4 research outputs found
Antimicrobial Peptide–Polymer Conjugates with High Activity: Influence of Polymer Molecular Weight and Peptide Sequence on Antimicrobial Activity, Proteolysis, and Biocompatibility
We report the synthesis,
characterization, activity, and biocompatibility of a novel series
of antimicrobial peptide–polymer conjugates. Using parent peptide
aurein 2.2, we designed a peptide array (∼100 peptides) with
single and multiple W and R mutations and identified antimicrobial
peptides (AMPs) with potent activity against <i>Staphylococcus
aureus</i> (S. aureus). These
novel AMPs were conjugated to hyperbranched polyglycerols (HPGs) of
different molecular weights and number of peptides to improve their
antimicrobial activity and toxicity. The cell and blood compatibility
studies of these conjugates demonstrated better properties than those
of the AMP alone. However, conjugates showed lower antimicrobial activity
in comparison to that of peptides, as determined from minimal inhibition
concentrations (MICs) against S. aureus, but considerably better than that of the available polymer–AMP
conjugates in the literature. In addition to measuring MICs and characterizing
the biocompatibility, circular dichroism spectroscopy was used to
investigate the interaction of the novel conjugates with model bacterial
biomembranes. Moreover, the novel conjugates were exposed to trypsin
to evaluate their stability. It was found that the conjugates resist
proteolysis in comparison with unprotected peptides. The peptide conjugates
were active in serum and whole blood. Overall, the results show that
combining a highly active AMP and low-molecular-weight HPG yields
bioconjugates with excellent biocompatibility, MICs below 100 μg/mL,
and proteolytic stability, which could potentially improve its utility
for in vivo applications
A Polymer Therapeutic Having Universal Heparin Reversal Activity: Molecular Design and Functional Mechanism
Heparins
are widely used to prevent blood clotting during surgeries
and for the treatment of thrombosis. However, bleeding associated
with heparin therapy is a concern. Protamine, the only approved antidote
for unfractionated heparin (UFH) could cause adverse cardiovascular
events. Here, we describe a unique molecular design used in the development
of a synthetic dendritic polycation named as universal heparin reversal
agent (UHRA), an antidote for all clinically used heparin anticoagulants.
We elucidate the mechanistic basis for the selectivity of UHRA to
heparins and its nontoxic nature. Isothermal titration calorimetry
based binding studies of UHRAs having different methoxypolyethylene
glycol (mPEG) brush structures with UFH as a function of solution
conditions, including ionic strength, revealed that mPEG chains impose
entropic penalty to the electrostatic binding. Binding studies confirm
that, unlike protamine or N-UHRA (a truncated analogue of UHRA with
no mPEG chains), the mPEG chains in UHRA avert nonspecific interactions
with blood proteins and provide selectivity toward heparins through
a combined steric repulsion and Donnan shielding effect (a balance
of <i>F</i><sub>el</sub> and <i>F</i><sub>steric</sub>). Clotting assays reveal that UHRA with mPEG chains did not adversely
affect clotting, and neutralized UFH over a wide range of concentrations.
Conversely, N-UHRA and protamine display intrinsic anticoagulant activity
and showed a narrow concentration window for UFH neutralization. In
addition, we found that mPEG chains regulate the size of antidote-UFH
complexes, as revealed by atomic force microscopy and dynamic light
scattering studies. UHRA molecules with mPEG chains formed smaller
complexes with UFH, compared to N-UHRA and protamine. Finally, fluorescence
and ELISA experiments show that UHRA disrupts antithrombin-UFH complexes
to neutralize heparin’s activity
Design of Polyphosphate Inhibitors: A Molecular Dynamics Investigation on Polyethylene Glycol-Linked Cationic Binding Groups
Inorganic polyphosphate
(polyP) released by human platelets has
recently been shown to activate blood clotting and identified as a
potential target for the development of novel antithrombotics. Recent
studies have shown that polymers with cationic binding groups (CBGs)
inhibit polyP and attenuate thrombosis. However, a good molecular-level
understanding of the binding mechanism is lacking for further drug
development. While molecular dynamics (MD) simulation can provide
molecule-level information, the time scale required to simulate these
large biomacromolecules makes classical MD simulation impractical.
To overcome this challenge, we employed metadynamics simulations with
both all-atom and coarse-grained force fields. The force field parameters
for polyethylene glycol (PEG) conjugated CBGs and polyP were developed
to carry out coarse-grained MD simulations, which enabled simulations
of these large biomacromolecules in a reasonable time scale. We found
that the length of the PEG tail does not impact the interaction between
the (PEG)<sub><i>n</i></sub>-CBG and polyP. As expected,
increasing the number of the charged tertiary amine groups in the
head group strengthens its binding to polyP. Our simulation shows
that (PEG)<sub><i>n</i></sub>-CBG initially form aggregates,
mostly with the PEG in the core and the hydrophilic CBG groups pointing
toward water; then the aggregates approach the polyP and sandwich
the polyP to form a complex. We found that the binding of (PEG)<sub><i>n</i></sub>-CBG remains intact against various lengths
of polyP. Binding thermodynamics for two of the (PEG)<sub><i>n</i></sub>-CBG/polyP systems simulated were measured by isothermal
titration calorimetry to confirm the key finding of the simulations
that the length PEG tail does not influence ligand binding to polyP
Toward Infection-Resistant Surfaces: Achieving High Antimicrobial Peptide Potency by Modulating the Functionality of Polymer Brush and Peptide
Bacterial
infection associated with indwelling medical devices
and implants is a major clinical issue, and the prevention or treatment
of such infections is challenging. Antimicrobial coatings offer a
significant step toward addressing this important clinical problem.
Antimicrobial coatings based on tethered antimicrobial peptides (AMPs)
on hydrophilic polymer brushes have been shown to be one of the most
promising strategies to avoid bacterial colonization and have demonstrated
broad spectrum activity. Optimal combinations of the functionality
of the polymer-brush-tethered AMPs are essential to maintaining long-term
AMP activity on the surface. However, there is limited knowledge currently
available on this topic. Here we report the development of potent
antimicrobial coatings on implant surfaces by elucidating the roles
of polymer brush chemistry and peptide structure on the overall antimicrobial
activity of the coatings. We screened several combinations of polymer
brush coatings and AMPs constructed on nanoparticles, titanium surfaces,
and quartz slides on their antimicrobial activity and bacterial adhesion
against Gram-positive and Gram-negative bacteria. Highly efficient
killing of planktonic bacteria by the antimicrobial coatings on nanoparticle
surfaces, as well as potent killing of adhered bacteria in the case
of coatings on titanium surfaces, was observed. Remarkably, the antimicrobial
activity of AMP-conjugated brush coatings demonstrated a clear dependence
on the polymer brush chemistry and peptide structure, and optimization
of these parameters is critical to achieving infection-resistant surfaces.
By analyzing the interaction of polymer-brush-tethered AMPs with model
lipid membranes using circular dichroism spectroscopy, we determined
that the polymer brush chemistry has an influence on the extent of
secondary structure change of tethered peptides before and after interaction
with biomembranes. The peptide structure also has an influence on
the density of conjugated peptides on polymer brush coatings and the
resultant wettability of the coatings, and both of these factors contributed
to the antimicrobial activity and bacterial adhesion of the coatings.
Overall, this work highlights the importance of optimizing the functionality
of the polymer brush to achieve infection-resistant surfaces and presents
important insight into the design criteria for the selection of polymers
and AMPs toward the development of potent antimicrobial coating on
implants