Model
Digital Document
Publisher
Florida Atlantic University
Description
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
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
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