The conjugation of the protein transduction domain (PTD)
from the HIV-1 Tat protein to shell crosslinked (SCK) nanoparticles
is reported as a method to facilitate cell surface binding and
transduction of SCK nanoparticles. Attaching increasing numbers
of peptide sequences to the SCK has been devised as a method
for increasing the efficiency of the cell-penetrating process.
This research provides an assessment of the effects of increasing
peptide derivatization on intracellular localization of the
SCKs as well as their effects on inflammation and cellular apoptosis.
Synthesis and Characterization
PTD was conjugated in (0.005, 0.01, and 0.02) molar ratios,
relative to the acrylic acid residues in the shell, to the SCK
nanoparticles resulting in SCK populations nominally possessing
52, 104, and 210 PTD peptides per particle, respectively.
Apoptosis Assay via Flow Cytometry
In apoptotic cells, phosphatidyl serine (PS) is translocated
from the inner to the outer leaflet of the plasma membrane,
thereby exposing PS to the external environment where Annexin
V can readily bind to it. The cell impermeant dye, 7-AAD is
included in the assay as an indicator of membrane structural
integrity. The respective binding amounts of Annexin V and 7-AAD
are quantified using a two dye fluorescent assay.
Although higher levels of peptide incorporation resulted in
decreased metabolic function as measured by MTT assay, significant
apoptosis was not observed below 500 mg/L for all the samples.
Although toxicity was determined to be negligible, the potential
immunogenic responses of the peptide derivatized SCKs are unknown
and warrants further investigation.
Publications
M. L. Becker, E. E. Remsen, D. Pan, K. L. Wooley
“Peptide Derivatized Shell Cross-linked Nanoparticles.
1. Synthesis and Characterization” Bioconjugate Chem. In
press.
M. L. Becker, L. O. Bailey, K. L. Wooley
“Peptide Derivatized Shell Cross-linked Nanoparticles.
2. Biocompatibility Evaluation” Bioconjugate Chem. In
press.
Biomaterials Group
Polymers Division
Materials Science and Engineering Laboratory
