Preparation Of Galactose Modified SiRNA Nanomedicine For Alzheimer's Disease Therapy

Alzheimer's disease(AD)is an irreversible,progressive neurodegenerative brain disease,while unfortunately there is no effective solution for its therapy.In recent years,small interfering RNA(si RNA)have attracted broad attentions in the treatment of diseases due to their excellent specific gene silencing effect.However,the challenges in si RNA delivery such as the low blood-brain barrier(BBB)penetration and poor blood stability limit the application of si RNA for AD therapy.Herein,we constructed a galactose-modified polymeric si RNA delivery systems for AD therapy in this thesis.The polymeric carrier can interact with si RNA to formulate blood-stable nanomedicine,by triple-interactions(electrostatic,hydrogen bond and hydrophobic)stabilization.Moreover,the galactose on the surface of the nanomedicine is able to recognize glucose transporter type 1(Glut1)of the BBB,and subsequently facilitate the delivered si RNA nanomedicines crossing of BBB.After the encapsulation of si RNA that can specific targets ?-secretase 1(BACE1),this galactose modified si RNA nanomedicine(Gal-NP@si RNA)shows promise in AD therapy.First,galactose modified poly(ethylene glycol)-block-poly[(N-(3-methacrylamidopropyl)guanidinium(Gal-PEG-b-P(Gu))and poly(ethylene glycol)-block-poly[(N-(3-methacrylamidopropyl)guanidinium-co-2,2,3,3-tetrafluoropropyl methacrylate](PEG-b-P(Gu F))were synthesized by reversible additionfragmentation chain transfer(RAFT)polymerization.This poymeric delivery vehicle interactedwith si RNA via guanidino-phosphate(Gu+/PO34-)salt bridge(electrostatic and hydrogen bonding interaction),and then further stabilized by hydrophobic interaction of fluorine group.Gel electrophoresis analysis showed that the Gal-PEG-b-P(Gu)/PEG-b-P(Gu F)polymer mixture can effectively encapsulate si RNA over the mole ratio of 2.5:1,while fluorine-free Gal-PEG-b-P(Gu)polymers can completely load si RNA at the mole ratio of 10:1.Moreover,fluorinated nanomedicines showed better performance in stability assays compared to fluorinefree counterparts in the negatively charged biomacromolecule heparin competition assay,signifying the importance of fluorination to improve the stability of si RNA nanomedicines.Dynamic light scattering(DLS)and transmission electron microscopy(TEM)characterizations showed that the Gal-NP@si RNA exhibited a spherical morphology with an average size of 118 nm and a low polydispersity index(PDI)of 0.13.Cell viability experiments showed that at the si RNA concentration of 400 n M,the cells incubated with nanoparticles exhibited good cell viability,indicating the synthesized polymer nanoparticles had good biocompatibility and no obvious toxicity.Flow cytometry and confocal laser microscope imaging demonstrated that,the nanoparticles can be efficiently taken up by cells.The q RT-PCR experiments at the cellular level showed that Gal-NP@si RNA loaded with si RNA targeting si BACE 1(si BACE1)exerted potent gene silencing effect,achieved 46% down-regulation at m RNA levels.The effective down-regulation was also abserved at protein levels.In contrast,nanoparticles loaded with control si RNA(Scramble si RNA,si Scr)failed to reduce BACE1 expression,confirming the sequence-specific gene silencing activity of si BACE1.In vivo pharmacokinetic experiments showed that fluorinated si RNA nanomedicine(Gal-NP@si RNA)had the longest blood circulation time with an elimination half-lifetime(t1/2)of 39.2 min,which was significantly longer than that of both the fluorine-free counterpart and free si RNA(t1/2 21.9 and 8.0 min,respectively),further confirming the excellent stability of fluorinated si RNA nanoparticles.Subsequently,the biodistribution of Gal-NP@Cy5-si RNA was quantified by fluorometry.These experiments demonstrated that the brain accumulation of Gal-NP@Cy5-si RNA nanomedicine was up to 5.8-fold higher than that of non-galactose modified NP@Cy5-si RNA nanocarriers,signifying the importance of galactose chemical modification for nanoparticles to cross the blood-brain barrier.To further assess the biocompatibility and systemic response to the nanomedicine,we assessed routine blood parameters and chemistry.These examinations demonstrated no significant difference between PBS and Gal-NP@si RNA treatment groups,indicating the biocompatibility and low toxicity of Gal-NP@si RNA nanomedicine.The therapeutic effect of the nanomedicine on the APP/PS1 double transgenic mouse model was evaluated by behavioral tests.Experimental nesting data showed that Gal-NP@si BACE1 treated APP/PS1 mice achieved a similar score to WT mice,which was much better than all other APP/PS1 control groups.Subsequently,after behavioural tests were completed,mice were sacrificed,and brain tissue was collected for analysis of BACE1 suppression by q RT-PCR and western blot experiments.The results showed that the BACE1 of hippocampus and cortex for Gal-NP@si BACE1 treated APP/PS1 mice were significantly decreased both in m RNA and protein levels.In summary,we developed a glycosylated “triple-interaction” stabilized polymeric si RNA nanomedicine(Gal-NP@si RNA),compared with the conventional si RNA nanomedicine which solely electrostatically stabilized,the “triple-interaction” stabilized nanomedicine has superior blood stability.By recognizing Glut1 on BBB,the si RNA nanomedicine can effectively penetrate the blood-brain barrier,effectively silence the BACE1 gene and reduce BACE1 protein expression.Moreover,the nanomedicine exhibits excellent biocompatibility and does not cause toxic or side effects in the kidney or liver.These results indicate that our Gal-NP@si RNA nanomedicine has great potential for AD treatment.In addition,the delivery vector can also be used to deliver therapeutic nucleic acids,including ASO and m RNA,for the treatment of a wide range of central nervous system diseases.