Jianli Ren

Ultrasound triggered drug release from 10-hydroxycamptothecin-loaded phospholipid microbubbles for targeted tumor therapy in mice.

Ultrasound targeted microbubble destruction (UTMD) was one of the most promising strategies to enhance drug delivery in cancer therapy. Microbubbles (MBs) serve as a vehicle to carry anti-tumor drugs and locally release them when exposed to therapeutic ultrasound, resulting in drug accumulation in tumor tissues and enhanced anti-tumor effect. However the ultrasound triggered drug delivery system has been seriously limited due to the poor loading capacity of MBs. Here we present a new strategy to overcome the low drug payload of MBs for ultrasound guided drug delivery. In this study, we developed a novel microbubble carrying 10-HCPT which only needs a particularly low single dose of injection (4-6 mg) for tumor therapy in clinical application, therefore, the required high dosing of drug loaded MBs for ultrasound mediated drug delivery is not necessary. We subsequently investigated the combination of ultrasound application with HLMs to achieve therapeutic effect on tumor at a feasible dose of MBs. HLMs were manufactured with a high drug encapsulation and loading content and simultaneously maintained the acoustic properties as an ultrasound contrast agent. After that, tumor-bearing mice were routinely and non-invasively administered with HLMs through the tail vein and were then exposed to ultrasound, resulting in a remarkable drug accumulation in tumor tissues and a significant increase in tumor inhibition rate (70.6%) compared with HLMs alone (47.8%) as well as commercial HCPT injection (49.4). In conclusion, HLMs are expected to improve the therapeutic efficacy of MBs and are worthy of further study for UTMD mediated drug delivery.

Experimental research on therapeutic angiogenesis induced by hepatocyte growth factor directed by ultrasound-targeted microbubble destruction in rats.

Objective. The purpose of this study was to explore the feasibility of therapeutic angiogenesis in myocardial infarction induced by hepatocyte growth factor (HGF) mediated by ultrasound‐targeted microbubble destruction. Methods. Forty Wistar rats were divided into 4 groups after the models of myocardial infarction were prepared: (1) HGF, ultrasound, and microbubbles (HGF+US/MB), (2) HGF and ultrasound, (3) HGF and microbubbles, and (4) surgery alone. Destruction of ultrasound‐targeted microbubbles loaded with the HGF gene with an electrocardiographic trigger mode was performed in the HGF+US/MB group. All the rats were killed after being transfected for 14 days. Enhanced green fluorescent protein expression was examined in the myocardium, liver, and kidney in all groups by fluorescence microscopy; CD34 expression was detected by immunohistochemistry, and microvessel density (MVD) was counted in the high‐power field on microscopy. Hepatocyte growth factor expression in the myocardium was detected by western blotting and an enzyme‐linked immunosorbent assay. Results. Enhanced green fluorescent protein expression was detected in the myocardium of the HGF+US/MB group, but a few areas of HGF expression were detected only in small vessels and the capillary endothelium, and no expression was found in the surgery‐alone and HGF and microbubbles groups. The results of MVD counting by microscopy showed that the MVD in the myocardium of the HGF+US/MB group was the highest among all the groups. The results of western blotting and the enzyme‐linked immunosorbent assay showed that the amount of HGF in the myocardium was highest in the HGF+US/MB group. Conclusions. Ultrasound‐targeted microbubble destruction could deliver HGF into the infracted myocardium and produce an angiogenesis effect, which could provide a novel strategy for gene therapy of myocardial infarction.

Transfection Efficiency of TDL Compound in HUVEC Enhanced by Ultrasound-Targeted Microbubble Destruction

The aim of the present study was to explore the gene transfection efficiency of Tat peptide/plasmid DNA/ liposome (TDL) compound combined with ultrasound-targeted microbubble destruction (UTMD) in human umbilical vein endothelial cell (HUVEC). Tat peptide, plasmid DNA (pIRES2-EGFP-HGF) and Lipofectamine 2000 were used to prepare the TDL compound. Microbubbles were prepared using mechanic vibration. The expression of the report gene enhanced green fluorescent protein (EGFP) was observed using fluorescent microscopy and flow cytometry. The viability of HUVEC was measured by MTT assay. mRNA and protein of HGF was analyzed by reverse transcription-polymerase chain reaction and Western Blot. The intensity of green fluorescence and the gene transfection efficiency of TDL compound + microbubbles + ultrasound group were higher than those of other groups, and no significantly different viability was found between TDL compound + microbubbles + ultrasound group and the other groups. The HGF mRNA and HGF protein of TDL compound + microbubbles + ultrasound group were higher than those of other groups. Our finding demonstrated that UTMD could enhance the transfection efficiency of TDL compound without obvious effects on the cell viability of HUVEC, suggesting that the combination of UTMD and TDL compound might be a useful tool for the gene therapy of ischemic heart disease.

AGT gene polymorphisms (M235T, T174M) are associated with coronary heart disease in a Chinese population

Objective: The angiotensinogen (AGT) gene has been shown to be involved in the development of coronary heart disease (CHD). However, the results have been inconsistent. In this study, the authors performed a meta-analysis to clarify the associations between AGT polymorphisms and CHD risk among the Chinese population. Methods: Published literature from PubMed, the China National Knowledge Infrastructure and Wanfang Data were searched. Pooled odds ratio (OR) and 95% confidence interval (CI) were calculated using a fixed- or random-effects model. Results: Fourteen studies (2540 cases and 2173 controls) for M235T polymorphism and five studies (655 cases and 815 controls) for T174M polymorphism were included in the meta-analyses. The results showed that M235T polymorphism was significantly associated with CHD risk under a recessive model (OR=1.65, 95% CI 1.22–2.25). There was also significant association between T174M polymorphism and CHD risk under a homogeneous co-dominant model (OR= 4.20, 95% CI 1.90–9.29) and a recessive model (OR=4.15, 95% CI 1.88–9.15). Further sensitivity analyses confirmed the significant association. Conclusions: The meta-analyses indicated the significant associations of two AGT polymorphisms (M235T, T174M) with CHD risk in the Chinese population.

Liquid-solid phase-inversion PLGA implant for the treatment of residual tumor tissue after HIFU ablation.

Background HIFU has been shown to be a more suitable alternative for the treatment of primary solid tumors and metastatic diseases than other focal heat ablation techniques due to its noninvasive and extracorporeal nature. However, similar to other focal heat ablation techniques, HIFU is still in need of refinements due to tumor recurrence. Methods In this work, we investigated the effectiveness of an adjunct treatment regimen using doxorubicin (DOX)-loaded, injectable, in situ-forming, and phase-inverting PLGA as the second line of defense after HIFU ablation to destroy detrimental residual tumors and to prevent tumor recurrence. All of the statistical analyses were performed using the Statistical Package for the Social Sciences 18.0(SPSS, Inc., Chicago, IL, USA), and p< 0.05 was considered statistically significant. All of the results are presented as the means ± STDEV (standard deviation). For multiple comparisons, ANOVA (differences in tumor volumes, growth rates, apoptosis, proliferation indexes, and Bcl-2 and Bax protein levels) was used when the data were normally distributed with homogenous variance, and rank sum tests were used otherwise. Once significant differences were detected, Student-t tests were used for comparisons between two groups. Results Our results revealed that DOX diffused beyond the ablated tissue regions and entered tumor cells that were not affected by the HIFU ablation. Our results also show that HIFU in concert with DOX-loaded PLGA led to a significantly higher rate of tumor cell apoptosis and a lower rate of tumor cell proliferation in the areas beyond the HIFU-ablated tissues and consequently caused significant tumor volume shrinkage (tumor volumes:0.26±0.1,1.09±0.76, and 1.42±0.9cm3 for treatment, sham, and no treatment control, respectively). Conclusions From these results, we concluded that the intralesional injection of DOX-loaded PLGA after HIFU ablation is significantly more effective than HIFU alone for the treatment of solid tumors.

Low-intensity focused ultrasound (LIFU)-induced acoustic droplet vaporization in phase-transition perfluoropentane nanodroplets modified by folate for ultrasound molecular imaging

The commonly used ultrasound (US) molecular probes, such as targeted microbubbles and perfluorocarbon emulsions, present a number of inherent problems including the conflict between US visualization and particle penetration. This study describes the successful fabrication of phase changeable folate-targeted perfluoropentane nanodroplets (termed FA-NDs), a novel US molecular probe for tumor molecular imaging with US. Notably, these FA-NDs can be triggered by low-intensity focused US (LIFU) sonication, providing excellent US enhancement in B-mode and contrast-enhanced US mode in vitro. After intravenous administration into nude mice bearing SKOV3 ovarian carcinomas, 1,1′-dioctadecyl-3,3,3′,3′ -tetramethylindotricarbocya-nine iodide-labeled FA-NDs were found to accumulate in the tumor region. FA-NDs injection followed by LIFU sonication exhibited remarkable US contrast enhancement in the tumor region. In conclusion, combining our elaborately developed FA-NDs with LIFU sonication provides a potential protocol for US molecular imaging in folate receptor-overexpressing tumors.

Cell-penetrating Peptide-modified Targeted Drug-loaded Phase-transformation Lipid Nanoparticles Combined with Low-intensity Focused Ultrasound for Precision Theranostics against Hepatocellular Carcinoma

Objective: Prepare a multifunctional ultrasound molecular probe, hyaluronic acid-mediated cell-penetrating peptide-modified 10-hydroxycamptothecin-loaded phase-transformation lipid nanoparticles (HA/CPPs-10-HCPT-NPs), and to combine HA/CPPs-10-HCPT-NPs with low-intensity focused ultrasound (LIFU) for precision theranostics against hepatocellular carcinoma (HCC). Methods: HA/CPPs-10-HCPT-NPs were prepared using thin-film dispersion, ultrasound emulsification, and electrostatic effects. HA/CPPs-10-HCPT-NPs were characterized for particle size, zeta potential, encapsulation efficiency and drug-loading efficiency. In vitro, HA/CPPs-10-HCPT-NPs were tested for acoustic droplet vaporization (ADV) at different time points/acoustic intensities; the ability of HA/CPPs-10-HCPT-NPs to target SMMC-7721 cells was detected by confocal laser scanning microscopy (CLSM); the penetrating ability of CG-TAT-GC-modified NPs was verified by CLSM in a 3D multicellular tumor spheroid (MCTS) model; the effect of HA/CPPs-10-HCPT-NPs combined with LIFU on killing SMMC-7721 cells was measured by CCK-8 and flow cytometry. In vivo, the tumor-target efficiency of HA/CPPs-10-HCPT-NPs was evaluated by a small-animal fluorescence imaging system and CLSM; the enhanced ultrasound imaging efficiency of HA/CPPs-10-HCPT-NPs combined with LIFU was measured by an ultrasound imaging analyzer; the therapeutic effect of HA/CPPs-10-HCPT-NPs combined with LIFU was evaluated by tumor volume, tumor inhibition rate, and staining (hematoxylin and eosin (H & E), proliferating cell nuclear antigen (PCNA) and TUNEL). Results: Mean particle size and mean zeta potential of HA/CPPs-10-HCPT-NPs were 284.2±13.3 nm and - 16.55±1.50 mV, respectively. HA/CPPs-10-HCPT-NPs could bind to SMMC-7721 cells more readily than CPPs-10-HCPT-NPs. Penetration depth into 3D MCTS of HA/CPPs-10-HCPT-NPs was 2.76-fold larger than that of NPs without CG-TAT-GC. HA/CPPs-10-HCPT-NPs could enhance ultrasound imaging by undergoing ADV triggered by LIFU. HA/CPPs-10-HCPT-NPs+LIFU group demonstrated significantly higher efficiency of anti-proliferation and apoptosis percentage than all other groups. In mouse liver tumor xenografts, HA/CPPs-10-HCPT-NPs could target tumor sites and enhance ultrasound imaging under LIFU. HA/CPPs-10-HCPT-NPs+LIFU group had a significantly smaller tumor volume, lower proliferative index (PI), and higher tumor inhibition and apoptotic index (AI) than all other groups. Conclusions: Combined application of HA/CPPs-10-HCPT-NPs and LIFU should be a valuable and promising strategy for precise HCC theranostics.

Right ventricular function in pulmonary hypertension due to left heart disease by two-dimensional speckle tracking and real time three-dimensional echocardiography.

Objective The aim of this study was to assess right ventricular (RV) function in pulmonary hypertension due to left heart disease (LHD-PH) by two-dimensional speckle tracking echocardiography (2D-STE) and real-time three-dimensional echocardiography (RT-3DE) and to compare them with conventional echocardiography in identifying reactive PH. Subjects and methods The study consisted of 40 controls and 65 patients with LHD-PH. According to pulmonary vascular resistance, patients were divided into a passive PH and a reactive PH group. RV fractional area change (FAC), myocardial performance index (MPI) and systolic velocity (S’) were acquired by conventional echocardiography. Longitudinal peak systolic strain (LS) was measured at the basal (LSbas), middle (LSmid) and apical (LSapi) segments of RV free wall by 2D-STE, and global free wall LS (LSfw) was calculated. RV end-diastolic volume (EDV), end-systolic volume (ESV), ejection fraction (EF) and stroke volume (SV) were acquired using RT-3DE. Results Compared with the passive PH group, LSbas, LSfw, EF, FAC and S’ decreased in reactive PH group (all P < 0.05). However, they showed no significant differences between controls and the passive PH group (all P> 0.05). EDV, ESV and MPI increased gradually as the disease progressed (all P < 0.05). By logistic regression analysis, only LSbas and EF were independently associated with reactive PH. By ROC analysis, they were indicative of reactive PH with sensitivity of 83% and 86%, and specificity of 89% and 81%. Conclusions RV presented gradual dilatation with deteriorated systolic function that occurred late. 2D strain and3D volumetric assessments were able to identity reactive PH better than conventional echocardiography.