Integration of PEG 400 into a self-nanoemulsifying drug delivery system improves drug loading capacity and nasal mucosa permeability and prolongs the survival of rats with malignant brain tumors.

Introduction: Kolliphor® EL (K-EL) is among the most useful surfactants in the preparation of emulsions. However, it is associated with low hydrophobic drug loading in the resulting emulsified formulation. Methods: In this study, a formulation for intranasal administration of butylidenephthalide (Bdph), a candidate drug against glioblastoma (GBM), was prepared. Physical characteristics of the formulation such as particle size, zeta potential, conductivity, and viscosity were assessed, as well as its cytotoxicity and permeability, in order to optimize the formulation and improve its drug loading capacity. Results: The optimized formulation involved the integration of polyethylene glycol 400 (PEG 400) in K-EL to encapsulate Bdph dissolved in dimethyl sulfoxide (DMSO), and it exhibited higher drug loading capacity and drug solubility in water than the old formulation, which did not contain PEG 400. Incorporation of PEG 400 as a co-surfactant increased Bdph loading capacity to up to 50% (v/v), even in formulations using Kolliphor® HS 15 (K-HS15) as a surfactant, which is less compatible with Bdph than K-EL. The optimized Bdph formulation presented 5- and 2.5-fold higher permeability and cytotoxicity, respectively, in human GBM than stock Bdph. This could be attributed to the high drug loading capacity and the high polarity index due to DMSO, which increases the compatibility between the drug and the cell. Rats bearing a brain glioma treated with 160 mg/kg intranasal emulsified Bdph had a mean survival of 37 days, which is the same survival time achieved by treatment with 320 mg/kg stock Bdph. This implies that the optimized emulsified formulation required only half the Bdph dose to achieve an efficacy similar to that of stock Bdph in the treatment of animals with malignant brain tumor.

n-Butylidenephthalide exhibits protection against neurotoxicity through regulation of tryptophan 2, 3 dioxygenase in spinocerebellar ataxia type 3.

Spinocerebellar ataxia type 3 or Machado-Joseph disease (SCA3/MJD) is characterized by the repetition of a CAG codon in the ataxin-3 gene (ATXN3), which leads to the formation of an elongated mutant ATXN3 protein that can neither be denatured nor undergo proteolysis in the normal manner. This abnormal proteolysis leads to the accumulation of cleaved fragments, which have been identified as toxic and further they act as a seed for more aggregate formation, thereby increasing toxicity in neuronal cells. To date, there have been few studies or treatment strategies that have focused on controlling toxic fragment formation. The aim of this study is to develop a potential treatment strategy for addressing the complications of toxic fragment formation and to provide an alternative treatment strategy for SCA3. Our preliminary data on anti-aggregation and toxic fragment formation using an HEK (human embryonic kidney cells) 293T-84Q-eGFP (green fluorescent protein) cell model identified n-butylidenephthalide (n-BP) as a potential drug treatment for SCA3. n-BP decreased toxic fragment formation in both SCA3 cell and animal models. Moreover, results showed that n-BP can improve gait, motor coordination, and activity in SCA3 mice. To comprehend the molecular basis behind the control of toxic fragment formation, we used microarray analysis to identify tryptophan metabolism as a major player in controlling the fate of mutant ATXN3 aggregates. We also demonstrated that n-BP functions by regulating the early part of the kynurenine pathway through the downregulation of tryptophan 2, 3-dioxygenase (TDO2), which decreases the downstream neurotoxic product, quinolinic acid (QA). In addition, through the control of TDO2, n-BP also decreases active calpain levels, an important enzyme involved in the proteolysis of mutant ATXN3, thereby decreasing toxic fragment formation and associated neurotoxicity. Collectively, these findings indicate a correlation between n-BP, TDO2, QA, calpain, and toxic fragment formation. Thus, this study contributes to a better understanding of the molecular interactions involved in SCA3, and provides a novel potential treatment strategy for this neurodegenerative disease.

Genetic and epigenetic instability of stem cells.

Recently, research on stem cells has been receiving an increasing amount of attention, both for its advantages and disadvantages. Genetic and epigenetic instabilities among stem cells have been a recurring obstacle to progress in regenerative medicine using stem cells. Various reports have stated that these instabilities can transform stem cells when transferred in vivo and thus have the potential to develop tumors. Previous research has shown that various extrinsic and intrinsic factors can contribute to the stability of stem cells. The extrinsic factors include growth supplements, growth factors, oxygen tension, passage technique, and cryopreservation. Controlling these factors based on previous reports may assist researchers in developing strategies for the production and clinical application of "safe" stem cells. On the other hand, the intrinsic factors can be unpredictable and uncontrollable; therefore, to ensure the successful use of stem cells in regenerative medicine, it is imperative to develop and implement appropriate strategies and technique for culturing stem cells and to confirm the genetic and epigenetic safety of these stem cells before employing them in clinical trials.

Cells and materials for liver tissue engineering.

Liver transplantation is currently the most efficacious treatment for end-stage liver diseases. However, one main problem with liver transplantation is the limited number of donor organs that are available. Therefore, liver tissue engineering based on cell transplantation that combines materials to mimic the liver is under investigation with the goal of restoring normal liver functions. Tissue engineering aims to mimic the interactions among cells with a scaffold. Particular materials or a matrix serve as a scaffold and provide a three-dimensional environment for cell proliferation and interaction. Moreover, the scaffold plays a role in regulating cell maturation and function via these interactions. In cultures of hepatic lineage cells, regulation of cell proliferation and specific function using biocompatible synthetic, biodegradable bioderived matrices, protein-coated materials, surface-modified nanofibers, and decellularized biomatrix has been demonstrated. Furthermore, beneficial effects of addition of growth factor cocktails to a flow bioreactor or coculture system on cell viability and function have been observed. In addition, a system for growing stem cells, liver progenitor cells, and primary hepatocytes for transplantation into animal models was developed, which produces hepatic lineage cells that are functional and that show long-term proliferation following transplantation. The major limitation of cells proliferated with matrix-based transplantation systems is the high initial cell loss and dysfunction, which may be due to the absence of blood flow and the changes in nutrients. Thus, the development of vascular-like scaffold structures, the formation of functional bile ducts, and the maintenance of complex metabolic functions remain as major problems in hepatic tissue engineering and will need to be addressed to enable further advances toward clinical applications.

Adipose-derived stem cells can abrogate chemical-induced liver fibrosis and facilitate recovery of liver function.

Adipose-derived stem cells (ADSCs) are easy to harvest and have the ability for self-renewal and to differentiate into various cell types, including those of the hepatic lineage. Studies on the use of ADSCs for liver transplantation are, however, limited. The objective of this study was to investigate the feasibility of using human ADSCs and to better understand their mechanism of action for the repair of liver damage in a thioacetamide (TAA)-induced model of chronic liver damage in the rat. To induce liver damage, 200 mg/kg TAA was injected intraperitoneally into Wistar rats every 3 days for 60 days. For cell therapy, 1 × 10(6) human ADSCs suspended in 300 µl of phosphate-buffered saline were transplanted into each experimental rat by direct liver injection. Immunohistochemistry showed that the transplanted ADSCs differentiated into albumin- and α-fetoprotein-secreting liver-like cells 1 week after transplantation. In addition, liver function recovered significantly, as determined by biochemical analyses that analyzed total bilirubin, prothrombin time, and albumin levels. The Metavir score, derived from histopathological analysis, also showed a significant decrease in liver fibrosis and inflammatory activity after ADSC transplantation. Finally, we found a reduction in the expression of α-smooth muscle actin, a marker of hepatic stellate cells, which produce collagen fiber, and an increase in the expression of matrix metalloproteinase-9, which degrades collagen fiber, after ADSC transplantation. These findings are consistent with abrogation of liver fibrosis in the ADSC therapy group. Consequently, these results suggest that ADSC transplantation may facilitate recovery from chronic liver damage and thus may have clinical applications.

Synthesis and evaluation of a new series of tri-, di-, and mono-N-alkylcarbamylphloroglucinols as bulky inhibitors of acetylcholinesterase.

1,3,5-Tri-N-alkylcarbamylphloroglucinols (1-4) are synthesized as a new series of bulky inhibitors of acetylcholinesterase that may block the catalytic triad, the anionic substrate binding site, and the entrance of the enzyme simultaneously. Among three series of phloroglucinol-derived carbamates, tridentate inhibitors 1,3,5-tri-N-alkylcarbamylphloroglucinols (1-4), bidentate inhibitors 3,5-di-N-n-alkylcarbamyloxyphenols (5-8), and monodentate inhibitors 5-N-n-alkylcarbamyloxyresorcinols (9-12), tridentate inhibitors 1-4 are the most potent inhibitors of mouse acetylcholinesterase. When different n-alkylcarbamyl substituents in tridentate inhibitors 1-4 are compared, n-octylcarbamate 1 is the most potent inhibitor of the enzyme. All inhibitors 1-12 are characterized as the pseudo substrate inhibitors of acetylcholinesterase. Thus, tridentate inhibitors 1-4 are supposed to be hydrolyzed to bidentate inhibitors 5-8 after the enzyme catalysis. Subsequently, bidentate inhibitors 5-8 and monodentate inhibitors 9-12 are supposed to yield monodentate inhibitors 9-12 and phloroglucinol, respectively, after the enzyme catalysis. This means that tridentate inhibitors 1-4 may act as long period inhibitors of the enzyme. Therefore, inhibitors 1-4 may be considered as a new methodology to develop the long-acting drug for Alzheimer's disease. Automated dockings of inhibitor 1 into the X-ray crystal structure of acetylcholinesterase suggest that the most suitable configuration of inhibitor 1 to the enzyme binding is the (1,3,5)- (cis,trans,trans)-tricarbamate rotamer. The cis-carbamyl moiety of this rotamer does not bind into the acetyl group binding site of the enzyme but stretches out itself to the entrance. The other two trans-carbmayl moieties of this rotamer bulkily block the tryptophan 86 residue of the enzyme.

Local interstitial delivery of z-butylidenephthalide by polymer wafers against malignant human gliomas.

We have shown that the natural compound z-butylidenephthalide (Bdph), isolated from the chloroform extract of Angelica sinensis, has antitumor effects. Because of the limitation of the blood-brain barrier, the Bdph dosage required for treatment of glioma is relatively high. To solve this problem, we developed a local-release system with Bdph incorporated into a biodegradable polyanhydride material, p(CPP-SA; Bdph-Wafer), and investigated its antitumor effects. On the basis of in vitro release kinetics, we demonstrated that the Bdph-Wafer released 50% of the available Bdph by the sixth day, and the release reached a plateau phase (90% of Bdph) by the 30th day. To investigate the in situ antitumor effects of the Bdph-Wafer on glioblastoma multiforme (GBM), we used 2 xenograft animal models-F344 rats (for rat GBM) and nude mice (for human GBM)-which were injected with RG2 and DBTRG-05MG cells, respectively, for tumor formation and subsequently treated subcutaneously with Bdph-Wafers. We observed a significant inhibitory effect on tumor growth, with no significant adverse effects on the rodents. Moreover, we demonstrated that the antitumor effect of Bdph on RG2 cells was via the PKC pathway, which upregulated Nurr77 and promoted its translocation from the nucleus to the cytoplasm. Finally, to study the effect of the interstitial administration of Bdph in cranial brain tumor, Bdph-Wafers were surgically placed in FGF-SV40 transgenic mice. Our Bdph-Wafer significantly reduced tumor size in a dose-dependent manner. In summary, our study showed that p(CPP-SA) containing Bdph delivered a sufficient concentration of Bdph to the tumor site and effectively inhibited the tumor growth in the glioma.

Pulsed electromagnetic fields accelerate proliferation and osteogenic gene expression in human bone marrow mesenchymal stem cells during osteogenic differentiation.

Osteogenesis is a complex series of events involving the differentiation of mesenchymal stem cells to generate new bone. In this study, we examined the effect of pulsed electromagnetic fields (PEMFs) on cell proliferation, alkaline phosphatase (ALP) activity, mineralization of the extracellular matrix, and gene expression in bone marrow mesenchymal stem cells (BMMSCs) during osteogenic differentiation. Exposure of BMMSCs to PEMFs increased cell proliferation by 29.6% compared to untreated cells at day 1 of differentiation. Semi-quantitative RT-PCR indicated that PEMFs significantly altered temporal expression of osteogenesis-related genes, including a 2.7-fold increase in expression of the key osteogenesis regulatory gene cbfa1, compared to untreated controls. In addition, exposure to PEMFs significantly increased ALP expression during the early stages of osteogenesis and substantially enhanced mineralization near the midpoint of osteogenesis. These results suggest that PEMFs enhance early cell proliferation in BMMSC-mediated osteogenesis, and accelerate the osteogenesis.

Effect of pulsed electromagnetic field on the proliferation and differentiation potential of human bone marrow mesenchymal stem cells.

Pulsed electromagnetic fields (PEMFs) have been used clinically to slow down osteoporosis and accelerate the healing of bone fractures for many years. The aim of this study is to investigate the effect of PEMFs on the proliferation and differentiation potential of human bone marrow mesenchymal stem cells (BMMSC). PEMF stimulus was administered to BMMSCs for 8 h per day during culture period. The PEMF applied consisted of 4.5 ms bursts repeating at 15 Hz, and each burst contained 20 pulses. Results showed that about 59% and 40% more viable BMMSC cells were obtained in the PEMF-exposed cultures at 24 h after plating for the seeding density of 1000 and 3000 cells/cm2, respectively. Although, based on the kinetic analysis, the growth rates of BMMSC during the exponential growth phase were not significantly affected, 20-60% higher cell densities were achieved during the exponentially expanding stage. Many newly divided cells appeared from 12 to 16 h after the PEMF treatment as revealed by the cell cycle analysis. These results suggest that PEMF exposure could enhance the BMMSC cell proliferation during the exponential phase and it possibly resulted from the shortening of the lag phase. In addition, according to the cytochemical and immunofluorescence analysis performed, the PEMF-exposed BMMSC showed multi-lineage differentiation potential similar to the control group.