Jinchao Zhang

Gold nanoparticles promote osteogenic differentiation of mesenchymal stem cells through p38 MAPK pathway.

Understanding the interaction mechanisms between nanomaterials and biological cells is important for the control and manipulation of these interactions for biomedical applications. In this study, we investigated the cellular effects of gold nanoparticles (AuNPs) on the differentiation of mesenchymal stem cells (MSCs) and the associated molecular mechanisms. The results showed that AuNPs promoted the differentiation of MSCs toward osteoblast cells over adipocyte cells by inducing an enhanced osteogenic transcriptional profile and an attenuated adipogenic transcriptional profile. AuNPs exerted the effects by interacting with the cell membrane and binding with proteins in the cytoplasm, causing mechanical stress on the MSCs to activate p38 mitogen-activated protein kinase pathway (MAPK) signaling pathway, which regulates the expression of relevant genes to induce osteogenic differentiation and inhibit adipogenic differentiation.

Inhibition of proliferation and differentiation of mesenchymal stem cells by carboxylated carbon nanotubes.

Multipotent mesenchymal stem cells (MSCs) have attracted substantial attention in stem cell therapy and tissue engineering due to their ability to be cultured for successive passages and multilineage differentiation. Carbon nanotubes (CNTs) have been proposed to be used as potential biomedical structures for bone formation. Therefore, it is important to study the mechanisms of interaction between MSCs and CNTs. We demonstrated that carboxylated single-walled carbon nanotubes (SWCNTs) and carboxylated multiwalled carbon nanotubes (MWCNTs) inhibited the proliferation, osteogenic differentiation, adipogenic differentiation, and mineralization of MSCs. Oxidative stress assay indicated that reactive oxygen species (ROS) may not be responsible for the observed cytotoxicity of carboxylated CNTs. Quantitative real-time polymerase chain reaction (Q-PCR) experiments confirmed that the expression of osteoblast specific genes and adipocyte differentiation specific genes was greatly attenuated during the differentiation of MSCs in the presence of carboxylated CNTs. TEM images revealed that CNTs might interact with proteins located on the cell membrane or in the cytoplasm, which have a further impact on subsequent cellular signaling pathways. Q-PCR results and Western blot analysis together verified that the inhibition of proliferation and osteogenic differentiation of MSCs may be modulated through a Smad-dependent bone morphogenetic protein (BMP) signaling pathway.

Ultrasmall Gold Nanoparticles as Carriers for Nucleus-Based Gene Therapy Due to Size-Dependent Nuclear Entry

The aim of this study was to determine the size-dependent penetration ability of gold nanoparticles and the potential application of ultrasmall gold nanoparticles for intranucleus delivery and therapy. We synthesized gold nanoparticles with diameters of 2, 6, 10, and 16 nm and compared their intracellular distribution in MCF-7 breast cancer cells. Nanoparticles smaller than 10 nm (2 and 6 nm) could enter the nucleus, whereas larger ones (10 and 16 nm) were found only in the cytoplasm. We then investigated the possibility of using ultrasmall 2 nm nanoparticles as carriers for nuclear delivery of a triplex-forming oligonucleotide (TFO) that binds to the c-myc promoter. Compared to free TFO, the nanoparticle-conjugated TFO was more effective at reducing c-myc RNA and c-myc protein, which resulted in reduced cell viability. Our result demonstrated that the entry of gold nanoparticles into the cell nucleus is critically dependent on the size of the nanoparticles. We developed a strategy for regulating gene expression, by directly delivering TFOs into the nucleus using ultrasmall gold nanoparticles. More importantly, guidelines were provided to choose appropriate nanocarriers for different biomedical purposes.

Hybrid Mesoporous Silica-Based Drug Carrier Nanostructures with Improved Degradability by Hydroxyapatite

Potential bioaccumulation is one of the biggest limitations for silica nanodrug delivery systems in cancer therapy. In this study, a mesoporous silica nanoparticles/hydroxyapatite (MSNs/HAP) hybrid drug carrier, which enhanced the biodegradability of silica, was developed by a one-step method. The morphology and structure of the nanoparticles were characterized by TEM, DLS, FT-IR, XRD, N2 adsorption-desorption isotherms, and XPS, and the drug loading and release behaviors were tested. TEM and ICP-OES results indicate that the degradability of the nanoparticles has been significantly improved by Ca(2+) escape from the skeleton in an acid environment. The MSNs/HAP sample exhibits a higher drug loading content of about 5 times that of MSNs. The biological experiment results show that the MSNs/HAP not only exhibits good biocompatibility and antitumor effect but also greatly reduces the side effects of free DOX. The as-synthesized hybrid nanoparticles may act as a promising drug delivery system due to their good biocompatibility, high drug loading efficiency, pH sensitivity, and excellent biodegradability.

Neuropilin-1-Targeted Gold Nanoparticles Enhance Therapeutic Efficacy of Platinum(IV) Drug for Prostate Cancer Treatment

Platinum-based anticancer drugs such as cisplatin, oxaliplatin, and carboplatin are some of the most potent chemotherapeutic agents but have limited applications due to severe dose-limiting side effects and a tendency for cancer cells to rapidly develop resistance. The therapeutic index can be improved through use of nanocarrier systems to target cancer cells efficiently. We developed a unique strategy to deliver a platinum(IV) drug to prostate cancer cells by constructing glutathione-stabilized (Au@GSH) gold nanoparticles. Glutathione (GSH) has well-known antioxidant properties, which lead to cancer regression. Here, we exploit the advantages of both the antioxidant properties and high surface-area-to-volume ratio of Au@GSH NPs to demonstrate their potential for delivery of a platinum(IV) drug by targeting the neuropilin-1 receptor (Nrp-1). A lethal dose of a platinum(IV) drug functionalized with the Nrp-1-targeting peptide (CRGDK) was delivered specifically to prostate cancer cells in vitro. Targeted peptide ensures specific binding to the Nrp-1 receptor, leading to enhanced cellular uptake level and cell toxicity. The nanocarriers were themselves nontoxic, but exhibited high cytotoxicity and increased efficacy when functionalized with the targeting peptide and drug. The uptake of drug-loaded nanocarriers is dependent on the interaction with Nrp-1 in cell lines expressing high (PC-3) and low (DU-145) levels of Nrp-1, as confirmed through inductively coupled plasma mass spectrometry and confocal microscopy. The nanocarriers have effective anticancer activity, through upregulation of nuclear factor kappa-B (NF-κB) protein (p50 and p65) expression and activation of NF-κB-DNA-binding activity. Our preliminary investigations with platinum(IV)-functionalized gold nanoparticles along with a targeting peptide hold significant promise for future cancer treatment.

Cerium oxide nanoparticles protect endothelial cells from apoptosis induced by oxidative stress.

Oxidative stress is well documented to cause injury to endothelial cells (ECs), which in turn trigger cardiovascular diseases. Previous studies revealed that cerium oxide nanoparticles (nanoceria) had antioxidant property, but the protective effect of nanoceria on ROS injury to ECs and cardiovascular diseases has not been reported. In the current study, we investigated the protective effect and underlying mechanisms of nanoceria on oxidative injury to ECs. The cell viability, lactate dehydrogenase release, cellular uptake, intracellular localization and reactive oxygen species (ROS) levels, endocytosis mechanism, cell apoptosis, and mitochondrial membrane potential were performed. The results indicated that nanoceria had no cytotoxicity on ECs but had the ability to prevent injury by H2O2. Nanoceria could be uptaken into ECs through caveolae- and clathrin-mediated endocytosis and distributed throughout the cytoplasma. The internalized nanoceria effectively attenuated ROS overproduction induced by H2O2. Apoptosis was also alleviated greatly by nanoceria pretreatment. These results may be helpful for more rational application of nanoceria in biomedical fields in the future.

The effects of carbon nanotubes on the proliferation and differentiation of primary osteoblasts

This chapter provides a detailed protocol for studying the effects of carbon nanotubes (CNTs) on the proliferation, differentiation, adipocytic transdifferentiation, and mineralization of primary osteoblasts. SWNTs, DWNTs, and MWNTs with the same mean length and various diameters were shown to reduce the viability of osteoblasts and inhibit the adipocytic transdifferentiation in both time- and dose-dependent manners. The order of inhibition effect is SWNTs > DWNTs > MWNTs. CNTs were found to inhibit the formation of mineralized nodules greatly and dose-dependently during the final stage of osteoblast differentiation, causing a 50% decrease in the formation of mineralized nodules at the concentration of 50 microg/mL. The expression of important proteins such as Runx-2 and Col-I in osteoblasts was also greatly inhibited by the CNTs. TEM results revealed that the effects on cellular behavior may be exerted by the CNTs from in- and outside of the cells.

Self-assembled Peptide nanofibers designed as biological enzymes for catalyzing ester hydrolysis.

The structural arrangement of amino acid residues in a native enzyme provides a blueprint for the design of artificial enzymes. One challenge of mimicking the catalytic center of a native enzyme is how to arrange the essential amino acid residues in an appropriate position. In this study, we designed an artificial hydrolase via self-assembly of short peptides to catalyze ester hydrolysis. When the assembled hydrolase catalytic sites were embedded in a matrix of peptide nanofibers, they exhibited much higher catalytic efficiency than the peptide nanofibers without the catalytic sites, suggesting that this well-ordered nanostructure is an attractive scaffold for developing new artificial enzymes. Furthermore, the cytotoxicity of the assembled hydrolase was evaluated with human cells, and the novel artificial biological enzyme showed excellent biocompatibility.

TGF-β/BMP signaling pathway is involved in cerium-promoted osteogenic differentiation of mesenchymal stem cells.

The extensive applications of cerium (Ce) increased the chance of human exposure to Ce and its compounds. It was reported that Ce was mainly deposited in the bone after administration. However, the potential effect and mechanism of Ce on bone metabolism are not well understood. In this study, we investigated the cellular effects of Ce on the differentiation of mesenchymal stem cells (MSCs) and the associated molecular mechanisms. The results indicated that Ce promoted the osteogenic differentiation and inhibited the adipogenic differentiation of MSCs at cell level. Genes involved in transforming growth factor‐β/bone morphogenetic proteins (TGF‐β/BMP) signaling pathway were significantly changed when the MSCs were exposed to 0.0001 µM Ce by RT2Profiler™ PCR Array analysis. The expression of genes and proteins related to pathways, osteogenic, and adipogenic biomarkers of MSCs upon interaction with Ce was further confirmed by quantitative real‐time reverse transcriptase polymerase chain reaction (Q‐PCR) and Western blot analysis. The results suggest that Ce exerts the effects by interacting with bone morphogenetic protein receptor (BMPR) and activates TGF‐β/BMP signaling pathway, leads to the up‐regulation of the osteogenic master transcription factor, runt‐related transcription factor 2 (Runx 2), and the down‐regulation of the adipocytic master transcription factor, peroxisome proliferator‐activated receptor gamma 2 (PPARγ2). Runx2, which subsequently up‐regulates osteoblast (OB) marker genes collagen I (Col I) and BMP2 at early stages, alkaline phosphatase (ALP), and osteocalcin (OCN) at later stages of differentiation, thus driving MSCs to differentiate into OBs. The results provide novel evidence to elucidate the mechanisms of bone metabolism by Ce. J. Cell. Biochem. 114: 1105–1114, 2013.

Effect of yttrium ion on the proliferation, differentiation and mineralization function of primary mouse osteoblasts in vitro

Abstract A series of experimental methods including MTT test, alkaline phosphatase (ALP) activity measurement, oil red O stain and measurement and mineralized function were employed to assess the effects of Y 3+ on the proliferation, differentiation, adipogenic transdifferentiation and mineralization function of primary mouse osteoblasts (OBs) in vitro . The results indicated that Y 3+ (1×10 −9 , 1×10 −8 , 1×10 −7 , 1×10 −6 , 1×10 −5 , and 1×10 −4 mol/L) promoted the proliferation of OBs on day 1, 2 and 3. Y 3+ had no effect on the differentiation of OBs at concentrations of 1×10 −9 and 1×10 −8 mol/L, promoted the differentiation of OBs at concentration of 1×10 −7 mol/L, but turned to inhibit the differentiation of OBs at other tested concentrations on day 1. On day 2, Y 3+ inhibited the differentiation of OBs at all tested concentrations. On day 3, Y 3+ promoted the differentiation of OBs at lower concentrations of 1×10 −9 and 1×10 −8 mol/L, but turned to inhibit the differentiation of OBs at other concentrations. Y 3+ promoted the adipocytic transdifferentiation of OBs at most tested concentrations on day 9 and 12. On day 15, Y 3+ promoted the adipocytic transdifferentiation of OBs at lower concentrations of 1×10 −9 and 1×10 −8 mol/L, turned to inhibit at other tested concentrations. Y 3+ inhibited the formation of mineralized matrix nodules of OBs at concentrations of 1×10 −9 , 1×10 −8 and 1×10 −6 mol/L, but turned to promote the formation of mineralized matrix nodules of OBs at other tested concentrations. These findings suggested that the effects of Y 3+ on the proliferation, differentiation, adipocytic transdifferentiation and mineralization function of primary OBs depended on the concentration and culture time, moreover, concentration and culture time were pivotal factors for switching the biological effects of Y 3+ from toxicity to activity, from damage to protection, or from down-regulation to up-regulation.