Cellulose nanocrystals for targeted drug delivery applications
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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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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. 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


Outline

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


Objective

Objective

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


Cellulose molecular structure

Cellulose Molecular Structure

  • Polysaccharide of a linear chain of linked glucose units


Cellulose nanocrystals

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


Kb cells

KB Cells

  • Human nasopharyngeal epidermal carcinoma cell line

  • Overexpress surface receptor for folic acid as positive control


Synthesis of cnc fitc fa

Synthesis of CNC-FITC/FA


Cnc conjugates preparation

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


Cnc conjugates preparation cont d

CNC Conjugates Preparation (cont’d)

  • CNCs were measured based on the weight concentration

  • Weight concentrations were calculated by gravimetric analysis


Cnc characterization

CNC Characterization

  • UV-vis spectroscopy

    • Measured number of covalently attached FITC and FA


Uv vis spectroscopy

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


Cnc characterization1

CNC Characterization

  • Atomic Force Microscopy

    • Analyzed shape and size of the functionalized CNCs

CNC

CNC-NH2

CNC-FITC

CNC-FITC/FA


Cellular binding uptake assay

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


Fluorescent microscopy fm

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


Fluorescent microscopy results

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


Zeiss lsm 510 nlo

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


Confocal microscopy results

Confocal Microscopy Results

  • CNC-FITC

  • CNC-FITC/FA


Confocal microscopy results1

Confocal Microscopy Results

  • CNC-FITC

  • CNC-FITC/FA


Results

Results

  • CNC-FITC was not uptaken by the cell

  • Cellular uptake of CNC-FITC/FA

    • Folate mediated endocytosis has occured


Conclusions

Conclusions

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


Acknowledgements

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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