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Cellulose Nanocrystals for Targeted Drug Delivery Applications

Cellulose Nanocrystals for Targeted Drug Delivery Applications. S. Oh , 1 S. Dong, 1 Y. W. Lee, 2 M. Roman 1 1 Macromolecules and Interfaces Institute 2 VT–WFU School of Biomedical Engineering and Sciences Virginia Tech, Blacksburg, VA 24061. Research and Investigation in Science

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Cellulose Nanocrystals for Targeted Drug Delivery Applications

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  1. Cellulose Nanocrystals for Targeted Drug Delivery Applications S. Oh,1 S. Dong,1 Y. W. Lee,2 M. Roman1 1Macromolecules and Interfaces Institute 2VT–WFU School of Biomedical Engineering and Sciences Virginia Tech, Blacksburg, VA 24061 Research and Investigation in Science Assessment #7

  2. Outline • Research Objectives • Background • CNCs • KB Cells • CNC Preparation • Chemicals • Reaction Scheme • Characterization of CNC and CNC conjugates • Cellular Binding/Uptake Assay • Fluorescent Microscopy • Zeiss LSM 510 NLO • Results • Conclusions • Future work • Acknowledgements

  3. Objective • This study evaluates the potential of Cellulose Nanocrystals (CNCs) in targeted drug delivery applications

  4. Cellulose Molecular Structure • Polysaccharide of a linear chain of linked glucose units

  5. Cellulose Nanocrystals • Rod-like nanoparticles of cellulose • Average lengths between 100-150 nm • Average diameters between 3-5 nm • Have properties for applications as drug carriers in cancer therapy • Biocompatible, nontoxic, produced from renewable resources

  6. KB Cells • Human nasopharyngeal epidermal carcinoma cell line • Overexpress surface receptor for folic acid as positive control

  7. Synthesis of CNC-FITC/FA

  8. CNC Conjugates Preparation • Sulfuric acid hydrolysis of bleached wood pulp • Surface hydroxyl groups were activated with epichlorohydrin, then aminated with aqueous ammonia • Aminated CNCs were labeled by FITC (fluoresceinisothiocyanate) as fluorescent tracing agent and functionalized with folic acid as targeting agent

  9. CNC Conjugates Preparation (cont’d) • CNCs were measured based on the weight concentration • Weight concentrations were calculated by gravimetric analysis

  10. CNC Characterization • UV-vis spectroscopy • Measured number of covalently attached FITC and FA

  11. UV-vis Spectroscopy • Beer-Lambert Law • A=− • A=measured absorption • I0= incident light at a given wavelength • I= transmitted intensity • Є= constant (molar absorptivity) • C= concentration of the absorbing species • L= pathlength through the sample • All the variables were kept under the same condition except the ε, to compare four different kinds of CNCs

  12. CNC Characterization • Atomic Force Microscopy • Analyzed shape and size of the functionalized CNCs CNC CNC-NH2 CNC-FITC CNC-FITC/FA

  13. Cellular Binding/Uptake Assay • Cells were • Cultured in chamber slide for 24 hours • Treated with nanoparticles for 4, 6 hours • CNC-FITC, CNC-5,6 Carboxyfluorescein, CNC-FITC/FA • Cell membranes were stained with AlexaFluor 633 • Fixed with ethanol • Nuclei of cells were stained with Mounting Medium containing DAPI • Laser Scanning Microscopy was used to analyze uptake of untargeted and targeted CNCs by KB cells

  14. Fluorescent Microscopy (FM) • Light microscope exciting sample at specific wavelength • Sample could be labeled with fluorophore or naturally fluoresceing • Fluorophores in sample absorbs the illuminated light and sample emits longer wavelengths of light (diff. color) • With emission filter, desired wavelength that matches the fluorescence material could be seen

  15. Fluorescent Microscopy Results Nuclei Fluorescence Cell Membrane Overlay Grp 1: CNC-FITC Grp 2: CNC-5/6 CarboxyFluorescein Grp 3: CNC-FITC/FA Grp 4: CNC-FITC/FA

  16. Zeiss LSM 510 NLO • Using pulsed infrared laser to excite a fluorophore in the sample • Overcame the limitations of regular FM • Spatial pinhole eliminates out-of-focus light in specimens • Provides 3-D image from scans at different depth of specimen • In this research, will be used to confirm the location of CNC particles in cell

  17. Confocal Microscopy Results • CNC-FITC • CNC-FITC/FA

  18. Confocal Microscopy Results • CNC-FITC • CNC-FITC/FA

  19. Results • CNC-FITC was not uptaken by the cell • Cellular uptake of CNC-FITC/FA • Folate mediated endocytosis has occured

  20. Conclusions • From this result, folic acid conjugate of CNCs could be used as application for anticancer drug carriers

  21. Acknowledgements • Dr. Maggie Bump • Angie Flynn • Won Hee Lee • Hyung Joon Cho • Summer Undergraduate Research Program (SURP) • National Science Foundation (NSF)

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