9 research outputs found
Poly(N-vinylpyrrolidone)-Poly(dimethylsiloxane)-Based Polymersome Nanoreactors for Laccase-Catalyzed Biotransformations
Laccases (Lac) are oxidizing enzymes with a broad range of applications, for example, in soil remediation, as bleaching agent in the textile industry, and for cosmetics. Protecting the enzyme against degradation and inhibition is of great importance for many of these applications. Polymer vesicles (polymersomes) from poly(N-vinylpyrrolidone)-block-poly(dimethylsiloxane)-block-poly(N-vinyl pyrrolidone) (PNVP-b-PDMS-b-PNVP) triblock copolymers were prepared and investigated as intrinsically semipermeable nanoreactors for Lac. The block copolymers allow oxygen to enter and reactive oxygen species (ROS) to leave the polymersomes. EPR spectroscopy proved that Lac can generate ROS. They could diffuse out of the polymersome and oxidize an aromatic substrate outside the vesicles. Michaelis-Menten constants K-m between 60 and 143 mu M and turn over numbers k(cat) of 0.11 to 0.18 s(-1) were determined for Lac in the nanoreactors. The molecular weight and the PDMS-to-PNVP ratio of the block copolymers influenced these apparent Michaelis-Menten parameters. Encapsulation of Lac in the polymersomes significantly protected the enzyme against enzymatic degradation and against small inhibitors: proteinase K caused 90% less degradation and the inhibitor sodium azide did not affect the enzyme`s activity. Therefore, these polymer nanoreactors are an effective means to stabilize laccase
Amino acid composition of nanofibrillar self-assembling peptide hydrogels affects responses of periodontal tissue cells in vitro
Background: The regeneration of tissue defects at the interface between soft and hard tissue, eg, in the periodontium, poses a challenge due to the divergent tissue requirements. A class of biomaterials that may support the regeneration at the soft-to-hard tissue interface are self-assembling peptides (SAPs), as their physicochemical and mechanical properties can be rationally designed to meet tissue requirements.
Materials and methods: In this work, we investigated the effect of two single-component and two complementary β-sheet forming SAP systems on their hydrogel properties such as nanofibrillar architecture, surface charge, and protein adsorption as well as their influence on cell adhesion, morphology, growth, and differentiation.
Results: We showed that these four 11-amino acid SAP (P11-SAP) hydrogels possessed physicochemical characteristics dependent on their amino acid composition that allowed variabilities in nanofibrillar network architecture, surface charge, and protein adsorption (eg, the single-component systems demonstrated an ~30% higher porosity and an almost 2-fold higher protein adsorption compared with the complementary systems). Cytocompatibility studies revealed similar results for cells cultured on the four P11-SAP hydrogels compared with cells on standard cell culture surfaces. The single-component P11-SAP systems showed a 1.7-fold increase in cell adhesion and cellular growth compared with the complementary P11-SAP systems. Moreover, significantly enhanced osteogenic differentiation of human calvarial osteoblasts was detected for the single-component P11-SAP system hydrogels compared with standard cell cultures.
Conclusion: Thus, single-component system P11-SAP hydrogels can be assessed as suitable scaffolds for periodontal regeneration therapy, as they provide adjustable, extracellular matrix-mimetic nanofibrillar architecture and favorable cellular interaction with periodontal cells
Stabilizing Enzymes within Polymersomes by Coencapsulation of Trehalose
Enzymes are essential biocatalysts and very attractive as therapeutics. However, their functionality is strictly related to their stability, which is significantly affected by the environmental changes occurring during their usage or long-term storage. Therefore, maintaining the activity of enzymes is essential when they are exposed to high temperature during usage or when they are stored for extended periods of time. Here, we stabilize and protect enzymes by coencapsulating them with trehalose into polymersomes. The anhydrobiotic disaccharide preserved up to about 81% of the enzymeâEurotms original activity when laccase/trehalose-loaded nanoreactors were kept desiccated for 2 months at room temperature and 75% of its activity when heated at 50 °C for 3 weeks. Moreover, the applicability of laccase/trehalose-loaded nanoreactors as catalysts for bleaching of the textile dyes orange G, toluidine blue O, and indigo was proven. Our results demonstrate the advantages of coencapsulating trehalose within polymersomes to stabilize enzymes in dehydrated state for extended periods of time, preserving their activity even when heated to elevated temperature
Direct Write Protein Patterns for Multiplexed Cytokine Detection from Live Cells Using Electron Beam Lithography
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
Poly(<i>N</i>‑vinylpyrrolidone)-Poly(dimethylsiloxane)-Based Polymersome Nanoreactors for Laccase-Catalyzed Biotransformations
Laccases (Lac) are oxidizing enzymes
with a broad range of applications,
for example, in soil remediation, as bleaching agent in the textile
industry, and for cosmetics. Protecting the enzyme against degradation
and inhibition is of great importance for many of these applications.
Polymer vesicles (polymersomes) from polyÂ(<i>N-</i>vinylpyrrolidone)-<i>block</i>-polyÂ(dimethylsiloxane)-<i>block</i>-polyÂ(<i>N-</i>vinylpyrrolidone) (PNVP-<i>b</i>-PDMS-<i>b</i>-PNVP) triblock copolymers were prepared and investigated
as intrinsically semipermeable nanoreactors for Lac. The block copolymers
allow oxygen to enter and reactive oxygen species (ROS) to leave the
polymersomes. EPR spectroscopy proved that Lac can generate ROS. They
could diffuse out of the polymersome and oxidize an aromatic substrate
outside the vesicles. Michaelis–Menten constants <i>K</i><sub>m</sub> between 60 and 143 μM and turn over numbers <i>k</i><sub>cat</sub> of 0.11 to 0.18 s<sup>–1</sup> were
determined for Lac in the nanoreactors. The molecular weight and the
PDMS-to-PNVP ratio of the block copolymers influenced these apparent
Michaelis–Menten parameters. Encapsulation of Lac in the polymersomes
significantly protected the enzyme against enzymatic degradation and
against small inhibitors: proteinase K caused 90% less degradation
and the inhibitor sodium azide did not affect the enzyme’s
activity. Therefore, these polymer nanoreactors are an effective means
to stabilize laccase
Combination of Integrin-Binding Peptide and Growth Factor Promotes Cell Adhesion on Electron-Beam-Fabricated Patterns
Understanding and controlling cell adhesion on engineered
scaffolds
is important in biomaterials and tissue engineering. In this report
we used an electron-beam (e-beam) lithography technique to fabricate
patterns of a cell adhesive integrin ligand combined with a growth
factor. Specifically, micron-sized polyÂ(ethylene glycol) (PEG) hydrogels
with aminooxy- and styrene sulfonate-functional groups were fabricated.
Cell adhesion moieties were introduced using a ketone-functionalized
arginine-glycine-aspartic acid (RGD) peptide to modify the <i>O</i>-hydroxylamines by oxime bond formation. Basic fibroblast
growth factor (bFGF) was immobilized by electrostatic interaction
with the sulfonate groups. Human umbilical vein endothelial cells
(HUVECs) formed focal adhesion complexes on RGD- and RGD and bFGF-immobilized
patterns as shown by immunostaining of vinculin and actin. In the
presence of both bFGF and RGD, cell areas were larger. The data demonstrate
confinement of cellular focal adhesions to chemically and physically
well-controlled microenvironments created by a combination of e-beam
lithography and “click” chemistry techniques. The results
also suggest positive implications for addition of growth factors
into adhesive patterns for cell-material interactions