Studies On Glioma By Drug Loaded Polybutylcyanoacrylate Nanoparticles

PartⅠPreparation and drug loading of doxorubicin–polybutylcyano -acrylate nanoparticlesObjective To prepare the doxorubicin–polybutylcyanoacrylate nanoparticles. Methods The DOX-PBCA-NP was prepared using the emulsion polymerization method under the optimal conditions of orthogonal design.The size and size distribution of nanoparticles were measured using Malven laser mastersizer 3000HS, nanoparticle morphology observed by transmission electron microsope.The Drud loading and entrapment efficiency of doxorubicin in the nanoparticles were measured by means of UV spectra at weave length of 481 nm.Results The nanoparticles were discrete and uniform spheres with average diameter of 120nm and 100nm for bare and drug loaded nanoparticles respectively.Conclusion Nanoparticles obtained by emulsion polymerization method were steady and the same size and morphology, which can be provided for the further in vitro and in vivo tests.PartⅡInhibition of SHG44 cells by polybutylcyanoacrylate nanoparticles loaded with Doxorubicin Objective To investigate the effect of polybutylcyanoacrylate nanoparticles loaded with Doxorubicin on the SHG44 cells.Methods The polybutylcyanoacrylate nanoparticles loaded with Doxorubicin were prepared by emulsion polymerization process .The inhibitory rate of SHG44 cells were detected by MTT assay, cell modalities were observed using inverse microscope and the cell cycles determined by flow cytometry. The expression of telomerase protein was measured by immunocytochemical method.Results 1. At three concentration levels of DOX, DOX-PBCA-NP, the inhibitory rate of SHG44 cells were (32.3±0.2)%,(48.7±0.7)%,(67.1±0.4)%% for DOX groups and(45.7±0.2)%,(61.6±0.4)%,(76.2±0.8)% for DOX-PBCA-NP groups, when the concentration of DOX were 2μg/ml, 5μg/ml and 10μg/ml for each group,with PBCA-NP group for (1.6±0.7)%.2. The telomerase protein expression were (86.4±3.7)%,75.6±2.1)% and(69.7±1.3)% for DOX groups, (72.3±1.6)%,63.2±3.1)% and(55.2±0.8)% for DOX-PBCA-NP groups , with the concentration of DOX were 2μg/ml, 5μg/ml and 10μg/ml for each group. 3. Compared with other groups, DOX-PBCA-NP groups displayed higher inhibitory rate, obviously cell modality change and cell cycle variety, reduced telomerase protein expression. Their differences are evidently remarkable(P<0.05).Conclusion Polybutylcyanoacrylate nanoparticles loaded with doxorubicin were more effective on the SHG44 cells than doxorubicin as to cell inhibition .PartⅢPreparation and drug loading of polybutylcyanoacrylate nanoparticles loaded with antisense oligodeoxynucleotide of hTERT mRNA Objective To prepare the hTERT-ASODN-polybutylcyanoacrylate nanoparticles. Methods The hTERT ASODN-PBCA-NP was prepared using the emulsion polymerization method under the optimal conditions of orthogonal design.The size , size distribution and Zeta potential of nanoparticles were measured using Malven laser Mastersizer 3000HS, nanoparticle morphology observed by transmission electron microsope.The drud loading and entrapment efficiency of ASODN in the nanoparticles were measured by means of HPLC method at weave length of 260 nm.Results The nanoparticles were discrete and uniform spheres with average diameter of 120nm PBCA nanoparticles, with drud loading and entrapment efficiency for 71.17% and 95.20%, and +41.3mV for Zeta potential.Conclusion Nanoparticles obtained by emulsion polymerization method were steady and the same size and morphology,with higher drud loading and entrapment efficiency of oligodeoxynucletide in the nanoparticles, and a high positive Zeta potential.PartⅣInhibition of SHG44 cells by polybutylcyanoacrylate nanoparticles loaded with antisense oligodeoxynucleotide of hTERT mRNAObjective: To investigate the effect of polybutylcyanoacrylate nanoparticles loaded with antisense oligodeoxynucleotide of hTERT mRNA on SHG44 cells.Methods: The polybutylcyanoacrylate nanoparticles loaded with antisense oligodeoxynucleotide of hTERT mRNA were prepared using the emulsion polymerization process .The inhibition rate of SHG44 cells were detected by MTT assay, cell modalities were observed using inverse microscope and the cell cycles and intracellular fluorescence intensity determined by flow cytometry after transfection . The expression of hTERT mRNA was measured using RT-PCR method, and expression telomerase protein was measured by immunocytochemical method. Results: Nanoparticles obtained by emulsion polymerizations process were the same size and morphology. Compared with FASODN group ( NP-free ) , the intracellular fluorescence intensity of FASODN-NP group was obviously stronger after transfection of 48h(P<0.01). The telomerase protein expression were (53.1±1.8)%, (56.7±1.5)% and(59.6±4.3)% for ASODN1-NP group, ASODN2-NP group and ASODN3-NP group, with control group for(94.6±1.3)%. The hTERT mRNA expression were 2.23±0.12,2.31±0.14,2.26±0.19 for ASODN1-NP group, ASODN2-NP group and ASODN3-NP group, with control group for 1.45±0.11.The ASODN-NP groups displayed higher inhibitory rate, obviously cell modality change and cell cycle variety, reduced hTERT mRNA and telomerase protein expression than that of ASODN groups(NP-free), SODN group , NP group and control group, their differences are evidently remarkable.Conclusion: Polybutylcyanoacrylate nanoparticles loaded with antisense oligodeoxynucleotide of hTERT mRNA can enter the inside of SHG44 cells, preventing the expression of hTERT mRNA, and inhibit cell proliferation .PartⅤChemotheraphy of glioma nude mice using polybutylcyanoacrylate nanoparticles loaded with antisense oligodeoxynucleotide of hTERT mRNAObjective: To investigate the chemotheraphic effect of polybutylcyanoacrylate nanoparticles loaded with antisense oligodeoxynucleotide of hTERT mRNA on SHG44 cell glioma nude mice.Methods: The polybutylcyanoacrylate nanoparticles loaded with antisense oligodeoxynucleotide of hTERT mRNA were prepared by emulsion polymerization process . The glioma nude mice models were established by means of tumor tissue pieces of SHG44 cells inoculated into BALB/c-nu nude mice. The preparation of drugs were injected i.v. into the tail vein.The expression of hTERT mRNA was measured using RT-PCR method,the telomerase protein and the pathology of the tumor tissue were measured by immunohistochemical method.Results: The glioma models in nude mice were successfully established by means of tumor tissue pieces inoculation.The telomerase protein expression were (92.6±2.7)%,(91.2±3.4)%,(94.7±1.3)%,(90.5±2.6)%,(65.2±2.1)% for control group, NP group, SODN-NP group, ASODN group and ASODN-NP group, and the hTERT mRNA expression were 2.34±0.15, 2.46±0.22, 2.41±0.35, 2.39±0.24, 1.56±0.39 respectively . Compared with other groups , the ASODN-NP group displayed reduced hTERT mRNA and telomerase protein expression,smaller tumor size, their differences are evidently remarkable(P<0.05). The above differences between control group, NP group, SODN-NP group and ASODN group were no statistical meaning.Conclusion: Polybutylcyanoacrylate nanoparticles loaded with antisense oligodeoxynucleotide of hTERT mRNA were chemotheraphic effective to SHG44 glioma nude mice .