The unique chemical, optical and physical properties of inorganic nanocrystals have generated tremendous interest to develop a variety of applications, most importantly as novel probes in biology. Recent developments have advertised them as promising platforms for sensing, drug delivery, imaging cells and tissue, and as diagnostic tools. However, one impediment to achieve these goals has remained the lack of effective means to deliver them into the cytosol of live cells. A variety of techniques have been explored to achieve this goal including receptor mediated internalization, lipid-based transfection, electroporation and viral peptide-mediated delivery. Nevertheless, most if not all those approaches tend to sequester the nanoparticles within endocytic vesicles. This prevents them from reaching intracellular targets, thus limiting their use in cellular studies. Despite several strategies and efforts there is still a need for an easy and reproducible approach to deliver exogenous nanomaterials to cells directly via membrane translocation. In this dissertation, we summarize different approaches to overcome this issue by employing two distinct peptides to deliver nanoparticles into the cell cytoplasm through the plasma membrane. We also describe the use of gold nanoparticles to develop a potential platform for thiol sensing. In Chapter 1, we introduce the optical and physical properties of inorganic nanomaterials including, quantum dots (QDs), gold nanoparticles (AuNPs) and gold nanorods (AuNRs). We further briefly describe their syntheses approach followed by an overview of surface functionalization and bio-conjugation strategies employed to assemble colloidally stable and biocompatible nanoparticle-biomolecule conjugates. This followed by a brief discussion about various recent applications of nanoparticles in biology and biomedicine focused on imaging, biosensing, drug delivery and photothermal therapy. Finally, we conclude by presenting several approaches that have been applied for intracellular delivery of nanoparticles into the cell cytoplasm. In Chapter 2, we characterize the energy transfer quenching of mCherry fluorescent proteins immobilized on AuNPs via metal-imidazole coordination, where parameters such as NP size and number of attached proteins were varied. Using steady-state and time-resolved fluorescence measurements, we recorded very high mCherry quenching, with efficiency reaching ≈ 95-97%, independent of the NP size or number of bound fluorophores (i.e., conjugate valence). We further describe the use of this system to develop a solution phase sensing platform targeting thiolate compounds. This is based on the use of Energy Transfer (ET) as a transduction mechanism to monitor the competitive displacement of mCherry from the Au surface upon the introduction of varying amounts of thiolates with different size and coordination numbers. We then demonstrate that the competitive displacement of mCherry depends on the thiolate concentration, time of reaction and type of thiol derivatives used and also provide a measure for the equilibrium dissociation constant (Kd-1) for these compounds. In Chapter 3, we describe a new quantum dot (QD)-conjugate prepared with a lytic peptide, derived from a non-enveloped virus capsid protein, capable of bypassing the endocytotic pathways and delivering large amounts of QDs to living cells. The polypeptide, derived from the Nudaurelia capensis Omega virus, was fused onto the C-terminus of maltose binding protein that contained a hexa-HIS tag at its N-terminus, allowing spontaneous self-assembly of controlled numbers of the fusion protein per QD via metal-HIS interactions. We illustrate how the efficacy of QD-peptide conjugate uptake by several mammalian cell lines was substantial even for small concentrations (10-100 nM). Upon internalization the QDs were primarily distributed outside the endosomes/lysosomes. We further provide evidence indicating an entry mechanism that does not involve endocytosis, but rather the perforation of the cell membrane by the lytic peptide on the QD surfaces. In Chapter 4, we propose the use of a chemically-synthesized anticancer peptide, SVS-1, as an efficient vehicle to promote the rapid delivery of ligated quantum dots across the cell membrane and directly into the cytoplasm of live cells. We describe the assembly of QD-SVS-1 bioconjugates by functionalizing the QD surface with maleimide groups, which were then subsequently reacted with the N-terminal thiol of a cysteine containing SVS-1 analogue (CGG-SVS-1) to form a stable thioether linkage. We provide epi-fluorescence, confocal microscopy, and flow cytometry data, combined with specific endocytosis inhibition measurements to demonstrate that conjugates stain the cytoplasm, without interactions with endosomes or the nuclei. We have also provided QD-conjugate internalization data collected by live cell imaging as supplemental files. Finally, in Chapter 5, we demonstrate the use of SVS-1 to promote non-endocytic uptake of both small size gold nanoparticles (AuNPs) and larger size gold nanorods (AuNRs) into mammalian cells. We describe the preparation of colloidally stable AuNPs and AuNRs with an amine-functionalized polymer, His-PIMA-PEG-OCH3/NH2, as their capping ligand. Subsequently, the amine groups were utilized for covalent attachment of cysteine terminated SVS-1 (via a thioether linkage) and NHS-ester-Texas-Red dye onto the nanocrystal surface. We further demonstrate nanocrystal staining throughout the cytoplasmic volume of the cells incubated with these conjugates via fluorescence microscopy. We further provide additional endocytosis inhibition experiment results to confirm that physical translocation of these conjugates takes place through the cell membrane independent of endocytosis. / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester 2018. / May 9, 2018. / Includes bibliographical references. / Hedi Mattoussi, Professor Directing Dissertation; Samuel C. Grant, University Representative; Brian Miller, Committee Member; Hong Li, Committee Member; Joseph B. Schlenoff, Committee Member.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_650242 |
| Contributors | Kapur, Anshika (author), Mattoussi, Hedi (professor directing dissertation), Grant, Samuel C. (university representative), Miller, Brian G (committee member), Li, Hong (committee member), Schlenoff, Joseph B. (committee member), Florida State University (degree granting institution), College of Arts and Sciences (degree granting college), Department of Chemistry and Biochemistry (degree granting departmentdgg) |
| Publisher | Florida State University |
| Source Sets | Florida State University |
| Language | English, English |
| Detected Language | English |
| Type | Text, text, doctoral thesis |
| Format | 1 online resource (158 pages), computer, application/pdf |
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