaceutical composition of claim 7, wherein the active pharmaceutical ingredient, the diagnostic marker, or the combination thereof is released from the polymeric nanoparticle upon exposure to a pH below about 6.5, exposure to a reactive oxygen species, or a combination thereof.

9. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition comprises an active pharmaceutical ingredient comprising a neuroprotective agent.

10. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition comprises a diagnostic marker for detecting oxidative stress.931623347761.1PATENT ATTORNEY DOCKET NO. JHU4770-1WO11. The pharmaceutical composition of claim 7, wherein the block copolymer comprises a structure of Formula II:Formula II12. The pharmaceutical composition of claim 7, wherein the block copolymer comprises a structure of Formula III:Formula III13. A method of diagnosing or treating a radiotherapy -induced brain injury in a patient in need thereof, comprising: administering to the patient a therapeutically effective amount of a pharmaceutical composition, comprising:941623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO an active pharmaceutical ingredient, a diagnostic marker, or a combination thereof encapsulated in a polymeric nanoparticle comprising a block copolymer which comprises a structure of Formula I:Formula I wherein o is an integer between 60 and 100; wherein p is an integer between 20 and 60; wherein m is an integer between 10 and 20; and wherein n is an integer between 5 and 15; thereby diagnosing or treating the radiotherapy -induced brain injury in the patient.

14. The method of claim 13, wherein the pharmaceutical composition comprises an active pharmaceutical ingredient comprising a neuroprotective agent.

15. The method of claim 13, wherein the pharmaceutical composition comprises an active pharmaceutical ingredient which is released from the polymeric nanoparticle upon exposure to a pH below about 6.5, exposure to a reactive oxygen species, or a combination thereof, thereby treating the radiotherapy-induced brain injury in the patient.

16. The method of claim 13, wherein the pharmaceutical composition comprises a diagnostic marker for detecting oxidative stress in the patient.

17. The method of claim 13, wherein the pharmaceutical composition comprises a diagnostic marker which is released from the polymeric nanoparticle upon exposure to a pH below about 6.5, exposure to a reactive oxygen species, or a combination thereof, and further951623347761.1PATENTATTORNEY DOCKET NO. JHU4770-1WO comprising detecting the diagnostic marker in the patient, thereby diagnosing the radiotherapy-induced brain injury in the patient.

18. The method of claim 13, wherein the pharmaceutical composition comprises a diagnostic marker comprising an MRI-detectable biomarker, a PET-detectable biomarker, or a combination thereof, and further comprising evaluating the patient by MRI, PET, or a combination thereof.

19. The method of claim 13, wherein the block copolymer comprises a structure of Formula II:Formula II20. The method of claim 13, wherein the block copolymer comprises a structure of FormulaIII:Formula Ill961623347761.y

Images