| Blood-brain barrier (BBB) restricts brain entry of drugs such as chemical compounds, peptides and proteins and gene drugs. Therefore, the research of enhancement of brain drug delivery, especially development of novel drug delivery system for brain drug targeting, is on focusing in pharmaceutics. Thereinto, lipidization, chemical delivery system and carrier-mediated transport were applied to enhance the brain drug uptake. However, these methods were required to directly modify the drug structure. Therefore, it was faced with stringency in physico-chemical characteristics. A murine anti-rat transferrin receptor monocloncal antibody was used as brain targetor, which was coupled with drug carrier such as liposome. This immunoliposome penetrated into brain via receptor-mediated transcytosis. Nevertheless, it required genetic engineering techniques to make humanized fusion antibody, which was very complicated.So solve this problem, a novel brain drug targeting system, cationic bovine serum albumin conjugated stealthy nanoparticulate system (CBSA-NP), was developed. The surface modified cationized bovine serum albumin (CBSA) can enhance the nanoparticle's brain entrance via absorptive-mediated transcytosis; the nanoparticle served as drug carrier, which can increase the drug delivery capacity; the preparation selected as double-emulsion and solvent evaporation method facilitated macromolecules such as proteins, peptides and genes, which can overshade physico-chemical their characteristics instead of nanoparticle properties; the stealthy pegylated nanoparticle can avoid the uptake by mononuclear phagocytic system, and prolong their plasma half-life, and enhance area under the concentration-time curve (AUC).The first part described the construction and characterization of CBSA-NP. The pegylated polylactic acid (PLA) nanoparticle whose surface was co-modified with maleimide-poly(ethyleneglycol) (Maleimide-PEG) and methoxy- poly(ethyleneglycol) (MPEG), was made by double-emulsion and solvent evaporation method and covalently conjugated with thiolated CBSA via its maleimide functional group. The number based particle size of CBSA-NP was below 100 nm, its zeta potential wasaround -10 mV. X-ray photoelectron spectroscopy and immuno-gold staining result showed that CBSA was covalently coupled to the nanoparticle's surface. Isotope techniques detected the surface CBSA number per nanoparticle with its component ratio of maleimide-PEG-PLA to MPEG-PLA 1: 20, 1: 10 and 1: 5, was 33.2, 109.9 and 372.0, respectively,To evaluate the brain delivery characteristics and toxicity of CBSA-NP, the second part described that a coculture of brain capillary endothelial cells (BCEC) and astrocytes both from rat was established as an in vitro BBB model. Scanning electron micrograph and transmission electron micrograph showed that BCEC formed tight junction and the astrocyte spread its foot-process across the membrane pore to contact BCEC in consistence with BBB structure morphologically. Transendothelial electric resistance and permeability coefficient of I4C-sucrose were 313 + 23 Q-cm2 and 0.94><10"3 cm/min, respectively, accordingly.The third part described a lipophilic fluorescent dye, 6-coumarin, serving as nanoparticle probe, was incorporated in CBSA-NP to investigate its brain drug delivery characteristics. The uptake result demonstrated that the uptake amount of CBSA-NP was significantly more than that of neutral bovine serum albumin (BSA) conjugated nanoparticle (BSA-NP) at 30, 60 and 120 min. The time-, concentration-and temperature-dependant uptake of CBSA-NP showed the uptake amount of CBSA-NP was 2 times higher than that of BSA-NP. The transendothelial experiment in BBB model showed that the permeability of 10 ug/ml CBSA-NP was 7.76 times higher than that of BSA-NP. However, its transport can be inhibited by free CBSA, which proved CBSA-NP can transport across BBB via the absorptive mediated transcytosis attributed to its surface CBSA. MTT assay illustrated that there were no significant difference between CBSA-NP and pegylated nanoparticle (NP) without CBSA. The unchanged transendothelial permeability coefficient of l4C-sucrose in appearance of 200 ug/ml CBSA-NP validated that CBSA-NP cannot impact the BBB tight junction.The fourth part described that NP serving as control, the bio-distribution result of CBSA-NP in mice revealed the AUC of CBSA-NP in cerebrum was 2.26 times higher than that of BSA-NP, while the blood AUC of CBSA-NP was its 80%. The brain drug targeting index of CBSA-NP, compared with NP, was 2.8 times higher. The pharmacokinetic parameters showed CBSA-NP preferentially penetrated into brain. Thirty minutes after a dose of 60 mg/kg CBSA-NP or BSA-NP injection in micecaudal vein, fluorescent microscopy of brain coronal sections revealed a higher accumulation of CBSA-NP in the lateral ventricle, third ventricle and periventricular region than that of BSA-NP. Immunostaining of monocyte-macrophage demonstrated that high dose of CBSA-NP cannot induce the increase amount of macrophage in cerebrum, cerebellum, heart and lung in BALB/c mice, only had light toxicity to liver, spleen and kidney, and its acute toxicity to liver and kidney was transient. Derived from the above in vitro and in vivo experimental results, we can conclude that CBSA-NP was a novel drug delivery system for brain delivery and with low toxicity.To achieve the targeting therapy to glioma, CBSA-NP served as brain drug delivery carrier. The chemotherapeutical drug, aclarubicin, and gene drug, pORF-hTRAIL plasmid were incorporated in CBSA-NP, respectively, and its preliminary evaluation was also made.The fifth part described that CBSA-NP was used as non-viral gene carrier to encapsulate the plasmid pORF-hTRAIL, termed as CBSA-NP-hTRAIL gene therapy system. The characteristic examination proved its number-based particle size was 115.7±13.9 nm; zeta potential was -15.83±1.25 mV; drug loading efficiency was 0.46% and drug encapsulation efficiency was 57.5%. The preparation procedure cannot cause significant fragmentation of plasmid. By using 6-coumarin as the fluorescent probe, the investigation result of uptake of CBSA-NP and NP by C6 rat glioma cells revealed that the uptake of CBSA-NP was mainly mediated by caveolae-coated pits and can be concentration-dependently inhibited by polycationic substances such as CBSA and polylysine. With the fluorescent tracking of intracellular pathway, CBSA-NP can enter into the cytoplasm via endo-lysosomal escape mechanism and quickly deliver hTRAIL plasmid into the nuclei. Enzyme linked-immnuno-sorbent assay result showed high C6 transfection efficiency of CBSA-NP-hTRAIL; Fluorescence activated cell sorting results shew that apoptosis ratio of CBSA-NP-hTRAIL to C6 cell was similar to lipofectamine 2000/hTRAIL, and about 5 times higher than NP-hTRAIL. Twenty-four hours after intravenous administration of CBSA-NP-hTRAIL, the interested mRNA was transducted in the area of lateral ventricle, cerebrum cortex and hippocampus; Forty-eight hours after administration, hTRAIL protein was specifically expressed in the area of cerebrum cortex, hippocampus and third ventricle and periventricular region. Immunostaining of active Caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling results showed that intravenous injection of CBSA-NP-hTRAIL... |
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