The Cellular and Molecular Mechanisms Underlying Silver Nanoparticle/Chitosan Oligosaccharide/Poly(vinyl alcohol) Nanofiber-Mediated Wound Healing.

Wound healing is a complex pathophysiological process that occurs frequently in everyday pathology and remains a challenge during the treatment of trauma. Previously, we prepared silver nanoparticle/chitosan oligosaccharide/poly(vinyl alcohol) (PVA/COS-AgNP) nanofibers via an electrospinning technique. These nanofibers promoted the proliferation of human skin fibroblasts (HSFs) and the expression of transforming growth factor TGF-ß1 in the early stage of wound repair, although the specific mechanisms remain unclear. Therefore, considering that TGF-ß1 has emerged as a major modulator of wound healing, the objective of this study was to further understand whether the molecular mechanisms responsible for PVA/COS-AgNP nanofiber-mediated wound healing include the TGF-ß1/Smad signal transduction pathway. In this study, we used human skin fibroblasts (HSFs) to investigate the molecular and cellular mechanisms underlying PVA/COSAgNP nanofiber-mediated wound healing. Cell adhesion and proliferation experiments, immunofluorescence staining, hydroxyproline content measurements, flow cytometry, quantitative real-time PCR (qRT-PCR), and western blotting (WB) were used to analyze the wound healing mechanisms of human skin fibroblasts treated with various concentrations of PVA/COS-AgNP nanofibers and the combined application of silver nanofibers and SB431542 (an inhibitor of the TGF-ß1 receptor kinase). Our study showed that PVA/COS-AgNP nanofibers markedly promoted fibroblast proliferation, collagen synthesis, and cell adherence. We also found that treating fibroblasts with PVA/COS-AgNP nanofibers stimulated cell cycle progression from G1 into the S and G2 phases, reducing the proportion of cells in the G0/G1 phase and inducing S and G2/M arrest. Importantly, the cell factors associated with the TGF-ß1/Smad signal transduction pathway, such as TGF-ß1, TGFßRI, TGFßRII, pSmad2, pSmad3, collagen I, collagen III, and fibronectin were also up-regulated. Moreover, this enhancing effect was markedly inhibited by the TGFßRI receptor inhibitor, SB431542. Therefore, the PVA/COS-AgNP nanofibers used to accelerate wound healing do so by activating the TGF-ß1/Smad signal transduction pathway.

Silver nanoparticles/chitosan oligosaccharide/poly(vinyl alcohol) nanofiber promotes wound healing by activating TGFß1/Smad signaling pathway.

Wound healing occupies a remarkable place in everyday pathology and remains a challenging clinical problem. In our previous study, we prepared a silver nanoparticle/chitosan oligosaccharide/poly(vinyl alcohol) (PVA/COS-AgNPs) nanofiber via electrospinning and revealed that it could promote wound healing; however, the healing mechanism remained unknown. Therefore, we aimed to clarify the mechanism underlying the accelerated healing effect of the PVA/COS-AgNPs nanofiber. The TGFß1/Smad signaling pathway is actively involved in wound healing. Considering the key role of this signaling pathway in wound healing, our preliminary study showed that the TGFß1 level was significantly increased during the early stage of wound healing. Thus, in this study, hematoxylin-eosin, Masson's trichrome, immunofluorescent staining, hydroxyproline content, quantitative real-time polymerase chain reaction, and Western blot analyses were used to analyze the wound healing in a rat model treated with gauze, the PVA/COS-AgNPs nanofiber, and the nanofiber plus SB431542 (an inhibitor of TGFß1 receptor kinase). The results showed that the PVA/COS-AgNPs nanofiber promoted wound healing and upregulated the expression levels of cytokines associated with the TGFß1/Smad signaling pathway such as TGFß1, TGFßRI, TGFßRII, collagen I, collagen III, pSmad2, and pSmad3. Inhibiting this pathway with SB431542 resulted in prevention of the PVA/COS-AgNPs nanofiber-associated salutary effects on the early stage of wound healing and relative cytokines expression. In conclusion, the wound healing effect of the PVA/COS-AgNPs nanofiber involves activation of the TGFß1/Smad signaling pathway.

Hypericin-photodynamic therapy induces human umbilical vein endothelial cell apoptosis.

The conventional photosensitizers used in photodynamic therapy (PDT), such as haematoporphyrin (HP), have not yet reached satisfactory therapeutic effects on port-wine stains (PWSs), due largely to the long-term dark toxicity. Previously we have showed that hypericin exhibited potent photocytotoxic effects on Roman chicken cockscomb model of PWSs. However, the molecular mechanism of hypericin-mediated photocytotoxicity remains unclear. In this study, we employed human umbilical vein endothelial cells (HUVECs) to investigate the hypericin-photolytic mechanism. Our study showed that hypericin-PDT induced reactive oxygen species (ROS), resulting in cell killings and an activation of the inflammatory response. Importantly, we have also discovered that photoactivated hypericin induced apoptosis by activating the mitochondrial caspase pathway and inhibiting the activation of the vascular endothelial growth factor-A (VEGF-A)-mediated PI3K/Akt pathway. Notably, we found that hypericin exhibited a more potent photocytotoxic effect than HP, and largely addressed the inconvenience issue associated with the use of HP. Thereby, hypericin may be a better alternative to HP in treating PWSs.

[The complexes of adenovirus and anionic liposomes: preparation and in vitro characterization].

This study is to report the preparation of complexes of Ad5 and anionic liposomes (AL-Ad5), the amplification of adenoviruses with enhanced green fluorescent protein (eGFP) reporter gene performed by HEK 293 cells, the adenoviral vectors purified by cesium chloride gradient centrifugation, and the titer of adenovirus determined by cytopathic effect (CPE) method, hexon capsid immunoassay and quantitative-PCR (Q-PCR), separately. The prescription and experiment conditions were optimized by central composite design (CCD). The complexes of Ad5 and AL-Ad5 were formulated by the calcium-induced phase change method. The morpholopy, particle size and zeta potential were detected by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. Additionally, the bicolourable fluoresce-labeled complexes (F(labeled)-AL-Ad5) were prepared and their intracellular location in MDCK cells was detected by confocal laser scanning microscopy (CLSM). The results indicate that the complexes of AL-Ad5 exhibited a uniform distribution with a particle size of 211 +/- 10 nm and a zeta potential of -41.2 +/- 2.2 mV. The result of CLSM demonstrates that the intracellular location of red fluoresce-labeled adenovirus was consistent with that of green fluoresce-labeled liposomes suggesting that the naked adenovirus was well encapsulated by the anionic liposomes in complexes of AL-Ad5.

Solid lipid nanoparticles loaded with anti-microRNA oligonucleotides (AMOs) for suppression of microRNA-21 functions in human lung cancer cells.

PURPOSE: Literature has highlighted the practical use of solid lipid nanoparticles (SLNs) in research, but few reports have combined SLNs with miRNA-based therapy. We aimed to prepare SLNs to load anti-miRNA oligonucleotide (AMO) for miRNA-based therapy in vitro. METHODS: SLNs were employed to encapsulate AMO by a solvent diffusion method, and then the properties of AMO-CLOSs (cationic lipid binded oligonucleotide (AMO)-loaded SLNs) were characterized. We studied cellular uptake and activation properties of AMO-CLOSs in A549 cells, including antisense efficiency, cell migration and invasion. RESULTS: AMO-CLOSs were 187 nm in size and 46.6 mV in zeta potential with an approximately toroid morphology in the TEM image. AMO-CLOSs uptake by A549 cells was increased significantly higher and more effective than free AMO. Further results demonstrated that AMO-CLOSs showed high antisense efficiency of microRNA-21 and subsequently decreased the proliferation, migration and invasion of tumor cells. CONCLUSIONS: These findings suggest that AMO-CLOSs represent a potential new approach for carrying anti-miRNA inhibitors for cancer therapy.

Synthesis, transport and mechanism of a type I prodrug: L-carnitine ester of prednisolone.

Aerosol glucocorticoid medications have become more and more important in treating BA (bronchial asthma). Although these agents are dosed to directly target airway inflammation, adrenocortical suppression and other systematic effects are still seen. To tackle this problem in a novel way, two L-carnitine ester derivatives of prednisolone (as the model drug), namely, PDC and PDSC, were synthesized to increase the absorption of prednisolone across the human bronchial epithelial BEAS-2B cells by the organic cation/carnitine transporter OCTN2 (SLC22A5) and then to slowly and intracellularly release prednisolone. The transport of prednisolone, PDC and PDSC into the human bronchial epithelial BEAS-2B cells was in the order PDSC > prednisolone > PDC at 37 °C. It was found that PDSC displayed 1.79-fold increase of uptake compared to prednisolone. Transport of PDSC by BEAS-2B was temperature-, time-, and Na(+)-dependent and saturable, with an apparent K(m) value of 329.74 µM, suggesting the involvement of carrier-mediated uptake. An RT-PCR study showed that organic cation/carnitine transporters OCTN1 and OCTN2 are expressed in BEAS-2B cells, but little in HEK293T cells. The order of uptake by HEK293T was prednisolone > PDC > PDSC. In addition, the inhibitory effects of organic cations such as L-carnitine, ergothioneine, TEA(+) and ipratropium on PDSC uptake in BEAS-2B cells were in the order L-carnitine > ipratropium > TEA(+) > ergothioneine, whereas their inhibitory effects on PDSC uptake in HEK293T cells were negligible. Finally, in vitro LPS-induced IL-6 production from BEAS-2B was more and longer suppressed by PDSC than prednisolone and PDC. All of these results suggested PDSC may be an attractive candidate for asthma treatment.

Lipid nanoparticles loaded with 10-hydroxycamptothecin-phospholipid complex developed for the treatment of hepatoma in clinical application.

For the purpose of clinical intravenous injection of 10-hydroxycamptothecin, a novel formulation of lipid nanoparticles loaded with 10-hydroxycamptothecin-phospholipid complex (HCPT-PC-LNs) was prepared by solvent evaporation and high-pressure homogenization methods. Spherical particles with a mean particle size of 200 nm and high encapsulation efficiency of 97.39 +/- 0.91% could be achieved under optimal conditions. In vitro release profile showed that the release pattern of HCPT from nanoparticles was retarded with neglectable initial burst release and well-fitted to Ritger-Peppas equation. In vivo distribution studies in mice showed that HCPT-PC-LNs were mainly localized in liver. Besides, in situ mouse hepatoma model was established and tumor uptake of HCPT-PC-LNs was much higher than that of free HCPT (more than 18-fold). It was found that HCPT-PC-LNs exhibited high growth inhibitory effect on human liver cancer cells by MTT assay. The cellular uptake of HCPT was 24.33 +/- 1.30 x 10(-7) microg/cell in HCPT-PC-LNs treated group at 12 h, which was almost 14-fold higher than that of free HCPT (1.72 +/- 0.57 x 10(-7) microg/cell). This study suggested that the HCPT-PC-LNs could be utilized as a novel formulation for liver tumors therapy, which might be applied in clinic in the near future.