Studies On Construction And Anti-epileptic Evaluation Of Electro-responsive Hydrogel Nanoparticles

Epilepsy is one of the most prevalent neurological disorders. For most patients, antiepileptic drugs are the mainstay of the management of epilepsy. However, the lack of targeting ability of the drug, which results in systemic side effects and drug resistance, makes the complete seizure control failure. Epileptic seizure is the paroxysmal abnormalities synchronization electrical activity in brain neurons. Therefore, constraining the discharge of epilepsy in the local site and suppressing it timely is the key to prevent the seizure. In this dissertation, the angiopep-2-modified electro-responsive hydrogel nanoparticles were prepared based on electrophysiology together with active targeting technology. With the functionalization of homing peptide, the hydrogel nanoparticles targeted to the brain efficiently, and at the beginning of the seizure, the epileptiform discharges were weaken and drug was released quickly, leading to an efficient treatment of epilepsy.The main content of this dissertation are described as below:1. Synthesis and evaluation of electro-responsive hydrogel nanoparticles. The electro-responsive hydrogel nanoparticles were obtained from the soap-free emulsion copolymerization using 2-dimethylamino ethyl methacrylate, sodium 4-vinylbenzene sulfonate (NaSS), styrene as the monomers and N,N’-methylene bisacrylamide as the cross-linker. The obtained hydrogel nanoparticles were positive charged and had an average diameter of about 50 nm to 146 nm. The hydrogel nanoparticles had the characteristics of quickly response to the electric field in water, with the hydrogel nanoparticles swelling and average diameter enlarged, and the degree of enlarged was related to the content of NaSS. With phenytoin sodium (PHT) as a model drug, the PHT-loaded hydrogel nanoparticles were prepared by dry hydrogel nanoparticles swelling in PHT solution. PHT release rate from PHT-loaded hydrogel nanoparticles in vitro was related to average diameter of nanoparticles, and a relatively slow release rate was obtained in PHT-loaded hydrogel nanoparticles of average diameter greater than 100 nm. At the same time, the drug release from the hydrogel nanoparticles had the same characteristics of electric field response. Drug release amount was increased by 0.71-fold at 0.5 h after electric field with 100 μA was applied for 1 min, and the increased ratio was affected by content of NaSS in hydrogel nanoparticles. After the evaluation of the hydrogel nanoparticles, the ideal electro-responsive hydrogel nanoparticles were obtained for further study.2. Synthesis and evaluation of brain targeting electro-responsive hydrogel nanoparticles. To facilitate brain delivery of hydrogel nanoparticles, the angiopep-2 was used as a brain targeting peptide to modify the hydrogel nanoparticles. With acrylate-poly (ethylene glycol)-N-hydroxysuccinimadyl-ester (ACLT-PEG-NHS) added, polyethylene glycol modified electro-responsive hydrogel nanoparticles (ERHNPs) were synthesized. Angiopep-2 modified ERHNPs (ANG-ERHNPs) were synthesized by further modify the hydrogel nanoparticles by coupling the N-hydroxysuccinimadyl-ester of the ACLT-PEG-NHS on the surface of ERHNPs to the amino groups of the angiopep-2. ANG-ERHNPs also responzed to electric field rapidly. ANG-ERHNPs showed low toxicity in bEnd.3 cells and the IC50 was 449.0 μg/mL. ANG-ERHNPs had the characteristics of penetrating the BBB easily, the cumulative transport percent in 4 h was increased by 100%, and this was probably attributed to the combination angiopep-2 of ANG-ERHNPs with specific protein on bEnd.3 cell surface, thus rapid uptaken was obtained on bEnd.3 cells. ANG-ERHNPs were accumulated in rat brain and brain regions after intraperitoneal injection. The pharmacokinetic study showed that the relative bioavailability of PHT-loaded ANG-ERHNPs (ANG-PHT-ERHNPs) vs PHT solution was 1.91 times after intraperitoneal injection in rats, and circulation time of PHT in vivo was significantly prolonged. The tissue distribution results showed that ANG-PHT-ERHNPs had the ability of delivery to brain, and its brain targeting index was 2.56. Moreover, after intraperitoneal injection of ANG-PHT-ERHNPs, PHT was found in epilepsy related brain regions such as cortex, hippocampus, amygdala, cerebellum and brainstem, and the Cmax of PHT in these areas were increased by nearly 1 times vs PHT solution, indicating that the dose of PHT can be reduced when administrated with ANG-PHT-ERHNPs.3. Anti-epileptic evaluation of brain targeting electro-responsive hydrogel nanoparticles Compared with PHT-loaded non-electro-responsive hydrogel nanoparticles (ANG-PHT-HNPs) and PHT solution, an improved anti-epileptic efficacy was obtained in ANG-PHT-ERHNPs in the rat models of electric shock, amygdala kindling and the model induced by pentylenetetrazol (PTZ). The reduced therapeutic dose and extended antiepileptic drug treatment time window were achieved in ANG-PHT-ERHNPs, which have important clinical significance for the treatment of epilepsy. The result of free PHT concentration by microdialysis in epilepsy model induced by PTZ displayed the fast release of PHT from ANG-PHT-ERHNPs and the increased release was positively correlated with the severity of seizure EEG.