Multimodal phototherapy agents target lipid droplets for near-infrared imaging-guided type I photodynamic/photothermal therapy.

The construction and optimization of a single phototherapeutic agent with photoluminescence, type I photodynamic therapy (PDT), and photothermal therapy (PTT) functions remain challenging. In this study, we aimed to design and synthesize four donor-acceptor (D-A) type aggregation-induced emission molecules: PSI, TPSI, PSSI, and TPSSI. We employed phenothiazine as an electron donor and 1,3-bis(dicyanomethylidene)indan as a strong electron acceptor in the synthesis process. Among them, TPSSI exhibited efficient type I reactive oxygen species generation, high photothermal conversion efficiency (45.44 %), and near-infrared emission. These observations can be attributed to the introduction of a triphenylamine electron donor group and a thiophene unit, which resulted in increased D-A strengths, a reduced singlet-triplet energy gap, and increased free intramolecular motion. TPSSI was loaded into bovine serum albumin to prepare biocompatible TPSSI nanoparticles (NPs). Our results have indicated that TPSSI NPs can target lipid droplets with negligible dark toxicity and can efficiently generate O2•- in hypoxic tumor environments. Moreover, TPSSI NPs selectively targeted 4T1 tumor tissues and exhibited a good PDT-PTT synergistic effect in vitro and in vivo. We believe that the successful preparation of multifunctional phototherapeutic agents will promote the development of efficient tumor diagnosis and treatment technologies. STATEMENT OF SIGNIFICANCE: The construction of a single phototherapeutic agent with photoluminescence, type I photodynamic therapy, and photothermal therapy functions, and its optimization remain challenging. In this study, we construct four donor-acceptor aggregation-induced emission molecules using phenothiazine as an electron donor and 1,3-Bis(dicyanomethylidene)indan as a strong electron acceptor. By optimizing the molecular structure, an integrated phototherapy agent with fluorescence imaging ability and high photodynamic / photothermal therapy performance was prepared. We believe that the successful preparation of multifunctional phototherapeutic agents will promote the development of efficient tumor diagnosis and treatment technology.

Aminophosphate precursors for the synthesis of near-unity emitting InP quantum dots and their application in liver cancer diagnosis.

InP quantum dots (QDs) are a promising and environment-friendly alternative to Cd-based QDs for in vitro diagnostics and bioimaging applications. However, their poor fluorescence and stability severely limit their biological applications. Herein, we synthesize bright (∼100%) and stable InP-based core/shell QDs by using cost-effective and low-toxic phosphorus source, and then aqueous InP QDs are prepared with quantum yield over 80% by shell engineering. The immunoassay of alpha-fetoprotein can be detected in the widest analytical range of 1-1000 ng ml-1 and the limit of detection of 0.58 ng ml-1 by using those InP QDs-based fluorescent probes, making it the best-performing heavy metal-free detection reported so far, comparable to state-of-the-art Cd-QDs-based probes. Furthermore, the high-quality aqueous InP QDs exhibit excellent performance in specific labeling of liver cancer cells and in vivo tumor-targeted imaging of live mice. Overall, the present work demonstrates the great potential of novel high-quality Cd-free InP QDs in cancer diagnosis and image-guided surgery.

Evaluation of the Biological Activity of Folic Acid-Modified Paclitaxel-Loaded Gold Nanoparticles.

PURPOSE: Gold nanoparticles (AuNPs) with good physical and biological properties are often used in medicine, diagnostics, food, and similar industries. This paper explored an AuNPs drug delivery system that had good target selectivity for folate-receptor overexpressing cells to induce apoptosis. METHODS: A novel drug delivery system, Au@MPA-PEG-FA-PTX, was developed carrying paclitaxel (PTX) on folic acid (FA) and polyethylene glycol (PEG)-modified AuNPs. The nanomaterial was characterized by transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), and ultraviolet-visible spectroscopy (UV-Vis). Also, the biological activity of the AuNPs drug delivery system was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in HL-7702, Hela, SMMC-7721, and HCT-116 cells. Furthermore, apoptotic activity using annexin V-FITC, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) levels was estimated by flow cytometry and fluorescence microscopy. RESULTS: Au@MPA-PEG-FA-PTX exhibited a distinct core-shell structure with a controllable size of 28±1 nm. Also, the AuNPs maintained good dispersion and spherical shape uniformity before and after modification. The MTT assay revealed good antitumor activity of the Au@MPA-PEG-FA-PTX against the Hela, SMMC-7721, and HCT-116 cells, while Au@MPA-PEG-FA-PTX produced better pharmacological effects than PTX in isolation. Further mechanistic investigation revealed that effective internalization of AuNPs by folate-receptor overexpressing cancer cells induced cell apoptosis through excessive production of intracellular ROS. CONCLUSION: The AuNPs drug delivery system showed good target selectivity for folate-receptor overexpressing cancer cells to induce target cell-specific apoptosis. These AuNPs may have great potential as theranostic agents such as in cancer.

Mesoporous silica nanoparticles loaded with fluorescent coumarin-5-fluorouracil conjugates as mitochondrial-targeting theranostic probes for tumor cells.

In this study, a nanodrug carrier (mesoporous silica nanoparticle (MSN)-SS-cysteamine hydrochloride (CS)-hyaluronic acid (HA)) for targeted drug delivery was prepared using MSNs, in which HA was used as a targeting ligand and blocking agent to control drug release. Coumarin is a fluorescent molecule that targets mitochondria. Two conjugates (XDS-DJ and 5-FUA-4C-XDS) were synthesized by chemically coupling nitrogen mustard and 5-fluorouracil with coumarin, which was further loaded into MSN-SS-CS-HA nanocarriers. MTT analysis demonstrated that the nanocomposite MSN-SS-CS@5-FUA-4C-XDS/HA displayed stronger cytotoxicity toward HCT-116 cells than HeLa or QSG-7701 cells. Furthermore, MSN-SS-CS@5-FUA-4C-XDS/HA was able to target the mitochondria of HCT-116 cells, causing decreased mitochondrial membrane potential and excessive production of reactive oxygen species. These results indicate that MSN-SS-CS@5-FUA-4C-XDS/HA has the potential to be a nanodrug delivery system for the treatment of colon cancer.

Hyaluronic Acid-Modified and Doxorubicin-Loaded Gold Nanoparticles and Evaluation of Their Bioactivity.

Functionalized gold nanoparticles (AuNPs) have been successfully used in many fields as a result of having low cytotoxicity, good biocompatibility, excellent optical properties, and their ability to target cancer cells. Here, we synthesized AuNP carriers that were modified by hyaluronic acid (HA), polyethylene glycol (PEG), and adipic dihydrazide (ADH). The antitumor drug doxorubicin (Dox) was loaded into AuNP carriers and attached chemically. The Au nanocomposite AuNPs@MPA-PEG-HA-ADH-Dox was able to disperse uniformly in aqueous solution, with a diameter of 15 nm. The results of a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay indicated that AuNP carriers displayed very little toxicity toward cells in high doses, although the antitumor properties of Au nanocomposites were significantly enhanced. Cellular uptake experiments demonstrated that AuNPs modified with hyaluronic acid were more readily ingested by HepG2 and HCT-116 cells, as they have a large number of CD44 receptors. A series of experiments measuring apoptosis such as Rh123 and annexin V-FITC staining, and analysis of mitochondrial membrane potential (MMP) analysis, indicated that apoptosis played a role in the inhibition of cell proliferation by AuNPs@MPA-PEG-HA-ADH-Dox. Excessive production of reactive oxygen species (ROS) was the principal mechanism by which the Au nanocomposites inhibited cell proliferation, leading to apoptosis. Thus, the Au nanocomposites, which allowed cell imaging in real-time and induced apoptosis in specific cell types, represent theragnostic agents with potential for future clinical applications in bowel cancer.

Association of polymorphisms in vascular endothelial growth factor gene with the age of onset of amyotrophic lateral sclerosis.

This study investigated the association between polymorphisms in vascular endothelial growth factor (VEGF) gene (-1558C-T, -1190A-G and -1154A-G) and age at onset of amyotrophic lateral sclerosis (ALS). Between July 2000 and June 2004 we conducted a clinical genetic study at Peking University Third Hospital, China. The analyses included a total of 93 ALS patients. Genotyping was performed by using the 5'-nuclease assay technology (Applied Biosystems) with TaqMan allele-specific fluorogenic oligonucleotide probes. We used multivariate linear regression modelling and haplotype-based association test to analyse the association of VEGF gene polymorphisms with the age of onset, adjusting for initial symptoms and sex. The results indicated that patients with the -1190A/G and -1190G/G genotypes exhibited about a 4.1- and 9.4-years earlier onset of ALS than the patients with the -1190A/A genotype. A similar pattern emerged when the VEGF -1154A-G gene was considered: the beta was -7.9(p<0.001) years and -11.7(p<0.001) years for -1154A/G and -1154G/G genotypes, respectively. The VEGF -1558C-T had a positive effect in the -1558C/T group (p = 0.007, beta = 7.0) and -1558T/T (p<0.001, beta = 9.6) compared to the -1558C/C group. We neither observed an interaction nor haplotype association with age onset among -1558C-T, -1190A-G and -1154A-G. In conclusion, our results indicate, for the first time, that there was an important association between the polymorphism of the VEGF gene and age of ALS onset. This suggests a possible role for VEGF variability in the aetiology of individual differences in ALS onset.

Dedifferentiation of adult human myoblasts induced by ciliary neurotrophic factor in vitro.

Ciliary neurotrophic factor (CNTF) is primarily known for its important cellular effects within the nervous system. However, recent studies indicate that its receptor can be highly expressed in denervated skeletal muscle. Here, we investigated the direct effect of CNTF on skeletal myoblasts of adult human. Surprisingly, we found that CNTF induced the myogenic lineage-committed myoblasts at a clonal level to dedifferentiate into multipotent progenitor cells--they not only could proliferate for over 20 passages with the expression absence of myogenic specific factors Myf5 and MyoD, but they were also capable of differentiating into new phenotypes, mainly neurons, glial cells, smooth muscle cells, and adipocytes. These "progenitor cells" retained their myogenic memory and were capable of redifferentiating into myotubes. Furthermore, CNTF could activate the p44/p42 MAPK and down-regulate the expression of myogenic regulatory factors (MRFs). Finally, PD98059, a specific inhibitor of p44/p42 MAPK pathway, was able to abolish the effects of CNTF on both myoblast fate and MRF expression. Our results demonstrate the myogenic lineage-committed human myoblasts can dedifferentiate at a clonal level and CNTF is a novel regulator of skeletal myoblast dedifferentiation via p44/p42 MAPK pathway.

Increased heme oxygenase-1 expression in infarcted rat hearts following human bone marrow mesenchymal cell transplantation.

Heme oxygenase-1 (HO-1) plays an important role in oxidative stress and recent studies indicate that it is a graft survival protein in cardiac and liver transplant models. In this study, we investigated the relation between the expressions of HO-1 and the effects of human bone marrow mesenchymal cells (MSCs) transplantation to xenogenic rat hearts with experimental myocardial infarction (MI). A total of 5 x 10(6) cells in 100 microl PBS or equal volume PBS alone were injected into the ischemic zones immediately post-MI. At 1, 3, and 7 days post-MI, cardiac function was evaluated by echocardiography, the expression of HO-1 was assessed by real-time PCR and Western blot, the localization of HO-1 protein was determined under immunofluorescence microscopy. The infarct size was examined by histology. The numbers of Hoechst-33342 positive MSCs were evaluated under immunofluorescence microscopy and also by flow cytometry after isolation from the host hearts. The results indicated that the HO-1 expressions were markedly increased at both mRNA and protein levels in comparison with injection of PBS at each time point post MSCs transplantation (P < 0.01). HO-1 was revealed both in transplanted MSCs and recipient cardiomyocytes by immunofluorescence. Up-regulated HO-1 expression was accompanied by increase of the numbers of Hoechst-33342 positive MSCs, the reduction of infarct size, and the improvement of cardiac function. Transplantation of human MSCs could up-regulate HO-1 expression in infarct rat hearts, which might play an important role in protecting transplanted MSCs, cardiomyocytes survival, and cardiac function improvement during the early stage after MI.

Purified human bone marrow multipotent mesenchymal stem cells regenerate infarcted myocardium in experimental rats.

Recent findings suggest the feasibility of cardiac repair by transplantation of bone marrow mesenchymal stem cell (MSCs). However, it remains controversial regarding which cell type is the best source for transplanting into the ischemic heart because of lack of well-defined cell markers. In this study, we investigated the in vitro and in vivo effects of the novel multipotent marrow mesenchymal stem cells (MMSCs) from human bone marrow. Pluripotent markers (Oct4, Bmi1, and Abcg2) and vascular endothelial growth factor (VEGF) were detected by RT-PCR and immunofluorescence in MMSCs. Myocardial differentiation was induced in the expanded MMSC cultures by treatment with 5-azacyline. Expressions of VEGF in the animals transplanted with MMSCs were markedly increased in comparison with the animals injected with fibroblasts or saline at both mRNA and protein levels. VEGF expression was observed in both transplanted MMSCs and recipient cardiomyocytes by immunofluorescence. Confocal immunofluorescence microscopy revealed the specific markers for cardiomyocytes and endothelial cells in transplanted MMSCs 14 days after transplantation. Vessel count was increased and left ventricular function improved post-MMSC transplantation. These results indicate that transplantation of purified MMSCs from human bone marrow upregulated VEGF expression, enhanced angiogenesis, and improved the functional recovery following myocardial infarction in rats.

Fetal human neural progenitors can be the target for tumor transformation.

The hypothesis that stem cells may seed cancer has emerged from the cancer stem cells concept. However, the experimental systems necessary to provide more direct evidence to support the hypothesis have been lacking. We have used fetal neural progenitor cells (hNPC) transduced with the telomerase hTERT gene to investigate the neoplastic potential of hNPCs. The hTERT-transduced line, hNPCs-G3 lost normal diploid karyotype, showed loss of contact inhibition, anchorage independence, and formed neuroblastoma-like tumours in all of 10 mice. These data suggest that hNPCs have the potential for neoplastic transformation. These data have implications for providing a novel tool to test the feasibility of new anticancer treatment strategies and raise the possibility of a risk for the use of hNPCs in cell transplantation.

Distinct efficacy of pre-differentiated versus intact fetal mesencephalon-derived human neural progenitor cells in alleviating rat model of Parkinson's disease.

Neural progenitor cells have shown the effectiveness in the treatment of Parkinson's disease, but the therapeutic efficacy remains variable. One of important factors that determine the efficacy is the necessity of pre-differentiation of progenitor cells into dopaminergic neurons before transplantation. This study therefore investigated the therapeutic efficacy of mesencephalon-derived human neural progenitor cells with or without the pre-differentiation in alleviating a rat model of Parkinson's disease. We found that a combination of 50 ng/ml fibroblast growth factor 8, 10 ng/ml glial cell line-derived neurotrophic factor and 10 microM forskolin facilitated the differentiation of human fetal mesencephalic progenitor cells into dopaminergic neurons in vitro. More importantly, after transplanted into the striatum of parkinsonian rats, only pre-differentiated grafts resulted in an elevated production of dopamine in the transplanted site and the amelioration of behavioral impairments of the parkinsonian rats. Unlike pre-differentiated progenitors, grafted intact progenitors rarely differentiated into dopaminergic neurons in vivo and emigrated actively away from the transplanted site. These data demonstrates the importance of pre-differentiation of human progenitor cells before transplantation in enhancing therapeutic potency for Parkinson's disease.

Elevation of NMDAR after transplantation of neural stem cells.

Cognitive deficits could be alleviated by transplantation of neural stem cells in animals. Grafted cells may differentiate into neurons, thereby improving animal cognition. Alternatively, grafted cells may provide neurotrophic factors to modify neuronal functions and to alleviate cognitive deficits. To test which mechanism is underlying this recovery process, senescence-accelerated mice were transplanted with human neural stem cells into the hippocampus. The effect of cell transplantation was assessed in the Morris water maze. The survival and differentiation of grafted cells and the expression of NMDA receptors were examined. The data suggested that in addition to the neural differentiation of grafted neural stem cells, up-regulation of NMDA receptors after transplantation also contributed to the alleviation of cognitive deficits in this animal model.

Telomerase immortalization of human neural progenitor cells.

It is necessary to expand human neural progenitor cells in vitro to obtain large numbers for research purposes and cell transplantation. A potential obstacle to in vitro expansion, however, is that neural progenitor cells have a limited replication life-span and gradually lose their differentiation potential. We report here that ectopic expression of the catalytic subunit of human telomerase (hTERT) gene in neural progenitor cells could induce telomerase activity, stabilize telomeres and extend their replicative life-spans. The telomerase-immortalized cells (hNPC-TERT) maintained the normal diploid karyotype, expressed the markers of human neural progenitor cells and meanwhile held the differentiation potential in vitro for up to 120 population doublings. This study provides a new approach for obtaining unlimited quantities of normal phenotypic and homogeneous human neural progenitor cells in vitro.

Long-term effects of bone marrow mononuclear cell transplantation on left ventricular function and remodeling in rats.

This study was performed to evaluate the long-term effect on left ventricular function and remodeling in a rat model of bone marrow cell transplantation (BMT) into acute infarcted myocardium. After myocardial infarction was induced in inbred Lewis rats by left anterior descending artery ligation, the ischemic area was directly injected with saline, peripheral blood mononuclear cells (PB-MNCs) or bone marrow mononuclear cells (BM-MNCs). Cardiac function and structure were evaluated by echocardiography before the operation, and on day 1 and 2 months post-infarct. The collagen content, the number of vessels and the vasculogenesis were examined by histology and immunohistochemistry. We found at 2 months post-infarct, BMT significantly improved cardiac systolic function and recovered diastolic function. Transplantation of BM-MNCs, but not PB-MNCs, reversed remodeling and reduced collagen density. Vessel counts showed greater angiogenesis occurred in the animals transplanted with BM-MNCs. Furthermore, a vascular endothelial cell-specific marker was detected in the transplanted bone marrow cells. Our data suggest that BM-MNC transplantation results in long-term improvement in left ventricular function-especially diastolic function- and remodeling, possibly related with the reduction of the amount of the collagen and enhancement of neovascularization.

Enhanced cytoprotection and angiogenesis by bone marrow cell transplantation may contribute to improved ischemic myocardial function.

OBJECTIVES: Heat shock proteins (HSPs) are cytoprotective proteins. Vascular endothelial growth factor (VEGF) is the most potent angiogenic factor. This study aimed to elucidate the possible role of cytoprotection and angiogenesis on cardiac function after bone marrow cell transplantation (BMT). METHODS: Myocardial infarction was induced in inbred Lewis rats by left anterior descending artery ligation. A total of 5 x 10(6) bone marrow-mononuclear cells were transplanted into the ischemic zone by direct injection. At 1, 3, 7, 14 and 28 days post-transplantation, cardiac function was evaluated by echocardiography. The expressions of HSP32, HSP70 and VEGF were assessed by immunofluorescence and RT-PCR. The number of vessels was examined by immunohistochemistry. The differentiation of the transplanted cells was determined by immunofluorescence. RESULTS: Echocardiography showed BMT led to sustained improvement in cardiac function, as assessed by left ventricle ejection fraction and fraction of shortening. Immunofluorescence revealed that the expressions of HSP32, HSP70 and VEGF were promoted in both transplanted bone marrow cells and recipient cardiomyocytes. RT-PCR showed that the mRNA expression levels of HSP32, HSP70 and VEGF in the BMT group were markedly higher in comparison with injection of peripheral blood cells or saline (P<0.01) by day 7. Seven days later, the vessel count showed that angiogenesis had been induced to a significantly greater degree in the BMT groups. Fourteen days later, specific markers for myocardial or vascular endothelial cells were detected in the transplanted bone marrow cells. CONCLUSIONS: BMT upregulated the expressions of HSP32, HSP70 and VEGF in both transplanted bone marrow cells and recipient endogenous cardiomyocytes in the early phase post-transplantation. This enhanced cytoprotection and angiogenesis, and contributed to the functional recovery following cardiac infarction. In the late phase, the transplanted bone marrow cells might differentiate into both myocardial and vascular endothelial cells that enhanced the ischemic cardiac function further.