Effects of hydrophilic fullerene nanoarchitectured structures on the behaviour of neural stem cells.

The interaction between nanoarchitectonic fullerenes and cells is essential for their applications in the biological field. Herein we reported the preparation and investigation of the function of different types of water-dispersible self-assembled fullerenes. The hydrophobic self-assembled fullerenes were either surface-modified or chemically etched to become water dispersible. Different types of fullerenes were then examined for their effects on the behavior of neural stem cells (NSCs). Our results indicated that only the hydrophilic fullerene nanotubes (FNTs, diameter ∼480 nm) created by chemically etching were endocytosed by NSCs, which showed a spindle-like morphology after the uptake. Meanwhile, the FNTs did not increase the reactive oxygen species (ROS) production of the cells. The expression levels of neural-related genes (CNPase and ß-tubulin) were upregulated 1.5-fold in the presence of FNTs. The differentiation of NSCs depended on the size, shape, and surface functional group of various fullerenes. Besides, the addition of FNTs in a chitosan self-healing hydrogel did not influence the integrity, injectability, and self-healing properties of the composite hydrogel. These results revealed that FNTs induced the neural differentiation of NSCs in the composite hydrogel. The addition of FNTs at a low concentration (50 µg mL-1) was enough to create such effects in the composite hydrogel. The expression levels of the oligodendrocytic marker gene CNPase and the neuronal marker gene ß-tubulin were increased remarkably by ∼14.5- and ∼8.4-fold, respectively, by the composite self-healing hydrogel containing 50 µg mL-1 FNTs. The fullerene nanoarchitectured structures may have potential for use as nanovehicles and in neural tissue engineering in the future.

Effect of chitosan-alginate nanoparticles and ultrasound on the efficiency of gene transfection of human cancer cells.

BACKGROUND: Gene therapy has been used to treat a variety of health problems, but transfection inefficiency and the lack of safe vectors have limited clinical progress. Fabrication of a vector that is safe and has high transfection efficiency is crucial for the development of successful gene therapy. The present study aimed to synthesize chitosan-alginate nanoparticles that can be used as carriers of the pAcGFP1-C1 plasmid and to use these nanoparticles with an ultrasound protocol to achieve high efficiency gene transfection. METHODS: Chitosan was complexed with alginate and the pAcGFP1-C1 plasmid at different charge ratios to create chitosan-alginate-DNA nanoparticles (CADNs). The average particle size and loading efficiency were measured. Plasmid DNA retardation and integrity were analysed on 1% agarose gels. The effect of CADNs and ultrasound on the efficiency of transfection of cells and subcutaneous tumors was evaluated. RESULTS: In the CADNs, the average size of incorporated plasmid DNA was 600-650 nm and the loading efficiency was greater than 90%. On the basis of the results of the plasmid DNA protection test, CADNs could protect the transgene from DNase I degradation. The transgene product expression could be enhanced efficiently if cells or tumor tissues were first given CADNs and then treated with ultrasound. CONCLUSIONS: The use of CADNs combined with an ultrasound regimen is a promising method for safe and effective gene therapy.

Folic acid-conjugated chitosan nanoparticles enhanced protoporphyrin IX accumulation in colorectal cancer cells.

Folic acid can be covalently conjugated to chitosan molecules via its gamma-carboxyl moiety and thus retain a high affinity for colorectal cancer cells bearing folate receptor overexpression. Colorectal cancer is one of the leading causes of malignant death and often goes undetected with current colonoscopy practices. Improved methods of detecting dysplasia and tumors during colonoscopy will improve mortality. A folic acid conjugated chitosan nanoparticle as a suitable vehicle for carrying 5-aminolaevulinic acid (5-ALA) is developed to enhance the detection of colorectal cancer cells in vivo after a short-term uptake period. Chitosan can be successfully conjugated with folic acid to produce folic acid-chitosan conjugate, which is then loaded with 5-ALA to create nanoparticles (fCNA). The loading efficiency of 5-ALA in fCNA particles and the z-average diameter were in the range 35-40% and 100 nm, respectively. The zeta-potential for fCNA was 20 mV, enough to keep the nanoparticle stable without aggregation. The fCNA is then incubated with HT29 and Caco-2 colorectal cancer cell lines overexpressing folate receptor on the surface of the cell membrane to determine the rate of accumulation of protoporphyrin IX (PpIX). The results show that fCNA can be taken up more easily by HT29 and Caco-2 cell lines after short-term uptake period, most likely via receptor-mediated endocytosis, and the PpIX accumulates in cancer cells as a function of the folate receptor expression and the folic acid modification. Therefore, the folic acid-chitosan conjugate appears to be an ideal vector for colorectal-specific delivery of 5-ALA for fluorescent endoscopic detection.

Time dependency of ultrasound-facilitated gene transfection.

BACKGROUND: The use of ultrasound (US)-facilitated gene therapy is increasing rapidly as a result of its high specificity and non-invasiveness. However, the acoustic parameters that produce the most efficient transfection have not been established. The present study investigated the effects of time parameters [including pulsing strategy (on- and off-times), exposure duration, pore opening time and expression duration] of US-facilitated gene transfection. METHODS: Cervical cancer cells (HeLa cells) cultured with pCMViLUC plasmids were exposed to 1-MHz pulsed US, and gene transfection efficiency and cell viability were assessed. The ability of in vivo transfection by ultrasound using different pulsing strategy was also evaluated. RESULTS: For a constant total exposure time, the transfection was always better for a longer on-time (on-off ratio = 1, or 50% duty cycle) than a shorter one (ratio = 0.1, 9% duty cycle), whether performed in vitro or in vivo. However, for a fixed on-time, there was a strong inverse correlation between transfection efficiency and off-time (r(2) = 0.89). Multiple regression analysis showed that the on-time, off-time and on-off ratio are all independent variables for US-facilitated gene transfection. Furthermore, transfection increased with the total exposure time, but plateaued for long exposure times. More than half of the opened pores closed within 15 s after the cessation of US exposure. The half-life of the transfected DNA was 7.43 days. CONCLUSIONS: Time is an important determinant of US-facilitated gene transfection. Methods for increasing the transfected number of plasmids, such as increasing the concentration of plasmids or the duration of pore opening, will be critical for future research and clinical applications.

Differences in gene expression between sonoporation in tumor and in muscle.

BACKGROUND: Ultrasound (US) is a novel and effective tool for the local delivery of genes into target tissues. US can temporarily change the permeability of a cell membrane and thus enhance the delivery of naked DNA into cells. In the present study, the efficiencies of gene expression mediated by US delivery in orthotopic liver tumor, subcutaneous tumor and muscle tissue were evaluated by changing the contrast agent concentrations and US exposure durations. METHODS: Plasmid DNA coding for luciferase, interleukin-12 or enhanced green fluorescence protein was mixed with SonoVue and injected intratumorally or intramuscularly. The injection sites were then exposed to US (20% duty cycle and 0.4 W/cm(2) intensity). RESULTS: The results obtained showed that the optimal condition was 50% SonoVue for tumors and 30% for muscle, with 10 min of US exposure. The expression levels of the transfected DNAs were in the order: muscle > subcutaneous tumor > orthotopic liver tumor. CONCLUSIONS: The present study indicates that muscle tissue is a good target site for producing large amounts of gene products for the purpose of gene therapy.

Enhancement of chitosan nanoparticle-facilitated gene transfection by ultrasound both in vitro and in vivo.

In recent years, inefficiency of transfection and the lack of safe gene vectors have limited the feasibility of gene therapy. Fabrication of a vector that is safe and has high transfection efficiency is crucial for the development of successful gene therapies. Herein, we complexed chitosan to plasmids at various N/P ratios, the molar ratios of the amino groups of chitosan to the phosphate groups of DNA, to create chitosan-DNA nanoparticles (CDNs), and then measured CDNs size, zeta-potential, efficiency of plasmid complexation, and plasmid integrity from enzyme digestion. We also used flow cytometry and fluorescence microscopy to examine the effect of an ultrasound (US) regimen on the efficiency of transfection of HeLa cells. The results revealed that the average size, zeta-potential, and loading efficiency of plasmid DNA in CDNs were 180-200 nm, 26-35 mV, and greater than 80%, respectively. Moreover, the transgene expression could be enhanced efficiently while HeLa cells or tumor tissues were given CDNs and then treated with US. Therefore, the use of chitosan nanoparticles and an US regimen shows great promise as an effective method of gene therapy.