Rongqin Zheng

Nanobubbles for enhanced ultrasound imaging of tumors

The fabrication and initial applications of nanobubbles (NBs) have shown promising results in recent years. A small particle size is a basic requirement for ultrasound contrast-enhanced agents that penetrate tumor blood vessel pores to allow for targeted imaging and therapy. However, the nanoscale size of the particles used has the disadvantage of weakening the imaging ability of clinical diagnostic ultrasound. In this work, we fabricated a lipid NBs contrast-enhanced ultrasound agent and evaluated its passive targeting ability in vivo. The results showed that the NBs were small (436.8 ± 5.7 nm), and in vitro ultrasound imaging suggested that the ultrasonic imaging ability is comparable to that of microbubbles (MBs). In vivo experiments confirmed the ability of NBs to passively target tumor tissues. The NBs remained in the tumor area for a longer period because they exhibited enhanced permeability and retention. Direct evidence was obtained by direct observation of red fluorescence-dyed NBs in tumor tissue using confocal laser scanning microscopy. We have demonstrated the ability to fabricate NBs that can be used for the in vivo contrast-enhanced imaging of tumor tissue and that have potential for drug/gene delivery.

Tumor-penetrating codelivery of siRNA and paclitaxel with ultrasound-responsive nanobubbles hetero-assembled from polymeric micelles and liposomes

Drug resistance is a big problem in systemic chemotherapy of hepatocellular carcinoma (HCC), and nanomedicines loaded with both chemotherapeutic agents (e.g. paclitaxel, PTX) and siRNAs targeting antiapoptosis genes (e.g. BCL-2) possess the advantages to simultaneously overcome the efflux pump-mediated drug resistance and antiapoptosis-related drug resistance. However, tumor-penetrating drug delivery with this type of nanomedicines is extremely difficult due to their relatively big size compared to the single drug-loaded nanomedicines. Aiming at address this problem, US-responsive nanobubbles encapsulating both anti-cancer drug paclitaxel (PTX) and siRNA (PTX-NBs/siRNA) for HCC treatment were developed by hetero-assembly of polymeric micelles and liposomes in the present study. Utilizing an external low-frequency US force imposed to the tumor site, effective tumor-penetrating codelivery of siRNA and PTX was achieved via tail vein injection of PTX-NBs/siRNA into nude mice bearing human HepG2 xerografts. Consequently, the PTX treatment-inducible antiapoptosis in HepG2 cells was effectively suppressed by the codelivered siRNA targeting an antiapoptosis gene (BCL-2 siRNA) during chemotherapy. Owing to the synergistic anti-cancer effect of two therapeutic agents, tumor growth was completely inhibited using low-dose PTX in animal study. Our results highlight the great potential of this type of US-responsive hetero-assemblies carrying both anti-cancer drug and siRNA as an effective nanomedicinal system for HCC therapy.

Ultrasound-sensitive siRNA-loaded nanobubbles formed by hetero-assembly of polymeric micelles and liposomes and their therapeutic effect in gliomas

Ultrasound (US)-sensitive nanobubble (NB) which may utilize the physical power of US exposure to improve delivery efficiency to target cells is emerging as one of the most promising nanocarriers for drug delivery. On the basis of successfully fabricating NBs with the ability of passively accumulating in tumor tissue, in this study we synthesized a US-sensitive NB bearing siRNA (siRNA-NB) for tumor therapy via a hetero-assembling strategy using the siRNA-complexed polymeric micelles and gas-cored liposomes. The US exposure-aided siRNA transfection effectively enhanced the gene silencing effect of siRNA-NBs both in vitro and in vivo, which resulted in much elevated level of cancer cell apoptosis. Consequently, significantly improved therapeutic effect was achieved in a nude mouse glioma model, using siRNA-NBs bearing siRNA to target the anti-apoptosis gene sirtuin 2 (SIRT2). These results show that, with the aid of US exposure, the US-sensitive siRNA-NB may be an ideal delivery vector to mediate highly effective RNA interference for tumor treatment.

Two-dimensional Shear-Wave Elastography and Conventional US: The Optimal Evaluation of Liver Fibrosis and Cirrhosis

PURPOSE To evaluate the individual and combined performances of two-dimensional (2D) shear-wave elastography (SWE) and conventional ultrasonography (US) in assessing liver fibrosis and cirrhosis to determine when 2D SWE should be added to routine US. MATERIALS AND METHODS This prospective study was approved by the institutional ethics committee, and the patients provided written informed consent. Between April 2012 and March 2013, conventional US and 2D SWE examinations were performed in 198 patients (mean age, 37.7 years; age range, 18-67 years) with chronic liver disease. Liver biopsy was used as a reference standard for 167 patients; the other 31 patients had decompensated liver cirrhosis. Receiver operating characteristic (ROC) curves were obtained to assess the diagnostic performance. Differences between the areas under the ROC curves (AUCs) were compared by using a Delong test. RESULTS Two-dimensional SWE was significantly superior to US in diagnosis of significant fibrosis (score of F2 or greater) (AUC, 0.862 vs 0.725; P = .001) and early cirrhosis (score of F4) (AUC, 0.926 vs 0.789; P = .007). Combining 2D SWE with US did not increase the performance of depicting either significant fibrosis or liver cirrhosis (P = .713 and 0.410, respectively) relative to 2D SWE alone. There was no significant difference between 2D SWE and US in diagnosis of decompensated cirrhosis (AUC, 0.878 vs 0.925; P = .323). In addition, combining 2D SWE with US did not increase the performance relative to that of US alone (P = .372). CONCLUSION Conventional US is sufficient to detect decompensated cirrhosis. Two-dimensional SWE is significantly superior to US in detecting liver fibrosis. Combining 2D SWE and US did not improve the diagnostic performance for either fibrosis or cirrhosis.

Enhanced apoptosis of ovarian cancer cells via nanocarrier-mediated codelivery of siRNA and doxorubicin

A folate conjugated ternary copolymer, FA–PEG–PEI–PCL, of poly(ethylene glycol) (PEG), poly(ethylene imine) (PEI), and poly(ɛ-caprolactone) (PCL) was synthesized. The copolymer self-assembled into cationic micelles capable of co-delivering siRNA and the anticancer drug doxorubicin (DOX). This dual functional nanocarrier demonstrated low cytotoxicity and high performance in drug/siRNA delivery. Upon the codelivery of siRNA, targeting the Bcl-2 gene, and DOX, using the folate-targeted nanocarrier, DOX-induced apoptosis in the skov-3 cells overexpressing folate receptor was significantly enhanced through a mechanism of downregulating the antiapoptotic protein Bcl-2, while simultaneously upregulating the proapoptotic protein Bax. This work suggested that the combination of Bcl-2 siRNA and DOX therapies is feasible, based on our dual functional nanocarrier, which set up a good basis for a future in vivo test.

Normal liver stiffness in healthy adults assessed by real-time shear wave elastography and factors that influence this method.

Real-time shear wave elastography (SWE) is a novel two-dimensional elastographic method that is used to estimate the severity of liver fibrosis. However, the normal range of liver stiffness (LS) and the possible factors that influence SWE are not well understood. The aims of the current study are to define the normal range of LS in healthy subjects and to explore the factors that may affect SWE. A total of 509 healthy subjects underwent SWE to determine the stiffness of their livers, and the effects of gender, age and body mass index (BMI) on LS were analyzed. The effects of different factors on SWE, including the testing position, measurement depth and size of the region of interest (ROI), were analyzed in 137 subjects. SWE imaging was successfully performed in 502 healthy subjects (98.6%, 502/509). The mean value of the SWE measurements in 502 individuals was 5.10 ± 1.02 kPa, and the 95% confidence interval was 5.02-5.19 kPa (range: 2.4-8.7 kPa). We found that the detective position within the liver had a significant impact on the liver stiffness measurement (LSM), and the lowest coefficient of variation (CV = 8%) was obtained for LSMs made at segment V. LS was greater at a depth >5 cm (5.78 ± 1.66 kPa) compared with depths ≤5 cm (4.66 ± 0.77 kPa, p < 0.001); LS was also greater in men than in women (5.45 ± 1.02 kPa vs. 4.89 ± 0.96 kPa, p < 0.001). However, there were no significant differences in the LS values regarding the size of the ROI, age or BMI (all p > 0.05). The mean LS value in all 502 healthy subjects was 5.10 ± 1.02 kPa. The mean LS value obtained by SWE was not influenced by the size of the ROI, age or BMI, but the mean value was significantly influenced by the different segments of the liver, the detection depth and gender.

Ultrasound-responsive microbubbles for sonography-guided siRNA delivery

RNA interfering is a gene therapeutic approach of great potential for cancer. However, tumor-targeted delivery of small interfering RNA (siRNA) solely based on the enhanced permeability and retention effect of nanocarriers is often insufficient. To address this challenge, siRNA encapsulated ultrasound-responsive microbubble (MB) was developed from polymeric siRNA micelles and liposomal MBs using hetero-assembling strategy. 1MHz low-frequency ultrasound exposure of the tumor site after intratumoral injection of XIAP siRNA/MBs led to enhanced permeability for much more siRNA delivery into deep tumor regions. Significant improvement of XIAP gene silencing and cleaved caspase-3 activation was achieved, resulting in good therapeutic effect on human cervical cancer xenograft model in nude mice. Moreover, real-time US monitoring of the tumor was also possible using the siRNA/MBs as a contrast agent during the therapeutic process. These results show that the multi-functional siRNA/MBs are a promising theranostic system for cancer gene therapy.

New Diagnosis and Therapy Model for Ischemic-Type Biliary Lesions following Liver Transplantation—A Retrospective Cohort Study

Ischemic-type biliary lesions (ITBLs) are a major cause of graft loss and mortality after orthotopic liver transplantation (OLT). Impaired blood supply to the bile ducts may cause focal or extensive damage, resulting in intra- or extrahepatic bile duct strictures or dilatations that can be detected by ultrasonography, computed tomography, magnetic resonance cholangiopancreatography, and cholangiography. However, the radiographic changes occur at an advanced stage, after the optimal period for therapeutic intervention. Endoscopic retrograde cholangio-pancreatography (ERCP) and percutaneous transhepatic cholangiodrainage (PTCD) are the gold standard methods of detecting ITBLs, but these procedures cannot be used for continuous monitoring. Traditional methods of follow-up and diagnosis result in delayed diagnosis and treatment of ITBLs. Our center has used the early diagnosis and intervention model (EDIM) for the diagnosis and treatment of ITBLs since February 2008. This model mainly involves preventive medication to protect the epithelial cellular membrane of the bile ducts, regular testing of liver function, and weekly monitor of contrast-enhanced ultrasonography (CEUS) to detect ischemic changes to the bile ducts. If the liver enzyme levels become abnormal or CEUS shows low or no enhancement of the wall of the hilar bile duct during the arterial phase, early ERCP and PTCD are performed to confirm the diagnosis and to maintain biliary drainage. Compared with patients treated by the traditional model used prior to February 2008, patients in the EDIM group had a lower incidence of biliary tract infection (28.6% vs. 48.6%, P = 0.04), longer survival time of liver grafts (24±9.6 months vs. 17±12.3 months, P = 0.02), and better outcomes after treatment of ITBLs.

Immediate evaluation and guidance of liver cancer thermal ablation by three-dimensional ultrasound/contrast-enhanced ultrasound fusion imaging

Abstract Purpose: To investigate the feasibility and value of three-dimensional ultrasound/contrast-enhanced ultrasound (3D US-CEUS) fusion imaging for the immediate evaluation of technical success and the guidance of supplementary ablation during the liver cancer thermal ablation procedure. Materials and methods: Patients diagnosed with malignant liver cancer intending to receive thermal ablation including radiofrequency ablation (RFA) or microwave ablation (MWA) were enrolled. 3D US-CEUS fusion imaging was used to immediately assess the technical success and guide supplementary ablation. Contrast-enhanced computed tomography/magnetic resonance imaging (CECT/CEMRI) was performed one month after ablation to assess the technique effectiveness of the ablation. The registration success rate, duration time of 3D US-CEUS fusion imaging, technique effectiveness rate and major complications were recorded. Results: In total, 76 patients with 95 tumours who underwent RFA or MWA and assessed by 3D US-CEUS fusion imaging were enrolled. The registration success rate of 3D US-CEUS fusion imaging was 93.7% (89/95), and the duration time was 4.0 ± 1.1 min. Thirty lesions received supplementary ablation immediately during the procedure. The technique effectiveness rate of the ablation was 98.8% (81/82). There were no major complications related to ablation. Conclusions: 3D US-CEUS fusion imaging is a feasible and valuable technique for the immediate evaluation and guidance of supplementary ablation during the liver cancer thermal ablation procedure.

Contrast-Enhanced Sonography for Diagnosing Collateral Transformation of the Hepatic Artery After Liver Transplantation

To determine the contrast‐enhanced sonographic features of hepatic artery collateral transformation in patients with hepatic artery complications after liver transplantation.