13 research outputs found
Encapsulated Hydrogels by E-beam Lithography and Their Use in Enzyme Cascade Reactions
Electron beam (e-beam) lithography was employed to prepare one protein immobilized hydrogel encapsulated inside another by first fabricating protein-reactive hydrogels of orthogonal reactivity and subsequently conjugating the biomolecules. Exposure of thin films of eight arm star poly(ethylene glycol) (PEG) functionalized with biotin (Biotin PEG), alkyne (Alkyne-PEG) or aminooxy (AO-PEG) end groups to e-beam radiation resulted in cross-linked hydrogels with the respective functionality. It was determined via confocal microscopy that a nominal size exclusion effect exists for streptavidin immobilized on Biotin-PEG hydrogels of feature sizes ranging from 5 to 40 mu m. AO-PEG was subsequently patterned as an encapsulated core inside a contiguous outer shell of Biotin-PEG. Similarly, Alkyne-PEG was patterned as a core inside an AO-PEG shell. The hydrogel reactive end-groups were conjugated to dyes or proteins of complementary reactivity, and the three-dimensional (3-D) spatial orientation was determined for both configurations using confocal microscopy. The enzyme glucose oxidase (GOX) was immobilized in the core of the, encapsulated Alkyne-PEG core/AO-PEG shell architecture, and horseradish peroxidase (HRP) was conjugated to the shell periphery. Bioactivity for the HRP-GOX enzyme pair was observed in this encapsulated configuration by demonstrating that the enzyme pair was capable of enzyme cascade reactions
Encapsulated Hydrogels by E-beam Lithography and Their Use in Enzyme Cascade Reactions
Electron beam (e-beam) lithography was employed to prepare one protein immobilized hydrogel encapsulated inside another by first fabricating protein-reactive hydrogels of orthogonal reactivity and subsequently conjugating the biomolecules. Exposure of thin films of eight arm star poly(ethylene glycol) (PEG) functionalized with biotin (Biotin-PEG), alkyne (Alkyne-PEG) or aminooxy (AO-PEG) end-groups to e-beam radiation resulted in cross-linked hydrogels with the respective functionality. It was determined via confocal microscopy that a nominal size exclusion effect exists for streptavidin immobilized on Biotin-PEG hydrogels of feature sizes ranging from 5 to 40 ÎŒm. AO-PEG was subsequently patterned as an encapsulated core inside a contiguous outer shell of Biotin-PEG. Similarly, Alkyne-PEG was patterned as a core inside an AO-PEG shell. The hydrogel reactive end-groups were conjugated to dyes or proteins of complementary reactivity, and the three-dimensional (3-D) spatial orientation was determined for both configurations using confocal microscopy. The enzyme glucose oxidase (GOX) was immobilized in the core of the encapsulated Alkyne-PEG core/ AO-PEG shell architecture, and horseradish peroxidase (HRP) was conjugated to the shell periphery. Bioactivity for the HRP-GOX enzyme pair was observed in this encapsulated configuration by demonstrating that the enzyme pair was capable of enzyme cascade reactions
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 additionâfragmentation 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
Core/shell protein-reactive nanogels via a combination of RAFT polymerization and vinyl sulfone postmodification
Aim: A promising nanogel vaccine platform was expanded toward antigen conjugation.
Materials & methods: Block copolymers containing a reactive ester solvophobic block and a PEG-like solvophilic block were synthesized via reversible addition-fragmentation chain-transfer polymerization. Following self-assembly in DMSO, the esters allow for core-crosslinking and hydrophilization by amide bond formation with primary amines. Free thiols were accessed at the polymer chain ends through aminolysis of the reversible addition-fragmentation chain-transfer groups, and into the nanogel core by reactive ester conversion with cysteamine. Subsequently, free thiols were converted into vinyl sulfone moieties.
Results: Despite sterical constraints, nanogel-associated vinyl sulfone moieties remained well accessible for cysteins to enforce protein conjugation successfully.
Conclusion: Our present findings provide a next step toward well-defined vaccine nanoparticles that can co-deliver antigen and a molecular adjuvant
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
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Fibroblast growth factor 2 dimer with superagonist in vitro activity improves granulation tissue formation during wound healing
Site-specific chemical dimerization of fibroblast growth factor 2 (FGF2) with the optimal linker length resulted in a FGF2 homodimer with improved granulation tissue formation and blood vessel formation at exceptionally low concentrations. Homodimers of FGF2 were synthesized through site-specific linkages to both ends of different molecular weight poly(ethylene glycols) (PEGs). The optimal linker length was determined by screening dimer-induced metabolic activity of human dermal fibroblasts and found to be that closest to the inter-cysteine distance, 70 Ă
, corresponding to 2 kDa PEG. A straightforward analysis of the kinetics of second ligand binding as a function of tether length showed that, as the polymerization index (the number of monomer repeat units in the polymer, N) of the tether decreases, the mean time for second ligand capture decreases as âŒN(3/2), leading to an enhancement of the number of doubly bound ligands in steady-state for a given (tethered) ligand concentration. FGF2-PEG2k-FGF2 induced greater fibroblast metabolic activity than FGF2 alone, all other dimers, and all monoconjugates, at each concentration tested, with the greatest difference observed at low (0.1 ng/mL) concentration. FGF2-PEG2k-FGF2 further exhibited superior activity compared to FGF2 for both metabolic activity and migration in human umbilical vein endothelial cells, as well as improved angiogenesis in a coculture model in vitro. Efficacy in an in vivo wound healing model was assessed in diabetic mice. FGF2-PEG2k-FGF2 increased granulation tissue and blood vessel density in the wound bed compared to FGF2. The results suggest that this rationally designed construct may be useful for improving the fibroblast matrix formation and angiogenesis in chronic wound healing
Homodimeric ProteinâPolymer Conjugates via the Tetrazineâ<i>trans</i>-Cyclooctene Ligation
Tetrazine end-functionalized telechelic
polymers were synthesized
by controlled radical polymerization (CRP) and employed to generate
T4 lysozyme homodimers. Mutant T4 lysozyme (V131C), containing a single
surface-exposed cysteine, was modified with a protein-reactive <i>trans</i>-cyclooctene (T4L-TCO). Reversible additionâfragmentation
chain transfer (RAFT) polymerization yielded polyÂ(<i>N</i>-isopropylÂacrylamide) (pNIPAAm) with a number-average molecular
weight (<i>M</i><sub>n</sub> by <sup>1</sup>H NMR) of 2.0
kDa and a dispersity (<i>Ä</i> by GPC) of 1.05. pNIPAAm
was then modified at both ends by postpolymerization with 6-methylÂtetrazine.
For comparison, 2.0 kDa bis-tetrazine polyÂ(ethylene glycol) (PEG)
and 2.0 kDa bis-maleimide pNIPAAm were synthesized. Ligation of T4L-TCO
to bis-tetrazine pNIPAAm or bis-tetrazine PEG resulted in protein
homodimer in 38% yield and 37% yield, respectively, after only 1 h,
whereas bis-maleimide pNIPAAm resulted in only 5% yield of dimer after
24 h. This work illustrates the advantage of employing tetrazine ligation
over maleimide thiolâene chemistry for the synthesis of protein
homodimer conjugates