// Xiang Zhang 1, 2 , Jiangang Hu 1, 2 , Guanjian Zhao 1, 2 , Ning Huang 1 , Ying Tan 1 , Li Pi 1 , Qing Huang 1 , Feng Wang 1 , Zhigang Wang 2 , Zhibiao Wang 3 , Yuan Cheng 1 1 Department of Neurosurgery, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010, China 2 Chongqing Key Laboratory of Ultrasound Molecular Imaging, Institute of Ultrasound Imaging of Chongqing Medical University, Chongqing, 400016, China 3 Chongqing Key Laboratory of Biomedical Engineering, College of Biomedical Engineering of Chongqing Medical University, Chongqing, 400016, China Correspondence to: Yuan Cheng, email: chengyuan023@aliyun.com Keywords: blood brain barrier, focused ultrasound, acoustic droplet vaporization, perfluoropentane, PLGA Received: January 17, 2017 Accepted: April 05, 2017 Published: April 17, 2017 ABSTRACT Previous studies have shown that focused ultrasound (FUS) combined with systematic administration of microbubbles (MBs) can open the blood brain barrier (BBB) locally, transiently and reversibly. However, because of the micro size diameters, MBs are restricted in the intravascular space and cannot extravasate into diseased sites through the opened BBB. In this study, we fabricated one kind of nanoscale droplets which consisted of encapsulated liquid perfluoropentane cores and poly (ethyleneglycol) - poly (lactide-co-glycolic acid) shells. The nanodroplets had the capacity to realize liquid to gas phase shift under FUS. Significant extravasation of Evan's blue appeared when acoustic pressure reached 1.0 MPa. Intracerebral hemorrhages and erythrocyte extravasations were observed when the pressure was increased to 1.5 MPa. Prolonged sonication duration could enhance the level of BBB opening and broaden the time window simultaneously. Furthermore, compared with MBs, the distribution of EB extravasation was firmly confined within narrow region in the center of focal zone, suggesting the site of FUS induced BBB opening could be controlled with high precision by this procedure. Our results show the feasibility of serving PEGylated PLGA-based phase shift nanodroplet as an effective alternative mediating agent for FUS induced BBB opening.
Major histocompatibility complex class I C-related molecules A and B (MICA and MICB) are innate immune system ligands for the NKG2D receptor expressed by natural killer cells and activated CD8(+)T cells. Our previous study showed that 5-aza-2'-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, can induce the expression of MICB and sensitized cells to NKL-cell-mediated cytolysis. The aim of this study was to determine the expression level of MICA in HepG2 cells (an HCC cell line) and L02 cells ( a normal liver cell), and to investigate the effect of 5-aza-dC on MICA expression in HepG2 cells.Cells were treated with 5-aza-dC, caffeine and ATM-specific siRNA. The cell surface MICA protein on HepG2 cells and L02 cells was determined using flow cytometry. The mRNA level was detected using real time RT-PCR.MICA was undetectable on the surface of L02 cells, but was highly expressed on HepG2 cells. MICA expression was upregulated in response to 5-aza-dC treatment (P less than 0.05), and the upregulation of MICA was partially prevented by pharmacological or genetic inhibition of ataxia telangiectasia mutated (ATM) kinase (P less than 0.05).Our data suggest that 5-aza-dC induces the expression of MICA by a DNA damage-dependent mechanism.
To investigate the feasibility of using focused ultrasound (FUS) with microbubbles for targeted delivery of cytarabine to the brain. Sprague–Dawly rats (weighing 200–250 g) received focused ultrasound with intravenous injection microbubbles. At 0, 2, 4, 8, and 24 hours (n = 5 for each time point) after sonication, animals received intravenous administration of cytarabine at a normal dose of 4 mg/kg body weight. Additional five rats were given with a high dose (50 mg/kg body weight) of cytarabine alone. Blood–brain barrier (BBB) permeability and cerebral cytarabine were determined. FUS in conjunction with microbubbles caused a transient BBB opening. Sonication exposure promoted cytarabine accumulation at the sonicated site. Animals injected with a normal dose of cytarabine 2 hours after sonication had similar concentrations of cerebral cytarabine compared to those with higher cytarabine without sonication. FUS can temporarily open the BBB and thus facilitate the penetration of systemic cytarabine into the brain.
Objective To explore the dynamic changes of blood brain barrier(BBB) permeability after MRI guided targeted BBB disruption by low- intensity focused ultrasound combined with microbubble in rabbit brain.Method After ultrasound contrast-agent (SonoVue) was injected in vein,MRI-guided focused ultrasound was applied at target location in one hemisphere of rabbits' brains to induce BBB disruption.The opposite side was used as control group.MRI enhancedment scanning was applied before sonication at 0 h,2 h,4 h,6 h,8 h,24 h,1 week after sonication and the signal intensity was measured.BBB permeability in target was assessed at 0 h,2 hAh,6 h,8 h,24 h,1 week after sonication by fluoresence spectrophotometer for Evans Blue(EB) extravasation.Results BBB permeability change was occured immediately after sonication.The MRI signal intensity and the EB extravasation peaked at 2 h(P ＜0.05) and decreased to normal level at 8 h after sonication.The changes of MRI signal intensity was correlated with the EB extravasation.Their coefficient correlation was 0.964 (P＜0.05).Conclusions MRI-guided focused ultrasound could disrupt BBB selectively and reversibly.The changes of MRI signal intensity could evaluate the situation of BBB disruption quantitatively.
Key words:
Blood-brain barrier; Ultrasound; Capillary permeability; Magnetic resonance imaging
The multidrug resistance of glioma impedes chemo-radiotherapy and leads to adverse outcomes. In the present study, the doxorubicin (DOX)-resistant glioma SHG44/DOX cell line was established to investigate the effects and mechanisms of Salvia miltiorrhiza ligustrazine (SML), a traditional Chinese medicine, on the reversal of DOX-resistance. The SHG44/DOX cells grew continually in 0.1 µg/ml DOX and expressed increased levels of mRNA of multidrug resistance genes [multidrug resistance 1 (MDR1), multidrug resistance-associated protein 1 (MRP1) and lung resistance protein (LRP)] compared with the parental SHG44 cells. Treatment with DOX plus SML suppressed proliferation and promoted early apoptotic rates in SHG44/DOX cells, although treatment with DOX alone failed to inhibit SHG44/DOX cells. In addition, the levels of MDR1, MRP1 and LRP were downregulated by this traditional Chinese medicine, coupled with increased intracellular DOX concentrations. Tumor-bearing nude mouse models were also established using SHG44/DOX cells, and it was demonstrated that the tumor volumes and proliferation indexes were lower in the DOX plus SML group compared with the DOX alone group. The present data demonstrated that treatment with SML was able to reverse the DOX resistance of SHG44/DOX cells, which suggests that SML may be a potential adjuvant agent for glioma chemo-radiotherapy.
RNA interference is a promising therapy in glioma treatment. However, the application of RNA interference has been limited in glioma therapy by RNA instability and the lack of tumor targeting. Here, we report a novel DNA tetrahedron, which can effectively deliver small interfering RNA to glioma cells and induce apoptosis. siRNA, a small interfering RNA that can suppress the expression of survivin in glioma, was loaded into the DNA tetrahedron (TDN). To enhance the ability of active targeting of this nanoparticle, we modified one side of the DNA nanostructure with aptamer as1411 (As-TDN-R), which can selectively recognize the nucleolin in the cytomembrane of tumor cells. The modified nanoparticles were characterized by agarose gel electrophoresis, dynamic light scattering, and transmission electron microscopy. The serum stability was evaluated by agarose gel electrophoresis. Nucleolin was detected by Western blot and immunofluorescence, and targeted cellular uptake was examined by flow cytometry. The TUNEL assay, flow cytometry, and Western Blot were used to detect apoptosis in U87 cells. The gene silencing of survivin was examined by qPCR, Western Blot, and immunofluorescence. As-TDN-R alone showed better stability towards siRNA, indicating that TDN was a good siRNA protector. Compared with TDN alone, there was increased intercellular uptake of As-TDN-R by U87 cells, evidenced by overexpressed nucleolin in glioma cell lines. TUNEL assay, flow cytometry, and Western Blot revealed increased apoptosis in the As-TDN-R group. The downregulation of survivin protein and mRNA expression levels indicated that As-TDN-R effectively silenced the target gene. The novel nanoparticle can serve as a good carrier for targeting siRNA delivery in glioma. Further exploration of the DNA nanostructure can greatly promote the application of DNA-based drug systems in glioma.
Abstract Glioma is one of the most deadly tumors due to invasive growth. The treatment effect is poor due to the presence of the blood brain barrier (BBB) and blood tumor barrier (BTB) and insufficient drug targeting. DNA tetrahedron (TDN) is considered to have great potential for drug delivery and maybe a novel therapeutic strategy for glioma. In this study, we have developed a doxorubicin loaded-DNA tetrahedron modified by a Gint4.T aptamer(DOX@Apt-TDN) that could target PDGFRβ for therapy of glioma. The TDN and Apt-TDN were self-assembled by one-step synthesis. The characterization and stability were detected using gel electrophoresis analysis, dynamic light scatting and atomic force microscopy. The cytotoxicity of TDN in vitro was determined by CCK-8 assays. Fluorescence spectrophotometry was used to measure the drug loading capacity. Fluorescence microscopy imaging and flow cytometry were used to survey the cellular uptake. The cell viability, cell cycle and early apoptosis were estimated when the U87MG cells were treated with DOX, DOX@TDN and Dox@Apt-TDN. The rusults showed that a single Apt-TDN could carry 60 doxorubicin molecules. The 3D structure of the DNA tetrahedron remarkedly enhanced drug stability in fetal bovine serum, remaining intact for 7h leastwise. The CCK-8 assay showed that the activity of the U87 cells was not affected after co-cultured with different concentrations(10–500 nM) of TDN for 24 h and 48 h. The aptamer Gint4.T enhanced the targeted cellular uptake of TDN by U87MG cells. Dox-loaded Apt-TDN inhibited cell viability and induced early apoptosis. In conclusion this study establishes that the Gint4.T-modified DNA tetrahedron could target PDGFRβ and provide a novel therapy with promising clinical application for gliomas.
Objective. The purpose of this study was to investigate the effects of targeted and reversible disruption of the blood-brain barrier (BBB) by magnetic resonance imaging (MRI)-guided focused ultrasound (FUS) and delivery of methotrexate (MTX) to the rabbit brain. Methods. The brains of 20 rabbits were sonicated by MRI-guided FUS at different exposure times, and then Evans blue extravasation, contrast-enhanced MRI, and histologic examination were performed to determine the optimal exposure time for reversible BBB disruption with minimal damage. Five rabbits were sonicated at the optimal exposure time after MTX was injected intravenously (IV); the targeted locations were included in the sonicated group, and the nontargeted contralateral counterparts were included in the IV control group. Five other rabbits were not subjected to sonication and were administered internal carotid artery (ICA) injections of MTX; the specimens of the counterpart brain tissue were harvested as the ICA group. The MTX concentration in all of the specimens was determined by high-performance liquid chromatography. Results. The MTX concentration in the sonicated group (mean ± SD, 7.412 ± 1.471 μg/g of tissue) was notably higher than that in both the IV control group (0.544 ± 0.084 μg/g) and ICA group (1.984 ± 0.65 μg/g; P <.01). Conclusions. Magnetic resonance imaging–guided FUS can disrupt the BBB reversibly and deliver IV administered MTX to targeted brain locations; it brings about a greater than 10-fold increase in the drug level and is much more effective (≈3.7-fold) than drug delivery through the ICA without sonication. This may facilitate the development of improved treatment methods for central nervous system disorders.
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