PEG-b-(PELG-g-PLL) nanoparticles as TNF-α nanocarriers: potential cerebral ischemia/reperfusion injury therapeutic applications.

Brain ischemia/reperfusion (I/R) injury (BI/RI) is a leading cause of death and disability worldwide. However, the outcome of pharmacotherapy for BI/RI remains unsatisfactory. Innovative approaches for enhancing drug sensitivity and recovering neuronal activity in BI/RI treatment are urgently needed. The purpose of this study was to evaluate the protective effects of tumor necrosis factor (TNF)-α-loaded poly(ethylene glycol)-b-(poly(ethylenediamine L-glutamate)-g-poly(L-lysine)) (TNF-α/PEG-b-(PELG-g-PLL)) nanoparticles on BI/RI. The particle size of PEG-b-(PELG-g-PLL) and the loading and release rates of TNF-α were determined. The nanoparticle cytotoxicity was evaluated in vitro using rat cortical neurons. Sprague Dawley rats were preconditioned with free TNF-α or TNF-α/PEG-b-(PELG-g-PLL) polyplexes and then subjected to 2 hours ischemia and 22 hours reperfusion. Brain edema was assessed using the brain edema ratio, and the antioxidative activity was assessed by measuring the superoxide dismutase (SOD) activity and the malondialdehyde (MDA) content in the brain tissue. We further estimated the inflammatory activity and apoptosis level by determining the levels of interleukin-4 (IL-4), IL-6, IL-8, IL-10, and nitric oxide (NO), as well as the expression of glial fibrillary acidic protein (GFAP), intercellular adhesion molecule-1 (ICAM-1), and cysteine aspartase-3 (caspase-3), in the brain tissue. We provide evidence that TNF-α preconditioning attenuated the oxidative stress injury, the inflammatory activity, and the apoptosis level in I/R-induced cerebral injury, while the application of block copolymer PEG-b-(PELG-g-PLL) as a potential TNF-α nanocarrier with sustained release significantly enhanced the bioavailability of TNF-α. We propose that the block copolymer PEG-b-(PELG-g-PLL) may function as a potent nanocarrier for augmenting BI/RI pharmacotherapy, with unprecedented clinical benefits. Further studies are needed to better clarify the underlying mechanisms.

Human surfactant protein A2 gene mutations impair dimmer/trimer assembly leading to deficiency in protein sialylation and secretion.

Surfactant protein A2 (SP-A2) plays an essential role in surfactant metabolism and lung host defense. SP-A2 mutations in the carbohydrate recognition domain have been related to familial pulmonary fibrosis and can lead to a recombinant protein secretion deficiency in vitro. In this study, we explored the molecular mechanism of protein secretion deficiency and the subsequent biological effects in CHO-K1 cells expressing both wild-type and several different mutant forms of SP-A2. We demonstrate that the SP-A2 G231V and F198S mutants impair the formation of dimmer/trimer SP-A2 which contributes to the protein secretion defect. A deficiency in sialylation, but not N-linked glycosylation, is critical to the observed dimmer/trimer impairment-induced secretion defect. Furthermore, both mutant forms accumulate in the ER and form NP-40-insoluble aggregates. In addition, the soluble mutant SP-A2 could be partially degraded through the proteasome pathway but not the lysosome or autophagy pathway. Intriguingly, 4-phenylbutyrate acid (4-PBA), a chemical chaperone, alleviates aggregate formation and partially rescued the protein secretion of SP-A2 mutants. In conclusion, SP-A2 G231V and F198S mutants impair the dimmer/trimer assembly, which contributes to the protein sialylation and secretion deficiency. The intracellular protein mutants could be partially degraded through the proteasome pathway and also formed aggregates. The treatment of the cells with 4-PBA resulted in reduced aggregation and rescued the secretion of mutant SP-A2.