Delivery Of SiRNA Using Folate Receptor-targeted PH-sensitive Polymeric Nanoparticles For Rheumatoid Arthritis Therapy

Rheumatoid arthritis（RA）is an autoimmune disease,which seriously affects the living quality of patients,even threatens the life of patients.Small interfering RNA（siRNA）is able to specifically silence gene expression via RNA interference（RNAi）.RNAi-based therapy offers a novel approach for the treatment of RA.However,siRNA is hard to enter the cell due to its structural characteristics.Moreover,siRNA is easily degraded by serum nucleases during delivery.Therefore,a safe and efficient delivery system is vital for clinical application of siRNA.In this paper,the cationic phospholipid（DOTAP）was used to form the siRNA/DOTAP lipoplexes by combining with Mcl-1 siRNA through electrostatic interactions,whichwasdesignedfortheprotectionofsiRNA.Poly((cyclohexane_86.7%,1,5-pentanediol_13.3%)-1,4-diyl acetone dimethylene ketal)（PK3）was selected to achieve the acid sensitivity of polymeric nanoparticles,shield the positive charge of DOTAP and further protect the siRNA,which was synthesized by our laboratory.Due to the folate receptorβoverexpressed on the surface of macrophages in RA inflamed site,folate-polyethyleneglycol-poly（lactide-co-glycolide）（FA-PEG-PLGA）was selected to achieve the folate receptor targetability of polymeric nanoparticles.Targetability and inflammatory treatment effects were also investigated.The main content includes the following parts:1.Synthesis and characterization of PK3PK3 was synthesized through the ketal reaction among 1,4-cyclohexanedimethanol（CDM）,1,5-pentadiol（1,5-PeD）and 2,2-dimethoxypropane（DMP）by controlling the molar ratio of CDM and 1,5-PeD as 86.7%and 13.3%.Properties of the synthesized polymer were determined by nuclear magnetic resonance hydrogen spectroscopy（¹H NMR）,fourier transform infrared spectroscopy（FTIR）,gel permeation chromatography（GPC）and differential scanning calorimeter（DSC）.2.Establishment of in vitro analytical method of siRNAFor establishing the in vitro analytical method of siRNA,Cy5 labeled siRNA was used and fluorescence intensity was determined for analyzing the content of siRNA.The excitation wavelength and emission wavelength for fluorescence detection were 650 nm and 670 nm,respectively.The method had an accuracy of98%¹02%and RSD less than 2%,which met the requirements of accuracy and precision.3.Preparation and evaluation of folate PK3 polymeric nanoparticlesPK3,PLGA,and FA-PEG-PLGA were selected to prepare the polymeric nanoparticles.The size,polydispersity index（PDI）and zeta potential were chosen as the evaluation index for optimizing the formulations of nanoparticles.Polymeric nanoparticles had a particle size of 142.6±0.61 nm,a PDI of 0.112±0.020 and a zeta potential of 3.6±0.43 mV.In vitro stability and release studies indicated that the nanoparticles had good stability and pH-sensitivity.Cellular uptake and cytotoxicity studies indicated that the nanoparticles significantly enhanced uptake efficiency and cytotoxicity of siRNA to lipopolysaccharides（LPS）activated RAW264.7 cells（P<0.001）,compared with siRNA group.In vivo distribution studies showed that nanoparticles effectively delivered siRNA to inflamed site and prolonged in vivo circulation time of siRNA,compared with rats injected with siRNA.In vivo pharmacodynamic study indicated that nanoparticles significantly relieved the inflammation of rats（P<0.001）,compared with rats injected with siRNA.In conclusion,folate receptor-targeted and pH-sensitive nanoparticles are a promising drug carrier for siRNA delivery for rheumatoid arthritis therapy.