Chuanshan Xu

pH-responsive polymer-drug conjugates: Design and progress.

Polymer-drug conjugates are becoming established as a shining platform for drug delivery. Incorporation of pH-responsive linker between drug and polymer is expected to realize triggered release of bioactive agents from conjugates in specific sites, either in mildly acidic extracellular matrices of tumor tissues or, after cellular internalization, in acidic endosomes and lysosomes. As an emerging drug delivery system, such pH-responsive polymer-drug conjugates are able to selectively deliver and activate drug molecules while reducing their systemic side-effects. In this review, we present the recent advances in pH-responsive polymer-drug conjugates with different chemical structures and architectures, and attempt to clarify their mechanism of action, synthesis and characterization technology. Furthermore, several promising approaches for the future will also be suggested.

Antitumor Effect of Docetaxel-Loaded Lipid Microbubbles Combined With Ultrasound-Targeted Microbubble Activation on VX2 Rabbit Liver Tumors

Objective. The purpose of the study was to explore the antitumor effect of docetaxel‐loaded lipid microbubbles combined with ultrasound‐targeted microbubble activation (UTMA) on VX2 rabbit liver tumors. Methods. Docetaxel‐loaded lipid microbubbles were made by a mechanical vibration technique. VX2 liver tumor models were established in 90 rabbits, which were randomly divided into 6 groups, including control, docetaxal‐loaded lipid microbubbles alone, docetaxal alone, docetaxal combined with ultrasound, pure lipid microbubbles combined with ultrasound, and docetaxel‐loaded lipid microbubbles combined with ultrasound (DOC+MB/US). The tumor volume and inhibition rate (IR) of tumor growth were calculated and compared. Apoptosis was detected by terminal deoxyuridine nick end labeling. Proliferating cell nuclear antigen and matrix metalloproteinase 2 (MMP2) protein expression was detected by immunohistochemistry. Caspase 3 and MMP2 messenger RNA (mRNA) expression was detected by in situ hybridization histochemistry. The tumor metastasis rate and survival time of the animals were compared. Results. The IR and apoptotic index of the DOC+MB/US group were the highest among all groups, and the proliferating labeling index was the lowest. Matrix metalloproteinase 2 protein and mRNA expression in the DOC+MB/US group was the lowest among all groups, and caspase 3 mRNA expression in the DOC+MB/US group was the highest. The extensive metastasis rate in the DOC+MB/US group was the lowest, and the survival time of the animals in the DOC+MB/US group was the longest. Conclusions. Docetaxel‐loaded lipid microbubbles combined with UTMA could inhibit the growth of VX2 rabbit liver tumors by deferring proliferation and promoting apoptosis, which may provide a novel targeted strategy for chemotherapy of liver carcinoma.

Apoptosis of ovarian cancer cells induced by methylene blue-mediated sonodynamic action

OBJECTIVE The present study aims to investigate apoptosis of ovarian cancer cells induced by methylene blue (MB)-mediated sonodynamic therapy (SDT). METHODS The MB concentration was kept constant at 100μM and ovarian cancer HO-8910 cells were exposed to ultrasound therapy for 5s with an intensity of 0.46W/cm(2). The cytotoxicity was investigated 24h after MB-mediated sonodynamic action. Apoptosis was analyzed using a flow cytometer with Annexin V-FITC and propidium iodine (PI) staining as well as fluorescence microscopy with Hoechst 33258 staining. Intracellular reactive oxygen species (ROS) level was measured by flow cytometer with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. RESULTS The cytotoxicity of MB-mediated SDT on HO-8910 cells after MB-mediated SDT was significantly higher than those of other treatments including ultrasound alone, MB alone and sham treatment. Flow cytometric analysis showed a significant increase in the early and late apoptotic cell populations by MB-mediated SDT of HO-8910 cells. Nuclear condensation and increased ROS levels were also found in HO-8910 cells treated by MB-mediated SDT. CONCLUSIONS Our findings demonstrated that MB-mediated sonodynamic action significantly induced apoptosis of HO-8910 cells and an increase in intracellular ROS level. This indicates that apoptosis is an important mechanism of cell death induced by MB-mediated SDT. Thus, MB-mediated SDT might be a potential therapeutic strategy for combating ovarian cancer.

HYPOCRELLIN B ENHANCES ULTRASOUND-INDUCED CELL DEATH OF NASOPHARYNGEAL CARCINOMA CELLS

Hypocrellin B, a natural pigment from a traditional Chinese herb, has been attracting extensive attention. The present study aims to investigate whether hypocrellin B can enhance cell death induced by ultrasound sonification on nasopharyngeal carcinoma cells in vitro. The sonodynamic action of hypocrellin B was investigated on nasopharyngeal carcinoma cell line CNE2 cells as tumor model cells. In the experiments, the hypocrellin B concentration was kept constant at 2.5 microM and the cells were subject to ultrasound exposure for 15 s at an intensity of 0.65 W/cm(2). Cytotoxicity was investigated 24 h after ultrasound sonification. Apoptosis was evaluated using flow cytometry with annexin V-FITC and propidium iodine staining and nuclear staining with Hoechst 33258. Cell ultrastructure morphology was observed using transmission electron microscopy (TEM). No significant dark cytotoxicity of hypocrellin B in the CNE2 cells was observed at the concentration of 2.5 microM. The cell death rate induced by ultrasound sonification was significantly higher in the presence of hypocrellin B than in the absence of hypocrellin B. Flow cytometry showed that ultrasound exposure in the presence of hypocrellin B significantly increased the early and late apoptotic rate, 18.64% and 22.57%, respectively, compared with the controls. Nuclear condensation was observed in the nuclear staining and swollen mitochondria and more vacuolar and broken cell membrane were found in TEM after the treatment of hypocrellin B and ultrasound. Our findings demonstrated that the presence of hypocrellin B significantly enhanced the cytotoxicity of ultrasound radiation in CNE2 cells, suggesting that hypocrellin B is a novel sonosensitizer and hypocrellin B-mediated sonodynamic therapy is a potential therapeutic modality in the management of malignant tumors.

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.

HYPERECHO AS THE INDICATOR OF TISSUE NECROSIS DURING MICROBUBBLE-ASSISTED HIGH INTENSITY FOCUSED ULTRASOUND: SENSITIVITY, SPECIFICITY AND PREDICTIVE VALUE

The purpose of this study was to determine the sensitivity, specificity and predictive values of hyperecho in grayscale ultrasonic images as the indicator of tissue necrosis in microbubble-assisted high-intensity focused ultrasound (HIFU) exposure in vivo. Livers and kidneys of the rabbit were exposed to HIFU (control group) or microbubble-assisted HIFU (experimental group); a continuous line of ablation, viz. linear scan, was performed to destruct tissues. Tissue responses were evaluated macroscopically and microscopically 24 h after HIFU. The cases of positive (hyperecho occurred and tissue necrotized), false positive (tissue was unaffected although hyperecho appeared), negative (echo was not changed and tissue was intact) and false negative (tissue was destructed despite the lack of hyperecho) were counted, and then the sensitivity, specificity and positive and negative predictive values of hyperecho were calculated. The sensitivity, specificity, positive predictive value and negative predictive value were 49.25% vs. 79.63% (p < 0.001), 45.45% vs. 30.00%, 84.62% vs. 86.00% and 12.82% vs. 21.43% for liver and 76.06% vs. 81.25%, 26.53% vs. 41.67%, 60.00% vs. 82.28% (p = 0.002) and 43.33% vs. 40.00% for kidney, in control and experimental groups, respectively. Rates varied between tissue types in control group. These findings indicated that the use of microbubble during HIFU improved the sensitivity in liver and the positive predictive value in kidney. The specificity and negative predictive value were poor. Hyperecho could only be used as the indicator of tissue necrosis in some tissue types.

Mitochondrial Damage in Nasopharyngeal Carcinoma Cells Induced by Ultrasound Radiation in the Presence of Hypocrellin B

Objective. The mitochondrion is an important target of ultrasound‐induced cell death. This study aimed to investigate the mitochondrial damage in nasopharyngeal carcinoma (NPC) cells induced by ultrasound radiation in the presence of hypocrellin B (HB). Methods. The NPC cell line CNE2 was used to investigate the effect of HB on ultrasonic action with an HB concentration of 2.5 μmol/L and ultrasound exposure for 15 seconds at an intensity of 0.65 W/cm2. Cytotoxicity was investigated 24 hours after ultrasound exposure. Mitochondrial structure changes were observed by transmission electron microscopy. The mitochondrial membrane potential was evaluated by confocal laser‐scanning microscopy with rhodamine 123 staining. Results. The mean death rates of the CNE2 cells ± SD were 25.14% ± 1.50% after ultrasound radiation alone and 76.72% ± 1.13% after ultrasound radiation in the presence of HB. Transmission electron microscopy showed that slightly enlarging mitochondria were found in the ultrasound‐treated cells. After treatment with ultrasound and HB together, some cells had seriously damaged mitochondria, namely, obvious swollen mitochondria and mitochondria in which cristae had almost completely disappeared. The mitochondrial membrane potential was more significantly collapsed when the CNE2 cells were exposed to HB for 5 hours and then ultrasound at 0.65 mW/cm2 than with ultrasound radiation alone (P < .05). Conclusions. Hypocrellin B significantly enhanced the cytotoxicity of ultrasound radiation in the CNE2 cells. The damage to the mitochondrial structure and function might be an important cause of death in the CNE2 cells induced by treatment with ultrasound radiation and HB together.

Hematoporphyrin monomethyl ether enhances the killing action of ultrasound on osteosarcoma in vivo.

Objectives. Osteosarcoma is one of the most common malignant cancers afflicting young adults. Ultrasound is a new therapeutic modality for controlling malignant cancers. Enhancing the efficacy of ultrasound treatment will improve clinical outcomes. The aim of this study was to investigate the killing action of ultrasound on osteosarcoma enhanced by hematoporphyrin monomethyl ether (HMME) in vivo. Methods. An animal model of an osteosarcoma xenograft was set up to investigate the inhibitory effect of sonoactivating HMME on osteosarcoma. High‐performance liquid chromatography was used to analyze the time course of HMME in the osteosarcoma xenograft. Three hours after intravenous (IV) administration of HMME, ultrasound radiation was administered in the osteosarcoma xenograft. On day 7 after ultrasound radiation, the tumor volume and weight were measured and calculated for effect assessment, hematoxylin‐eosin staining for histopathologic examination, immunohistochemical staining for proliferating cell nuclear antigen (PCNA) expression, and terminal deoxyuridine nick end‐labeling (TUNEL) staining for apoptosis examinations. Results. The peak value of HMME in osteosarcoma tissues increased with time after IV administration of HMME and reached a plateau at 3 hours. The increasing rates of the tumor volume and weight in the control group were very fast, but the increasing rates in the ultrasound‐alone group were slower, and those in the ultrasound‐HMME group were the slowest throughout the observation period. There was a significant difference between the HMME‐ultrasound, ultrasound‐alone, and control groups (P < .01). Hematoxylin‐eosin staining showed that some cells had typical cell death such as pyknosis and nuclear fragmentation after ultrasound radiation alone. More cells with pyknosis, nuclear fragmentation, and even karyolysis were found after HMME‐ultrasound treatment. Immunohistochemical staining showed that the percentage of PCNA‐positive cells decreased and that of TUNEL‐positive cells increased after ultrasound treatment alone, and the changes in the PCNA‐ and TUNEL‐positive percentages were significantly enhanced by pretreatment with HMME (20 mg/kg, IV) for 3 hours and ultrasound radiation (10.5 MHz) for 120 seconds at an intensity of 0.8 W/cm2 (P < .05). Conclusions. Hematoporphyrin monomethyl ether pretreatment could substantially enhance the growth inhibition of ultrasound on osteosarcoma, which suggests that HMME is an efficient sonosensitizer, and ultrasound radiation with HMME could be developed as a new modality for treating osteosarcoma.

Hypocrellin B-mediated sonodynamic action induces apoptosis of hepatocellular carcinoma cells

OBJECTIVE The present study aims to investigate apoptosis of hepatocellular carcinoma cells induced by hypocrellin B-mediated sonodynamic action. METHODS The hypocrellin B concentration was kept constant at 2.5 μM and cells from the hepatocellular carcinoma HepG2 cell line were exposed to ultrasound with an intensity of 0.46 W/cm(2) for 8s. Cell cytotoxicity was quantified using an MTT assay 24 h after sonodynamic therapy (SDT) of hypocrellin B. Apoptosis was investigated using a flow cytometry with Annexin V-FITC and propidium iodine staining. Intracellular reactive oxygen species (ROS) levels were detected using a flow cytometry with 2,7-dichlorodihydrofluorecein diacetate (DCFH-DA) staining. RESULTS The cytotoxicity of hypocrellin B-mediated sonodynamic action on HepG2 cells was significantly higher than those of other treatments including ultrasound alone, hypocrellin B alone and sham treatment. Flow cytometry showed that hypocrellin B-induced sonodynamic action markedly enhanced the apoptotic rate of HepG2 cells. Increased ROS was observed in HepG2 cells after being treated with hypocrellin B-mediated sonodynamic action. CONCLUSIONS Our data demonstrated that hypocrellin B-mediated sonodynamic action remarkably induced apoptosis of HepG2 cells, suggesting that apoptosis is an important mechanism of cell death induced by hypocrellin B-mediated SDT.

Microbubbles improve the ablation efficiency of extracorporeal high intensity focused ultrasound against kidney tissues

ObjectiveThe necrosis rate is low when ablating kidney tissues with extracorporeal high intensity focused ultrasound (HIFU), and this drawback has been limiting the application of ultrasonic therapy. The aim of the present study was to determine whether microbubbles increased the ablation efficiency in vivo.MethodsGoat kidneys were exposed to HIFU (control group) or microbubble-assisted HIFU (experimental group). Microbubbles were intravenously injected before focused ultrasound exposure. The linear scan was employed and tissue ablation was performed in manner of a clinical regime. The necrosis rate was determined 24 h after HIFU. Pathological examinations were performed to confirm tissue necrosis and to determine whether there were unaffected tissues within the exposed volume.ResultsThe necrosis rate was increased in experimental group (4.17 ± 1.33 vs. 9.32 ± 2.27 mm3/s, P = 0.0007). Ablated tissues formed a hemorrhagic volume on gross examinations, and the boundary between treated and untreated areas was sharp. There was no intact tissue within the exposed volume. Hemorrhage frequently occurred in insonated parenchymas. Destructed ghost cells just inside the demarcation were full of vacuoles, when introducing microbubbles. In control group, the volumes of ablated tissues varied drastically between animals despite a same treatment template.ConclusionMicrobubbles increased the ablation efficiency of HIFU against kidney tissues. A preoperative regime might poorly predict the therapeutic outcome.