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Autophagy is found to serve as a surviving mechanism for cancer cells. Inhibiting autophagy has been considered as an adjuvant anti-cancer strategy. In this study, we investigated the anti-tumor effect of combining pulsed-wave ultrasound hyperthermia (pUH) enhanced PEGylated liposomal doxorubicin (PLD) delivery with an autophagy inhibitor chloroquine (CQ). BALB/c mice bearing subcutaneous 4T1 tumor received intravenous injection of PLD (10 mg/kg) plus 15-minute on-tumor pUH on Day 5 after tumor implantation and were then fed with CQ (50 mg/kg daily) thereafter. Prolonged suppression of tumor growth was attained with PLD + pUH + CQ treatment, whereas in PLD + pUH group tumors quickly recurred after an initial inhibition. Treatment with CQ monotherapy had no benefit compared to the control group. Immunohistochemical staining and Western blotting showed that autophagy of cancer cells was blocked for the mice receiving CQ. It indicates that PLD + pUH + CQ is a promising strategy to treat cancer for a long-term inhibition.

Purpose In this study, we hypothesized that systemic antitumor immunity might be enhanced by combining pulsed-wave ultrasound hyperthermia (pUSHT) with OK-432 and that the induced antitumor immunity could confer protection against tumorigenesis. These hypotheses were tested in bilateral and rechallenged tumor models. Methods and Materials Bilateral and rechallenged tumor models were applied in the studies. In the bilateral tumor model, BALB/c mice were inoculated in both flanks with CT26-luc tumor cells. The tumors in the right flank were treated with 4 courses of pUSHT with or without OK-432. In the rechallenged tumor model, tumor cells were implanted into the right flank. Once formed, the tumors were treated with pUSHT with OK-432, followed by surgical resection. New tumor cells were then implanted into the contralateral flank. The antitumor response was evaluated via infiltrated immune cells and the severity of necrosis/apoptosis in tumors. Results In the bilateral tumor model, the tumor growth rate and growth activity of both treated (100% reduction) and untreated tumors (90.5% reduction) were significantly inhibited with the combination treatment compared with the sham control group, and the systemic antitumor effect was prolonged. The survival rate was significantly enhanced (sham control, 8 days; OK plus pUSHT, >20 days). IFNγ+ CD4 (treated tumor, 8.6-fold; untreated tumor, 4-fold), IFNγ+ CD8 (treated tumor, 6.7-fold; untreated tumor, 2.6-fold), and T cell and NK cell (treated tumor, 4-fold; untreated tumor, 2.5-fold) infiltration was increased in the combination group compared with the control group. In the rechallenged tumor model, new tumors failed to form with the combination treatment. Conclusion This experimental study combining pUSHT and OK-432 explored a new therapeutic strategy for controlling colon cancer metastasis. The results show that the combination treatment may produce an effective antitumor immune response.

The clinical application of chemotherapy for brain cancer tumors remains a challenge due to difficulties in the transport of therapeutic agents across the blood–brain barrier/blood–tumor barrier (BBB/BTB). In this study, we developed des-octanoyl ghrelin-conjugated microbubbles (GMB) loaded with TGFβ1 inhibitor (LY364947) (GMBL) to induce BBB/BTB disruption for ultrasound (US) sonication with GMBL. The in-vitro stability study showed that GMB was pretty stable over one month. The in-vivo study showed that the accumulation of superparamagnetic iron oxide nanoparticles (SPION) in the sonicated tumor was significantly higher for focused US sonication in the presence of GMBL, indicating that GMBL/US can locally disrupt BBB/BTB to promote vascular permeability of nanoparticles. In addition, the combination of folate-conjugated polymersomal doxorubicin (FPD) and GMBL/US (FPD + GMBL/US) achieved the best anti-glioma effect and significant improvement in the overall survival time for brain tumor-bearing mice. When combined with focused US, GMBL facilitated local BBB/BTB disruption and simultaneously released LY364947 to decrease the pericyte coverage of the endothelium at the targeted brain tumor sites, resulting in enhanced accumulation and antitumor activity of FPD. The overall results indicate that GMBL/US owns a great potential for non-invasive targeting delivery of nanomedicine across the BBB to treat central nervous system (CNS) diseases.

Chemotherapy for brain cancer tumors remains a big challenge for clinical medicine due to the inability to transport sufficient drug across the blood-brain barrier (BBB) and the poor penetration of drug into the tumors. To effectively treat brain tumors and reduce side effects on normal tissues, both des-octanoyl ghrelin and folate conjugated with polymersomal doxorubicin (GFP-D) was developed in this study to help transport across the BBB and target the tumor as well. The size measurements revealed that this BBB-penetrating cancer cell-targeting GFP-D was about 85 nm. In-vitro experiments with a BBB model and C6 glioma cells demonstrated that GFP-D owned a robust penetrating-targeting function for drug delivery. In C6 cell viability tests, GFP-D exhibited an inhibitory effect significantly different from the unmodified polymersomal doxorubicin (P-D). In-vivo antitumor experiments showed that GFP-D performed a much better anti-glioma effect and presented a significant improvement in the overall survival of the tumor-bearing mice as compared to the treatments with free doxorubicin (Dox), liposomal doxorubicin (L-D), P-D, or single ligand conjugated P-D. In addition, Cy 5.5 was used as a probe to investigate the delivery property of this penetrating-targeting delivery system. The overall experimental results indicate that this BBB-penetrating cancer cell-targeting GFP is a highly potential nanocarrier for the treatment of brain tumors.

Significant cooling effect by blood vessels, particularly in treated tumor region, during hyperthermia treatment has been recognized by researchers. The present study investigated a heating strategy, using a preheating zone and adaptive optimization, to effectively reduce the cooling effect as thermally significant blood vessels flowed through treated region during hyperthermia treatment. The preheating zone is located in a vessel's entrance region adjacent to treated tumor and the heating strategy attempted to elevate blood temperature before blood flowing into the treated region. We numerically calculated blood and tissue temperatures using 3-D models and the goal of treatment was to reach a uniform therapeutic temperature in the tumor region using the proposed heating strategy. Results showed first, for large blood vessels, the heating strategy effectively elevated blood temperature at the entrance of treated tumor and reduced total tumor power deposition. Consequently, it helped to reach the ideal treated temperature on tumor more effectively, and avoided extreme power deposition due to the cooling effect of blood vessels entering the treated region. For small blood vessels, the preheating zone could further improve the treatment result. Secondly, heating flowing blood with adaptive optimization results in a unique phenomenon along blood flow paths. That is a strong convective nature of blood flow, which creates high thermal gradients in the treated region. Thus, it plays a different and significant role in adaptive optimization process as compared to thermal diffusion of solid tissues.

The clinical application of chemotherapeutics for brain tumors remains a challenge due to limitation of blood-brain barrier/blood-tumor barrier (BBB/BTB). In this study, we investigated the effects of low-dose focused ultrasound hyperthermia (UH) on the delivery and therapeutic efficacy of pegylated liposomal doxorubicin (PLD) for brain metastasis of breast cancer. Murine breast cancer cells (4T1-luc2) expressing firefly luciferase were implanted into mouse striatum as a brain tumor model. The mice were intravenously injected with PLD with/without transcranial pulsed-wave/continuous-wave UH (pUH/cUH) treatment on day-6 after tumor implantation. pUH (frequency: 500kHz, PRF: 1000Hz, duty cycle: 50%) was conducted under equal acoustic power (2.2-Watt) and sonication duration (10-min) as cUH. The amounts of doxorubicin accumulated in the normal brain and tumor tissues were measured with fluorometry. The tumor growth responses for the control, pUH, PLD, PLD+cUH, and PLD+pUH groups were evaluated with IVIS. The PLD distribution and cell apoptosis were assessed with immunofluorescence staining. The results showed that pUH significantly enhanced the PLD delivery into brain tumors and the tumor growth was further inhibited by PLD+pUH without damaging the sonicated normal brain tissues. This indicates that low-dose transcranial pUH is a promising method to selectively enhance nanodrug delivery and improve the brain tumor treatment.

Po-Chin Liang,1,2,* Yung-Chu Chen,1,3,* Chi-Feng Chiang,1 Lein-Ray Mo,4 Shwu-Yuan Wei,2 Wen-Yuan Hsieh,3 Win-Li Lin1,5 1Institute of Biomedical Engineering, College of Medicine, College of Engineering, 2Department of Medical Imaging, National Taiwan University Hospital, Taipei, 3Biomedical Technology and Device Research Labs, Industrial Technology Research Institute, Hsinchu, 4Division of Gastroenterology, Department of Internal Medicine, E-Da Hospital, Kaohsiung, 5Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, Miaoli, Taiwan *These authors contributed equally in this work Abstract: In this study, we developed functionalized superparamagnetic iron oxide (SPIO) nanoparticles consisting of a magnetic Fe3O4 core and a shell of aqueous stable polyethylene glycol (PEG) conjugated with doxorubicin (Dox) (SPIO-PEG-D) for tumor magnetic resonance imaging (MRI) enhancement and chemotherapy. The size of SPIO nanoparticles was ~10nm, which was visualized by transmission electron microscope. The hysteresis curve, generated with vibrating-sample magnetometer, showed that SPIO-PEG-D was superparamagnetic with an insignificant hysteresis. The transverse relaxivity (r2) for SPIO-PEG-D was significantly higher than the longitudinal relaxivity (r1) (r2/r1 >10). The half-life of Dox in blood circulation was prolonged by conjugating Dox on the surface of SPIO with PEG to reduce its degradation. The in vitro experiment showed that SPIO-PEG-D could cause DNA crosslink more serious, resulting in a lower DNA expression and a higher cell apoptosis for HT-29 cancer cells. The Prussian blue staining study showed that the tumors treated with SPIO-PEG-D under a magnetic field had a much higher intratumoral iron density than the tumors treated with SPIO-PEG-D alone. The in vivo MRI study showed that the T2-weighted signal enhancement was stronger for the group under a magnetic field, indicating that it had a better accumulation of SPIO-PEG-D in tumor tissues. In the anticancer efficiency study for SPIO-PEG-D, the results showed that there was a significantly smaller tumor size for the group with a magnetic field than the group without. The in vivo experiments also showed that this drug delivery system combined with a local magnetic field could reduce the side effects of cardiotoxicity and hepatotoxicity. The results showed that the developed SPIO-PEG-D nanoparticles own a great potential for MRI-monitoring magnet-enhancing tumor chemotherapy. Keywords: superparamagnetic iron oxide, polyethylene glycol, doxorubicin, MRI monitoring, magnet enhancing, chemotherapy

A pregnant woman's body is a very sophisticated dynamic system that is sensitive to whole-body vibrations in a sitting posture. While much effort has been devoted to vibration effects on non-pregnant seated persons, little research has been devoted to the biodynamic responses of seated pregnant women exposed to vertical vibrations, especially in driving conditions. In this study a thorough survey of lumped-parameter models for non-pregnant seated persons is implemented. According to the literature the mathematical models adequate for representing the increase in mass due to pregnancy are proposed and evaluated systematically. On the basis of an analytical study and experimental validation, the six-degree-of-freedom model modified from the non-linear model proposed by Muksian and Nash is recommended. In addition, the recommended pregnant model is further integrated with two-dimensional half-car and three-dimensional full-car models to assess the responses of pregnant drivers or passengers exposed to vertic...

Humans are most sensitive to whole body vibration under low-frequency excitation in seated posture. As a result, biodynamics of seated human subjects has been a topic of interest over the years, and a number of mathematical models have been established. While much research has been performed on building up specific biodynamic models based on certain experimental data under prescribed testing conditions, a thorough investigation of mathematical human models in seated posture has not yet received the same level of attention. In this paper, a complete study on lumped-parameter models for seated human subjects without backrest support under vertical vibration excitation has been carried out. As part of the study, all models have been analyzed systematically, and validated by the synthesis of various experimental data from published literature. Based on the analytical study and experimental validation, the four-degree-of-freedom (DOF) model developed by Wan, Y. and Schimmels, J.M. [1995. A simple model that captures the essential dynamics of a seated human exposed to whole body vibration. Advances in Bioengineering, ASME, BED 31 333–334] is found to be best fitted to the existing test results, and is recommended for the study of biodynamic responses of seated human subjects under vertical whole body vibration. In addition, for the cases such as the female pregnant, more mass segments might be needed in the analysis. The six-DOF linear model from the modification of nonlinear model established by Muksian, R. and Nash, C.D. [1974. A model for the response of seated human to sinusoidal displacements of the seat, Journal of Biomechanics 7, 209–215] will be suggested.

Sheng-Kai Wu,1 Chi-Feng Chiang,1 Yu-Hone Hsu,1,4 Tzu-Hung Lin,2 Houng-Chi Liou,2 Wen-Mei Fu,2 Win-Li Lin1,3 1Institute of Biomedical Engineering, College of Medicine and College of Engineering, 2Institute of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan; 3Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, Miaoli, Taiwan; 4Department of Neurosurgery, Cheng-Hsin General Hospital, Taipei, Taiwan Abstract: The blood–brain/tumor barrier inhibits the uptake and accumulation of chemotherapeutic drugs. Hyperthermia can enhance the delivery of chemotherapeutic agent into tumors. In this study, we investigated the effects of short-time focused ultrasound (FUS) hyperthermia on the delivery and therapeutic efficacy of pegylated liposomal doxorubicin (PLD) for brain metastasis of breast cancer. Murine breast cancer 4T1-luc2 cells expressing firefly luciferase were injected into female BALB/c mice striatum tissues and used as a brain metastasis model. The mice were intravenously injected with PLD (5 mg/kg) with/without 10-minute transcranial FUS hyperthermia on day 6 after tumor implantation. The amounts of doxorubicin accumulated in the normal brain tissues and tumor tissues with/without FUS hyperthermia were measured using fluorometry. The tumor growth for the control, hyperthermia, PLD, and PLD + hyperthermia groups was measured using an IVIS spectrum system every other day from day 3 to day 11. Cell apoptosis and tumor characteristics were assessed using immunohistochemistry. Short-time FUS hyperthermia was able to significantly enhance the PLD delivery into brain tumors. The tumor growth was effectively inhibited by a single treatment of PLD + hyperthermia compared with both PLD alone and short-time FUS hyperthermia alone. Immunohistochemical examination further demonstrated the therapeutic efficacy of PLD plus short-time FUS hyperthermia for brain metastasis of breast cancer. The application of short-time FUS hyperthermia after nanodrug injection may be an effective approach to enhance nanodrug delivery and improve the treatment of metastatic cancers. Keywords: hyperthermia, focused ultrasound (FUS), pegylated liposomal doxorubicin (PLD), brain metastasis of breast cancer

The effective protection of the blood-brain barrier (BBB) from tight junctions and efflux transport systems ultimately results in the limited entry of 95% of drug/gene candidates, which are potentially beneficial for central nervous system (CNS) diseases. In order to enhance the brain-specific delivery, in this study we developed a targeting carrier system, which consists of poly(carboxyl ethylene glycol-g-glutamate)-co-poly(distearin-g-glutamate) (CPEGGM-PDSGM) polymersomes with the conjugation of des-octanoyl ghrelin. Des-octanoyl ghrelin across the BBB was reported to be unidirectional (blood-to-brain direction). However, there is no report about the conjugation of des-octanoyl ghrelin to a drug carrier system to confer the BBB targeting property through des-octanoyl ghrelin binding sites mediated endocytosis. To qualitatively and quantitatively investigate this carrier's properties, coumarin 6, Cy5.5 and met-enkephalin were individually encapsulated in these polymersomes. The experimental results showed that the cellular uptake was significantly higher for des-octanoyl ghrelin-conjugated polymersomes (GPs) than unconjugated polymersomes when co-incubated with the BBB cells. In addition, an enhanced accumulation in brain together with a reduced accumulation in liver and spleen was observed in animal study, indicating better brain selectivity for the GPs. In a hot-plate test, a significant inhibition of nociceptive response could be achieved for an intravenous injection of GPs encapsulated with met-enkephalin. The overall results demonstrated that GPs own a great potential for targeting delivery of drug across the BBB to treat CNS diseases.

Although much research has been devoted to constructing specific models or to measuring the response characteristics of seated subjects, investigations on a mathematical human model on a seat with a backrest to evaluate vehicular riding comfort have not yet attracted the same level of attention. For the responses of a seated body to vertical vibrations, mathematical models of the mechanisms must be at least two-dimensional in the sagittal plane. In describing the motions of a seated body, two multibody models representative of the automotive postures found in the literature were investigated, one with and the other without a backrest support. Both models were modified to suitably represent the different automotive postures with and without backrest supports, and validated by various experimental data from the published literature pertaining to the same postural conditions. On the basis of the analytical study and the experimental validation, the fourteen-degrees-of-freedom model proposed in this research was found to be best fitted to the test results; therefore, this model is recommended for studying the biodynamic responses of a seated human body exposed to vertical vibrations in various automotive postures.