Construction Of PLGA-based Brain Targeted Nanoparticles For Co-delivery Of Doxorubicin And SiRNA And Their Application In Anti-glioma Therapy

Drug combination therapy is one of the new strategies for tumor treatment.Doxorubicin is a traditional chemotherapy drug, but also the substrate ofP-glycoprotein, and the exsitence of P-gp on the brain capillary endothelial cellsurface prevent doxorubicin from penetrating the blood brain barrier and enteringbrain tissues. Gene therapy is a relatively promising stragetry. Many tumors, such asbrain, lung, breast, head and neck cancer, overexpressed epidermal growth factorreceptor (EGFR). The EGFR expression level is closed with the tumor grade, poorprognosis and resistenace to chemotherapy. In this thesis, a multifuncionalPLGA-based nanocarrier capable of co-delivering small interfering RNA (siRNA)aganist EGFR and doxorubicn specifically to glioma was developted andcharacterized. The main content are listed as following:1. Synthesis and characterization of ANG-CHI. Angiopep-2, a brain-targetingligand, was conjugated with chitosan through3-maleimide acid N-hydroxysuccinimide ester (Pr-NHS-Mal). Angiopep-2was succefully grafted to chitosan asqualitatively and quntitatively measured by fourier transform infrared spectroscopy(FT-IR), nuclear magnetic resonance (NMR) and BCA protein assay.2. Preparation of ANG/PLGA/DOX/siRNA targeted drug delivery system.Firstly, Preparation technology of ANG/PLGA/DOX nanoparticles drug system wasopimized through single-factor test, and then it was systemly characterized by usingtransmission electron microscopy (TEM), nano size and potential analyzer,UV-visible spectrophotometer to understand and study the nanoparticle morphology,size, potential, drug loading, encapsulation efficiency, in vitro release characteristics.Finally, ANG/PLGA/DOX/siRNA targeted drug delivery system was obtained byincubating ANG/PLGA/DOX nanoparticels with negative charge EGFR siRNA, andagarose gel electrophoresis showed that EGFR siRNA could be well loaded on thenano-complex.3. Investigation of ANG/PLGA/DOX/siRNA targeted drug delivery system in human astrocytic glioblastoma U87MG. Benzene sulfonic rhodamine B (SRB)method was used to examine the effects of the drug system on U87MG cell viability,and cell uptake of nanocarriers carrying siRNA and DOX was evaluated using laserscanning confocal microscope and flow cytometry. The transfection of the drugsystem, cell cycle and cell apoptosis were analysized by flow cytometriy (FCM). Theresults showed that the drug delivery system exhibited significant antitumor activitythan did a single system, and siRNA and doxorubicin can be simultaneously deliveredinto U87MG cells, resulting in more cell apoptosis.4. Evaluation of the pharmacodynamics and tissue distribution ofANG/PLGA/DOX drug system in mice and its abilty of penetrating the blood brainbarrier. Pharmacodynamics and tissue distribution of the drug system in vivo showedthat the loaded drug could be effectively delivered into the brain tissue.This study involves in the preparation a multifuncional PLGA-based nanocarriercapable of co-delivering small interfering RNA (siRNA) against EGFR anddoxorubicn. After investigation on the preparation technology and characterization ofnanocarrier, in vitro anti-tumor activity, and the pharmacokinetics of nanoparticlesand tissue distribution, it was confirmed that the system was capable of deliveringdrugs into the brain.