(293g) Endotoxin Binding By the Cationic Amphiphilic Peptide WLBU2 in Relation to Polymyxin B
AIChE Annual Meeting
2013
2013 AIChE Annual Meeting
Materials Engineering and Sciences Division
Biomaterials II
Tuesday, November 5, 2013 - 2:18pm to 2:36pm
Introduction:
Sepsis is a
blood infection that in the US alone affects about 750,000 people each year,
killing 28-50% of them.1-3 Passing blood through a sorbent
device (hemoperfusion) to specifically remove targets such as endotoxin and
bacterial cells holds promise for rapid treatment of acute sepsis. Clinical use
of hemoperfusion in this context is based on immobilized polymyxin B (PMB),4,5 but remains limited owing to ineffective
endotoxin removal, and serious complications (e.g., nephrotoxicity,
neurotoxicity, monocyte stimulation, substantial
protein loss) associated with PMB and PMB-based devices4,6-9. The synthetic, cationic
amphiphilic peptide (CAP) WLBU2 has greater
antimicrobial activity than PMB, works against a much broader spectrum of
Gram-positive and Gram-negative bacteria, and shows higher selectivity
for pathogenic entities over host cells.10,11 In this research WLBU2 was
compared to PMB with respect to its ability to bind endotoxin in the presence and
absence of blood proteins, while in solution or immobilized at surfaces.
Endotoxin binding of WLBU2 while covalently tethered to a surface was also
investigated.
Materials and
Methods: Optical
waveguide lightmode spectroscopy (OWLS) was used to detect adsorption on hydrophilic
and silanized (hydrophobic) SiO2 coated OW2400c waveguides under
steady flow conditions. Vapor-phase silanization was carried out with
trichlorovinylsilane (TCVS). Peptide (WLBU2 or PMB) and endotoxin
(lipopolysaccharide, LPS, isolated from Pseudomonas aeruginosa), in PBS
or diluted human plasma, were introduced to the OWLS flow cell in sequence or
in mixed suspensions. Each adsorption step was followed by an elution step in
peptide-free, LPS-free PBS buffer. LPS vesicle adsorption and spreading behavior
under conditions representative of the OWLS experiments were also recorded, using
interfacial tensiometry.
Results and
Discussion: The
antimicrobial effect of PMB and many CAPs is attributed to membrane disruption
in susceptible bacteria. Assuming a multi-step mechanism for binding and
disruption, our results strongly indicate that in contrast to PMB, WLBU2 binding
is not accompanied by rapid disruption of LPS bilayer vesicles. In particular, competitive
adsorption results showed adsorption of WLBU2 to hydrophobic surfaces from
peptide-LPS mixtures is reduced by an order of magnitude relative to WLBU2 in
the absence of LPS. Peptide-LPS and LPS-peptide sequential adsorption results were
consistent with this outcome. Figure 1 shows that after LPS layering at a
hydrophobic surface, WLBU2 binding was entirely irreversible with respect to
buffer dilution.
Figure
1: Sequential
adsorption of LPS and WLBU2 (solid line) or PMB (dashed line). After
introduction of peptide, the resistance to elution by PBS is much more
pronounced in the case of WLBU2, indicating peptide ?entrapment? within the LPS
layer. This is consistent with binding affinity of WLBU2 for LPS.
Conclusions:
Over the
timescales of these experiments, we have seen that WLBU2 tightly binds LPS in a
fashion which leaves the LPS largely intact. In contrast, PMB binding to LPS is
associated with vesicle disruption and evolution of disruption products. Use of
WLBU2 to capture pathogenic entities in a hemoperfusion device would avoid adverse
clinical effects due to reintroduction of such entities to circulating blood. These
early investigations of use of tethered WLBU2 to capture LPS are promising.
Further investigation will be conducted using tethered constructs in the
presence of blood proteins, as well as against whole bacterial cells.
References
8. Ikeda T. Hemoadsorption in critical care.
Therapeutic Apheresis 2002;6(3):189-192.