Highly Sensitive Detection of Caspase‑3 Activities via a Nonconjugated Gold Nanoparticle–Quantum Dot Pair Mediated by an Inner-Filter Effect

We describe here a simple fluorometric assay for the highly sensitive detection of caspase-3 activities on the basis of the inner-filter effect of gold nanoparticles (AuNPs) on CdTe quantum dots (QDs). The method takes advantage of the high molar absorptivity of the plasmon band of gold nanoparticles as well as the large absorption band shift from 520 to 680 nm upon nanoparticle aggregation. When labeled with a peptide possessing the caspase-3 cleavage sequence (DEVD), the monodispersed Au-Ps (peptide-modified AuNPs) exhibited a tendency to aggregate when exposed to caspase-3, which induced the absorption band transition from 520 to 680 nm and turned on the fluorescence of the CdTe QDs for caspase-3 sensing. Under optimum conditions, a high sensitivity towards caspase-3 was achieved with a detection limit as low as 18 pM, which was much lower than the corresponding assays based on absorbance or other approaches. Overall, we demonstrated a facile and sensitive approach for caspase-3 detection, and we expected that this method could be potentially generalized to design more fluorescent assays for sensing other bioactive entities

6‑<i>O</i>‑Sulfated Chitosan Promoting the Neural Differentiation of Mouse Embryonic Stem Cells

Embryonic stem cells (ESCs) can be induced to differentiate into nerve cells, endowing them with potential applications in the treatment of neurological diseases and neural repair. In this work, we report for the first time that sulfated chitosan can promote the neural differentiation of ESCs. As a type of sulfated glycosaminoglycan analog, sulfated chitosan with well-defined sulfation sites and a controlled degree of sulfation (DS) were prepared through simple procedures and the influence of sulfated glycosaminoglycan on neural differentiation of ESCs was investigated. Compared with other sulfation sites, 6-<i>O</i>-sulfated chitosan showed the most optimal effects. By monitoring the expression level of neural differentiation markers using immunofluorescence staining and PCR, it was found that neural differentiation was better enhanced by increasing the DS of 6-<i>O</i>-sulfated chitosan. However, increasing the DS by introducing another sulfation site in addition to the 6-<i>O</i> site to chitosan did not promote neural differentiation as much as 6-<i>O</i>-sulfated chitosan, indicating that compared with DS, the sulfation site is more important. Additionally, the optimal concentration and incubation time of 6-<i>O</i>-sulfated chitosan were investigated. Together, our results indicate that the sulfate site and the molecular structure in a sulfated polysaccharide are very important for inducing the differentiation of ESCs. Our findings may help to highlight the role of sulfated polysaccharide in inducing the neural differentiation of ESCs

Regulation of Protein Binding Capability of Surfaces via Host–Guest Interactions: Effects of Localized and Average Ligand Density

The protein binding capability of biomaterial surfaces can significantly affect subsequent biological responses, and appropriate ligand presentation is often required to guarantee the best functions. Herein, a new facile method for regulating this capability by varying the localized and average ligand density is presented. Binding between lysine and plasminogen relevant to a fibrinolysis system was chosen as a model. We integrated different lysine-modified β-cyclodextrin (β-CD) derivatives onto bioinert copolymer brushes via host–guest interactions. The localized and average lysine density can be conveniently modulated by changing the lysine valency on β-CD scaffolds and by diluting lysine-persubstituted β-CD with pure β-CD, respectively. Both the plasminogen adsorption and the plasminogen binding affinity were enhanced by lysine-persubstituted β-CD compared with those of lysine-monosubstituted β-CD, which is possibly due to the higher localized lysine density and the multivalent binding of plasminogen on lysine-persubstituted β-CD surfaces. With a change in the ratio of lysine-persubstituted β-CD to β-CD, the average lysine density can be tuned, leading to the linear regulation of the adsorption of plasminogen on surfaces

Cell Adhesion on a POEGMA-Modified Topographical Surface

It is well known that adsorbed proteins play a major role in cell adhesion. However, it has also been reported that cells can adhere to a protein-resistant surface. In this work, the behavior of L02 and BEL-7402 cells on a protein-resistant, 3D topographical surface was investigated. The topographical gold nanoparticle layer (GNPL) surfaces were prepared by chemical gold plating, and the topography was described by roughness parameters acquired from a multiscale analysis. Both smooth Au and GNPL surfaces were modified with POEGMA polymer brushes using surface-initiated ATRP. The dry and hydrated thicknesses of POEGMA brushes on both smooth and rough surfaces were measured by AFM using a nanoindentation method. Protein adsorption experiments using ¹²⁵I radiolabeling revealed similarly low levels of protein adsorption on smooth and GNPL surfaces modified with POEGMA, thus allowing an investigation of the effects of topography on cell behavior under conditions of minimal protein adsorption. The roles of VN and FN adsorption in both L02 cells and BEL-7402 cells adhesion were investigated using cell culturing with and without a serum supplement. It was found that initial cell adhesion occurred via proteins adsorbed from the cell culture medium, whereas subsequent durable cell adhesion could be attributed to the topographical structure of the surface. Although cell spreading on protein-resistant surfaces was constrained because of the lack of adsorbed proteins, we found that cells adherent to topographical surfaces were more firmly attached and thus were more durable compared to those on smooth surfaces. In general, however, we conclude that topography is more important for cell adhesion on a protein-resistant surface

VEGF-Mediated Proliferation of Human Adipose Tissue-Derived Stem Cells

<div><p>Human adipose tissue-derived stem cells (ADSCs) are an attractive multipotent stem cell source with therapeutic applicability across diverse fields for the repair and regeneration of acute and chronically damaged tissues. In recent years, there has been increasing interest in ADSC for tissue engineering applications. However, the mechanisms underlying the regulation of ADSC proliferation are not fully understood. Here we show that 47 transcripts are up-regulated while 23 are down-regulated in ADSC compared to terminally differentiated cells based on global mRNA profiling and microRNA profiling. Among the up-regulated genes, the expression of vascular endothelial growth factor (VEGF) is fine-tuned by miR-199a-5p. Further investigation indicates that VEGF accelerates ADSC proliferation whereas the multipotency of ADSC remains stable in terms of adipogenic, chondrogenic and osteogenic potentials after VEGF treatment, suggesting that VEGF may serve as an excellent supplement for accelerating ADSC proliferation during <i>in vitro</i> expansion.</p></div

MiR-199a-5p-mediated VEGF down-regulation in ADSCs.

<p>(A) Targetscan indicates that miR-199a-5p binds the 3′UTR of VEGF mRNA; (B) Lentiviral infection efficiency was evaluated by ZsGreen intensity; (C) Dual-luciferase reporter assay indicated that miR-199a-5p interacted with the 3′UTR of VEGF mRNA by using lentiviral infection (n = 3); (D) qRT-PCR assay indicated that the lentiviruses overexpressed miR-199a-5p (n = 3); (E) qRT-PCR assay indicated that miR-199a-5p overexpression in ADSCs reduced VEGF mRNA levels by approximately 42% (n = 3). (F) ELISA assay indicated that miR-199a-5p overexpression in ADSCs reduced VEGF protein levels by approximately 16% (n = 5). The values were mean ± SEM (n = 5). **<i>P</i><0.01.</p

Up-regulated and down-regulated genes in both arrays.

<p>P Value<0.01, fold change >2 or fold change <0.5.</p

Global expression profiles of mRNAs and microRNAs.

<p>(A) Hot map of mRNA levels. (B) Hot map of miRNA levels. A set of mRNAs and miRNA were significantly differentially expressed in human endothelial cells, human fibrobast cells and ADSCs. Red color indicated high expression whereas yellow color indicated low expression. H1, human endothelial cell; S1, human fibroblast cell; E1–E6, human adipose tissue-derived stem cells (ADSCs).</p

VEGF-mediated proliferation of ADSCs.

<p>(A) MTT assay indicated that proliferation of ADSCs was accelerated by supplementation of a gradient concentration of VEGF (n = 5); (B) Trypan blue staining confirmed that the effect of VEGF on ADSC proliferation was dose-dependent (n = 3, p<0.01); (C) ELISA assay indicated that VEGF expression was reduced by approximately 40% in ADSCs after shRNA silencing (n = 5, p<0.01); (D) Cell number was significantly reduced when ADSCs were cultured in FBS-containing medium for 24 or 48 hours after VEGF silencing (n = 3, P<0.01); (D) The cell number reduction was also confirmed when ADSCs were cultured for 24 or 48 hours in KSR-containing medium after VEGF silencing (n = 3, P<0.01); (E) ADSC proliferation was also evaluated in FBS medium by trypan blue assay when VEGF expression was knocked down, it was demonstrated that VEGF reduction resulted in inhibition of ADSC proliferation (n = 5). * <i>P</i><0.05, **<i>P</i><0.01.</p

Down-regulated microRNAs.

<p>Down-regulated microRNAs.</p