Active-targeted cancer imaging and therapy of glioma has attracted much attention in theranostic nanomedicine. As a promising tumor-targeting ligand, holo-transferrin (holo-Tf) has been applied for enhancing delivery of nanotheranostics. However, holo-Tf-based nanoassemblies for active targeting mediated multimodal imaging and therapeutics have not been previously reported. Here, we develop a one-step method for the preparation of holo-Tf-indocyanine green (holo-Tf-ICG) nanoassemblies for fluorescence (FL) and photoacoustic (PA) dual-modal imaging and photothermal therapy (PTT) of glioma. The nanoassemblies are formed by hydrophobic interaction and hydrogen bonds between holo-Tf and ICG, which exhibit excellent active tumor-targeting and high biocompability. The brain tumor with highly expressed Tf receptor can be clearly observed with holo-Tf-ICG nanoassemblies base on FL and PA dual-modal imaging in subcutaneous and orthotopic glioma models. Under the near-infrared laser irradiation, the holo-Tf-ICG nanoassemblies accumulated in tumor regions can efficiently convert laser energy into hyperthermia for tumor ablation. The novel theranostic nanoplatform holds great promise for precision diagnosis and treatment of glioma.Keywords: fluorescence imaging; glioma; holo-Transferrin; Indocyanine green; photoacoustic imaging; photothermal therapy;

Targeting drug delivery remains a challenge in various disease treatment including cancer. The local drug deposit could be greatly enhanced by some external stimuli-responsive systems. Here we develop pluronic P123/F127 polymeric micelles (M) encapsulating curcumin (Cur) that are permeabilized directly by focused ultrasound, in which ultrasound triggers drug release. Tumor preferential accumulation and site-specific sonochemotherapy were then evaluated. Cur-loaded P123/F127 mixed micelles (Cur-M) exhibited longer circulating time and increased cellular uptake compared to free Cur. With the assistance of focused ultrasound treatment, Cur-M showed tumor-targeting deposition in a time-dependent manner following systemic administration. This was due to enhanced permeabilization of tumor regions and increased penetration of Cur-M in irradiated tumor cells by ultrasound sonoporation. Furthermore, Cur-M self-assembly could be regulated by ultrasound irradiation. In vitro Cur release from mixed micelles was greatly dependent on ultrasound intensity but not on duration, suggesting the cavitational threshold was necessary to initiate subsequent sonochemotherapy. In vivo site-specific drug release was demonstrated in dual-tumor models, which showed spatial-temporal release of entrapped drugs following intratumoral injection. The sonoporation-assisted site-specific chemotherapy significantly inhibited tumor growth and the decrease in tumor weight was approximately 6.5-fold more than without exposure to ultrasound irradiation. In conclusion, the established ultrasound-guided nanomedicine targeting deposit and local release may represent a new strategy to improve chemotherapy efficiency.Keywords: local release; mixed micelles; sonoporation-assisted chemotherapy; targeting accumulation; ultrasound-responsive;

•Tumor inhibition effect caused by DVDMS-SPDT was compared with monotherapy of SDT and PDT both in vivo and in vitro.•DVDMS-SPDT can efficiently suppress cell migration in vitro and lung metastasis in 4T1 in the highly metastatic 4T1 mouse xenograft model.•The excess level of ROS may plays a crucial role in cell death, cavitation effect and changes of membrane permeability also work on the enhancement of combination therapy.Sono-photodynamic therapy (SPDT) is a promising anti-cancer strategy. Briefly, SPDT combines ultrasound and light to activate sensitizers that produce mechanical, sonochemical and photochemical activities. Sinoporphyrin sodium (DVDMS) is a newly identified sensitizer that shows great potential in both sonodynamic therapy (SDT) and photodynamic therapy (PDT). In this study, we primarily evaluated the combined effects of SDT and PDT by using DVDMS on breast cancer both in vitro and in vivo. In vitro, DVDMS-SPDT elicits much serious cytotoxicity compared with either SDT or PDT alone by MTT and colony formation assays. 2′,7′-Dichlorodihydrofluo-rescein-diacetate (DCFH-DA) and dihydroethidium (DHE) staining revealed that intracellular reactive oxygen species (ROS) were significantly increased in groups given combined therapy. Terephthalic acid (TA) method and FD500-uptake assay reflected that cavitational effects and cell membrane permeability changes after ultrasound irradiation were also involved in the enhancement of combination therapy. In vivo, DVDMS-SPDT markedly inhibits the tumor volume and tumor weight growth. Hematoxylin-eosin staining and immunohistochemistry analysis show DVDMS-SPDT greatly suppressed tumor proliferation. Further, DVDMS-SPDT significantly inhibits tumor lung metastasis in the highly metastatic 4T1 mouse xenograft model, which is consistent well with the in vitro findings evaluated by transwell assay. Moreover, DVDMS-SPDT did not produces obvious effect on body weight and major organs in 4T1 xenograft model. The results suggest that by combination SDT and PDT, the sensitizer DVDMS would produce much better therapeutic effects, and DVDMS-SPDT may be a potential strategy against highly metastatic breast cancer.

•LPUS–MBs enhanced cytomembrane permeability and intracellular uptake of curcumin.•LPUS–MBs aggravated curcumin-induced apoptotic effect on MDA-MB-231 cells.•Curcumin with LPUS–MBs damaged cytoskeleton to reduce motility of MDA-MB-231 cells.Ultrasound and microbubbles-mediated drug delivery has become a promising strategy to promote drug delivery and its therapeutic efficacy. The aim of this research was to assess the effects of microbubbles (MBs)-combined low-intensity pulsed ultrasound (LPUS) on the delivery and cytotoxicity of curcumin (Cur) to human breast cancer MDA-MB-231 cells. Under the experimental condition, MBs raised the level of acoustic cavitation and enhanced plasma membrane permeability; and cellular uptake of Cur was notably improved by LPUS–MBs treatment, aggravating Cur-induced MDA-MB-231 cells death. The combined treatment markedly caused more obvious changes of cell morphology, F-actin cytoskeleton damage and cell migration inhibition. Our results demonstrated that combination of MBs and LPUS may be an efficient strategy for improving anti-tumor effect of Cur, suggesting a potential effective method for antineoplastic therapy.

•Membrane damage caused by ultrasound was compared between MCF-7 and MCF-7/ADR cells.•The ultrasound induced cell membrane damage showed an intensity-dependent manner.•MCF-7/ADR was more sensitive to ultrasound than MCF-7 at the same conditions.•The decline of membrane fluidity in MCF-7/ADR might contribute to its susceptibility.ObjectivesThe aim of this study was to compare the cell membrane damage induced by ultrasound at different intensities between MCF-7/ADR cells and MCF-7 cells.MethodsTumor cells in the culture dishes (35 mm diameter) were exposed to planner ultrasound at intensities range from 0.25 W/cm2 to 0.75 W/cm2 for 60 s. The viability of cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and Guava Viacount assay. The cell membrane integrity was estimated by flow cytometry using propidium iodide (PI) staining and cellular uptake of fluorescein isothiocyanate-dextran (FD500). The membrane lipid peroxidation and membrane fluidity were also specially compared between two cell lines in this paper using spectrophotometry. Ultrastructural alterations on membrane surface were observed by scanning electron microscopy.ResultsThe ultrasound produced cytotoxicity in both cell lines increased with the irradiation intensity increased from 0.25 W/cm2 to 0.75 W/cm2. Cell membrane permeability and the level of lipid peroxidation were remarkably enhanced after ultrasound application. In addition, relatively severe cell damage was observed under scanning electron microscopy after 0.75 W/cm2 ultrasound treatment.ConclusionsUltrasound exposure decreased MCF-7 and MCF-7/ADR cell viability in an intensity-dependent manner and MCF-7/ADR cells were more sensitive to ultrasound exposure than MCF-7 cells at the same experimental conditions. The declined membrane fluidity in MCF-7/ADR cell may be one of the reasons for its increased membrane damage.

Most cancer cells have the specially increased glycolytic phenotype, which makes this pathway become an attractive therapeutic target. Although glycolytic inhibitor 2-deoxyglucose (2-DG) has been demonstrated to potentiate the cytotoxicity of photodynamic therapy (PDT), the impacts on cell migration after the combined treatment has never been reported yet. The present study aimed to analyze the influence of glycolytic inhibitors 2-DG and 3-bromopyruvate (3-BP) combined with Ce6-PDT on cell motility of Triple Negative Breast Cancer MDA-MB-231 cells. As determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium-bromide-Tetraz-olium (MTT) assay, more decreased cell viability was observed in 2-DG + PDT and 3-BP + PDT groups when compared with either monotherapy. Under optimal conditions, synergistic potentiation on cell membrane destruction and the decline of cell adhesion and cells migratory ability were observed in both 2-DG + PDT and 3-BP + PDT by electron microscope observation (SEM), wound healing and trans-well assays. Besides, serious microfilament network collapses as well as impairment of matrix metalloproteinases-9 (MMP-9) were notably improved after the combined treatments by immunofluorescent staining. These results suggest that 2-DG and 3-BP can both significantly potentiated Ce6-PDT efficacy of cell migration inhibition.

Malignant cells are highly dependent on aerobic glycolysis, which differs significantly from normal cells (the Warburg effect). Interference of this metabolic process has been considered as an innovative method for developing selective cancer therapy. A recent study demonstrated that the glycolysis inhibitor 2-deoxyglucose (2-DG) can potentiate PDT efficacy, whereas the possible mechanisms have not been carefully investigated. This study firstly proved the general potentiation of PDT efficacy by 2-DG and 3-bromopyruvate (3-BP) in human breast cancer MDA-MB-231 cells, and carefully elucidated the underlying mechanism in the process. Our results showed that both 2-DG and 3-BP could significantly promote a PDT-induced cell cytotoxic effect when compared with either monotherapy. Synergistic potentiation of mitochondria- and caspase-dependent cell apoptosis was observed, including a mitochondrial membrane potential (MMP) drop, Bax translocation, and caspase-3 activation. Besides, ROS generation and the expression of oxidative stress related proteins such as P38 MAPK phosphorylation and JNK phosphorylation were notably increased after the combined treatments. Moreover, when pretreated with the ROS scavenger N-acetylcysteine (NAC), the ROS generation, the MMP drop, cell apoptosis and cytotoxicity were differently inhibited, suggesting that ROS was vertical in the pro-apoptotic process induced by 2-DG/3-BP combined with PDT treatment. These results indicate that the combination of glycolytic antagonists and PDT may be a promising therapeutic strategy to effectively kill cancer cells.

•Both PDT and SDT exhibit cytotoxicity on MDA-MB-231 cells.•Synergistic damage of PDT and SDT are due to the mitochondria damage, excessive intracellular ROS generation and the MMP loss.•Cell membrane is an important target in SDT, but not in PDT on MDA-MB-231 cells.Photodynamic therapy (PDT) and sonodynamic therapy (SDT) are therapeutic modalities for tumors. In this study we investigated the combined cytotoxic effect of 0.36 W/cm2 and 0.72 W/cm2 ultrasound with various Ce6 concentrations (1, 2, 5, 10 μg/ml), and that of 1 μg/ml Ce6 with different laser light dose (650 nm; 10.4 mW/cm2; 0.3, 0.6, 1.2 and 2.5 J/cm2) on MDA-MB-231 cells. Both high reactive oxygen species (ROS) production and a decline in mitochondrial membrane potential (MMP) were detected with high Ce6 concentrations (5 and 10 μg/ml) combined with 0.72 W/cm2 ultrasound and 1.2, 2.5 J/cm2 laser light with 1 μg/ml Ce6. In addition, cell membrane integrity was evaluated by using propidium iodide (PI), revealing membrane damage was aggravated with the increasing ultrasound intensity, but no significant difference on cell membrane integrity could be observed after PDT treatment. These results suggest ROS may play an important role both in SDT and PDT. Besides, mitochondria may be an initial target in PDT while SDT can cause multi-site damages in MDA-MB-231 cells.

The main objective of this study was to evaluate the efficacy of administration of doxorubicin (DOX) in combination with protoporphyrin IX (PpIX)-assisted low-level therapeutic ultrasound (US) in K562/DOX cells as a potential strategy in cancer therapy. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to determine the cytotoxicity of different treatments. Apoptosis was analyzed using annexin V-PE/7-amino-actinomycin D staining. Changes in DNA fragmentation, intracellular reactive oxygen species production, cellular membrane permeability, P-glycoprotein expression and DOX uptake were analyzed with flow cytometry. Under optimal conditions, PpIX-US significantly aggravated DOX-induced K562/DOX cell death, compared with either monotherapy. Synergistic potentiation of DNA damage, generation of reactive oxygen species and P-glycoprotein inhibition were observed. Plasma membrane integrity changed slightly after US exposure, and DOX uptake was notably improved after PpIX-US exposure. The results indicate that PpIX-US could increase the susceptibility of tumors to antineoplastic drugs, suggesting a clinical potential method for sonodynamic therapy-mediated tumor chemotherapy.

•Ce6-PDT induced ROS production to activate p38MAPK in SW620 cells.•Activation of p38MAPK prevents cells from photodamage in SW620 cells.•Inhibitor SB203580 accelerates cell apoptosis and cell proliferation inhibition.•p38MAPK inhibitor SB203580 enhanced autophagy protects cells.•p38MAPK promotes survival of cells through modulation of apoptosis and autophagy.BackgroundPhotodynamic therapy (PDT) has been undergoing clinical evaluation for the treatment of colorectal cancer. But the molecular mechanism of photodynamic injury in human colorectal cancer cells still remains unclear.MethodsChlorin e6 (Ce6) was used to photosensitize SW620 cells. The inhibitory effect of PDT was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide tetrazolium) assay and colony forming assay. Apoptosis was determined by nuclear DAPI (4′-6-diamidino-2-phenylindole) staining and Annexin V-PE/7-AAD assay. Monodansylcadaverine (MDC) staining was used to evaluate the abundance of autophagic vacuoles in PDT treated cells. The apoptosis and autophagy associated proteins were analyzed by western blotting. Moreover, we applied siRNA p38MAPK and p38MAPK inhibitor SB203580 to dissect its effect on cellular response to PDT in SW620 cells.ResultsCe6 mediated PDT (Ce6-PDT) induced apparent autophagy and apoptosis with dependent on ROS (reactive oxygen species) generation. When p38MAPK was inhibited by siRNA or inhibitor SB203580, a marked enhancement of apoptosis and autophagy in SW620 cells was detected after PDT. Moreover, autophagy inhibitor 3-methyladenine/Bafilomycin A1 greatly aggravated PDT induced photodamage in SW620 cells.ConclusionCe6-PDT induced ROS production to activate p38MAPK probably to prevent SW620 cells from photodamage. Inhibition of p38MAPK activation accelerated cell apoptosis, meanwhile enhanced autophagy may act as a cytoprotective process in SW620 cells.