Drug Delivery System Targeted To Glioma Basing On Low Density Lipoprotein Recetor Related Protein (LRP)-mediated Transcytosis

Glioma is the most frequent primary central nervous system tumor, which represents 80% of brain tumor. Since glioma differs from the other cancers by its diffuse invasion of the surrounding normal tissue and usually placed in important domain of brain, it is impossible to make the complete removal of tumor by the conventional surgical method and tumor recurrence from residual tumors is very possible. Therefore, chemotherapy is very critical among the therapeutic regimens for the treatment of glioma, which directly influence the prognosis and life quality of patients. However, the therapeutic effects of present chemotherapy are very limited, but often causing systemic side effects, because almost all large molecule drugs and more than 98% small molecule candidate drugs are unable to reach the brain tissue due to their poor permeability across the Blood-brain Barrier (BBB). Based on cytobiology and molecular biology, it is promising to overcome the chemotherapy barrier via pharmaceutical method, using nano-size drug deliver system.The main objective of this study was to develop a paclitaxel (PTX)-loaded PEG-PCL nanoparticles (PEG-NP) as a passively targeted drug delivery system to glioma. Furthermore, PEG-NP was modified by conjugating Angiopep-2 (ANG) to form a dual-targeting drug delivery system (ANG-PEG-NP) in the treatment of glioma. ANG-PEG-NP could enhance drug delivery across the BBB as well as for targeting the tumor via low-density lipoprotein receptor-related protein (LRP)-mediated endocytosis, which would provide the theoretical basis for the future studies on targeting treatment of glioma.The five chapters of this dissertation are as follows:(1) PEGylated poly(ε-caprolactone) nanoparticles construction and characterization. (2) In vivo evaluation of PEG-PCL copolymer nanoparticles. (3) Construction of Angiopep-2 conjugated PEG-NP and its brain delivery characteristics evaluation. (4) The glioma target performance study of Angiopep-2 conjugated PEG-NP. (5) The study of dual targeting effect and primary safe of Angiopep-2 conjugated PEG-NP.At first, The PEG-PCL block copolymers were synthesized by ring-opening polymerization ofε-CL and PEG-PCL block copolymers nanoparticles were prepared using the O/W emulsion and evaporation technique. The preparation protocol was investigated on the responses of particle size, drug-loading coefficient (DL%) and encapsulation ratio (ER%). The optimized formulation as following:25 mg/mL block copolymers concentration in dichlormethane,0.5% sodium cholate in water as surfactant, ratio of o/w 1:10,10% PTX administration. The optimized formulation showed a particle size of about 70 nm with neutral potential. The ER% and DL% was 90.4% and 8.2%, respectively. PEG-NP-PTX kept a sustained release behavior. Nanoparticles formation was confirmed by DSC that PTX formulated in nanoparticles was in a disorder crystalline phase of a molecular dispersion or a solid solution state in the polymer matrix. In addition, stability studies implied that introduction of PEG on the surface of nanoparticles significantly increased the stability of nanoparticles. The in vitro cytotoxicity toward C6 glioma cells determined by MTT assay indicated that it was higher or comparable effectiveness of PEG-NP-PTX versus Taxols.Secondly, In vivo evaluation of PEG-PCL copolymer nanoparticles was studied. The pharmacokinetic parameters for PTX in plasma were estimated by compartmental method. The long-circulating property of PEG-NP-PTX was superior to NP-PTX and Taxol. In the case of PEG-NP-PTX, the AUC0â†'t were 1.7 times and 3.8 times higher than that of Taxol and NP-PTX. Compared to the Taxol and NP-PTX, MRT for the formulations of PEG-NP-PTX were significantly increased and CL was significantly decreased. The biodistribution of fluorescence labeled nanoparticles in intracranial C6 glioblastoma bearing mice through non-invasive NIRF imaging in live animals indicated that they could accumulate in brain tumor tissue via EPR effect and the semi-quantitative AUC of PEG-NP group was 4 times that of the NP group. As to the PTX accumulation in plasma and glioma tissue of intracranial C6 glioblastoma bearing mice, PEG-NP-PTX group accumulated significantly increased than NP-PTX group and Taxol group. However, compared with NP-PTX and Taxol, PEG-NP-PTX group did not accumulated significantly increased in normal brain tissue. PEG-NP-PTX treatment effectively prolonged survival in intracranial C6 glioblastoma bearing mouse model. Thirdly, Angiopep-2 modified PEG-NP (ANG-PEG-NP) was prepared by incorporating maleimide into one end of the PEG-PCL copolymer and taking advantage of its thiol group-binding activity to conjugate with Angiopep. The preparation protocol was optimized based on particle size and ANG density on the nanoparticle surface, through which the optimal ratio of Male-PEG-PCL to Me-PEG-PCL around 1:9, maleimide:Angiopep3:1 and the incubation time of 8 h were obtained. The modification of Angiopep did not evidently influence the physico-chemical property of nanoparticles. RBITC was used as fluorescent probe to investigate the in vitro and in vivo brain targeted function of ANG-PEG-NP. The BCEC cellular uptake of ANG-PEG-NP evidently increased and exhibited an energy-dependent mode. The internalization of ANG-PEG-NP was mediated by LRP and then delivered to endosome, which was through clathrin-mediated endocytosis and caveolae-mediated endocytosis. It also did not display evidently cytotoxicity to BCEC. Mouse brain tissue sections revealed a higher accumulation of ANG-PEG-NP in cerebral, lateral cerebral ventricle, third ventricle and hippocampus than PEG-NP, which was significantly competitively inhibited by Angiopep-2 equilibrium in advanced. It indicated that ANG-PEG-NP could enhance drug deliver to brain and showed the target activity to brain.Fourthly, the active targeting to glioma of ANG-PEG-NP was investigated. The U87 MG cellular uptake of ANG-PEG-NP evidently increased and exhibited a time-dependent, energy-dependent and concentration-dependent mode, and could be competitively inhibited by ligands of LRP. The internalization of ANG-PEG-NP was mediated by LRP and then delivered to endosome, which was mainly through caveolae-mediated endocytosis. A number of cellular experimental works such as in vitro cytotoxicity, cell apoptosis, cellular uptake and cell cycle analysis were developed in U87 MG. In vitro and in vivo three diamensions solid U87 MG glioma were also adopted to evaluate the target activity to glioma. These results indicated that ANG-PEG-NP possess obvious glioma active targeting potential through LRP receptor-mediated endocytosis.Fifthly, BCEC-U87 MG co-culture model demonstrated that PTX-loaded nanoparticles modified with Angiopep exhibited a significant potency of transporting across the BBB and targeting the U87 MG cells, which called "dual-targeting effect" in vitro. PEG-PCL nanoparticles modified with Angiopep not only accumulated in glioma bed via EPR effect but also transported across the BBB and then targeted glioma infiltrating margin via LRP receptor-mediated endocytosis on the BBB and glioma cells. Hence, ANG-PEG-NP exhibit potential dual-targeting effect in vivo in intracranial U87 MG glioma tumor-bearing model. PTX-loaded nanoparticles modified with Angiopep treatment effectively prolonged survival in intracranial U87 MG glioblastoma bearing mouse model, which further validate the dual-targeting effect of ANG-PEG-NP. Primary safe study revealed that ANG-PEG-NP did not display any side effects. We suggested that the dual-targeting nanoparticles drug delivery system will be beneficial in treatment of brain glioma.