The Neuroprotective Effects Of Astaxanthin-loaded Nanoparticles In Early Brain Injury After Experimental Subarachnoid Hemorrhage

Background and purpose: Extensive research have demonstrated that early brain injury(EBI)after subarachnoid hemorrhage(SAH)plays a key role for high mortality and poor prognosis in SAH patients.Thus,timely and effective intervention and treatment in EBI period becomes vital to improve survival and prognosis of SAH patients.Astaxanthin(ATX)is a carotenoid with formidable anti-oxidant and antiinflammatory properties due to its highly unsaturated molecular structure.In basic studies,nowadays,amounts of evidence proved that ATX could alleviate the inflammatory and apoptotic injury significantly after SAH.However,the low stability and solubility of ATX results in poor bioavailability,which markedly hampers its application as therapeutic agent in clinic advancement.To further enhance the application of ATX in clinic,this study proposed to prepare nanoparticles(NPs)coated with ATX and coupled with ligand for targeted delivery,improving the efficiency of medicine.Meanwhile,we conducted comprehensive evaluation for possible neuroprotective mechanism from ATX-NPs.It may provide ATX with theoretical and practical basis,in order for better transformation for advancement of SAH therapy.Methods: The Fe3O4 NPs were firstly synthesized by thermal decomposition method.For the formulation of ATX-NPs,ATX was directly absorbed on the particle surfaces and encapsulated with polyethylene glycol(PEG)by hydrophobic interaction.In order to target neurons,transferrin was effectively conjugated with ATX-NPs by carbodiimide method.In cellular experiments,primary neurons were stimulated by oxyhemoglobin(Oxy Hb)to mimic the SAH environment in vivo for establishing SAH model in vitro and ATX-NPs were employed as treatment regimen.The toxicity of ATX-NPs to primary neurons was detected by spectrophotometric colorimetry.Transmission electron microscopy,flow cytometry and immunofluorescence were used to observe the targeted delivery of ATX-NPs to neurons from each group.Western blot and flow cytometry were used to explore the neuroprotective effects of ATX-NPs on Oxy Hb-stimulated neuronal apoptosis in each group,and compared with that of free ATX.Finally,small living animal imaging technology was used to detect the targeted delivery of ATX-NPs to the blood-brain barrier in mice by tail vein injection.Results: ATX-NPs conjugated with transferrin molecules were successfully prepared with suitable size and stable property by thermal decomposition and carbodiimide method.In cortical neurons in vitro,ATX-NPs exhibited no significant toxicity on neurons after drug release,and ATX-NPs coupled with transferrin could target and accumulate in the cytoplasm of neurons at 12 h after release.In in vitro SAH model,ATX-NPs significantly inhibited neuronal apoptosis after Oxy Hb stimulation,and ATX-NPs conjugated with transferrin at low doses presented the same neuroprotective effects as free ATX at high doses.Finally,in mice after SAH in vivo,intravenous application of NPs coupled with transferrin could assemble in the brain.Conclusions: The results revealed that transferrin-containing ATX-NPs significantly improved the survival of neurons and reduced apoptotic markers after Oxy Hb incubation.Furthermore,compared with free ATX,the neuroprotective effects obtained from ATX-NPs conjugated with transferrin occurred for a better capacity,providing insights for advancement of SAH therapy.With the successful application of ATX-NPs targeting brain in vivo,further studies need to be validated and the underlying mechanism of its exact role in the central nervous system needs to be addressed in the future.