Cationic Albumin Conjugated PLGA Nanoparticles For C-Myc SiRNA Gene Therapy Curing C6 Glioma

Blood-brain barrier(BBB) is significantly important for maintaining the stability of the CNS inner environment.Its unique structure could selectively transport most of the nutrition,exclude harmful and metabolic substances.However,this self-protection mechanism restricts CNS entry of therapeutic drugs for medical treatment.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.In order to solve this problem,realizing gene therapy for brain tumor,a novel brain drug targeting system,cationic bovine serum albumin conjugated nanoparticle (CBSA-NP),was developed.The surface modified cationized bovine serum albumin can enhance the nanoparticle brain entrance via absorptive-mediated transcytosis;the nanoparticle served as drug carrier,which can increase the drug delivery capacity and prevent therapeutic drugs from degradation;the preparation selects double-emulsion and solvent evaporation method facilitated macromolecules encapsulation,such as proteins,peptides and genes.The first part described the optimization and preparation of CBSA,and the construction and characterization of CBSA-NP.Single factor and orthogonal experiments were carried out and then defined the albumin cationic extent standard to be pI value around 8.7～9.0,and optimized the preparation formulation of CBSA,the pI value shifted to 8.9 after optimization,higher than the previous pI value 8.5. MAL-PEG-PLGA and MPEG-PLGA were mixed at ratio 1:10,double-emulsion and solvent evaporation method was used to prepare PLGA-NP;this PLGA-NP was covalently conjugated with thiolated CBSA via its maleimide functional group to form the resulted CBSA-NP.The volume based particle size of CBSA-NP was about 150 nm,its Zeta potential was around +10 mV,which is significantly higher than our previous Zeta data(-10 mV).Immuno-gold staining result showed that CBSA was covalently coupled to the nanoparticle's surface.The second part described that brain capillary endothelial cells(BCEC) was selected as the in vitro cell model,the lipophilic fluorescent dye,6-coumarin,serving as nanoparticle probe,was incorporated in CBSA-NP to investigate its brain drug delivery characteristics,the results would be compared with the ones of unmodified NP.The qualitative uptake results demonstrated that the uptake amount of CBSA-NP was significantly more than that of NP.The uptake of two types nanoparticles by BCEC was concentration,time and energy-dependent within certain concentration range.In 37℃,the saturated concentration of both CBSA-NP and NP is 400μg/ml, the saturation time is around 4 h,and to lower the temperature(4℃)would depress cell uptake.Cationic reagents such as free CBSA and PLL would have an impact on uptaking but not significant difference,which indicated the absorptive-mediated transcytosis did not have a site-affinity and could combine to a rather wide range of sections.MTT assay illustrated that there were no significant difference among the CBSA-NP,NP test groups and the normal control group in terms of cell viability, indicating the optimized CBSA-NP was highly safe.6-coumarine using as fluorescent probe of CBSA-NP,NP serving as control,the brain delivery characteristics and systemic bio-distribution have been evaluated. Results of CBSA-NP in mice revealed the C_max of CBSA-NP in cerebrum was 2.3 times higher than that of NP;the brain AUC of CBSA-NP was 1.6 times compared with that of NP;the brain drug targeting index of CBSA-NP,compared with NP,was 2.5 times higher,demonstrating that CBSA-NP processed certain brain targeting ability;the brain clearance rate of CBSA-NP was slower than that of NP,which indicated long retention time in cerebrum.In the mice heart,lungs and kidneys,no substantial accumulation of CBSA-NP and NP was observed and no significantly difference was noticed between the two formulations,however,the distribution of CBSA-NPs in spleen and liver featured a significant reduction when maintained brain targeted function.Thirty min after i.v.6-coumarin-loaded NP and CBSA-NP via mouse caudal vein,most green fluorescence was observed in the cerebral cortex caudate putamen region.In the same visual field,more green fluorescence was detected on the samples from mouse treated with CBSA-NP than that from the NP group.These results suggested that CBSA-NP preferentially crossed the brain capillary endothelium and accumulated in brain parenchyma,meanwhile,did not bring more toxicity and side effect;it was an idle nano-delivery system for brain targeting.To achieve the gene therapy to glioma,in the third part we preferentially designed and constructed plasmid DNAs(pDNA) containing three different siRNA sequences.Lipofectamin 2000 was used to transfect C6 glioma cells in vitro, fluorescent microscopy,Hoechst 33258 and PI double stain and flow cytometry were served to measure the interference efficacy and cell apoptosis rate of the three siRNA sequences.B3:GAATTTCTATCACCAGCAA was selected as the most effective siRNA sequence,and then the corresponding interference pDNA was constructed.The results showed that the B3-siRNA,specifically targeting c-Myc,could effectively induce C6 glioma cell apoptosis.CBSA-NP served as brain targeted drug delivery carrier,encapsulated siRNA interference pDNA targeting c-Myc oncogene,the particle size of CBSA-NP was around 147.7±46.0 nm,Zeta potential was 6.87±0.5 mV,drug loading efficiency was 1.92%,and encapsulated efficiency was 76.7%.Agarose gel electrophoresis analysis indicated that the preparation formulation did not destroy the structure of pDNA;an acid environment caused by PLGA degradation could not affect pDNA stability; CBSA-NP could protect pDNA from enzyme degradation.CBSA-NP-siRNA gene therapy system was conducted to transfect C6 glioma cells in vitro,which revealed higher transfected efficiency and control released ability comparing with those of NP-siRNA system.Laser scanning confocal microscope observation indicated CBSA-NP could rapidly escape from late-endosomes and lysosomes to the cytoplasm, mediating pDNA enter nuclei.All the facts demonstrated CBSA-NP served as gene delivery carrier could enhance brain entry and targeting glioma cells,siRNA technology was used at the same time to silence c-Myc oncogene,inducing tumor cell apoptosis and necrosis.We established the brain glioma model of C6 lines in mice,and measured the tumor volume,the expression of glioma specifically representative protein S-100βand tumor cell apoptosis rate in order to evaluate the CBSA-NP-siRNA gene therapy efficacy and the performance in inhibiting cancer cell proliferation.Results showed that,after a week tail vein injection treatment,the tumor volume of CBSA-NP-siRNA group was 9.64±14.97 mm³,significantly smaller than the volume of control group, which was 97.62±20.12 mm³;the amount of S-100βexpression of CBSA-NP-siRNA group reduced dramatically;flow cytometry detection for the cancer cell apoptosis rate of the CBSA-NP-siRNA group was 29.77±3.05%,much higher than the rate of the two control groups and NP-siRNA group.All results revealed that CBSA-NP-siRNA was a much more effective gene delivery system for brain glioma targeting;CBSA-NP supplied an alternative and efficient method to realize the brain targeted delivery of therapeutic gene for curing glioma disease.