Development and testing of nanoparticles delivery for P7C3 small molecule using injury models.

The use of nanoparticles (NPs) has emerged as a potential tool for safe and effective drug delivery. In the present study, we developed small molecule P7C3-based NPs and tested its efficacy and toxicity along with the tissue specific aptamer-modified P7C3 NPs. The P7C3 NPs were prepared using poly (D, L-lactic-co-glycolic acid) carboxylic acid (PLGA-COOH) polymer, were conjugated with skeletal muscle-specific RNA aptamer (A01B P7C3 NPs) and characterized for its cytotoxicity, cellular uptake, and wound healing in vitro. The A01B P7C3 NPs demonstrated an encapsulation efficiency of 30.2 ± 2.6%, with the particle size 255.9 ± 4.3 nm, polydispersity index of 0.335 ± 0.05 and zeta potential of + 10.4 ± 1.8mV. The FTIR spectrum of P7C3 NPs displayed complete encapsulation of the drug in the NPs. The P7C3 NPs and A01B P7C3 NPs displayed sustained drug release in vitro for up to 6 days and qPCR analysis confirmed A01B aptamer binding to P7C3 NPs. The C2C12 cells viability assay displayed no cytotoxic effects of all 3 formulations at 48 and 72 h. In addition, the cellular uptake of A01B P7C3 NPs in C2C12 myoblasts demonstrated higher uptake. In vitro assay mimicking wound healing showed improved wound closure with P7C3 NPs. In addition, P7C3 NPs significantly decreased TNF-α induced NF-κB activity in the C2C12/NF-κB reporter cells after 24-hour treatment. The P7C3 NPs showed 3-4-fold higher efficacy compared to P7C3 solutions in both wound-closure and inflammation assays in C2C12 cells. Furthermore, the P7C3 NPs showed 3-4-fold higher efficacy in reducing the infarct size and protected mouse hearts from ex vivo ischemia-reperfusion injury. Overall, this study demonstrates the safe and effective delivery of P7C3 NPs.

Honokiol-Loaded Methoxy Poly (Ethylene Glycol) Polycaprolactone Micelles for the Treatment of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD), a multifactorial age-related retinal hypoxic disorder resulting in irreversible loss of vision, is the foremost cause of blindness in the United States. Current treatment strategies involve multiple intraocular injections of antivascular endothelial growth factor (VEGF) agents into the vitreous of eye. In addition to the challenges of drug localization and targeted delivery, the need of frequent injections into the eye raises patient compliance issues, and thus call for development of sustained drug delivery systems. In this study, a sustained drug delivery system was prepared by loading an antihypoxia-induced factor (HIF) agent, honokiol (HON), into methoxy poly (ethylene glycol) polycaprolactone (MPEG-PCL) polymer. These HON-MPEG-PCL micelles were characterized by evaluating size, ζ potential, in vitro drug release profile, and morphology by transmission electron microscopy. The cytotoxic nature of developed micelles was assessed on human retinal pigment epithelial cell line (ARPE-19) cells by cytotoxicity assay. The cellular uptake and HIF and VEGF expression levels were determined in in vitro settings. Micelles formed had a particle size of 30.8 ± 0.8 nm with the poly dispersity index of 0.19 ± 0.0004 and ζ potential was found to be -5.46 ± 0.49 mv. Entrapment efficiency was calculated to be 64 ± 0.135%. In vitro drug release showed sustained release of drug from the formulation. Result from in vitro cytotoxicity study confirmed noncytotoxic nature of HON-MPEG-PCL micelles compared to HON drug solution. Furthermore, enzyme-linked immunosorbent assay studies performed showed the periodic downregulation of HIF and VEGF, which are major growth factors involved in underlying mechanism of AMD. The results showed successful development of HON-MPEG-PCL micelles, which may be useful for the effective treatment of AMD.

Hypusination of Eif5a regulates cytoplasmic TDP-43 aggregation and accumulation in a stress-induced cellular model.

TAR DNA-binding protein 43 (TDP-43) is a nuclear RNA/DNA binding protein involved in mRNA metabolism. Aberrant mislocalization to the cytoplasm and formation of phosphorylated/aggregated TDP-43 inclusions remains the hallmark pathology in a spectrum of neurodegenerative diseases, including frontotemporal disorders and Alzheimer's disease. Eukaryotic Translation Initiation Factor 5A undergoes a unique post-translation modification of lysine to hypusine (K50), which determines eIF5A binding partners. We used a sodium arsenite-induced cellular stress model to investigate the role of hypusinated eIF5A (eIF5AHypK50) in governing TDP-43 cytoplasmic mislocalization and accumulation in stress granule. Our proteomics and functional data provide evidence that eIF5A interacts with TDP-43 in a hypusine-dependent manner. Additionally, we showed that following stress TDP-43 interactions with eIF5AHypK50 were induced both in the cytoplasm and stress granules. Pharmacological reduction of hypusination or mutations of lysine residues within the hypusine loop decreased phosphorylated and insoluble TDP-43 levels. The proteomic and biochemical analysis also identified nuclear pore complex importins KPNA1/2, KPNB1, and RanGTP as interacting partners of eIF5AHypK50. These findings are the first to provide a novel pathway and potential therapeutic targets that require further investigation in models of TDP-43 proteinopathies.

Overexpression of human wtTDP-43 causes impairment in hippocampal plasticity and behavioral deficits in CAMKII-tTa transgenic mouse model.

AIMS: The current study utilizes the adeno-associated viral gene transfer system in the CAMKIIα-tTA mouse model to overexpress human wild type TDP-43 (wtTDP-43) and α-synuclein (α-Syn) proteins. The co-existence of these proteins is evident in the pathology of neurodegenerative disorders such as frontotemporal lobar degeneration (FTLD), Parkinson disease (PD), and dementia with Lewy bodies (DLB). METHODS: The novel bicistronic recombinant adeno-associated virus (rAAV) serotype 9 drives wtTDP-43 and α-Syn expression in the hippocampus via "TetO" CMV promoter. Behavior, electrophysiology, and biochemical and histological assays were used to validate neuropathology. RESULTS: We report that overexpression of wtTDP-43 but not α-Syn contributes to hippocampal CA2-specific pyramidal neuronal loss and overall hippocampal atrophy. Further, we report a reduction of hippocampal long-term potentiation and decline in learning and memory performance of wtTDP-43 expressing mice. Elevated wtTDP-43 levels induced selective degeneration of Purkinje cell protein 4 (PCP-4) positive neurons while both wtTDP-43 and α-Syn expression reduced subsets of the glutamate receptor expression in the hippocampus. CONCLUSIONS: Overall, our findings suggest the significant vulnerability of hippocampal neurons toward elevated wtTDP-43 levels possibly via PCP-4 and GluR-dependent calcium signaling pathways. Further, we report that wtTDP-43 expression induced selective CA2 subfield degeneration, contributing to the deterioration of the hippocampal-dependent cognitive phenotype.