20 research outputs found
Shape-Shifting Micro- and Nanopatterns Controlled by Temperature
Herein, features that alter their shape to form a different
pattern
upon an external trigger are described. Electron-beam lithography
was used to fabricate micrometer- and nanometer-sized surface immobilized
polyÂ(triethylene glycol methacrylate) (pTEGMA) that exhibits significant
thermal responsivity; the resulting hydrogels collapsed by up to 95%
of their height upon addition of heat. Multicomponent features composed
of both the thermoresponsive polymer and nonresponsive polyÂ(ethylene
glycol) (PEG) were then prepared. Upon increase in temperature, only
the thermally responsive component of the pattern collapsed, causing
a significant and predictable alteration in the overall pattern. Reversible
micrometer- and nanometer-sized square-to-triangles, squares-to-checkerboards,
smiles-to-neutral face, and zeros-to-ones shapes were shown
Trehalose Glycopolymers for Stabilization of Protein Conjugates to Environmental Stressors
Herein, we report the synthesis of trehalose side chain
polymers
for stabilization of protein conjugates to environmental stressors.
The glycomonomer 4,6-<i>O</i>-(4-vinylbenzylidene)-α,α-trehalose
was synthesized in 40% yield over two steps without the use of protecting
group chemistry. Polymers containing the trehalose pendent groups
were prepared via reversible addition–fragmentation chain transfer
(RAFT) polymerization using two different thiol-reactive chain transfer
agents (CTAs) for subsequent conjugation to proteins through disulfide
linkages. The resulting glycopolymers were well-defined, and a range
of molecular weights from 4200 to 49 500 Da was obtained. The
polymers were conjugated to thiolated hen egg white lysozyme and purified.
The glycopolymers when added or covalently attached to protein significantly
increased stability toward lyophilization and heat relative to wild-type
protein. Up to 100% retention of activity was observed when lysozyme
was stressed ten times with lyophilization and 81% activity when the
protein was heated at 90 °C for 1 h; this is in contrast to 16%
and 18% retention of activity, respectively, for the wild-type protein
alone. The glycopolymers were compared to equivalent concentrations
of trehalose and polyÂ(ethylene glycol) (PEG) and found to be superior
at stabilizing the protein to lyophilization and heat. In addition,
the protein–glycopolymer conjugates exhibited significant increases
in lyophilization stability when compared to adding the same concentration
of unconjugated polymer to the protein
Protein–Polymer Conjugation via Ligand Affinity and Photoactivation of Glutathione <i>S</i>‑Transferase
A photoactivated,
site-selective conjugation of polyÂ(ethylene glycol)
(PEG) to the glutathione (GSH) binding pocket of glutathione <i>S</i>-transferase (GST) is described. To achieve this, a GSH
analogue (GSH-BP) was designed and chemically synthesized with three
functionalities: (1) the binding affinity of GSH to GST, (2) a free
thiol for polymer functionalization, and (3) a photoreactive benzophenone
(BP) component. Different molecular weights (2 kDa, 5 kDa, and 20
kDa) of GSH-BP modified PEGs (GSBP-PEGs) were synthesized and showed
conjugation efficiencies between 52% and 76% to GST. Diazirine (DA)
PEG were also prepared but gave conjugation yields lower than for
GSBP-PEGs. PEGs with different end-groups were also synthesized to
validate the importance of each component in the end-group design.
End-groups included glutathione (GS-PEG) and benzophenone (BP-PEG).
Results showed that both GSH and BP were crucial for successful conjugation
to GST. In addition, conjugations of 5 kDa GSBP-PEG to different proteins
were investigated, including bovine serum albumin (BSA), lysozyme
(Lyz), ubiquitin (Ubq), and GST-fused ubiquitin (GST-Ubq) to ensure
specific binding to GST. By combining noncovalent and covalent interactions,
we have developed a new phototriggered protein–polymer conjugation
method that is generally applicable to GST-fusion proteins
Fluorous Comonomer Modulates the Reactivity of Cyclic Ketene Acetal and Degradation of Vinyl Polymers
Fluorine-containing
polymers have potential for use in medicine
and other applications, but the synthesis of degradable fluorous polymers
is underexplored. In this report, we present a facile route to degradable
fluorinated polymers and characterize the effect of fluorous comonomer
identity on the polymerization as well as the degradation kinetics
of the resulting polymer. Copolymers of polyÂ(ethylene glycol methyl
ether methacrylate) (PEGMA), fluorous methacrylate (1<i>H</i>,1<i>H</i>,2<i>H</i>,2<i>H</i>-perfluorooctyl
or 1<i>H</i>,1<i>H</i>,2<i>H</i>,2<i>H</i>,3<i>H</i>,3<i>H</i>-perfluoropentyl
methacrylate), and cyclic ketene acetal 5,6-benzo-2-methylene-1,3-dioxepane
(BMDO) were synthesized via ruthenium-catalyzed living radical polymerization.
It was observed that increasing the fluorous monomer content led to
enhanced BMDO incorporation in the resulting polymer. Density functional
theory calculations suggest that this is due to the decreased energy
gap between the singly occupied molecular orbital (SOMO) of the methacrylate
radical and the highest occupied molecular orbital (HOMO) of BMDO.
Moreover, polymers with higher fluorous monomer content were more
hydrolytically stable, with a degradation rate constant 100-fold smaller
for the polymer with highest fluorous content compared to the nonfluorous
polymer. This work provides easy access to degradable fluorous polymers
using vinyl monomers. In addition, the insights gained into modulation
of reactivity of cyclic ketene acetals and polymer degradation will
be useful in applying fluorous polymers for a variety of biomedical
applications
Aminooxy and Pyridyl Disulfide Telechelic Poly(poly(ethylene glycol) acrylate) by RAFT Polymerization
An efficient method to synthesize telechelic, bioreactive
polymers is described. Homotelechelic polymers were synthesized by
reversible addition–fragmentation chain transfer (RAFT) polymerization
in one step by employing bifunctional chain transfer agents (CTAs).
A bis-carboxylic acid CTA was coupled to <i>N</i>-Boc-aminooxy
ethanol or pyridyl disulfide ethanol, resulting in a bis-<i>N</i>-Boc-aminooxy CTA and a bis-pyridyl disulfide CTA, respectively.
RAFT polymerization of polyÂ(ethylene glycol) (PEG) acrylate in the
presence of both CTAs resulted in a series of polymers over a range
of molecular weights (∼8.4–35.2 kDa; polydispersity
indices, PDIs, of 1.11–1.44) with retention of end-groups postpolymerization.
The polymers were characterized by <sup>1</sup>H NMR spectroscopy
and gel permeation chromatography (GPC). Conjugations of small molecules
and peptides resulted in homotelechelic polymer conjugates
A Heparin-Mimicking Block Copolymer Both Stabilizes and Increases the Activity of Fibroblast Growth Factor 2 (FGF2)
Fibroblast growth factor 2 (FGF2)
is a protein involved in cellular
functions in applications such as wound healing and tissue regeneration.
Stabilization of this protein is important for its use as a therapeutic
since the native protein is unstable during storage and delivery.
Additionally, the ability to increase the activity of FGF2 is important
for its application, particularly in chronic wound healing and the
treatment of various ischemic conditions. Here we report a heparin
mimicking block copolymer, polyÂ(styrenesulfonate-<i>co</i>-polyÂ(ethylene glycol) methyl ether methacrylate)-<i>b</i>-vinyl sulfonate) (pÂ(SS-<i>co</i>-PEGMA)-<i>b</i>-VS, that contains a segment that enhances the stability of FGF2
and one that binds to the FGF2 receptor. The FGF2 conjugate retained
activity after exposure to refrigeration (4 °C) and room temperature
(23 °C) for 7 days, while unmodified FGF2 was inactive after
these standard storage conditions. A cell study performed with a cell
line lacking native heparan sulfate proteoglycans indicated that the
conjugated block copolymer facilitated binding of FGF2 to its receptor
similar to the addition of heparin to FGF2. A receptor-based enzyme-linked
immunosorbant assay (ELISA) confirmed the results. The conjugate also
increased the migration of endothelial cells by 80% compared to FGF2
alone. Additionally, the FGF2-pÂ(SS-<i>co</i>-PEGMA)-<i>b</i>-VS stimulated endothelial cell sprouting 250% better than
FGF2 at low concentration. These data verify that this rationally
designed protein-block copolymer conjugate enhances receptor binding,
cellular processes such as migration and tube-like formation, and
stability, and suggest that it may be useful for applications in biomaterials,
tissue regeneration, and wound healing
Imine Hydrogels with Tunable Degradability for Tissue Engineering
A shortage of available organ donors
has created a need for engineered
tissues. In this context, polymer-based hydrogels that break down
inside the body are often used as constructs for growth factors and
cells. Herein, we report imine cross-linked gels where degradation
is controllable by the introduction of mixed imine cross-links. Specifically,
hydrazide-functionalized polyÂ(ethylene glycol) (PEG) reacts with aldehyde-functionalized
PEG (PEG-CHO) to form hydrazone linked hydrogels that degrade quickly
in media. The time to degradation can be controlled by changing the
structure of the hydrazide group or by introducing hydroxylamines
to form nonreversible oxime linkages. Hydrogels containing adipohydrazide-functionalized
PEG (PEG-ADH) and PEG-CHO were found to degrade more rapidly than
gels formed from carbodihydrazide-functionalized PEG (PEG-CDH). Incorporating
oxime linkages via aminooxy-functionalized PEG (PEG-AO) into the hydrazone
cross-linked gels further stabilized the hydrogels. This imine cross-linking
approach should be useful for modulating the degradation characteristics
of 3D cell culture supports for controlled cell release
Poly(vinyl sulfonate) Facilitates bFGF-Induced Cell Proliferation
Heparin is a highly sulfated polysaccharide
and is useful because
of its diverse biological functions. However, because of batch-to-batch
variability and other factors, there is significant interest in preparing
biomimetics of heparin. To identify polymeric heparin mimetics, a
cell-based screening assay was developed in cells that express fibroblast
growth factor receptors (FGFRs) but not heparan sulfate proteoglycans.
Various sulfated and sulfonated polymers were screened, and polyÂ(vinyl
sulfonate) (pVS) was identified as the strongest heparin-mimicking
polymer in its ability to enhance binding of basic fibroblast growth
factor (bFGF) to FGFR. The results were confirmed by an ELISA-based
receptor-binding assay. Different molecular weights of pVS polymer
were synthesized by reversible addition–fragmentation chain
transfer (RAFT) polymerization. The polymers were able to facilitate
dimerization of FGFRs leading to cell proliferation in FGFR-expressing
cells, and no size dependence was observed. The data showed that pVS
is comparable to heparin in these assays. In addition, pVS was not
cytotoxic to fibroblast cells up to at least 1 mg/mL. Together this
data indicates that pVS should be explored further as a replacement
for heparin
Direct Write Protein Patterns for Multiplexed Cytokine Detection from Live Cells Using Electron Beam Lithography
Simultaneous
detection of multiple biomarkers, such as extracellular
signaling molecules, is a critical aspect in disease profiling and
diagnostics. Precise positioning of antibodies on surfaces, especially
at the micro- and nanoscale, is important for the improvement of assays,
biosensors, and diagnostics on the molecular level, and therefore,
the pursuit of device miniaturization for parallel, fast, low-volume
assays is a continuing challenge. Here, we describe a multiplexed
cytokine immunoassay utilizing electron beam lithography and a trehalose
glycopolymer as a resist for the direct writing of antibodies on silicon
substrates, allowing for micro- and nanoscale precision of protein
immobilization. Specifically, anti-interleukin 6 (IL-6) and antitumor
necrosis factor alpha (TNFα) antibodies were directly patterned.
Retention of the specific binding properties of the patterned antibodies
was shown by the capture of secreted cytokines from stimulated RAW
264.7 macrophages. A sandwich immunoassay was employed using gold
nanoparticles and enhancement with silver for the detection and visualization
of bound cytokines to the patterns by localized surface plasmon resonance
detected with dark-field microscopy. Multiplexing with both IL-6 and
TNFα on a single chip was also successfully demonstrated with
high specificity and in relevant cell culture conditions and at different
times after cell stimulation. The direct fabrication of capture antibody
patterns for cytokine detection described here could be useful for
biosensing applications
Biocompatible Hydrogels by Oxime Click Chemistry
Oxime Click chemistry was used to form hydrogels that
support cell
adhesion. Eight-armed aminooxy polyÂ(ethylene glycol) (PEG) was mixed
with glutaraldehyde to form oxime-linked hydrogels. The mechanical
properties, gelation kinetics, and water swelling ratios were studied
and found to be tunable. It was also shown that gels containing the
integrin ligand arginine-glycine-aspartic acid (RGD) supported mesenchymal
stem cell (MSC) incorporation. High cell viability and proliferation
of the encapsulated cells demonstrated biocompatibility of the material