Dissertation > Excellent graduate degree dissertation topics show

Preparation and Characterization of Anti-HER2 Fab’ Modified PE38KDEL-loaded Nanoparticles for Antitumor Experimental Study in Vitro and Vivo

Author: ChenHuaiWen
Tutor: ZhongYanQiang；LuYing；ZhangZuo
School: Second Military Medical University
Course: Pharmacy
Keywords: PLGA nanoparticles toxin protein HER2 monoclonal antibody targeted delivery
CLC: R730.5
Type: Master's thesis
Year: 2008
Downloads: 157
Quote: 0
Read: Download Dissertation

Abstract


The clinical use of immunotoxins is severely limited by nonspecific toxicity of its toxin protein.To overcome this limitation,PE38KDEL was used as a model protein toxin to prepare PE38KDEL-Ioaded poly(lactic-co-glycolic acid)(PLGA) nanoparticles(PE-NP) by double-emulsion evaporation method.The PE38KDEL-loaded anti-HER2 targeted nanoparticles(PE-NP-HER)were fabricated by conjugation of Fab’ fragments of a humanized anti-HER2 humanized monoclonal antibody(rhHER2-mAb) onto the surface of nanoparticles according to carbodiimide method.The processing parameters of NP preparation were optimized by single factor study and orthogonal design.The pharmaceutical characterization of nanoparticles made with optimized prescription and technic,such as particle size,zeta potential,drug loading,encapsulation efficiency,Fab’ conjugation efficiency and drug release were evaluated.Particle size,zeta potential and morphology of PE-NP-HER and PE-NP were observed by dynamic light-scattering detector(DLS) and transmission electron microscope(TEM).The results showed both NPs were quiet round and homogenous with a 100～130nm average size distribution.And it could be speculated that Fab’ had presented on the PE-NP surface due to the increase in particle size(106nm→125nm) and the change of zeta potential(-35 mV→12 mV).Micro BCA assay was used to determine the drug content,encapsulation efficiency, drug release in vivo of PE-NP and Fab’ conjugation efficiency on the PE-NP-HER.The drug content and encapsulation efficiency of optimized prescription were 7.2±0.3μg/mg and 30.1±1.5%(n=9) respectively,and the accumulative drug release had a burst of 28.3%within 24 h and approached 50%in the first 96 h,the conjugation efficiency was about 19.4μg Fab’/mg NP.The biological activity of the PE38KDEL during the PLGA NP preparation procedure was evaluated by MTT assay.The results indicated that the activity of PE38KDEL was well-protected by protectant in the inner water phase during preparation.Recognition property,cytotoxicity against targeted cell and the antitumor mechanism of PE-NP-HER were studied in vitro experiment.The binding ability of PE-NP-HER(biological activity of Fab’) was demonstrated by flow cytometry(FCM).The binding and internalization of PE-NP-HER to BT-474 cells were analyzed by laser scanning confocal microscopy (LSCM).Cytotoxicity against HER2-over expressed breast cancer BT-474 cells was analyzed using cellular cytotoxicity assays.The FCM result showed PE-NP-HER could recognize and bind to the targeted cell,the biological activities of Fab’ on the NP were well protected.The binding and internalization of targeted NP were detected on the BT-474 cell membrane only after a 5-minute incubation and in the cytoplasm of cells after a 2 h incubation by LSCM. PE-NP-HER had remarkable in vitro cytotoxicity against HER2-overexpressing breast cancer BT-474 cells(Cell viability of 26±3%).Progressively,in developed HER2-overexpressing tumor xenograft model,administration of PE-NP-HER(0.9 mg/kg) showed a much better therapeutic efficacy in inhibiting tumor growth compared with PE-HER and other controls:the final mean tumor volume was 13±6 mm~3(mean±SD;n=8)(P＜0.01).In addition,PE-NP-HER were well tolerated in mice with a much higher MTD(maximally tolerated dose) over the control immunotoxin PE-HER constructed by chemically coupling PE38KDEL to rhHER2-mAb(2.92 mg/kg vs.0.92 mg/kg).Conclusion:The PE38KDEL-loaded HER2-targeted nanoparticles had dramatically reduced systematically nonspecific toxicity(especially hepatoxicity) of immunotoxin PE-HER by PLGA encapsulation.Comparing with PE-HER,PE-NP-HER showed a more remarkable antitumor efficacy both in vitro and in vivo experiment.Targeted NP provided a selective drug delivery to tumor which was conformed in the HER2-overexpressing tumor xenograft model therapeutic experiment.

Related Dissertations

  1. Study on Bispecific Monoclonal Antibody to Pesticide Imidacloprid and Parathion-Methyl,S482.2
  2. Expression and Purification of Influenza a Virus M2 Protein and Detection of Its Antigenicity,R392
  3. Studies on Dynamic Regularity of Antibody Against G. Anatis and Development of Hybridoma Cell Lines Secreting Monoclonal Antibodies Against G. Anatis Lps,S858.32
  4. Development of Hybridoma Cell Lines Secreting Monoclonal Antibodies Against Lps Antigen of B. Melitensis,R392
  5. Epidemiologic Studies on Duck Circovirus Infection in East China and Development of Monoclonal Antibody Against Duck Circovirus,S858.32
  6. Purification of Recombinant Schistosoma Japonicum Protein SjGST and Preparation and Characterization of the Monoclonal Antibody,R392
  7. Cloning, Identification and Eukaryotic Expression of Variable Region of Monoclonal Antibodies Against Chelated Mercury, Copper and Zinc and Three Dimentional Modeling of Recombinant Antibody,X171.5
  8. Development of the Colloidal Gold Immuno-Chromatographyic Strips for the Early Pregnancy Diagnosis of Hu Sheep,S858.26
  9. The Study on the Preparation of McAb to Anti-Melemine and Establishment of Ic-ELISA for Detetion of Melamine,S859.84
  10. Epidermal growth factor in breast carcinoma and its clinical significance 1,2,R737.9
  11. Study on pH-sensitive Liposomes Loaded with Lactosyl-norcantharitin Phospholipids Complex,R943
  12. Preparation of Type HPV16 E7 Protein Monoclonal Antibody,R737.33
  13. 1. TmTNF-α-mediated Apoptotic Signaling Via TNFR2 2. Antibody Preparation and Identification,R392
  14. Monoclonal 18A4 Against AGR2 Inhibit Human Breast Adenocarcinoma Cell Growth and Migration,R737.9
  15. Study on the Epidemiological Characteristics of Angiostrongylus Cantonesis and Protective Effects of Monoclonal Antibody 12D5 Against Angiostrongyliasis,R532.1
  16. ~ (18) F-SFB-Annexin B1 probe Experimental evaluation of apoptosis,R817.4
  17. Preparation and Application of Immunodepleted Plasma for FⅦ: C Assays,R944
  18. New antibody used for immunohistochemical evaluation of breast cancer Met4 Met expression levels of human anti- Met genetically engineered antibody scFv transformation characteristics of,R737.9
  19. Expression and Its Significance of Fatty Acid Synthase (FAS) and HER2 in Esophageal Carcinoma,R735.1
  20. HER2 and EGFR Expression in Breast Cancers and Corresponding Loco-regional Recurrences: Therapeutic Implications,R737.9
  21. Study on the Expression of MUC4 and Its Relationship with Her2/neu, CA125 in Epithelial Ovarian Carcinoma,R737.31

CLC: > Medicine, health > Oncology > General issues > Tumor Therapy
© 2012 www.DissertationTopic.Net  Mobile