Preparation Of Novel Brain-targeting Cyanoacrylate Nanoparticles For Gene Delivery In Glioma

Objective Novel nano-drug delivery system is an important research area in the development of nucleic acid drugs.Many works aimed to:reduce the phagocytiosis of the nanoparticles by the reticuloendothelial system,prolong the circulation time of the drug,enhance the targeting,et al.In this paper,we propose a novel type of brain-targeting cationic nanospheres based on alkoxy cyanoacrylate.The alkoxy side chain might endow the nanospheres with good hydrophilic properties,together with the polysorbate-80 modification,this work aimed to develop novel"invisible"nano-microspheres.Our nano-microspheres were supposed to slow down the drug release,extend the systemic circulation time and reduce reticuloendothelial system phagocytosis,and improve drug bioavailability,et al.Two alkoxy cyanoacrylate were synthesized,whichare2-methoxyethyl-2-cyanoacrylateand2-?2-methoxyethoxy?ethyl 2-cyanoacrylate,a traditional n-butyl cyanoacrylate was used as control.Totally six homopolymeric or co-polymeric nanospheres(NS_1-6)were prepared.An antisense oligonucleotide?ASON?targeting to tumor cells transforming growth factor-?2 were used as model drug to investigate:the protection and delivery effect for ASON,anti-glioma activity,and ability to pass through the blood-brain barrier.Methods Six kinds of blank poly cyanoacrylate nanospheres(NS_1-6)were prepared by the optimized emulsification polymerization method using DEAE-Dextran as cationic stabilizer.ASON-NS_1-6-6 was prepared by adsorption method,and polysorbate-80 was used as a brain-targeting modification.The morphology was observed by transmission electron microscopy?TEM?.The average particle size and zeta potential were determined by dynamic light scattering?DLS?.The ultraviolet spectrophotometry was used to determine the entrapment efficiency and drug loading.Agarose gel electrophoresis was used to analyze the optimal loading ratio of ASON-NS,and also the protection of ASON in Dnase I and serum containing environment.The release rate of ASON was determined by dialysis.The cytotoxicity on L929 cells and the anti-tumor activity in vitro on A172 and U251 cells were evaluated by MTS.The apoptosis rate and the Cellular uptake of ASON-NS_1-6-6 on A172 and U251 cells was detected by flow cytometry?FCM?and confocal scanning laser microscope?CSLM?.The phagocytosis by macrophage RAW264.7 was detected by FCM and CSLM.The expression of TGF-?2 m RNA was detected by RT-PCR.The animal model of zebrafish was used to investigate the ability of NS_1-6 to transport ASON through the blood-brain barrier?BBB?.Results TEM showed that NS_1-6 was in round shape with no adhesions to each other,and the average particle size was about 50-80 nm.The average particle size of NS_1-6 by DLS was?49.71±3.56?nm,?110.50±2.01?nm,?79.04±4.33?nm,?67.53±8.29?nm,?91.48±5.60?nm and?136.60±1.93?nm respectively.The Zeta potential of NS_1-6-6 was about 30mV and the potential was about 20mV after ASON loading.The entrapment efficiencies of NS_1-6were?78.84±1.50?%,?81.16±1.51?%,?83.14±1.90?%,?85.82±1.35?%,?89.34±2.35?%,?89.21±2.82?%,?11.59±0.56?%,?15.19±0.72?%,?12.46±1.18?%and?14.74±0.58?%,respectively.The results of stability test of serum and DNase I showed that NS_1-6 had a certain protective effect on ASON.The results of cellular uptake by A172 and U152 showed that ASON-NS_1-6 can achieve well cell uptake,and the ASON-NS₃ was the best one.ASON-NS₃ also showed the lowest phagocytosis by RAW264.7 cell.ASON-NS_1-6 has well anti-tumor activity and low L929 cytotoxicity,in which ASON-NS₃ showed the best tumor inhibition.Fluorescence quantitative PCR results showed that ASON-NS_1-3 reduced the expression of TGF-?2 mRNA.The result of zebrafish BBB model test showed that ASON-NS_1-3 passed through the blood brain barrier effectively,and ASON-NS_2-3 were better than ASON-NS₁.Conclusions In this study,brain-targeting nanospheres based on alkoxy cyanoacrylates were successfully prepared.The structure of alkoxyesters not only reduced the phagocytosis of nanospheres by the reticuloendothelial system,but also endowed nanospheres with good tumor uptake and slow ASON release.This work provide a novel category of brain-targeting vector for clinical delivery of nucleic acid drugs.