Epirubicin-loaded superparamagnetic iron-oxide nanoparticles for transdermal delivery: cancer therapy by circumventing the skin barrier.

The transdermal administration of chemotherapeutic agents is a persistent challenge for tumor treatments. A model anticancer agent, epirubicin (EPI), is attached to functionalized superparamagnetic iron-oxide nanoparticles (SPION). The covalent modification of the SPION results in EPI-SPION, a potential drug delivery vector that uses magnetism for the targeted transdermal chemotherapy of skin tumors. The spherical EPI-SPION composite exhibits excellent magnetic responsiveness with a saturation magnetization intensity of 77.8 emu g(-1) . They feature specific pH-sensitive drug release, targeting the acidic microenvironment typical in common tumor tissues or endosomes/lysosomes. Cellular uptake studies using human keratinocyte HaCaT cells and melanoma WM266 cells demonstrate that SPION have good biocompatibility. After conjugation with EPI, the nanoparticles can inhibit WM266 cell proliferation; its inhibitory effect on tumor proliferation is determined to be dose-dependent. In vitro transdermal studies demonstrate that the EPI-SPION composites can penetrate deep inside the skin driven by an external magnetic field. The magnetic-field-assisted SPION transdermal vector can circumvent the stratum corneum via follicular pathways. The study indicates the potential of a SPION-based vector for feasible transdermal therapy of skin cancer.

[Establishment of optimized neuronal differentiation-promoting model derived from P19 embryonic carcinoma cells].

OBJECTIVE: To establish an optimized primary drug screen model of neuronal differentiation using P19 embryonal carcinoma cells. METHODS: The final concentration of retinoid acid (RA), days of suspension culture, manner of adherent culture, suitable cell density and adherent culture medium were tested, respectively. Two stages of neuronal differentiation were examined based on morphological changes and immunocytochemistry analysis of neuronal specific protein ß-tubulin III. RESULTS: On d 8 of differentiation culture, neuron-like cells were observed with final concentration of 1 µmol/L RA. Neuron-like network was formed on d 16 of neuronal differentiation. ß-tubulin III was positively stained on both stages, indicating P19 cells were differentiated into neurons. CONCLUSION: The model using RA to induce P19 embryonic carcinoma cells to differentiate into neuron-like cells has been successfully established, which may provide a rapid, phenotypic cell-based platform for primary screening of neurogenesis-promoting drugs.

[Expression of Junctophilin 1 during cardiogenesis of mouse embryonic stem cells and rat embryos].

OBJECTIVE: To investigate the expression of Junctophilin 1 (JP1) in cardiogenesis of mammalian. METHODS: Cardiac differentiation of embryonic stem cells (ESCs) was generated by hanging drop method. Fetal heart was obtained from the rats aged d 14-20 of gestation. The expression of JP1 and JP2 during cardiogenesis of ESCs and rat embryos was analyzed by RT-PCR or Western blotting. Immunofluorescence staining was employed to reveal the distribution of JP1 and JP2 in embryoid body (EB), probing for merging of JP1 and JP2 and cardiac sarcomeric α-Actinin or Troponin-T. Percentage of JP1 and JP2-positive staining cells was analyzed quantitatively by FCS on d17. RESULTS: JP1 mRNA was up-regulated at the early stage (d 5-11) and then decreased. The expression of JP1 protein was up-regulated at the early stage (d 7-9), then decreased gradually and disappeared after d 15. While JP2 gene and protein expression increased in a time-dependent manner during cardiogenesis of rat embryos. The results of immunofluorescence staining showed that there was a parallel co-localization of JP2 with Troponin-T or α-Actinin on d17, while JP1 failed to express in the sarcomeric positive area at the same time point. Furthermore, FCS analysis showed that about 16.59% of cells were JP2-positive, while no cells were stained positively for JP1 in d17 EBs. CONCLUSION: JP1 gene is expressed during the whole process of cardiogenesis, while JP1 protein only appears on the early stage. The expression of JP1 in cardiogenesis of ESCs is consistent with that of rat embryos.

Embryonic stem cell application in drug discovery.

Embryonic stem (ES) cells and their differentiated progeny offer tremendous potential for regenerative medicine, even in the field of drug discovery. There is an urgent need for clinically relevant assays that make use of ES cells because of their rich biological utility. Attention has been focused on small molecules that allow the precise manipulation of cells in vitro, which could allow researchers to obtain homogeneous cell types for cell-based therapies and discover drugs for stimulating the regeneration of endogenous cells. Such therapeutics can act on target cells or their niches in vivo to promote cell survival, proliferation, differentiation, and homing. In the present paper, we reviewed the use of ES cell models for high-throughput/content drug screening and toxicity assessment. In addition, we examined the role of stem cells in large pharmaceutical companies' R&D and discussed a novel subject, nicheology, in stem cell-related research fields.

Co-transfection with BMP2 and FGF2 via chitosan nanoparticles potentiates osteogenesis in human adipose-derived stromal cells in vitro.

OBJECTIVE: To investigate if co-transfection of human bone morphogenetic protein 2 (BMP-2, BMP2) and human fibroblast growth factor 2 (FGF2, FGF2) via chitosan nanoparticles promotes osteogenesis in human adipose tissue-derived stem cells (ADSCs) in vitro. MATERIALS AND METHODS: Recombinant BMP2 and/or FGF2 expression vectors were constructed and packaged into chitosan nanoparticles. The chitosan nanoparticles were characterized by atomic force microscopy. Gene and protein expression levels of BMP-2 and FGF2 in ADSCs in vitro were evaluated by real-time polymerase chain reaction (PCR), western blot, and enzyme-linked immunosorbent assay. Osteocalcin (OCN) and bone sialoprotein (BSP) gene expression were also evaluated by real-time PCR to assess osteogenesis. RESULTS: The prepared chitosan nanoparticles were spherical with a relatively homogenous size distribution. The BMP2 and FGF2 vectors were successfully transfected into ADSCs. BMP-2 and FGF2 mRNA and protein levels were significantly up-regulated in the co-transfection group compared with the control group. OCN and BSP mRNA levels were also significantly increased in the co-transfection group compared with cells transfected with BMP2 or FGF2 alone, suggesting that co-transfection significantly enhanced osteogenesis. CONCLUSIONS: Co-transfection of human ADSCs with BMP2/FGF2 via chitosan nanoparticles efficiently promotes the osteogenic properties of ADSCs in vitro.

Physicochemical-property guided design of a highly sensitive probe to image nitrosative stress in the pathology of stroke.

In vivo real-time imaging of nitrosative stress in the pathology of stroke has long been a formidable challenge due to both the presence of the blood-brain barrier (BBB) and the elusive nature of reactive nitrogen species, while this task is also informative to gain a molecular level understanding of neurovascular injury caused by nitrosative stress during the stroke episode. Herein, using a physicochemical property-guided probe design strategy in combination with the reaction-based probe design rationale, we have developed an ultrasensitive probe for imaging nitrosative stress evolved in the pathology of stroke. This probe demonstrates an almost zero background fluorescence signal but a maximum 1000-fold fluorescence enhancement in response to peroxynitrite, the nitrosative stress marker. Due to its good physicochemical properties, the probe readily penetrates the BBB after intravenous administration, and quickly accumulates in mice brain to sense local vascular injuries. After accomplishing its imaging mission, the probe is easily metabolized and therefore won't cause safety concerns. These desirable features make the probe competent for the straightforward visualization of nitrosative stress progression in stroke pathology.

Conformational restraint as a strategy for navigating towards lysosomes.

Using the conformational restraint strategy, we developed a hydrazonate-derived coumarin into a lysosome targeting probe for imaging native formaldehyde at the subcellular level. Using this probe, we observed the overproduction of formaldehyde in lysosomes when cells were treated with endoplasmic reticulum (ER) stress inducers, suggesting the involvement of formaldehyde in protein misfolding.

Myt1l induced direct reprogramming of pericytes into cholinergic neurons.

OBJECTIVE: The cholinergic deficit is thought to underlie progressed cognitive decline in Alzheimer Disease. The lineage reprogramming of somatic cells into cholinergic neurons may provide strategies toward cell-based therapy of neurodegenerative diseases. METHODS AND RESULTS: Here, we found that a combination of neuronal transcription factors, including Ascl1, Myt1l, Brn2, Tlx3, and miR124 (5Fs) were capable of directly converting human brain vascular pericytes (HBVPs) into cholinergic neuronal cells. Intriguingly, the inducible effect screening of reprogramming factors showed that a single reprogramming factor, Myt1l, induced cells to exhibit similarly positive staining for Tuj1, MAP2, ChAT, and VAChT upon lentivirus infection with the 5Fs after 30 days. HBVP-converted neurons were rarely labeled even after long-term incubation with BrdU staining, suggesting that induced neurons were directly converted from HBVPs rather than passing through a proliferative state. In addition, the overexpression of Myt1l induced the elevation of Ascl1, Brn2, and Ngn2 levels that contributed to reprogramming. CONCLUSIONS: Our findings provided proof of the principle that cholinergic neurons could be produced from HBVPs by reprogramming factor-mediated fate instruction. Myt1l was a critical mediator of induced neuron cell reprogramming. HBVPs represent another excellent alternative cell resource for cell-based therapy to treat neurodegenerative disease.

A Fluorogenic Probe for Ultrafast and Reversible Detection of Formaldehyde in Neurovascular Tissues.

Formaldehyde (FA) is endogenously produced in live systems and has been implicated in a diverse array of pathophysiological processes. To disentangle the detailed molecular mechanisms of FA biology, a reliable method for monitoring FA changes in live cells would be indispensable. Although there have been several fluorescent probes reported to detect FA, most are limited by the slow detection kinetics and the intrinsic disadvantage of detecting FA in an irreversible manner which may disturb endogenous FA homeostasis. Herein we developed a coumarin-hydrazonate based fluorogenic probe (PFM) based on a finely-tailored stereoelectronic effect. PFM could respond to FA swiftly and reversibly. This, together with its desirable specificity and sensitivity, endows us to track endogenous FA in live neurovascular cells with excellent temporal and spatial resolution. Further study in the brain tissue imaging showed the first direct observation of aberrant FA accumulation in cortex and hippocampus of Alzheimer's mouse model, indicating the potential of PFM as a diagnostic tool.

A H2O2-Responsive Theranostic Probe for Endothelial Injury Imaging and Protection.

Overproduction of H2O2 causes oxidative stress and is the hallmark of vascular diseases. Tracking native H2O2 in the endothelium is therefore indispensable to gain fundamental insights into this pathogenesis. Previous fluorescent probes for H2O2 imaging were generally arylboronates which were decomposed to emissive arylphenols in response to H2O2. Except the issue of specificity challenged by peroxynitrite, boric acid by-produced in this process is actually a waste with unknown biological effects. Therefore, improvements could be envisioned if a therapeutic agent is by-produced instead. Herein, we came up with a "click-to-release-two" strategy and demonstrate that dual functional probes could be devised by linking a fluorophore with a therapeutic agent via a H2O2-responsive bond. As a proof of concept, probe AP consisting of a 2-(2'-hydroxyphenyl) benzothiazole fluorophore and an aspirin moiety has been prepared and confirmed for its theranostic effects. This probe features high specificity towards H2O2 than other reactive species including peroxynitrite. Its capability to image and ameliorate endothelial injury has been verified both in vitro and in vivo. Noteworthy, as a result of its endothelial-protective effect, AP also works well to reduce thrombosis formation in zebrafish model.

Stress-responsive heme oxygenase-1 isoenzyme participates in Toll-like receptor 4-induced inflammation during brain ischemia.

Toll-like receptors (TLRs) are involved in the progression of ischemic brain injury and hence vascular dementia; however, the underlying mechanisms are largely unknown. Here, we have investigated the interrelationship between stress-responsive heme oxygenase (HO)-1 isoenzyme and TLR4 during chronic brain hypoperfusion. The right unilateral common carotid artery occlusion was performed by ligation of the right common carotid artery in C57BL/6J mice. The brain cortex or hippocampus was removed for western blotting and confocal immunofluorescence analysis. The link between HO-1 and TLR4 was further examined by silencing TLR4 and pharmacological inhibition of HO-1 in primary cultured cortical neurons. Cognitive dysfunction and decrease in cerebral blood flow in mice were observed 4 weeks after the occlusion. Our data further show that common carotid artery occlusion induced an increase in TLR4 and HO-1 protein levels. Although the administration of CoPP (10 mg/kg), HO-1 agonist, improved the cognitive dysfunction in a mice model of occlusion, western blot analysis in primary cultured cortical neurons showed that HO-1 was upregulated after lipopolysaccharide treatment; this was partially abolished by the TLR4 siRNA interference. The flow cytometry analysis showed that pharmacological inhibition of HO-1 by ZnPP (100 µM) further exaggerated lipopolysaccharide-induced neuronal cell death. Hence, stress-responsive HO-1 isoenzyme participates in TLR4-induced inflammation during chronic brain ischemia. The pharmacological manipulation of TLR4 or the HO-1 antioxidant defense pathway may represent a novel treatment strategy for neuronal protection in vascular dementia.

Involvement of p38MAPK and reactive oxygen species in icariin-induced cardiomyocyte differentiation of murine embryonic stem cells in vitro.

We previously reported that treatment of icariin could significantly induce cardiomyocyte differentiation of murine embryonic stem (ES) cells in vitro. In the present study, the exact activity initiated by icariin was further confirmed and the underlying molecular mechanism was investigated. We found that cardiomyocyte differentiation was efficiently stimulated only if icariin was administrated between days 5 and 8 in differentiation course, which indicated with elevated percentage of embryoid bodies (EB) and with beating areas and up- regulated expression of alpha-actinin and troponin T. Exposure of icariin triggered intracellular reactive oxygen species (ROS) generation of EBs in 3 h, which was abolished in the presence of either NADPH oxidase inhibitor DPI or antioxidant Trolox. Meanwhile, expression of NOX4, a membrane combined enzyme responsible for ROS generation, was promoted by icariin in a dose-dependent manner. Although p38MAPK (mitogen-activated protein kinase), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal protein kinase (JNK) were spontaneously activated in early differentiation, only the phosphorylation of p38MAPK was enhanced and prolonged when icariin was present, whereas both ERK and JNK showed no response to icariin treatment. Moreover, the inducible effect of icariin was blunted by SB203580, a specific inhibitor of p38MAPK. On the contrary, neither UO126 nor SP600125, the specific inhibitor of ERK and JNK, could abolish icariin-stimulated differentiation. Nuclear location of MEF2C, which played a critical role in cardiomyocyte differentiation and could be activated by p38MAPK, was stimulated after icariin exposure. Taken together, these results suggest that ROS generation and the subsequent activation of p38MAPK are essential for the inducible function of icariin on cardiomyocyte differentiation of murine embryonic stem cells in vitro.

Time-dependence of cardiomyocyte differentiation disturbed by peroxisome proliferator-activated receptor alpha inhibitor GW6471 in murine embryonic stem cells in vitro.

AIM: To investigate the possible roles of peroxisome proliferator-activated receptor alpha(PPAR alpha) and the signal pathway regulating the transcription of PPAR alpha in the cardiomyocyte differentiation course of murine embryonic stem (ES) cells in vitro. METHODS: The expression of PPAR alpha during cardiomyocyte differentiation was analyzed using both Western blotting and immunofluorescence. Cardiac specific genes and sarcomeric proteins were evaluated when embryoid bodies were challenged with PPAR alpha specific inhibitor GW6471 at different time courses. The phosphorylation of p38 mitogen-activated protein kinase (MAPK) was studied in the differentiation process, and its specific inhibitor SB203580 was employed to study the function of p38 MAPK on cardiac differentiation and the expression of PPAR alpha. RESULTS: The expression of PPAR alpha was observed to be at a low level in undifferentiated ES cells and markedly induced with the appearance of beating clusters. The inhibition of PPAR alpha by its specific inhibitor GW6471 (1X10(-5) mol/L) significantly prevented cardiomyocyte differentiation and resulted in the reduced expression of cardiac sarcomeric proteins (ie alpha-actinin, troponin-T) and specific genes (ie alpha-MHC, MLC2v) in a time-dependent manner. In the differentiation course, p-p38 MAPK was maintained at a high level from d 3 followed by a decrease from d 10. The inhibition of the p38 MAPK pathway by SB203580 between d 3 and d 7 efficiently prevented cardiomyocyte differentiation and resulted in the capture of the upregulation of PPAR alpha. CONCLUSION: Taken together, these results showed a close association between PPAR alpha and cardiomyocyte differentiation in vitro, and p38 MAPK was partly responsible for the regulation of PPAR alpha.

Icariin promotes expression of PGC-1alpha, PPARalpha, and NRF-1 during cardiomyocyte differentiation of murine embryonic stem cells in vitro.

AIM: To investigate the effect of icariin on the expression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1alpha), peroxisome proliferator-activated receptor alpha (PPARalpha), and nuclear respiratory factor 1 (NRF-1) on cardiomyocyte differentiation of murine embryonic stem (ES) cells in vitro. METHODS: The cardiomyocytes derived from murine ES cells were verified by immunocytochemistry using confocal laser scanning microscopy. Cardiac-specific sarcomeric proteins (ie alpha-actinin, troponin T) were evaluated when embryoid bodies (EB) were treated with icariin or retinoid acid. The expression of PGC-1alpha, PPARalpha, and NRF-1 were analyzed using both semiquantitative RT-PCR and Western blotting in cardiomyocyte differentiation. The phosphorylation of the p38 mitogen-activated protein kinase (MAPK) was studied in the differentiation process, and its specific inhibitor SB203580 was employed to confirm the function of the p38 MAPK on icariin-induced cardiac differentiation. RESULTS: The application of icariin significantly induced the cardiomyocyte differentiation of EB as indicated by the promoted expression of alpha-actinin and troponin T. The expression of PGC-1alpha, PPARalpha, and NRF-1 increased coincidently in early differentiation and the increase was dose-dependently upregulated by icariin treatment. The phosphorylation of the p38 MAPK peaked on d 6 and decreased after d 8, and the activation was further enhanced and prolonged when the EB were subjected to icariin, which was concurrent with the elevation of PGC-1alpha, PPARalpha, and NRF-1. Moreover, the inhibition of the p38 MAPK pathway by SB203580 efficiently abolished icariin-stimulated cardiomyocyte differentiation and resulted in the capture of the upregulation of PGC-1alpha, PPARalpha, and NRF-1. CONCLUSION: Taken together, icariin promoted the expression of PGC-1alpha, PPARalpha, and NRF-1 during cardiomyocyte differentiation of murine ES cells in vitro and the effect was partly responsible for the activation of the p38 MAPK.