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Study of Cell Penetrating Peptide Uptake and Cancer Cell Discrimination with Raman Spectroscopy and Microscopy

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Date Issued:
2016
Summary:
Cell penetrating peptides (CPPs) are short sequences of amino acids that excel in crossing the cellular membrane without inducing cytotoxicity Interest in these peptides stem from their ability to be attached, and grant their penetrating properties to, a variety of cargo In this work we have combined the application of Confocal Raman Microscopy (CRM) and Atomic Force Microscopy for the first time to examine the interactions of unlabeled Transportan (TP), one of the most well studied CPPs, with mammalian cells CRM’s capability to discriminate control and treated cell groups was verified by principal component analysis (PCA) and linear discriminant analysis (LDA) and was 93-100% accurate We’ve determined that at a concentration of 20 μM TP enters cells through a non-endocytotic mechanism, has a high affinity for the cytoplasm and membranes, and results in a significant increase in cellular stiffness Our work provides the first direct evidence of this cell-stiffening phenomenon SFTI-1, the smallest member of a bicyclic, cysteine rich class of CPPs, was examined by CRM to determine the potential role of cyclic structure on cellular uptake The peptide, along with monocyclic and linear analogs was heavy isotope labeled and incubated with mammalian cells at numerous concentrations and timespans Our work is the first SFTI-1 uptake study forgoing the use of fluorophore conjugates, which have been linked to artificial cellular uptake We demonstrate herein the absence of any CRM detectable uptake, providing the first evidence that SFTI-1 may not be a CPP Finally, CRM was applied to the discrimination of normal and basal cell carcinoma cells obtained from the same donor The use of patient matched cells avoids the normal biochemical variations that exist among individuals, ensuring that discrimination is based solely on the cell’s diseased state CRM spectra, analyzed by PCA and LDA, were capable of spectral discrimination with 100% accuracy Major differences in the cancerous cells were an increase in lipids and nucleic acids, and an overall decrease in protein We also demonstrate an enhancement in Raman signal through the use of an aluminum foil substrate, providing a practical approach for measuring cells with thin morphologies
Title: Study of Cell Penetrating Peptide Uptake and Cancer Cell Discrimination with Raman Spectroscopy and Microscopy.
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Name(s): Cosme, Patrick Jason, author
Terentis, Andrew C., Thesis advisor
Florida Atlantic University, Degree grantor
Charles E Schmidt College of Science
Department of Chemistry and Biochemistry
Type of Resource: text
Genre: Electronic Thesis Or Dissertation
Date Created: 2016
Date Issued: 2016
Publisher: Florida Atlantic University
Place of Publication: Boca Raton, Fla
Physical Form: application/pdf
Extent: 124 p
Language(s): English
Summary: Cell penetrating peptides (CPPs) are short sequences of amino acids that excel in crossing the cellular membrane without inducing cytotoxicity Interest in these peptides stem from their ability to be attached, and grant their penetrating properties to, a variety of cargo In this work we have combined the application of Confocal Raman Microscopy (CRM) and Atomic Force Microscopy for the first time to examine the interactions of unlabeled Transportan (TP), one of the most well studied CPPs, with mammalian cells CRM’s capability to discriminate control and treated cell groups was verified by principal component analysis (PCA) and linear discriminant analysis (LDA) and was 93-100% accurate We’ve determined that at a concentration of 20 μM TP enters cells through a non-endocytotic mechanism, has a high affinity for the cytoplasm and membranes, and results in a significant increase in cellular stiffness Our work provides the first direct evidence of this cell-stiffening phenomenon SFTI-1, the smallest member of a bicyclic, cysteine rich class of CPPs, was examined by CRM to determine the potential role of cyclic structure on cellular uptake The peptide, along with monocyclic and linear analogs was heavy isotope labeled and incubated with mammalian cells at numerous concentrations and timespans Our work is the first SFTI-1 uptake study forgoing the use of fluorophore conjugates, which have been linked to artificial cellular uptake We demonstrate herein the absence of any CRM detectable uptake, providing the first evidence that SFTI-1 may not be a CPP Finally, CRM was applied to the discrimination of normal and basal cell carcinoma cells obtained from the same donor The use of patient matched cells avoids the normal biochemical variations that exist among individuals, ensuring that discrimination is based solely on the cell’s diseased state CRM spectra, analyzed by PCA and LDA, were capable of spectral discrimination with 100% accuracy Major differences in the cancerous cells were an increase in lipids and nucleic acids, and an overall decrease in protein We also demonstrate an enhancement in Raman signal through the use of an aluminum foil substrate, providing a practical approach for measuring cells with thin morphologies
Identifier: FA00004756 (IID)
Degree granted: Dissertation (PhD)--Florida Atlantic University, 2016
Collection: FAU Electronic Theses and Dissertations Collection
Note(s): Includes bibliography
Subject(s): Peptides--Analysis
Peptides--Therapeutic use
Peptides--Physiological transport
Cellular signal transduction
Raman spectroscopy
Infrared spectroscopy
Held by: Florida Atlantic University Libraries
Sublocation: Digital Library
Persistent Link to This Record: http://purl.flvc.org/fau/fd/FA00004756
Use and Reproduction: Copyright © is held by the author with permission granted to Florida Atlantic University to digitize, archive and distribute this item for non-profit research and educational purposes Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder
Use and Reproduction: http://rightsstatements.org/vocab/InC/1.0/
Host Institution: FAU
Is Part of Series: Florida Atlantic University Digital Library Collections.