Investigating Intracellular Pathways of Surface-Functionalized Cationic Lipid-DNA Nanoparticles using Quantitative Fluorescence Microscopy
- Degree Grantor:
- University of California, Santa Barbara. Physics
- Degree Supervisor:
- Cyrus R. Safinya
- Place of Publication:
- [Santa Barbara, Calif.]
- University of California, Santa Barbara
- Creation Date:
- Issued Date:
- Biophysics and Materials science
- Image Analysis,
- Dissertations, Academic and Online resources
- Ph.D.--University of California, Santa Barbara, 2015
The delivery of functional nucleic acids has the potential to treat the large number of diseases that stem from missing, defective or over expressed genes. A major challenge in the development of gene therapy-based treatments is the efficient delivery of nucleic acids to the proper tissue or cell type. One promising approach in gene therapy is the implementation of a vector or carrier capable of actively targeting and delivering the therapeutic nucleic acid to the afflicted cells.
In this work, the structural and biological properties of peptide-grafted, lipid-based nanoparticles for the delivery of nucleic acids are investigated using cryo-electron microscopy, light scattering and fluorescence microscopy coupled with software-assisted automated image analysis. Cryo-electron microscopy and light scattering show that lipid- DNA nanoparticles can coexist at equilibrium with unexpected structures such as thread-like micelles and DNA-tethered nanoparticles when mixed in the appropriate conditions. Quantitative image analysis of fluorescent micrographs shows that grafting of targeting peptides to lipid nanoparticles promotes their internalization in cells but the major bottleneck to efficient nanoparticle-mediated delivery of nucleic acids is endosomal entrapment. Endosomal pathways are a means for cells to internalize and deliver cargo to the appropriate intracellular location.
Using members of the Rab GTPase family to label various stages of the endocytic pathway, we investigated the properties of lipid-DNA nanoparticles that influence their intracellular trafficking and efficiency. Studies using wildtype Rab5 and mutant Rab5-Q79L show that escape from the endosomal pathway occurs downstream of early endosomes. Using markers for late and recycling endosomes, Rab 7/9 and Rab11, respectively, we show that nanoparticles with high membrane charge density and cholesterol are preferentially trafficked through the degradative pathway which is conducive to efficient nucleic acid delivery. This conclusion is further supported by the improved efficacy of pH-sensitive nanoparticles capable of shedding their PEG layer and escaping endosomes in the low pH environment of the late endosome.
Lastly, we used fluorescence microscopy and quantitative image analysis to investigate the effect of targeting peptide (iRGD, cRGDfK, linear RGD or RPARPAR) on the trafficking and efficiency of nanoparticles. These results should guide future design of lipid nanoparticles for nucleic acid delivery applications.
- Physical Description:
- 1 online resource (309 pages)
- UCSB electronic theses and dissertations
- Catalog System Number:
- Ramsey Majzoub, 2015
- In Copyright
- Copyright Holder:
- Ramsey Majzoub
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