| Antisense oligonucleotides(ODN) can undergo Waston-Crick or Hoogsteen hybridization to targeted mRNA or DNA,and inhibit or block the gene expression. In this way,they have the potential to be used as therapeutic agents to control the synthesis of a deleterious protein associated with viral,neoplastic,or other diseases. However,problems such as the poor stability of these oligonucleotides versus nuclease activity in vitro and in vivo and the low intracellular penetration have limited their use in therapeutics. In order to increase their stability,to improve intracellular penetration and to avoid non-specific effect,lots of researchers adopt various vehicles to deliver them. Among these carriers,nanoparticles,biodegradable or not,have shown interesting potentialities to bind and deliver ODN in a stable,high efficiency and low cytotoxicity manner.In the present paper,we prepared the cationic Eudragit RL100 and RSI00 nanoparticles as ODN carrier in order to:a)enhance ODN intracellular penetration;b) improve intracellular distribution;c)improve the stability against nuclease.We prepared the nanoparticles by solvent and non-solvent method. Briefly,Eudragit RL100 or RS 100 dissolved in ethanol,and the nanoparticles formed through adding water slowly during stirring. Further evaporated the ethanol and gotstable nanoparticles. Appropriate concentration ODN was slowly added to nanoparticles suspension,vortexed for 10min,then obtained ODN-NP. The transmission electron microscopy showed that the nanoparticles took on spheres orderly. The z average mean diameter of RL100-NP and RS100-NP determined by PCS were 127.0nm and 231.2nm respectively. The diameter was slightly altered in the present of ODN.The loading efficiency was very high. Mixed with 6.6 fold RL100-NP,almost ODN were adsorbed into nanoparticles. The loading efficiency did not alter in the present of sodium chloride and phosphoric buffer. However,the loading efficiency was reduced by serum and high moleceular weigh heparin,30% serum reduced by 7.2%,and the heparin reduced by 13%.The nanoparticles appeared perfect stability,and could undergo sterilization. Storaged under room temperature for 3 months,the RL100-NP showed no change,and the RS100-NP produced little deposit,but it can disperse homogenously after shaked,and after ultrasound for 5min,the morphology and size showed no difference with the fresh. However the nanoparticle aggregated in the present of salt,culture medium and serum.The ODN release from RL100-ODN-NP in PBS was very slow,release 90% ODN should take 5d. The release profile fit the Hixcon-crowell equation and Niebergull equation very well,the correlation coefficient were 0.9943 and 0.9937,respectively. In the initials stage,it showed obviously burst release phenomenon.Incubating M3 or MGC cells with free FAM-ODN for 8h,there was little intracellular fluorescence. But absorbed by cationic nanoparticles,intracellular fluorescence increased largely. The more NP added,the more intracellular fluorescence enhanced. When the concentration of nanoparticles was 20ug/ml,intracellular ODN was enhanced 456 fold for M3 myeloma cells and 683 fold for MGC cells in the help of RL100-NP,enhanced 395 fold for M3 myeloma cells and520 fold for MGC cells in the help of RS100-NP. The cells uptake FAN-ODN-NP well depended on energy,there was little intracellular fluorescence observed in the cells incubation at 4 ,but incubation at 37 ,there appeared very strong intracellular fluorescence. This suggest FAN-ODN-NP entranced to cell by endocytosis or phagocytosis. Additional,FAN-ODN-NP entrance to cell was influenced by serum. In the present of serum,intracellular fluorescence decrease sharply,10% serum decreased intracellular fluorescence by 10 fold,intracellular fluorescence in the present of 30% serum was 4.6% of serum absent. Furthermore,we inspected the effect of chloroquine on intracellular fluorescence,and found that there was no difference in the present of chloroquine or absent,suggesting after entranced to cells,FAM-ODN were not stay...
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