(K-426) Development of temporospatial sensitive nanoprobes for traumatic brain injury

Every year, 70 million people experience a traumatic brain injury (TBI) demanding $400 million expenditure on treatment while the patient’s quality of life may be significantly impacted. TBI may also trigger neurodegeneration processes increasing the risk of Alzheimer’s disease, Parkinson’s disease, encephalopathy, and dementia. Following the initial TBI event, a complex physiopathology ensues including the primary injury of mechanical damage to the tissue followed by a secondary injury phase with dysregulated cellular processes. Notably, the blood-brain barrier (BBB) integrity is disrupted allowing for the entrance of foreign molecules into the brain – including nanoparticles (NPs). Our group recently used in vivo phage display to identify TBI-specific peptides that bind exclusively to the injury penumbra at 1-day post-injury (dpi) and 7 dpi. These timepoints coincide with two critical secondary injury processes: rampant inflammation and emergence of neurodegenerative processes. The objective of this study was to generate and characterize NPs that display these temporospatial TBI peptides on the surface and ultimately target and accumulate in the injured brain at the desired timepoints after TBI. This study serves as the foundation for future target drug delivery systems for TBI.

Carboxylated fluorescent polystyrene NPs (100nm) served as the base NP. Surface modifications were accomplished with two conjugation reactions. First, a carbodiimide crosslinking where the NPs are mixed with N-ethyl-N′-(3-(dimethylamino)propyl)carbodiimide, sulfo-N-hydroxysuccinimide, and a 7:3 ratio of amine-mPEG_2k:amine-PEG_2k-azide. The reaction forms an amide bond between the NP and the PEG effectively PEGylating the surface and leaving azide groups available for conjugation. The second reaction is a copper-catalyzed alkyne-azide cycloaddition (CuAAC) where the azide groups react with an alkyne group present at the end of the temporospatial targeting peptides. In brief, the NPs are mixed with the desired peptide, copper sulfate, sodium ascorbate, and tris(benzyltriazolylmethyl)amine to form a triazole between the NPs and the desired peptide. A series of characterization assays were performed to assess the resulting physical properties of the NPs (DLS for size and zeta-potential, serum stability test). A Limulus Amebocyte Lysate (LAL) chromogenic assay assessed the Endotoxin Unit (EU)/mg of the NP suspension to ensure safety for injection. An in vivo pre-clinical study utilizing 9-week-old male C57BL/6 mice was performed. We utilize a closed cortical impact (CCI) injury TBI model. NPs conjugated with the relevant targeting peptides were injected retro-orbitally 1dpi or 7dpi. NPs conjugated with control peptides with cyclic or linear conformation, 1dpi peptides with cyclic or linear conformation, and 7dpi peptides with cyclic or linear conformation will be tested. NP accumulation and distribution will be tested via tissue homogenates, immunohistochemistry and biodistribution assays. All animal procedures were approved by ASU IACUC.

Results

We successfully developed functionalized fluorescent NPs with the two temporospatial TBI targeting peptides. The conjugation of the targeting peptides was confirmed by Fourier transform infrared spectroscopy (FTIR), which showed a peak at 1630cm^-1 after CuAAC, indicative of C=C, C=N, and N=N bonds indicating presence of the triazole groups. Characterization with dynamic light scattering indicates a hydrodynamic diameter average of 146±6nm, a polydispersity index of 0.01±0.008 and a zeta potential of 0.36±0.6mV. The diameter remained consistent after three hours incubation with horse serum at 37°C for all groups except the control peptides in a linear conformation. Thus far, all NP formulations were well below that endotoxin limit for mouse models (< 0.2EU/mg). For the in vivo portion of the study, we moved forward to test only the NP groups that passed all quality control assays. We completed preliminary in vivo experiments with naïve and 24h CCI injured mice where no adverse events were observed. Additional assessments in vivo are currently underway.  

Discussion

The conjugated NPs with targeting peptides exhibited consistent size and low polydispersity across the multiple steps of the conjugation process, suggesting stability and the lack of agglomeration. The PEGylated NPs surface adequately repels the small molecules present in the horse serum which further demonstrates the NPs stability. Additionally, the LAL chromogenic assay consistently showed endotoxin units well below the endotoxin limit recommended for mice pre-clinical models. Our in vivo study is underway to assess the targeting NP biodistribution and spatial accumulation at 1 and 7 dpi. The results from this study will lay the foundation for targeted drug delivery for TBI – delivering the right drug to the precise location at the right time.

Conclusion

The varied characterization techniques revealed a robust fabrication protocol that yields stable and biocompatible NPs. The preliminary animal testing further confirmed the compatibility of the NPs. Next, NPs conjugated with control, acute, and subacute peptides will be tested at the 1dpi and 7dpi timepoints.