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Development of traceable Rituximab modified polymeric micelles for active targeting of B-cell Lymphoma

  • Author / Creator
    Saqr, Asma Ali
  • Abstract B-cell Non-Hodgkin Lymphoma (NHL) accounts for more than 90% of all adult cases of lymphomas and in most cases it is CD20 positive. B-cell NHL is usually treated by combination of conventional chemotherapeutics and the monoclonal antibody against CD20, rituximab. This combination regimen leads to severe side effects reducing the quality of life in lymphoma patients and/or forcing clinicians to use suboptimal doses of the medication. With current treatment, the survival of lymphoma patients is only around 50%. There is a need for the development of more effective and less toxic therapies for NHL. Towards this goal, we pursued development and optimization of traceable CD20 targeted nano-carriers that can stay stable upon dilution in blood and potentially deliver a high payload of conventional chemotherapeutics preferentially to NHL cells, as compared to normal cells that do not express CD20. Such nano-carriers may be used for targeted therapy of CD20 positive malignant cells as well as following the progression of the disease. In this context, preparation of immune-mixed micelles was pursued through incubation of rituximab conjugated poly(ethylene glycol)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG-DSPE) and methoxy poly(ethylene glycol)-poly(ε-caprolactone) (PEG-PCL) with degree of polymerization (DP) of 15 or 22 in the PCL block, or methoxy poly(ethylene glycol)-poly(ε-benzyl carboxylate-ε-caprolactone) (PEO-PBCL) with DP of 22 in the PBCL block. The effect of the hydrophobic block (PCL15, PCL22, and PBCL22) on the thermodynamic and kinetic stability of mixed micelles was then studied by dynamic light scattering. The mixed micelles were made traceable, by introducing Cy5.5 to rituximab, and/or conjugating Cy3 to propagyl capped PEO-PCL or PEO-PBCL. The uptake of plain versus rituximab modified mixed micelles by KG-15 (CD20+) and SUP-M2 cells (CD20-) was then evaluated by flowcytometry at emission wavelengths of 570 and 707 nm, for Cy3 and Cy5, respectively. Paclitaxel was loaded in mixed micelles; and it’s in vitro release was evaluated. The in-vitro cytotoxicity of paclitaxel loaded micelles against KG-15 and SUP-M2 cells was measured by MTT assay. Our data showed mixed micelles composed of PCL22 or PBCL22 to be thermodynamically and kinetically more stable than those with PCL15. Accordingly, rituximab modified mixed micelles containing PCL22 or PBCL22 showed the highest percentage of Cy3/Cy5 double positive association with KG-15 cells. The interaction of the rituximab modified mixed micelles was significantly higher by KG-15 than SUP-M2 cells. Pre-incubation of KG-15 cells with free antibody eliminated this difference, confirming the involvement of CD20 mediated micellar uptake. Paclitaxel release from mixed micelles with PCL22 and PBCL22 core was slower than that of PCL15. The in-vitro cytotoxicity study showed no significant difference in the cytotoxic effect of paclitaxel loaded rituximab mixed micelles compared to paclitaxel loaded in plain mixed micelles in KG-15 and SUP-M2 cells at 24 and 72 h, however, perhaps reflecting the non-internalizing function of CD20 receptor. In summary, the results of this study showed successful development of traceable immune-mixed micelles for active targeting of B-cell NHL based on rituximab-PEG-DSPE and PEO-PCL22 or PEO-PBCL22. Mixed micelles of PEO-PCL15 were found to be not stable enough for this purpose.

  • Subjects / Keywords
  • Graduation date
    Fall 2017
  • Type of Item
    Thesis
  • Degree
    Master of Science
  • DOI
    https://doi.org/10.7939/R34747740
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.