Dual-function Single-walled Carbon Nanotubes As A ’Car’ For Dopamine Delivery Into Brain Of Parkinsonian Mice

Parkinson’s disease(PD)originated from insufficient dopamine(DA)in related brain areas is a common neurodegenerative disorder,second only to Alzheimer’s disease,with 1-1.5%of the population over 60 being affected.Current treatments for PD are limited,partly due to the difficulties posed by the blood brain barrier when delivering drugs including DA to the brain.Up to date,oral administration of levodopa,the precursor of DA,remains the gold standard for treatment of the disease,while it could lead to a decrease in therapeutic effect over the long-term and may result in some side effects of the treatment.Herein we explore the feasibility and efficacy of functional single-walled carbon nanotubes ’car’(SWCNT-PEGs-Lf)carries and target-delivers DA to brain in parkinsonian mice for treatment.This study aimed at the exploration of both feasibility and efficacy of DA carried into brain by single-walled carbon nanotubes(SWCNTs)from the in vitro and in vivo.PC 12 cell line was selected as a model to study 6-OHDA-induced neurotoxicity and PD.The metabolic activity of the PC 12 cells was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)analysis and cell counting kit-8(CCK-8)assay.Lactate dehydrogenase(LDH leakage assay was chosen to examine the integrity of cell membrane.The oxidative stress levels were determined by reactive oxygen species(ROS).For the in vivo part,biomarkers including ROS,GSH,MDA,NF-κB,TNF-α,and IL-1β were used to determine the toxicity to the brain.In addition,Morris Water Maze(MWM)was conducted in this study to investigate the cognitive ability of the mice,and histological observation was applied to determine the pathological change in the brain.SWCNTs are relatively flexible,so they can penetrate the cell-membrane remarkably,with the characteristics including high drug-loading,pH-dependent therapeutic unloading capacities and prolonged circulation half-lives.It has been reported that polyethylene glycol(PEG)-coated SWCNTs could increase the circulation time and thus prolong the concentration gradient of SWCNTs to the brain.Besides,lactoferrin(Lf),as a novel brain targeting ligand,has drawn more and more attentions in recent years.Since an obvious lactoferrin-nanoparticle(Lf-NP)accumulation in striatum,wherein the pharmacological target site of PD,has been reported,a dual modification of PEG and Lf onto SWCNTS was applied and thus a specific ’car’(SWCNT-PEGs-Lf)to carry DA.The results from in vitro study demonstrate that with 20 mol·L-1 DA loaded onto SWCNT-PEGs in addition to 100 μmol·L-1 6-OHDA,the activity of PC12 cells increase significantly(p<0.05),and that the LDH levels and ROS content also significantly decrease(p<0.01).Further,the level of oxidative stress,TNF-α and IL-1β are all reduced significantly in parkinsonian mice and CAR-25 mg·kg-1 DA group in comparison to that in 6-OHDA-lesioned mice with or without saline,as well as the density of tyrosine hydroxylase-immunoreactivity(TH-ir)increased(p<0.01).The toxicity of CAR was investigated in vitro and in vivo,demonstrating that the safe dose of SWCNT-PEGs exposure to PC12 cells was 6.25μg·μ-1 or lower with a higher metabolic activity in comparison to that in control group and the safe dose of SWCNT-PEGs-Lf in the mice experiments was 3.25 mg·kg-1 or less by intraperitoneal injection with a lower level of oxidative stress and inflammatory responses in comparison to that in control group.This study suggests that 25 mg·kg-1 DA loaded onto 3.25 mg·kg-1 CAR can alleviate the oxidative stress and inflammatory responses in parkinsonian mice and increase the TH-ir density in striatum.In conclusion,this study has successfully fabricated dual-modified SWCNTs by both PEG and Lf.A ’car’ thus formed was used to deliver DA across the BBB and target DA to the striatum in the brain of Parkinsonian mice.With DA delivery,the oxidative stress level and inflammation level in mice brain were both effectively decreased,as well as an increase in the expression of TH in striatum and the cognitive level of the mice.Therefore,formation of the ’car’ might open a new horizon for the in situ treatment with drug delivered to the target of certain brain diseases.