4 research outputs found
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 poly(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
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
Trehalose Glycopolymers as Excipients for Protein Stabilization
Herein,
the synthesis of four different trehalose glycopolymers
and investigation of their ability to stabilize proteins to heat and
lyophilization stress are described. The disaccharide, α,α-trehalose,
was modified with a styrenyl acetal, methacrylate acetal, styrenyl
ether, or methacrylate moiety resulting in four different monomers.
These monomers were then separately polymerized using free radical
polymerization with azobisisobutyronitrile (AIBN) as an initiator
to synthesize the glycopolymers. Horseradish peroxidase and glucose
oxidase were incubated at 70 and 50 °C, respectively, and β-galactosidase
was lyophilized multiple times in the presence of various ratios of
the polymers or trehalose. The protein activities were subsequently
tested and found to be significantly higher when the polymers were
present during the stress compared to no additive and to equivalent
amounts of trehalose. Different molecular weights (10 kDa, 20 kDa,
and 40 kDa) were tested, and all were equivalent in their stabilization
ability. However, some subtle differences were observed regarding
stabilization ability between the different polymer samples, depending
on the stress. Small molecules such as benzyl ether trehalose were
not better stabilizers than trehalose, and the trehalose monomer decreased
protein activity, suggesting that hydrophobized trehalose was not
sufficient and that the polymeric structure was required. In addition,
cytotoxicity studies with NIH 3T3 mouse embryonic fibroblast cells,
RAW 264.7 murine macrophages, human dermal fibroblasts (HDFs), and
human umbilical vein endothelial cells (HUVECs) were conducted with
polymer concentrations up to 8 mg/mL. The data showed that all four
polymers were noncytotoxic for all tested concentrations. The results
together suggest that trehalose glycopolymers are promising as additives
to protect proteins from a variety of stressors
Correction to Trehalose Glycopolymers as Excipients for Protein Stabilization
Correction to Trehalose Glycopolymers as Excipients
for Protein Stabilizatio