Date of Award

Spring 5-20-2023

Degree Type

Dissertation

Degree Name

PhD. Chemistry

Department

Chemistry and Biochemistry

Advisor

Gregory Wiedman, PhD

Advisor

Jessica Cottrell, PhD

Committee Member

Cosimo Antonacci, PhD

Committee Member

Stephen Kelty, PhD

Committee Member

Wyatt Murphy, PhD

Keywords

siRNA, peptides, osteoblast, cancer, cell-surface adhesion proteins, RNA interference

Abstract

Osteoblast-targeting peptides in the treatment of bone disease is a new and novel approach to offering effective treatment of various cancers and can be used in bio-medical, medicinal chemistry and biotechnology applications. By targeting adhesion proteins produced by osteoblast cells, certain cancers which migrate and metastasize to the bone may be more effectively treated. An osteoblast-targeting peptide composed of Ser-Asp-Ser-Ser-Asp (SDSSD) which selectively binds to osteoblast cells via periostin has recently been identified. This peptide was functionalized with polyurethane, generating nanomicelles which encapsulated RNA for the therapeutic treatment of osteoporosis. This study has served as the basis for the research presented in this thesis, where the SDSSD peptide was synthesized and functionalized with polyarginine tails of either 9 or 12 Arginine residues long. These synthesized peptides served to allow the selective osteoblast cell uptake of siRNA encoding for the cell surface adhesion protein, N-cadherin.

Chapter 2 of this thesis highlights the successful synthesis as well as characterization of the SDSSD peptide. Using Solid-Phase peptide synthesis as well as reverse phase high performance liquid chromatography and mass spectroscopy, the identity of each peptide was confirmed to have been made. UV-visible spectroscopy was used to quantify the peptide concentration in solution after the fluorescent tagging of each completed sequence. The synthesized peptide binds specifically to periostin, and this was tested and confirmed by incubating the peptide with osteoblast cell line MC3T3.E1 and testing against a negative control cell line (ATDC5). When incubated with the ATDC5 cells, no peptide binding was observed via flow cytometry, but when the MC3T3.E1 cells were incubated with the peptides binding was confirmed. On average about 30% of the MC3T3.E1 cells in a given cell population were shown to be expressing periostin on their surface. The cells were also run on flow cytometry after incubation with a periostin binding antibody, and similar results were found as compared to the cell populations incubated with peptide, confirming the expression of periostin on the cell surface as well as the success of the peptide in binding to its intended target. Finally, to examine uptake of the peptide into the cells, flow cytometry was once again performed after incubating cells with peptide, and splitting this population to incubate half with Trypan Blue and half without. The Trypan blue would quench any external FITC signal, while not affecting anything internalized by the MC3T3.E1 cells. The results showed obvious shifts between both samples, where the majority of FITC labeled peptides appeared to have been taken into the cells. These results indicated that the peptides should serve as suitable delivery vehicles for the siRNA.

In Chapter 3 of this thesis the synthesis of the siRNA sequence is examined as well as the success of peptide:siRNA complexation and stability. the N-cadherin siRNA was successfully synthesized as well as purified, but yields were unreliable, and the process was inefficient. As a result, the siRNA was purchased to ensure consistent yield and to save time, as there was no way to purify the siRNA on a large scale in-house. The proper ratio to successfully complex the peptide to the siRNA was tested and found to be a 1:1 mole ratio of peptide to RNA. This was tested by combing peptides in various amounts to siRNA held constant, running a gel and staining to detect the presence of free RNA. A 1:1 ratio was the lowest ratio where no free RNA was detected. This was then used moving forward for all other gels as well as complexations. The gel results to test stability and displacement of the siRNA from the complex showed that the peptide:siRNA complexes were very stable, even in the presence of FBS at various time points, and the results showed that the peptide bound very tightly to the siRNA. Furthermore, heparin displacement assays did indicate that the release of the RNA from the peptide was possible, sometimes at very low concentrations of 0.1:1 (heparin:complex), and consistently at ratios of 5:1 and 10:1. These results suggested that the complexes perhaps would release RNA into the cell as desired and remain stable long enough to do so. In addition, TEM and DLS results showed that the complexes formed aggregates and exhibited particle sizes slightly larger than desirable for cell entry for the RNA duplex as well as the FITC-Ahx-R9:siRNA samples but formed less aggregates and more defined particles for both FITC-Ahx-R12:siRNA and FITC-Ahx-SDSSD-Ahx-R9:siRNA samples. This was promising as the less aggregation with the addition of the periostin targeting portion (SDSSD) indicated that the peptide:siRNA complxes would have a good chance of being taken into the cells to induce RNAi.

In Chapter 4 the effectiveness of the complexes at silencing the expression of N-cadherin was examined. The results indicated that N-cadherin expression was being silenced as expected, when cells were incubated with the peptide:siRNA complexes. These results were confirmed by qPCR. In addition, the peptides exhibited low cytotoxicity when incubated with cells for 1 hour and examined by flow cytometry and Annexin V eFluor450/7-AAD.

Finally, Chapter 5 of this thesis will briefly conclude and summarize the results obtained and examine the option for future work pertaining to the experiments performed.

Download

Share

COinS