Biodegradable nanoparticles of mPEG-PLGA-PLL triblock copolymers as novel non-viral vectors for improving siRNA delivery and gene silencing.

Degradation of mRNA by RNA interference is one of the most powerful and specific mechanisms for gene silencing. However, insufficient cellular uptake and poor stability have limited its usefulness. Here, we report efficient delivery of siRNA via the use of biodegradable nanoparticles (NPs) made from monomethoxypoly(ethylene glycol)-poly(lactic-co-glycolic acid)-poly-l-lysine (mPEG-PLGA-PLL) triblock copolymers. Various physicochemical properties of mPEG-PLGA-PLL NPs, including morphology, size, surface charge, siRNA encapsulation efficiency, and in vitro release profile of siRNA from NPs, were characterized by scanning electron microscope, particle size and zeta potential analyzer, and high performance liquid chromatography. The levels of siRNA uptake and targeted gene inhibition were detected in human lung cancer SPC-A1-GFP cells stably expressing green fluorescent protein. Examination of the cultured SPC-A1-GFP cells with fluorescent microscope and flow cytometry showed NPs loading Cy3-labeled siRNA had much higher intracellular siRNA delivery efficiencies than siRNA alone and Lipofectamine-siRNA complexes. The gene silencing efficiency of mPEG-PLGA-PLL NPs was higher than that of commercially available transfecting agent Lipofectamine while showing no cytotoxicity. Thus, the current study demonstrates that biodegradable NPs of mPEG-PLGA-PLL triblock copolymers can be potentially applied as novel non-viral vectors for improving siRNA delivery and gene silencing.

Contrast-enhanced ultrasound as a valuable imaging modality for characterizing testicular lesions.

Contrast-enhanced ultrasound (CEUS) is a new form of ultrasound (US) that can dynamically display microvessels in a highly sensitive manner. The purpose of this study was to investigate the efficacy of CEUS for characterizing testicular lesions in comparison with conventional US. Forty-seven patients with testicular lesions were enrolled. The histopathology results revealed that 31 cases were neoplastic (11 cases of seminomas, 8 nonseminomatous germ cell tumors, 8 lymphomas, 2 Leydig cell tumors, and 2 nonspecific tumors), and 16 cases were nonneoplastic (8 cases of infarctions, 3 epidermoid cysts, and 5 inflammation). The indicators of shallow lobulated morphology and cystic-solid echogenicity on conventional US were suggestive of germ cell tumors. More indicators on CEUS were found to be useful for characterizing testicular lesions. All the neoplastic lesions showed hyperenhancement on CEUS. Moreover, germ cell tumors presented with heterogeneous enhancement (73.7%, 14/19), a twisted blood vessel pattern, rapid wash-in and wash-out, and peripheral rim hyperenhancement signs. Lymphoma was characterized by nonbranching linear vessel patterns (87.5%, 7/8), rapid wash-in and slow wash-out. In nonneoplastic lesions, infarction and epidermoid cysts showed no enhancement, and abscesses were observed with marginal irregular enhancement. The sensitivity, specificity, and accuracy of CEUS for differentiating between neoplastic and nonneoplastic lesions were 100%, 93.8%, and 97.9%, respectively, and these values were higher than those for conventional US (90.3%, 62.5%, and 80.9%, respectively). CEUS can sensitively reflect the microvascular perfusion in testicular lesions and offers high accuracy for characterizing them.

Enhanced delivery of monomethoxypoly(ethylene glycol)-poly(lactic-co-glycolic acid)-poly l-lysine nanoparticles loading platelet-derived growth factor BB small interfering RNA by ultrasound and/or microbubbles to rat retinal pigment epithelium cells.

BACKGROUND: A novel small interfering RNA (siRNA) delivery method based on the combined use of nanoparticles (NPs) with ultrasound (US) and/or microbubbles (MBs) was introduced in the present study. We investigated the efficacy and safety of US and/or MBs-enhanced delivery of monomethoxypoly(ethylene glycol)-poly(lactic-co-glycolic acid)-poly l-lysine (mPEG-PLGA-PLL) NPs loading platelet-derived growth factor BB (PDGF-BB) siRNA to rat retinal pigment epithelium (RPE)-J cells. METHODS: The effect of US and/or MBs on the delivery of NPs containing Cy3-labeled siRNA was evaluated by fluorescence microscopy and flow cytometry. Potential toxicity of NPs and cell viability under different conditions of US and/or MBs were assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. RESULTS: The results obtained showed that low intensity US or 15-20% MBs could increase the delivery efficiency of a lower concentration of mPEG-PLGA-PLL NPs loading siRNA to RPE-J cells, whereas the combination of US with MBs under the optimal conditions for the enhancement of NPs delivery did not further increase the cellular uptake of NPs compared to either US or MBs alone (p = 0.072 and p = 0.488, respectively). Under the optimal condition for US-enhanced NPs delivery, the enhanced PDGF-BB gene silencing with a combination of US and NPs encapsulating siRNA resulted in a significant decrease of mRNA and protein expression levels compared to NPs alone. CONCLUSIONS: US and/or MBs could be used safely to enhance the delivery of NPs loading siRNA to rat RPE-J cells. A combination of the chemical (mPEG-PLGA-PLL NPs loading siRNA) and physical (US) approaches could more effectively downregulate the mRNA and protein expression of PDGF-BB.

Imaging findings of bile duct hamartomas: a case report and literature review.

Bile duct hamartomas (BHs), also called von Meyenburg complex (VMC), are benign biliary malformations that originate from disorganization of the small intrahepatic bile ducts. This disorganization is often associated with the abnormal involution of embryonic ductal end plates in the liver. This is clinically significant, as the development of BHs can cause diagnostic confusion with liver metastases and small hepatocellular carcinoma (SHCC). Currently, we report a specific case of BHs and review the literature to better define and diagnose BHs. In the following case, a 37 year-old male bearing a lesion in his liver is presented and undergoes both radiological and pathological diagnosis. The lesion is preliminarily suspected to be a hepatic hemangioma by examination of conventional ultrasound (US), contrast enhanced ultrasound (CEUS), computerized tomographic scanning (CT) and magnetic resonance imaging (MRI). However, SHCC is suspected by follow-up analysis of US and CEUS, due to the patient's background history of hepatitis B and growth of the lesion and a tumor-feeding vessel in BHs via CEUS. However, BHs are finally diagnosed by biopsy pathology under the guidance of ultrasound. Therefore, we believe pathology is imperative for correct diagnosis of BHs over other similar diseases when the imaging findings are atypical. Here we report the novel and unique detection of a tumor-feeding vessel, which mimicked SHCC strongly, during the course of CEUS. We also present a comprehensive review of the previous reported radiological examination related to BHs.

Acoustic Radiation Force Impulse Technology in the Differential Diagnosis of Solid Breast Masses with Different Sizes: Which Features Are Most Efficient?

PURPOSE: To evaluate diagnostic performance of acoustic radiation force impulse (ARFI) technology for solid breast masses with different sizes and determine which features are most efficient. MATERIALS AND METHODS: 271 solid breast masses in 242 women were examined with ARFI, and their shear wave velocities (SWVs), Virtual Touch tissue imaging (VTI) patterns, and area ratios (ARs) were measured and compared with their histopathological outcomes. Receiver operating characteristic curves (ROC) were calculated to assess diagnostic performance of ARFI for small masses (6-14 mm) and big masses (15-30 mm). RESULTS: SWV of mass was shown to be positively associated with mass size (P < 0.001). For small masses, area under ROC (Az) of AR was larger than that of SWV (P < 0.001) and VTI pattern (P < 0.001); no significant difference was found between Az of SWV and that of VTI pattern (P = 0.906). For big masses, Az of VTI pattern was less than that of SWV (P = 0.008) and AR (P = 0.002); no significant difference was identified between Az of SWV and that of AR (P = 0.584). CONCLUSIONS: For big masses, SWV and AR are both efficient measures; nevertheless, for small masses, AR seems to be the best feature.

Regions of interest and parameters for the quantitative analysis of contrast-enhanced ultrasound to evaluate the anti-angiogenic effects of bevacizumab.

The aim of the present study was to identify effective regions of interest (ROIs) and parameters for the quantitative analysis of contrast-enhanced ultrasound (CEUS) to evaluate the anti-angiogenic effects of bevacizumab. Thirty mice were subcutaneously injected with CT26 cells and randomly divided into a bevacizumab­treated (Bev) group and a control group (normal saline-treated). CEUS and quantitative analysis were performed on days 7, 11, 14 and 21 following tumor establishment. ROItotal, which included the whole tumor, and ROIsmall, which included the most enhanced part of the tumor, were selected and outlined. Parameters including time to peak (TTP), maximum intensity (Imax) and area under the curve (AUC; in addition to rates of AUC1, AUC2, AUCfast and AUCslow) were recorded. The tumors were resected on day 21 for microvessel density (MVD) counting. Our results showed that the MVD in the Bev group was significantly lower compared with that in the control group (4.09 vs. 6.41; P=0.001). Additional parameters of ROIsmall were identified to be significantly different between the two groups, compared with those of ROItotal. No significant differences in TTP and Imax were observed between the two groups at the four time­points examined (P>0.05). For the AUC parameters in ROIsmall, AUC and the rates of AUC2, AUCfast and AUCslow were lower in the Bev group compared with those in the control group on days 7 and 11 (P<0.05). These findings indicate that ROIsmall and AUC parameters in the quantitative analysis of CEUS may be useful for the evaluation of changes in tumor angiogenesis following bevacizumab treatment.

Enhancing HSP70-ShRNA transfection in 22RV1 prostate cancer cells by combination of sonoporation, liposomes and HTERT/CMV chimeric promoter.

Gene therapy is a potentially viable approach for treating hormone-refractory prostate cancer (HRPC), it requires efficient delivery systems and a target gene. Inducing carcinoma cell apoptosis by inhibition of heat shock protein 70 (HSP70) overexpression has been emerging as an attractive strategy for cancer therapy. In our study, the high tumor-specificity of human telomerase reverse transcriptase (HTERT) expression prompted the use of an HTERT/cytomegalovirus (CMV) chimeric promoter to drive HSP70-ShRNA expression to induce HRPC 22RV1 cell apoptosis. At the same time, sonoporation induced by ultrasound-targeted microbubble destruction (UTMD) was utilized for delivery of plasmid loaded with HTERT/CMV promoter. Our results indicated the combination of sonoporation, low-dose liposomes and HTERT/CMV chimeric promoter as a delivery system has the potential to promote efficient gene transfer with lower cytotoxicity.

Targeted delivery of biodegradable nanoparticles with ultrasound-targeted microbubble destruction-mediated hVEGF-siRNA transfection in human PC-3 cells in vitro.

A potentially viable approach for treating late-stage prostate cancer is gene therapy. Successful gene therapy requires safe and efficient delivery systems. In this study, we report the efficient delivery of small interfering RNA (siRNA) via the use of biodegradable nanoparticles (NPs) made from monomethoxypoly(ethylene glycol)-poly(lactic-co-glycolic acid)-poly-l-lysine (mPEG-PLGA-PLL) triblock copolymers. On the basis of previous findings, cyclic Arg-Gly-Asp (cRGD) peptides were conjugated to NPs to recognize the target site, integrin αvß3, expressed in high levels in PC-3 prostate cancer cells. The suppression of angiogenesis by the downregulation of vascular endothelial growth factor (VEGF) expression has been widely used to inhibit the growth of malignant tumors. In our study, human VEGF (hVEGF)-siRNA was encapsulated in NPs to inhibit VEGF expression in PC-3 cells. Concurrently, sonoporation induced by ultrasound-targeted microbubble destruction (UTMD) was utilized for the delivery of siRNA-loaded NPs. Our results showed low cytotoxicity and high gene transfection efficiency, demonstrating that the targeted delivery of biodegradable NPs with UTMD may be potentially applied as new vector system for gene delivery.