(216al) Adsorption, Structural Alteration and Elution of Peptides At Pendant PEO Layers

Adsorption, structural
alteration and elution of poly-peptides at pendant PEO layers

Xiangming Wu, Matthew P Ryder, Joseph McGuire,
Karl F Schilke*

A more
quantitative understanding of peptide entrapment and elution from otherwise
protein-repellent polyethylene oxide (PEO) brush layers will provide direction
for development of new strategies for drug storage and delivery. In this work
we recorded selected effects of peptide structure and amphiphilicity
on peptide adsorption into PEO brush layers based on covalently stabilized Pluronic^® F108. In particular we evaluated the
adsorption of poly-L-arginine (PLR),
and the cationic amphiphilic peptide WLBU2 to PEO
brush layers. Each peptide is comprised of 24-30 amino acids with structure
readily controllable between disordered and more ordered forms by varying
solution conditions. Further, while WLBU2 is highly amphiphilic,
PLR is not amphiphilic.

Self-assembled
PEO brush layers were formed by suspension of hydrophobic silica nanoparticles
in F108 solution. Uncoated and F108-coated nanoparticles were then incubated
with PLR or WLBU2 under different solvent conditions for a desired period of
time. Peptide structure in the presence or absence of nanoparticles was
evaluated by circular dichroism (CD). In addition
peptide-nanoparticle suspensions were washed by centrifugation, resuspended in different solvents, and CD spectra recorded
again to detect changes in the amount of peptide remaining on the
nanoparticles, and determine conformational changes associated with the washing
step. Optical waveguide lightmode spectroscopy (OWLS)
was used for direct determination of peptide adsorption and desorption kinetics
on F108-coated sensors under selected solution conditions. In addition, pyrene fluorescence quenching was used to determine the
existence of a hydrophobic inner region (favors peptide adsorption) of
self-assembled PEO brush layers.

For
both PLR and WLBU2 in their disordered states the CD spectra were similar,
independent of the presence of F108-coated nanoparticles. Moreover, CD spectra
indicated complete removal of the peptide from each sample after washing the
F108-coated nanoparticle suspensions with water. On the other hand, when
peptide is in a more ordered form (i.e., possessing α-helix content), CD
spectra showed that considerable α-helical PLR remained on the F108-coated
nanoparticle surface after washing the suspension with the helix-promoting
solvent, while a completely removal of PLR was observed upon washing
suspensions with the solvent favored the disordered form of the peptide, as
shown in Figure 1. Interestingly, peptide α-helix content increased after
location within the brush. Retention of the amphiphilic
peptide in the brush was independent of whether the eluting solvent favored the
structured or disordered form of the peptide. Complementary OWLS evaluation of
adsorbed mass changes within the F108 layer upon washing was fully consistent
with the CD results.

Figure 1. CD spectra of: Poly-L-arginine (PLR) in HClO₄, and in suspension with
F108-coated nanoparticles before and after washing with HClO₄ or
water. The non-amphiphilic
peptide PLR entrapped in the PEO brush layer was partially elutable
when the helix-promoting solvent (HClO4) was used for elution. However, in contact with water
(which favors PLR's disordered, non-adsorbable
conformation), the adsorbed PLR was entirely elutable.

In summary,
an initially more ordered (α-helical)
structure promotes
peptide adsorption into the PEO layer. Further, a partially helical peptide
undergoes a cooperative increase in helicity after entry,
likely due to concomitant loss of capacity for peptide-solvent hydrogen
bonding.  Peptide interaction with the hydrophobic
inner region of PEO chains resulted in entrapment and conformational change
that was irreversible to elution with changing solution conditions in the case
of the amphiphilic peptide.  In contrast, the adsorption and
conformational change of the non-amphiphilic peptide
was reversible. These results indicate that responsive drug delivery systems
based on peptide-loaded PEO layers can be controlled by modulation of solution
conditions and peptide amphiphilicity.