Lymph node metastasis (LNM) has a significant impact on cancer prognosis, emphasizing the need for effective treatment strategies. This study investigated the potential use of high osmotic pressure drug solutions with low viscosity administration using a lymphatic drug delivery system (LDDS) to improve LNM treatment outcomes. The hypothesis was that injection of epirubicin or nimustine at high osmotic pressure but without altered viscosity would enhance drug retention and accumulation in LNs, thereby improving the efficacy of treatment. Biofluorescence analysis revealed enhanced drug accumulation and retention in LNs after administration using LDDS compared to intravenous (i.v) injection. Histopathological results demonstrated minimal tissue damage in the LDDS groups. Pharmacokinetic analysis revealed an improved treatment response with higher drug accumulation and retention in LNs. The LDDS approach offers the potential for greatly reduced side effects of chemotherapy drugs, lower dosage requirements and crucially increased drug retention in LNs. The results highlight the promise of high osmotic pressure drug solutions with low viscosity administrated using the LDDS for enhancing the treatment efficacy of LN metastasis. Further research and clinical trials are warranted to validate these results and optimize the clinical translation of this novel treatment technique.

Abstract The graphene-based materials have been used as a potential coating material for nanoparticles due to their excellent passivation. Herein, we report for the first time the colloidal stability, photothermal profile, thermal stability, cytotoxicity, and photo-cytotoxicity of graphene quantum dots (GQDs) coupled with the second infrared window (NIR-II) absorbing gold nanorods (AuNRs/GQDs) and compare it to graphene oxide (GO)-coated NIR-II absorbing AuNRs (AuNRs/GO). The composites were achieved by electrostatic interaction of the GO or GQDs with AuNRs. The results revealed that (i) AuNRs/GQDs were more stable in the aqueous phosphate buffer and cell culture media than AuNRs/GO and AuNRs; (ii) GO enhanced the photothermal efficiency of the AuNRs, whereas GQDs reduced it; (iii) GQDs enhanced the photothermal stability of AuNRs than GO; (iv) both AuNRs/GO and AuNRs/GQDs were biocompatible with mouse colon carcinoma (C26) cell lines and malignant fibrous histiocytoma‐like, expressing a fusion of the luciferase and enhanced green fluorescent protein genes (KM-Luc/GFP) cell lines; and (v) photo-cytotoxicity of AuNRs/GO and AuNRs/GQDs conducted against C26 cell lines showed significantly improved cell death compared to laser irradiation alone; however, AuNRs/GO exhibited high photo-toxicity than AuNRs/GQDs. This study shows that AuNRs/GO and AuNRs/GQDs composites possess unique properties to improve AuNRs and be utilised in photothermal applications.

BACKGROUND: Immune checkpoint blockade (ICB) elicits a strong and durable therapeutic response, but its application is limited by disparate responses and its associated immune-related adverse events (irAEs). Previously, in a murine model of lymph node (LN) metastasis, we showed that intranodal administration of chemotherapeutic agents using a lymphatic drug delivery system (LDDS) elicits stronger therapeutic responses in comparison to systemic drug delivery approaches, while minimizing systemic toxicity, due to its improved pharmacokinetic profile at the intended site. Importantly, the LN is a reservoir of immunotherapeutic targets. We therefore hypothesized that metastatic LN-targeted ICB can amplify anti-tumor response and uncouple it from ICB-induced irAEs. METHODS: To test our hypothesis, models of LN and distant metastases were established with luciferase expressing LM8 cells in MXH10/Mo-lpr/lpr mice, a recombinant inbred strain of mice capable of recapitulating ICB-induced interstitial pneumonia. This model was used to interrogate ICB-associated therapeutic response and immune related adverse events (irAEs) by in vivo imaging, high-frequency ultrasound imaging and histopathology. qPCR and flowcytometry were utilized to uncover the mediators of anti-tumor immunity. RESULTS: Tumor-bearing LN (tbLN)-directed CTLA4 blockade generated robust anti-tumor response against local and systemic metastases, thereby improving survival. The anti-tumor effects were accompanied by an upregulation of effector CD8T cells in the tumor-microenvironment and periphery. In comparison, non-specific CTLA4 blockade was found to elicit weaker anti-tumor effect and exacerbated ICI-induced irAEs, especially interstitial pneumonia. Together these data highlight the importance of tbLN-targeted checkpoint blockade for efficacious response. CONCLUSIONS: Intranodal delivery of immune checkpoint inhibitors to metastatic LN can potentiate therapeutic response while minimizing irAEs stemming from systemic lowering of immune activation threshold.

Magnetic hyperthermia with magnetic nanoparticles (MNPs) has been introduced to selective treatment of tumor and the MNPs also has demonstrated diagnosis. For non-invasive treatment, a therapeutic platform with temperature monitoring that can avoid overheating in normal tissues is of vital importance. In this study, we have developed a wireless temperature monitoring system by utilizing the combination of magnetic harmonic signals of the MNPs for magnetic hyperthermia treatment in laboratory experiments. We achieved an accurate measurement with an error of 0.18 °C. For practical use on breast/oral cancer, a detectable distance of at least 10 mm is required. To demonstrate the feasibility toward future biomedical applications, we investigated the dependency on the amount of Resovist^® and the error is less than 0.5 °C in a 10 mm distance. Our system can measure the correct temperature regardless of Resovist amount. The results indicate that our system can apply for monitoring temperature on magnetic hyperthermia treatment.

An electrochemical immunosensor for alpha-fetoprotein.

Treatment of metastatic lymph nodes (LNs) is challenging due to their unique architecture and biophysical traits. Systemic chemotherapy fails to impede tumor progression in LNs due to poor drug uptake and retention by LNs, resulting in fatal systemic metastasis. To effectively treat LN metastasis, achieving specific and prolonged retention of chemotherapy drugs in the tumor-draining LNs is essential. The lymphatic drug delivery system (LDDS) is an ultrasound-guided drug delivery methodology for administration of drugs to LNs that addresses these requirements. However, early-stage metastatic LNs have an additional set of drug transport barriers, such as elevated intranodal pressure and viscosity, that negatively impact drug diffusion. In the present study, using formulations of elevated osmotic pressure and viscosity relative to saline, we sought to favorably alter the LN's physical environment and study its impact on pharmacokinetics and consequently the therapeutic efficacy of carboplatin delivered using the LDDS. Our study confirmed the capability of a drug formulation with elevated osmotic pressure and viscosity to alter the architecture of LNs, as it caused notable expansion of the lymphatic sinus. Additionally, the study delineated an optimal range of osmotic pressure and viscosity, centered around 1,897 kPa and 11.5 mPa·s, above and below which therapeutic efficacy was found to decline markedly. These findings suggests that formulation osmotic pressure and viscosity are parameters that require critical consideration as they can both hinder and promote tumorigenesis. The facile formulation reported here has wide ranging applicability across cancer spectrums and is thus anticipated to be of great clinical benefit.

Delivery of chemotherapeutic agents into metastatic lymph nodes (LNs) is challenging as they are unevenly distributed in the body. They are difficult to access via traditional systemic routes of drug administration, which produce significant adverse effects and low accumulation of drug into the cancerous LNs. To improve the survival rate of patients with LN metastasis, a lymphatic drug delivery system (LDDS) has been developed to target metastatic LNs by delivering chemotherapy agents into sentinel LNs (SLNs) under ultrasound guidance. The LDDS is an advanced method that can be applied at the early stage of progression of tumor cells in the SLNs, before tumor mass formation has occurred. Here we investigated the optimal physicochemical ranges of chemotherapeutic agents' solvents with the aim of increasing treatment efficacy using the LDDS. We found that an appropriate osmotic pressure range for drug administration was 700 - 3,000 kPa, with a viscosity < 40 mPa⋅s. In these physicochemical ranges, expansion of lymphatic vessels and sinuses, drug retention and subsequent antitumor effects could be more precisely controlled. Furthermore, the antitumor effects depended on the tumor progression stage in the SLNs, the injection rate and volumes of administered drugs. We anticipate these optimal ranges to be a starting point for the development of more effective drug regimens to treat metastatic LNs with the LDDS.

Bioluminescence (BL) imaging is a powerful non-invasive imaging modality widely used in a broad range of biological disciplines for many types of measurements. The applications of BL imaging in biomedicine are diverse, including tracking bacterial progression, research on gene expression patterns, monitoring tumor cell growth/regression or treatment responses, determining the location and proliferation of stem cells, and so on. It is particularly valuable when studying tissues at depths of 1 to 2 cm in mouse models during preclinical research. Here we describe the protocols for the therapeutic evaluation of a lymphatic drug delivery system (LDDS) using an in vivo BL imaging system (IVIS) for the treatment of metastatic lymph nodes (LNs) with 5-fluorouracil (5-FU). The LDDS is a method that directly injects anticancer drugs into sentinel LNs (SLNs) and delivers them to their downstream LNs. In the protocol, we show that metastases in the proper axillary LN (PALN) are induced by the injection of luciferase-expressing tumor cells into the subiliac LN (SiLN) of MXH10/Mo-lpr/lpr (MXH10/Mo/lpr) mice. 5-FU is injected using the LDDS into the accessory axillary LN (AALN) to treat tumor cells in the PALN after the tumor cell growth is confirmed in the PALN. The tumor growth and therapeutic effects are evaluated by IVIS. This method can be used to evaluate tumor growth and efficacy of anticancer drugs/particles, radiotherapy, surgery, and/or a combination of these methods in various experimental procedures in the oncology field.

A perfusion defect (PD) in non-enlarged lymph nodes (LNs) of oral squamous cell carcinoma (OSCC) is the most reliable radiological criterion for the diagnosis of metastasis. However, conventional contrast-enhanced (CE) T1 weighted images using turbo spin echo (TSE) sequence is limited in detecting PD in non-enlarged LNs due to flow artifacts from cervical blood vessels. Vessel wall (VW) MR imaging with blood vessel flow suppression and high spatial resolution may provide new insights into the detection of PD. However, there are no reports in the literature on the usefulness of VW MR imaging for the diagnosis of LN metastasis. It is demonstrated that PD of non-enlarged LNs in CE VR MR imaging of OSCC patients is useful for the diagnosis of metastatic LNs. VW MR imaging was significantly more sensitive in detecting PD of non-enlarged metastatic LNs than conventional TSE imaging on visual evaluation. Furthermore, it was found that the image contrast between PD and surrounding intranodal tissue in CE VW MR images was higher than that in conventional CE TSE images. In the correlation between imaging and histopathological findings of metastatic LNs, all LNs that exhibited PD on CE VW MR images were at an advanced histopathological metastatic stage. The pathology of PD was necrotic tissue with keratinization. The results indicated that PD in CE VW imaging is useful in diagnosing non-enlarged LNs at an advanced metastasis stage. The addition of VW MR imaging to conventional MR examination achieves higher diagnostic performance for non-enlarged metastatic LNs.

The cancer mortality rate has increased, and conventional cancer treatments are known for having many side effects. Therefore, it is imperative to find a new therapeutic agent or modify the existing therapeutic agents for better performance and efficiency. Herein, a synergetic phototherapeutic agent based on a combination of photothermal and photodynamic therapy is proposed. The phototherapeutic agent consists of water-soluble cationic porphyrin (5,10,15,20-tetrakis(N-methylpyridinium-3-yl)porphyrin, TMePyP), and gold nanorods (AuNRs) anchored on graphene-oxide (GO) sheet. The TMePyP was initially synthesized by Adler method, followed by methylation, while GO and AuNRs were synthesized using Hummer’s and seed-mediated methods, respectively. The structural and optical properties of TMePyP were confirmed using UV-Vis, zeta analyzer, PL, FTIR and NMR. The formation of both GO and AuNRs was confirmed by UV-Vis-NIR, FTIR, TEM and zeta analyzer. TMePyP and AuNRs were anchored on GO to form GO@AuNRs-TMePyP nanocomposite. The as-synthesized nanocomposite was stable in RPMI and PBS medium, and, on irradiation, produced high heat than the bare AuNRs, with high photothermal efficiency. In addition, the nanocomposite produced higher singlet oxygen than TMePyP with high biocompatibility in the absence of light. These results indicated that the as-synthesized nanocomposite is a promising dual photodynamic and photothermal agent for cancer therapy.

The link between the microbiome and cancer has led researchers to search for a potential probe for intracellular targeting of bacteria and cancer. Herein, we developed near infrared-emitting ternary AgInSe/ZnS quantum dots (QDs) for dual bacterial and cancer imaging. Briefly, water-soluble AgInSe/ZnS QDs were synthesized in a commercial kitchen pressure cooker. The as-synthesized QDs exhibited a spherical shape with a particle diameter of 4.5 ± 0.5 nm, and they were brightly fluorescent with a photoluminescence maximum at 705 nm. The QDs showed low toxicity against mouse mammary carcinoma (FM3A-Luc), mouse colon carcinoma (C26), malignant fibrous histiocytoma-like (KM-Luc/GFP) and prostate cancer cells, a greater number of accumulations in Staphylococcus aureus, and good cellular uptake in prostate cancer cells. This work is an excellent step towards using ternary QDs for diagnostic and guided therapy for prostate cancer.

The excellent photothermal properties of gold nanorods (Au-NRs) make them one of the most researched plasmonic photothermal nanomaterials. However, their biological applications have been hampered greatly due to surfactant-induced cytotoxicity. We herein report a simple synthesis of highly biocompatible gelatin stabilized Au-NRs (gelatin@Au-NRs) to address this issue. The optical and structural properties of the as-synthesized gelatin@Au-NRs were investigated by Zetasizer, Ultraviolet-Visible-Near Infrared (UV-Vis-NIR) spectroscopy, high-resolution transmission electron microscopy (HR-TEM), and Fourier transform infrared spectroscopy (FTIR). The as-synthesized gelatin@Au-NRs were highly crystalline and rod-like in shape with an average length and diameter of 66.2 ± 2.3 nm and 10 ± 1.6 nm, respectively. The as-synthesized gelatin@Au-NRs showed high stability in common biological media (phosphate buffer saline and Dulbecco’s Modified Eagle’s Medium) compared to CTAB capped Au-NRs. Similarly, the gelatin@Au-NRs showed an improved heat production and outstanding cell viability against two different cancer cell lines; KM-Luc/GFP (mouse fibroblast histiocytoma cell line) and FM3A-Luc (breast carcinoma cell line) compared to CTAB capped Au-NRs and PEG@Au-NRs. An in vitro photothermal therapy study against KM-Luc/GFP showed that gelatin@Au-NRs effectively destroys the cancer cells.

A perfusion defect in a metastatic lymph node (LN) can be visualized as a localized area of low contrast on contrast-enhanced CT, MRI or ultrasound images. Hypotheses for perfusion defects include abnormal hemodynamics in neovascular vessels or a decrease in blood flow in pre-existing blood vessels in the parenchyma due to compression by LN tumor growth. However, the mechanisms underlying perfusion defects in LNs during the early stage of LN metastasis have not been investigated. We show that tumor mass formation with very few microvessels was associated with a perfusion defect in a non-enlarged LN at the early stage of LN metastasis in a LN adenopathy mouse (LN size circa 10 mm). We found in a mouse model of LN metastasis, induced using non-keratinizing tumor cells, that during the formation of the perfusion defect in a non-enlarged LN, the number of blood vessels ≤ 50 μm in diameter decreased, while those of > 50 μm in diameter increased. The methods used were contrast-enhanced high-frequency ultrasound and contrast-enhanced micro-CT imaging systems, with a maximum spatial resolution of > 30 μm. Furthermore, we found no tumor angiogenesis or oxygen partial pressure (pO2) changes in the metastatic LN. Our results demonstrate that the perfusion defect appears to be a specific form of tumorigenesis in the LN, which is a vascular-rich organ. We anticipate that a perfusion defect on ultrasound, CT or MRI images will be used as an indicator of a non-enlarged metastatic LN at an early stage.

Photothermal therapy has been established recently as a non-invasive treatment protocol for cancer metastatic lymph nodes. Although this treatment approach shows efficient tumour ablation towards lymph node metastasis, the monitoring and reporting of treatment progress using the lymphatic delivery channel still need to be explored. Herein, we investigated the anti-tumour effect of pegylated gold nanorods with a high aspect ratio (PAuNRs) delivered via the lymphatic route in a mouse model. In this study, breast carcinoma (FM3A-Luc) cells were inoculated in the subiliac lymph node (SiLN) to induce metastasis in the proper axillary lymph node (PALN). The treatment was initiated by injecting the PAuNRs into the accessory axillary lymph node (AALN) after tumour metastasis was confirmed in the PALN followed by external NIR laser irradiation under a temperature-controlled cooling system. The anti-tumour impact of the treatment was evaluated using an in vivo bioluminescence imaging system (IVIS). The results showed a time-dependent reduction in tumour activity with significant treatment response. Tumour growth was inhibited in all mice treated with PAuNRs under laser irradiation; results were statistically significant (** p &lt; 0.01) even after treatment was concluded on day 3. We believe that this non-invasive technique would provide more information on the dynamics of tumour therapy using the lymphatically administered route in preclinical studies.

Many studies of the autoimmune disease Sjögren's syndrome have been performed using spontaneous mouse models. In the present study, we describe the characteristics of McH/lpr-RA1 mice and propose their use as a novel murine model of autoimmune sialadenitis. The McH/lpr-RA1 mouse is a recombinant congenic strain derived from generation F54 or more of MRL-Faslpr x (MRL- Faslpr x C3H- Faslpr) F1. We show for the first time that this mouse spontaneously develops autoimmune sialadenitis and vasculitis in submandibular gland tissues. Sialadenitis was accompanied by extensive inflammatory cell infiltration and tissue destruction. Immunohistochemical studies revealed that the salivary gland lesions strongly expressed four sialadenitis-related molecules: SSA and SSB (autoantigens of Sjögren's syndrome), gp91phox (an accelerator of reactive oxygen species production) and single strand DNA (a marker of apoptotic cells). In contrast, expression of aquaporin-5 (AQP5), which stimulates salivary secretion was weak or negligible. Statistical correlation analyses indicated that the apoptosis of salivary gland cells provoked by oxidative stress contributed to the severe sialadenitis and reduced expression of AQP5. Our study has demonstrated that McH/lpr-RA1 mice spontaneously develop the pathognomonic features of autoimmune sialadenitis and thus could be used as a new animal model of Sjögren's syndrome.

Lymph node (LN) metastasis is thought to account for 20-30% of deaths from head and neck cancer. The lymphatic drug delivery system (LDDS) is a new technology that enables the injection of drugs into a sentinel LN (SLN) during the early stage of tumor metastasis to treat the SLN and secondary metastatic LNs. However, the optimal physicochemical properties of the solvent used to carry the drug have not been determined. Here, we show that the osmotic pressure and viscosity of the solvent influenced the antitumor effect of cisplatin (CDDP) in a mouse model of LN metastasis. Tumor cells were inoculated into the proper axillary LN (PALN), and the LDDS was used to inject CDDP solution into the subiliac LN (SiLN) to treat the tumor cells in the downstream PALN. CDDP dissolved in saline had no therapeutic effects in the PALN after it was injected into the SiLN using the LDDS or into the tail vein (as a control). However, CDDP solution with an osmotic pressure of ~ 1,900 kPa and a viscosity of ~ 12 mPa⋅s suppressed tumor growth in the PALN after it was injected into the SiLN using the LDDS. The high osmotic pressure dilated the lymphatic vessels and sinuses to enhance drug flow in the PALN, and the high viscosity increased the retention of CDDP in the PALN. Our results demonstrate that optimizing the osmotic pressure and viscosity of the solvent can enhance the effects of CDDP, and possibly other anticancer drugs, after administration using the LDDS.

Conventional treatment for lymph node (LN) metastasis such as systemic chemotherapy have notable disadvantages that lead to the development of unwanted effects. Previously, we have reported the lymphatic administration of drugs into metastatic LNs using a lymphatic drug delivery system (LDDS). However, prior studies of the LDDS have not attempted to optimize the conditions for efficient drug delivery. Here, we investigated the influence of several factors on the efficiency of drug delivery by a LDDS in conjunction with ultrasound (US). First, the effect of the injection rate on delivery efficiency was evaluated. Fluorescent molecules injected into an upstream LN were delivered more effectively into a downstream LN when a lower injection rate was used. Second, the influence of molecular weight on drug delivery efficiency was determined. We found that molecules with a molecular weight > 10,000 were poorly delivered into the LN. Finally, we assessed whether the administration route affected the delivery efficiency. We found that the delivery efficiency was higher when molecules were administered into an upstream LN that was close to the target LN. These findings revealed the importance of a drug's physical properties if it is to be administered by LDDS to treat LN metastasis.

Cancer metastasis to lymph nodes (LNs) almost certainly contributes to distant metastasis. Elevation of LN internal pressure (intranodal pressure, INP) during tumor proliferation is associated with a poor prognosis for patients. We have previously reported that a lymphatic drug delivery system (LDDS) allows the direct delivery of anticancer drugs into the lymphatic system and is a promising treatment strategy for early-stage LN metastasis. However, methods for evaluating the treatment effects have not been established. Here, we used a mouse model of MXH10/Mo-lpr/lpr, which develops a systemic swelling of LNs, and murine malignant fibrous histiocytoma-like (KM-Luc/GFP) cells or murine breast cancer (FM3A-Luc) cells inoculated into the subiliac LN of mice to produce a tumor-bearing LN model. The changes in INP during intranodal tumor progression and after treatment with cis-dichlorodiammineplatinum(II) (CDDP) using an LDDS were measured. We found that tumor progression was associated with an increase in INP that occurred independently of LN volume changes. The elevation in INP was suppressed by CDDP treatment with the LDDS when intranodal tumor progression was significantly inhibited. These findings indicate that INP is a useful parameter for monitoring the therapeutic effect in patients with LN metastasis who have been given drugs using an LDDS, which will serve to manage cancer metastasis treatment and contribute to an improved quality of life for cancer patients.

Therapies targeting tumor vasculature would improve the treatment of lung metastasis, although the early changes in vascular structure are incompletely understood. Here, we show that obstructive metastatic foci in lung arterioles decrease the pulmonary vascular network. To generate a mouse model of lung metastasis activation, luciferase-expressing tumor cells were inoculated into the subiliac lymph node (SiLN) of an MXH10/Mo-lpr/lpr mouse, and metastatic tumor cells in the lungs were activated by SiLN resection. Activation of metastases was monitored by in vivo bioluminescence imaging. Pulmonary blood vessel characteristics were analyzed using ex vivo micro-computed tomography. The enhanced permeability and retention (EPR) effect in neovasculature after tumor cell activation was evaluated from the accumulation of intravenously injected indocyanine green (ICG) liposomes. Metastatic foci in lung arterioles were investigated histologically. Micro-computed tomography revealed decreases in pulmonary blood vessel length, volume and number of branching nodes during the early stage of metastasis caused by metastatic foci. ICG liposome accumulation by the EPR effect was not detected. Histology identified metastatic foci in lung arterioles. The lack of an EPR effect after the formation of metastatic foci in lung arterioles makes conventional systemic chemotherapy ineffective for lung metastasis. Thus, alternative therapeutic methods of drug delivery are needed.

Lymph node (LN) metastasis through the lymphatic network is a major route for cancer dissemination. Tumor cells reach the marginal sinuses of LNs via afferent lymphatic vessels (LVs) and form metastatic lesions that lead to distant metastasis. Thus, targeting of metastatic cells in the marginal sinuses could improve cancer treatment outcomes. Here, we investigated whether lymphatic administration of a drug combined with sonoporation could be used to treat a LN containing proliferating murine FM3A breast cancer cells, which are highly invasive, in its marginal sinus. First, we used contrast-enhanced high-frequency ultrasound and histopathology to analyze the structure of LVs in MXH10/Mo-lpr/lpr mice, which exhibit systemic lymphadenopathy. We found that contrast agent injected into the subiliac LN flowed into the marginal sinus of the proper axillary LN (PALN) and reached the cortex. Next, we examined the anti-tumor effects of our proposed technique. We found that a strong anti-tumor effect was achieved by lymphatic administration of doxorubicin and sonoporation. Furthermore, our proposed method prevented tumor cells in the marginal sinus from invading the parenchyma of the PALN and resulted in tumor necrosis. We conclude that lymphatic administration of a drug combined with sonoporation could exert a curative effect in LNs containing metastatic cells in their marginal sinuses.

© 2019 The Author(s). Background: McH-lpr/lpr-RA1 mice are a new strain of mice which spontaneously develop destructive arthritis and enthesitis in the ankle. There is no published data that drug treatment has been trialed on these mice. This study examined the effect of the mouse anti-IL-6 receptor antibody, MR16-1, for the treatment of arthritis and enthesitis in McH-lpr/lpr-RA1 mice. Methods: Male McH-lpr/lpr-RA1 mice were randomly divided into control and treatment groups. MR16-1 was administered from 10 weeks of age for the treatment group. Saline was applied for the control group. The drug was administered once a week, at an initial dose of 2 mg, then maintained at 0.5 mg once per week thereafter. The effects were evaluated by the histopathological synovitis score, in vivo imaging using indocyanine green liposomes, and analysis of the gene expression of inflammatory cytokines. Results: Tissue analyses were carried out at 14, 17 and 20 weeks of age. The synovitis scores of treated groups were significantly lower compared with those of the control group at 14 and 17 weeks of age. The kappa coefficient was 0.77. However, progression of entheseal ossification persisted in the MR16-1 treated group. In vivo imaging using indocyanine green liposomes showed significant decreases in signal intensities of treated groups at week 14, but no significant differences were observed at week 18. Blood serum amyloid A levels in treated groups were significantly lower at 17 weeks of age. The gene expression levels of Tnf and Il17 were also significantly lower in MR16-1 treated groups. Conclusions: Administration of the anti-IL-6 receptor antibody is effective for the treatment of synovitis and bone destruction of McH-lpr/lpr-RA1 mice. McH-lpr/lpr-RA1 mice may be a suitable experimental model for the development of new treatments for destructive arthritis and enthesitis. IL-6 signal blockade could contribute to the treatment of destructive arthritis, and further studies should be carried out to confirm its potential in the prevention of enthesopathy developed to ossification.

Metastatic lymph nodes (LNs) may be the origin of systemic metastases. It will be important to develop a strategy that prevents systemic metastasis by treating these LNs at an early stage. False-negative metastatic LNs, which are found during the early stage of metastasis development, are those that contain tumor cells but have a size and shape similar to LNs that do not host tumor cells. Here, we show that 5-fluorouracil (5-FU), delivered by means of a novel lymphatic drug delivery system (LDDS), can treat LNs with false-negative metastases in a mouse model. The effects of 5-FU on four cell lines were investigated using in vitro cytotoxicity and cell survival assays. The therapeutic effects of LDDS-administered 5-FU on false-negative metastatic LNs were evaluated using bioluminescence imaging, high-frequency ultrasound (US), and histology in MHX10/Mo-lpr/lpr mice. These experimental animals develop LNs that are similar in size to human LNs. We found that all cell lines showed sensitivity to 5-FU in the in vitro assays. Furthermore, a concentration-dependent effect of 5-FU to inhibit tumor growth was observed in tumor cells with low invasive growth characteristics, although a significant reduction in metastatic LN volume was not detected in MHX10/Mo-lpr/lpr mice. Adverse effects of 5-FU were not detected. 5-Fluorouracil administration with a LDDS is an effective treatment method for false-negative metastatic LNs. We anticipate that the delivery of anticancer drugs by a LDDS will be of great benefit in the prevention and treatment of cancer metastasis via LNs.

Lymph node (LN) dissection is a crucial procedure for cancer staging, diagnosis and treatment, and for predicting patient survival. Activation of lung metastatic lesions after LN dissection has been described for head and neck cancer and breast cancer. Preclinical studies have reported that dissection of a tumor-bearing LN is involved in the activation and rapid growth of latent tumor metastases in distant organs, but it is also important to understand how normal (non-tumor-bearing) LN resection influences secondary cancer formation. Here, we describe how the resection of tumor-bearing and non-tumor-bearing LN affects distant metastases in MXH10/Mo-lpr/lpr mice. Tumor cells were administered intravenously and/or intranodally into the right subiliac lymph node (SiLN) to create a mouse model of lung metastasis. Luciferase imaging revealed that tumor cells in the lung were activated after resection of the SiLN, irrespective of whether it contained tumor cells. No luciferase activity was detected in the lungs of mice that did not undergo LN resection (excluding the intravenous inoculation group). Our results indicate that resection of an LN can activate distant metastases regardless of whether the LN contains tumor cells. Hence, lung metastatic lesions are suppressed while metastatic LN are present but activated after LN resection. If this phenomenon occurs in patients with cancer, it is likely that lung metastatic lesions may be activated by elective LN dissection in clinical N0 cases. The development of minimally invasive cancer therapy without surgery would help to minimize the risk of activation of distant metastatic lesions by LN resection.

PURPOSE: Lymph node (LN) metastasis is detected prior to distant metastasis in many types of cancer. Detecting early stage LN metastasis can improve treatment outcomes. However, there are few clinical imaging modalities capable of diagnosing metastatic LNs of clinical N0 status (i.e., before their volume increases) with high precision. Here, we report a new method for diagnosing metastatic LNs of clinical N0 status in a mouse model of LN metastasis. PROCEDURES: The method involved using intranodal lymphangiography with x-ray micro-computed tomography (micro-CT). Contrast agent was injected into an upstream LN to deliver it to a downstream LN, which was then removed and analyzed by micro-CT. RESULTS: We found that using an intranodal injection rate of 10-60 μl/min filled the lymphatic sinus of the downstream LN with contrast agent, although the accumulation of contrast agent in the upstream LN increased with a faster injection rate. Furthermore, breast cancer cells growing in the lymphatic sinus of the downstream LN (which was of clinical N0 status) impeded the flow of contrast agent from the upstream LN, resulting in areas deficient of contrast agent in the metastatic downstream LN. The formation of defect areas in the downstream LN manifested as a difference in position between the centroid of the entire LN area and the centroid of the region that filled with contrast agent. CONCLUSION: The present study indicates that intranodal lymphangiography with micro-CT has the potential to be used as a new method for diagnosing metastatic LNs of clinical N0 status.

The lymphatic drug delivery system (LDDS) is a new technique that permits the injection of drugs into a sentinel lymph node (SLN) at an early stage of tumor metastasis, thereby treating metastasis in the SLN and its secondary lymph nodes (LNs). The quantity of drug required for a LDDS is much smaller than that needed for systemic chemotherapy. However, the relationship between the rate of drug injection into a SLN and the amount of drug reaching the secondary LNs has not been investigated. In this study, we used an MXH10/Mo-lpr/lpr mouse model to show that the optimal rate for the injection of a fluorescent dye by a LDDS was 10 to 80 μL/min. An injection rate of 10 to 80 μL/min was able to fill the downstream LN. However, an injection rate of 100 μL/min drove the fluorescent dye into the efferent lymphatic vessels and thoracoepigastric vein, decreasing the amount of dye retained in the downstream LN. Bolus injection (defined as an injection rate of 2400 μL/min) was unable to deliver fluorescent dye into the downstream LN. These results agree with the impulse values calculated from the injection pressures in the upstream LN. We anticipate that our findings will facilitate the development of a LDDS for use in the clinic.

Conventional treatment of lymph node metastasis involves dissection of the tumor and regional lymph nodes, but this may cause activation of latent metastatic tumor cells. However, there are few reports on animal models regarding the activation of latent metastatic tumor cells and effective methods of treating activated tumor cells. Here, we report the use of a superselective drug delivery system in a mouse model of lung metastasis in which activated tumor cells are treated with doxorubicin-encapsulated liposomes (DOX-LP) and ultrasound. The axillary lymph node was injected with DOX-LP and exposed to ultrasound so that the released DOX would be delivered from the axillary lymph node to the metastatic lung via the subclavian vein, heart and pulmonary artery. The size of the DOX-LP was optimized to a diameter of 460 nm using indocyanine green-encapsulated liposomes, and the ultrasound intensity was 0.5 W/cm2. We found that compared with DOX or DOX-LP alone, the superselective drug delivery system was effective in the treatment of metastasis in both the lung and axillary lymph node. We anticipate that this superselective drug delivery system will be a starting point for the development of new techniques for treating lung metastasis in the clinical setting. Furthermore, the superselective drug delivery system may be used to screen novel drugs for the treatment of lung cancer and investigate the mechanisms of tumor cell activation after resection of a primary tumor or lymph nodes.

Background: Surgical removal of primary tumors can promote the incidence of tumor metastasis. However, molecular mechanisms underlying this process remain unclear. Methods: We inoculated tumor cells expressing luciferase gene into subiliac lymph node (SiLN) of the MXH10/Mo-lpr/lpr mice. The tumor-bearing SiLNs were surgically removed at a certain period of time after inoculation. Results: In vivo bioluminescence imaging system and histological staining revealed metastasis in lung, proper axillary lymph node (PALN) and liver. The lung metastasis rate in SiLN removal groups was significantly higher than in the control group using Fisher exact test. Mann-Whitney U-test indicated that the luciferase-positive tumor cells in the lung and liver were significantly higher than in the control groups. The lung samples in SiLN removal groups had strong expression of lysine oxidase (LOX). Moreover, the number of CD11b+ cells in the lung and liver in the SiLN removal groups was significantly increased, which was positively correlated with LOX expression level. In addition, the condition of LOX and CD11b in liver was similar to lung. In the SiLN surgical removal groups, the matrix metalloproteinase (MMP)-2 and VEGFA expression in the lung tissues was significantly higher than in the control groups the collagen fibers per area around the pulmonary vessels was quite significantly lower and negatively correlated with the expression of MMP-2 by Spearman's analysis. Our data indicated that the reticular fibers were deposited and disordered in the tumor tissues of the lungs in the removal groups, and the reticular fibers per area was higher than in the control groups. The tumor cells in the PALN of control groups were significantly higher than in the SiLN removal groups, and CD169+ and CD11c+ cells were also higher than in the SiLN removal groups. Conclusions: Altogether, surgical removal of the tumor-bearing lymph node promoted tumor metastasis through changing the niche in lung and liver. Treatment targeting the metastatic niche might be an effective strategy to prevent tumor metastasis, thereby possibly increasing the survival and reducing the incidence of metastasis in cancer patients.

The acidic environment at bacterial infection sites is a potential external stimulus for targeted antibiotic delivery. This paper reports new biocompatible pH-sensitive lipids (PSLs) with three hydrocarbon tails, and a head group with a secondary amine and carboxylate function for site-specific nano delivery of vancomycin (VCM). PSLs formed stable liposomes with mean vesicle diameters and polydispersity indices between 99.38 ± 6.59 nm to 105.60 ± 5.38 nm and 0.161 ± 0.003 to 0.219 ± 0.05 respectively. The zeta potential values were negative at physiological pH (7.4) and shifted towards positivity with a decrease in pH. The encapsulation efficiency and loading capacity were in the range of 29–45% and 2.8–4.5% respectively. The VCM release increased and was more sustained at acidic pH than at the physiological pH. The molecular modeling studies revealed that structural changes in lipids at acidic pH could have caused the deformation of liposome structure and subsequent fast release. In vitro antibacterial activity revealed that the minimum inhibitory concentrations (MICs) of prepared liposomes at pH 6.5 were lower than the MICs at pH 7.4 against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) respectively. In addition, in vivo antibacterial activity study performed on two of the most active formulations showed that log10 CFU/mL of MRSA recovered from TOAPA-VCM-Lipo and the TLAPA-VCM-Lipo treated mice were 1.5- and 1.8-fold lower than that found in bare VCM treated ones respectively.

Tumor metastasis to lymph nodes is an important contributory factor for cancer-related deaths despite recent developments in cancer therapy. In this study, we demonstrate that tumor in the proper axillary lymph node (PALN) of the mouse can be treated by the application of external laser light to trigger the unloading of doxorubicin (DOX) encapsulated in thermosensitive liposomes (TSLs) administered together with gold nanorods (GNRs). GNRs + DOX-TSLs were injected into a mouse lymph node containing cancer cells (malignant fibrous histiocytoma-like cells) and intranodal DOX release was activated using near-infrared (NIR) laser irradiation. The temperature changes arising from the laser-irradiated GNRs triggered the release of DOX from the TSLs. A greater degree of inhibition of tumor growth was found in the co-therapy group compared to the other groups. The treatment effect was achieved by a combination of chemotherapy and NIR-activated hyperthermia. In vivo bioluminescence imaging and histological analysis confirmed tumor necrosis in response to combined treatment. This work presents a theranostic approach with excellent treatment results that has the potential to be developed into an alternative to surgery for the treatment of breast cancer metastasis. [GRAPHICS] .

Quantum dots (QDs) have attracted great attention due to their superior optical properties compared to organic dyes. However, the cytotoxicity of the QDs has been a major source of concern for their biological applications thus, finding solution to this problem has become an interesting area of research. We herein report green synthesis of mercaptopropionic acid (MPA) capped CdTe/CdSe core/shell QDs at different pH and their characterization using UV-vis absorption and photoluminescence (PL) spectroscopy, transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM). By varying the pH of the reaction, the temporal evolution of the optical properties and cytotoxicity of the QDs were monitored. The optical analysis showed that, increase in the solution pH, produce CdTe/CdSe QDs with excellent optical properties such as red-emission with enhanced luminescence intensity and narrow full width at half maximum (FWHM). The cell viability assay of QDs using fibroblast histiocytoma (KM-Luc/GFP) cells indicated improved cell viability as the pH of the reaction increased. The improved cell viability as the pH increase is attributed to the higher stability of the material and hence lower release of Cd2+. (C) 2017 Elsevier B.V. All rights reserved.

Systemic administration of drugs into the blood circulation is standard treatment for prevention of metastasis. However, systemic delivery cannot maintain sufficiently high concentrations of anticancer drugs in lymph nodes (LN). Here, we show that giving cisplatin (CDDP) using a lymphatic drug delivery system (LDDS) has the potential to treat false-negative metastatic LN. We found that in MXH10/Mo-lpr/lpr mice, which develop systemic swelling of LN up to 10 mm in diameter, accumulation of indocyanine green (ICG), which has a similar molecular weight to CDDP, in a target LN was greater for lymphatic delivery of ICG than for systemic (i.v.) administration. Furthermore, CDDP administration with a LDDS inhibited tumor growth in false-negative metastatic LN and produced fewer adverse effects than systemically given CDDP. We anticipate that drug delivery using a LDDS will, in time, replace systemic chemotherapy for the treatment of false-negative metastatic LN.

Cancer cells metastasize to lymph nodes, with distant metastasis resulting in poor prognosis. The role of lymph node metastasis (LNM) in the spread of cancer to distant organs remain incompletely characterized. The visualization of flow dynamics in the lymphatic and blood vessels of MXH10/Mo-lprilpr mice, which develop systemic swelling of lymph nodes up to 10 mm in diameter, has revealed that lymph nodes have the potential to be a direct source of systemic metastasis. However, it is not known whether these fluid dynamics characteristics are universal phenomena present in other strains of laboratory mice. Here we show that the fluid dynamics observed in MXH10/Mo-lpr/Ipr mice are the same as those observed in C57BL/6J, BALB/cAJcl and NOD/ShiJic-scidJcl mice. Furthermore, when fluorescent solution was injected into a tumor-bearing lymph node, the flow dynamics observed in the efferent lymphatic vessels and thoracoepigastric vein depended on the type of tumor cell. Our results indicate that fluid dynamics in the lymphatic and blood vessels of MXH10/Mo-lpr/Ipr mice are generalized phenomena seen in conventional laboratory mice. We anticipate our results can facilitate studies of the progression of lymphatic metastasis to hematogenous metastasis via lymph nodes and the early diagnosis and treatment of LNM. (C) 2017 Elsevier B.V. All rights reserved.

Most solid cancers spread to new sites via the lymphatics before hematogenous dissemination. However, only a small fraction of an intravenously administered anti-cancer drug enters the lymphatic system to reach metastatic lymph nodes (LN). Here, we show that the enhanced permeability and retention (EPR) effect is not induced during the early stages of LN metastasis. Luciferase-expressing tumor cells were injected into the subiliac LN of the MXH10/Mo-lpr/lpr mouse to induce metastasis to the proper axillary LN (PALN). In vivo biofluorescence imaging was used to confirm metastasis induction and to quantify the EPR effect, measured as PALN accumulation of intravenously injected indocyanine green (ICG) liposomes. PALN blood vessel volume changes were measured by contrast-enhanced high-frequency ultrasound imaging. The volume and density of blood vessels in the PALN increased until day 29 after inoculation, whereas the LN volume remained constant. ICG retention was first detected on day 29 post-inoculation. While CD31-positive cells increased up to day 29 post-inoculation, alpha-smooth muscle actin-positive cells were detected on day 29 post-inoculation for the first time. These results suggest that the EPR effect was not induced in the early stages of LN metastasis; therefore, systemic chemotherapy would likely not be beneficial during the early stages of LN metastasis. The development of an alternative drug delivery system, independent of the EPR effect, is required for the treatment of LN metastasis.

Systemic delivery of an anti-cancer agent often leads to only a small fraction of the administered dose accumulating in target sites. Delivering anti-cancer agents through the lymphatic network can achieve more efficient drug delivery for the treatment of lymph node metastasis. We show for the first time that polymeric gold nanorods (PAuNRs) can be delivered efficiently from an accessory axillary lymph node to a tumor-containing proper axillary lymph node, enabling effective treatment of lymph node metastasis. In a mouse model of metastasis, lymphatic spread of tumor was inhibited by lymphatic-delivered PAuNRs and near-infrared laser irradiation, with the skin temperature controlled by cooling. Unlike intravenous injection, lymphatic injection delivered PAuNRs at a high concentration within a short period. The results show that lymphatic administration has the potential to deliver anti-cancer agents to metastatic lymph nodes for inhibition of tumor growth and could be developed into a new therapeutic method.

© 2016 World Federation for Ultrasound in Medicine & Biology We examined whether enhancement area ratios obtained by the new bubble detection method correlate with histologic microvessel density in invasive breast cancer. Forty consecutive patients with invasive breast cancer lesions underwent contrast-enhanced ultrasound. The ratio of enhanced area to manually segmented tumor area (enhancement area ratio) was obtained with the new method at peak and delayed phases (50–54, 55–59, 60–64 and 65–69 s). We also analyzed time–intensity curves to obtain peak intensity and area under curve. Enhancement area ratios in both peak and delayed phases (50–54, 55–59, 60–64 and 65–69 s) were significantly correlated with microvessel density (r = 0.57, 0.62, 0.68, 0.61 and 0.58; p = 0.0001, <0.0001, <.0001, <.0001 and 0.0001, respectively). In time–intensity curve analysis, peak intensity was significantly correlated (r = 0.43, p = 0.0073), whereas area under the curve was not (r = 0.29, p = 0.0769). Enhancement area ratios obtained by the new method were correlated with microvessel density in invasive breast cancer.

We herein report a simple, economical and green synthesis of highly fluorescent, water soluble and stable arginine functionalised CdTe/CdSe/ZnSe multi core-shell nanoparticles (NPs) with enhanced cell viability for cellular imaging. The synthesis of the CdTe/CdSe/ZnSe NPs was carried out under ambient conditions in the absence of an inert environment. The as-prepared NPs were characterised using UV-vis absorption and photoluminescence (PL) spectroscopy, energy dispersive spectroscopy (EDS) and high resolution transmission electron microscopy (HRTEM). The optical analyses showed an enhancement in the fluorescent intensity after the functionalisation with improved optical properties. The functionalised NPs (F-NPs) displayed higher cell viability compared to the bare NPs when investigated on KM-Luc/GFP cell line at different concentrations. The fluorescent image indicated that the as-synthesised functionalised NPs were taken up by the cells. (C) 2016 Elsevier B.V. All rights reserved.

<p>The Enhanced Permeability and Retention (EPR) effect is considered to be a landmark principle in systemic tumor-targeting chemotherapy. Chemotherapy is used for the treatment of lung metastasis in clinics. However, the EPR effect in the lung metastasis has not been fully investigated. In this study, we evaluate the EPR effect in a mouse model of lung metastasis.Luciferase expressing tumor cells were inoculated into MXH10/Mo/lpr mice to induce lung metastasis. Lung metastasis was activated by dissecting the subiliac lymph node. The EPR effect in the metastatic lung was quantified by accumulation of intravenously injected ICG liposomes. Luciferase activity as well as fluorescence intensity were measured using bioluminescence and biofluorescence imaging. The harvested tissues were analyzed by HE and anti-CD31 staining. Herein, we found that no ICG liposome accumulation was detected in the metastatic lung and the EPR effect was not observed in the mouse model of lung metastasis.</p>

Time-dependent alterations in the ultrasonography characteristics of lymph nodes during early-stage metastasis have not been compared with those of tumor-draining lymph nodes that do not develop tumor; this is partly due to the absence of an appropriate experimental model. In a previous study of lymph nodes with experimental early-stage metastasis, we used contrast-enhanced high-frequency ultrasound to demonstrate that an increase in lymph node blood vessel density preceded any changes in lymph node volume. In the present study, we used an experimental model of lymph node metastasis in which tumor cells metastasized from the subiliac lymph node to the proper axillary lymph node (the tumor-draining lymph node). We utilized contrast-enhanced high-frequency ultrasound to perform a longitudinal analysis of tumor-draining lymph nodes, comparing those at an early-stage of metastasis with those that did not develop detectable metastasis. We found that the normalized blood vessel density of an early-stage metastatic lymph node exhibited a progressive rise, whereas that of a tumor-draining lymph node not containing tumor began to increase later. For both types of lymph nodes, the normalized blood vessel density on the final day of experiments showed a trend towards being higher than that measured in controls. We further found that mice with an initially low value for lymph node blood vessel density subsequently showed a larger increase in the blood vessel density of the metastatic lymph node; this differed significantly from measurements in controls. The present study indicates that a longitudinal analysis of the blood vessel densities of tumor-draining lymph nodes, made using contrast-enhanced high-frequency ultrasound imaging, may be a potentially promising method for detecting early-stage lymph node metastasis in selected patients. Furthermore, our findings suggest that tumor in an upstream lymph node may induce alteration of the vascular structures in draining lymph nodes that do not contain tumor.

Intravenous chemotherapy has poor access to metastatic lymph nodes (LNs) and is limited by short-lived drug concentrations. Here, we describe the administration of chemotherapy via the lymphatic network as a new concept for the prevention and treatment of metastatic LNs. A metastatic LN can be treated by the injection of drugs into an upstream LN, either the sentinel LN (SLN) or another upstream LN. In a mouse model, tumor cells were inoculated into the subiliac LN (SiLN) to induce metastasis to the proper axillary LN (PALN). Two routes were used for drug delivery to the PALN, namely from the SiLN and from the accessory axillary LN (AALN). We found that tumor masses were formed in lymphatic vessels between the SiLN and PALN. The flow of fluorescent solution injected into the SiLN towards the PALN decreased with tumor mass formation. Delivery from the AALN (free of metastatic tumor cells) to the PALN was identified as an alternative route. Intranodal injection can deliver high concentrations of drugs to secondary metastatic LNs. The study advocates a new concept for the prevention and treatment of metastatic lymph nodes whereby drugs injected into upstream lymph nodes can reach metastatic lymph nodes via the lymphatic network.

Vasohibin-1 (VASH1) was isolated as a negative-feedback regulator of angiogenesis expressed in endothelial cells (ECs). There are two transcripts of VASH1, that is, the full-length VASH1A consisting of seven exons and the splicing variant VASH1B consisting of four exons. Here, we compared the effects of VASH1A and VASH1B on tumor angiogenesis. When ECs were transfected with VASH1A or VASH1B cDNAs, VASH1B transfectants, but not VASH1A Ones, induced autophagic cell death of ECs. With sonoporation, the VASH1A or VASH1B gene were transfected specifically in ECs of tumor vessels in mice.-Both VASH1A and VASH1B decreased tumor vessel density and inhibited tumor growth. VASH1A normalized the remaining tumor vessels, increased their rate of perfusion, decreased tumor hypoxia and enhanced the efficacy of anticancer chemotherapy, whereas VASH1B pruned tumor vessels without causing normalization, increased tumor hypoxia and tumor necrosis and did not enhance the efficacy of anticancer chemotherapy. The alternate transfection of mice with the VASH1A and VASH1B gene showed the highest effects on antitumor activity and normalization of tumor vessels. Our present findings on VASH1A and VASH1B should provide an innovative approach that would improve the efficacy of antiangiogenic cancer therapy by balancing vascular normalization and pruning.

Regional lymph node status is an important prognostic indicator of tumor aggressiveness. However, early diagnosis of metastasis using intranodal pressure, at a stage when lymph node size has not changed significantly, has not been investigated. Here, we use an MXH10/Mo-lpr/lpr mouse model of lymph node metastasis to show that intranodal pressure increases in both the subiliac lymph node and proper axillary lymph node, which are connected by lymphatic vessels, when tumor cells are injected into the subiliac lymph node to induce metastasis to the proper axillary lymph node. We found that intranodal pressure in the subiliac lymph node increased at the stage when metastasis was detected by in vivo bioluminescence, but when proper axillary lymph node volume (measured by high-frequency ultrasound imaging) had not increased significantly. Intravenously injected liposomes, encapsulating indocyanine green, were detected in solid tumors by in vivo bioluminescence, but not in the proper axillary lymph node. Basic blood vessel and lymphatic channel structures were maintained in the proper axillary lymph node, although sinus histiocytosis was detected. These results show that intranodal pressure in the proper axillary lymph node increases at early stages when metastatic tumor cells have not fully proliferated. Intranodal pressure may be a useful parameter for facilitating early diagnosis of lymph node metastasis.

ObjectivesTo evaluate whether visual assessment of T2-weighted imaging (T2WI) or an apparent diffusion coefficient (ADC) could predict lymphovascular invasion (LVI) status in cases with clinically node-negative invasive breast cancer. Materials and methodsOne hundred and thirty-six patients with 136 lesions underwent MRI. Visual assessment of T2WI, tumour-ADC, peritumoral maximum-ADC and the peritumour-tumour ADC ratio (the ratio between them) were compared with LVI status of surgical specimens. ResultsNo significant relationship was found between LVI and T2WI. Tumour-ADC was significantly lower in the LVI-positive (n = 77, 896 +/- 148 x 10(-6) mm(2)/s) than the LVI-negative group (n = 59, 1002 +/- 163 x 10(-6) mm(2)/s; p &lt; 0.0001). Peritumoral maximum-ADC was significantly higher in the LVI-positive (1805 +/- 355 x 10(-6) mm(2)/s) than the LVI-negative group (1625 +/- 346 x 10(-6) mm(2)/s; p = 0.0003). Peritumour-tumour ADC ratio was significantly higher in the LVI-positive (2.05 +/- 0.46) than the LVI-negative group (1.65 +/- 0.40; p &lt; 0.0001). Receiver operating characteristic curve analysis revealed that the area under the curve (AUC) of the peritumour-tumour ADC ratio was the highest (0.81). The most effective threshold for the peritumour-tumour ADC ratio was 1.84, and the sensitivity, specificity, positive predictive value and negative predictive value were 77 % (59/77), 76 % (45/59), 81 % (59/73) and 71 % (45/63), respectively. ConclusionsWe suggest that the peritumour-tumour ADC ratio can assist in predicting LVI status on preoperative imaging.

<p>The extended Bernoulli equation is formulated in an exact form for a microscopic and small Reynolds number Jeffery-Hamel flow in a two-dimensional convergent or divergent channel. The friction loss and the friction coefficient derived from the extended Bernoulli equation are also obtained for the purpose of engineering applications. The assumption of microscopic and low Reynolds number flow enables us to make the analysis simple, and the results obtained are expressed in forms easy to use. The zeroth- and first-order approximate solutions of velocity distribution in the channel are obtained by solving the nonlinear ordinary differential equation with the optimal homotopy asymptotic method. The zeroth-order solution is shown to be the same function form as that in the two-dimensional parallel flow, i.e., the two-dimensional Poiseuille flow. The extended Bernoulli equation, the friction loss, and the friction coefficient in a finite region of the channel, which are indispensable for applications, are reasonably derived along a stream line and also expressed by cross-sectional average quantities. The cross-sectional average formulae of the friction loss and the friction coefficient are expressed by the geometry of the channel, i.e., the convergent or divergent angle, the channel length, channel widths at inlet and exit. These formulae include the corresponding well-known ones for the two-dimensional parallel flow as a special case where the angle is zero. The friction coefficient drastically increases according to the increase in the angle, especially in a narrow channel region, and attains more than ten times of the friction coefficient for the parallel flow.</p>

An accurate method for detecting contrast agents using diagnostic ultrasound imaging systems is proposed. Contrast agents, such as microbubbles, passing through a blood vessel during ultrasound imaging are detected as blinking signals in the temporal axis, because their intensity value is constantly in motion. Ultrasound contrast agents are detected by evaluating the intensity variation of a pixel in the temporal axis. Conventional methods are based on simple subtraction of ultrasound images to detect ultrasound contrast agents. Even if the subject moves only slightly, a conventional detection method will introduce significant error. In contrast, the proposed technique employs spatiotemporal analysis of the pixel intensity variation over several frames. Experiments visualizing blood vessels in the mouse tail illustrated that the proposed method performs efficiently compared with conventional approaches. We also report that the new technique is useful for observing temporal changes in microvessel density in subiliac lymph nodes containing tumors. The results are compared with those of contrast-enhanced computed tomography. (C) 2015World Federation for Ultrasound in Medicine & Biology.

Photothermal therapy (PTT) using near-infrared (NIR) laser light and gold nanorods (GNRs) shows promise as a novel cancer treatment modality. However, the laser intensity required to destroy tumor cells located beneath the skin is greater than the threshold intensity that causes skin damage; thus, irradiation with laser light damages the skin as well as the tumor. Here, we show that a temperature control system allows metastatic lymph nodes (LNs) to be treated by PTT using NIR laser light and GNRs, without skin damage. A mouse model of LN metastasis was developed by injection of tumor cells, and the tumor-bearing proper axillary LN was treated with NIR laser light after injection of GNRs. The skin temperature was maintained at 45 A degrees C during irradiation by using a temperature control system. Bioluminescence imaging revealed that tumor progression was less in LNs exposed to NIR laser light and GNRs than in LNs exposed to NIR laser light alone or controls (no irradiation or GNRs). Furthermore, the skin and LN capsule were macroscopically intact on day 9 after irradiation with NIR laser light, whereas tumor cells within the LN showed apoptosis. A numerical analysis demonstrated that the high-temperature zone and the LN region showing damage were localized to an area up to 3 mm in depth. The proposed novel PTT technique, using NIR laser light and GNRs with controlled surface cooling, could be applied clinically to treat metastatic LNs located within or outside the area accessible for surgical dissection.

The lymphatic system in mice consists of lymphatic vessels and 22 types of lymph nodes. Metastatic tumor cells in the lymphatic system spread to distant organs through the venous system. However, the communication routes between the lymphatic and venous systems have not been fully elucidated. Here, we identify the communication routes between the lymphatic and venous systems in the axillary and subiliac regions of MXH10/Mo-lpr/Ipr inbred mice, which develop systemic swelling of lymph nodes up to 10 mm in diameter, allowing investigation of the topography of the lymph nodes and lymphatic vessels. Using a gross anatomy dissection approach, the efferent lymphatic vessels of the proper axillary lymph node were shown to communicate with the subclavian vein. Furthermore, we found that the thoracoepigastric vein, which connects the subdavian vein and inferior vena cava, runs adjacent to the subiliac and proper axillary lymph nodes, and receives venous blood from these lymph nodes routed through small branches. The direction of blood flow in the thoracoepigastric vein occurred in two directions in the intermediate region between the proper axillary lymph node and subiliac lymph node; one to the subclavian vein, the other to the inferior vena cava. This paper reveals the anatomy of the communication between the lymphatic and venous systems in the axillary and subiliac regions of the mouse, and provides new insights relevant to the investigation of the mechanisms of lymph node metastasis and cancer immunology, and the development of diagnostic and treatment methods for lymph node metastasis, including drug delivery systems. (C) 2015 Elsevier B.V. All rights reserved.

Recent basic and clinical studies have assessed the use of highly sensitive imaging modalities for visualizing transplanted islets. We investigated the utility of enhanced ultrasonography, combined with fluorescent acoustic liposome nano/microbubbles (FALs), for evaluating angiogenesis and the endocrine function of transplanted islets. BALB/c mice were classified into three groups: Diabetic mice that underwent syngeneic islet transplantation into the subrenal capsule and achieved normoglycemia (Tx group); those that failed to achieve normoglycemia (Tx-DM group); and those not receiving any treatment (DM group). Mice were examined by FAL-enhanced high frequency ultrasonography. The echogenicity of the islets increased rapidly within the first minute after injection of FALs and remained at a higher level in the Tx group, while small increases were observed in the other two groups. In histological assessments, fluorescently stained erythrocytes could be seen in and around the transplanted islets, indicating that the transplanted islets were enhanced by infusion of FALs via vessel networks between the engrafted islets and tissue. Furthermore, the echogenicity correlated significantly with endocrine parameters, including blood glucose (BG), serum insulin, and the BG change in the glucose tolerance test. In conclusion, the echogenicity of the islets under FAS-enhanced ultrasonosonography correlated with the endocrine status of transplanted islets.

Lymph node (LN) dissection is the primary option for head and neck cancer when imaging modalities and biopsy confirm metastasis to the sentinel LN. However, there are no effective alternative treatments to dissection for LN metastasis. Here, we describe a novel drug delivery system combining nano/microbubbles (NMBs) with ultrasound (US) that exhibits considerable potential for the delivery of exogenous molecules into LNs through the lymphatic vessels. A solution containing fluorophores (as a model of a therapeutic molecule) and NMBs was injected into the subiliac LNs of MXH10/Mo-lpr/lpr mice, which develop systemic swelling of LNs (up to 13 mm in diameter, similar to human LNs). It was found that the NMBs were delivered to the entire area of the proper axillary LN (proper-ALN) via the lymphatic channels and that these were retained there for more than 8 min. Furthermore, exposure to US in the presence of NMBs enhanced the delivery of fluorophores into the lymphocytes near the lymphatic channels, compared with exposure to US in the absence of NMBs. It is proposed that a system using US and NMBs to deliver therapeutic drugs via lymphatic vessels can serve as a new treatment method for LN metastasis. (C) 2015 World Federation for Ultrasound in Medicine & Biology.

latrogenic induction of regional and distant cancer metastases is a risk associated with clinical resection of tumor-positive sentinel lymph nodes. However, there have been no studies of this risk in a mouse model of cancer metastasis. Here, we report that resection of a tumor-bearing subiliac lymph node (SiLN) enhanced lung metastasis in a mouse model of lymph node metastasis. Bioluminescence imaging revealed that metastatic tumor cells in the secondary lymph node continued to grow after resection of the SiLN, and that the probability of metastasis to the lungs was increased when the interval between SiLN inoculation and resection was reduced. Futhermore, histological analysis demonstrated that latents in the lung were stimulated to grow after resection of the SiLN. Fluorescence imaging indicated that the route of tumor cell dissemination from SiLN to the lung was the venous system located over the SiLN. We speculate that our mouse model will be useful for studying the mechanisms of tumor cell latency, with a view to improving the detection and treatment of latent metastases. (C) 2015 Elsevier Inc. All rights reserved.

Direct injection of an anticancer agent into a metastatic lymph node (LN) has not been used as a standard treatment because evidence concerning the efficacy of local administration of a drug into a metastatic LN has not been established. Here we show that the combination of intralymphatic drug delivery with nano/microbubbles (NMBs) and ultrasound has the potential to improve the chemotherapeutic effect. We delivered cis-diamminedichloroplatinum (II) (CDDP) into breast carcinoma cells in vitro and found that apoptotic processes were involved in the antitumor action. Next, we investigated the antitumor effect of intralymphatic chemotherapy with NMBs and ultrasound in an experimental model of LN metastasis using MXH10/Mo-lpr/lpr mice exhibiting lymphadenopathy. The combination of intralymphatic chemotherapy with NMBs and ultrasound has the potential to improve the delivery of CDDP into target LNs without damage to the surrounding normal tissues. The present study indicates that intralymphatic drug delivery with NMBs and ultrasound will potentially be of great benefit in the clinical setting.

Curing/preventing micrometastasis to lymph nodes (LNs) located outside the surgically resected area is essential for improving the morbidity and mortality associated with breast cancer and head and neck cancer. However, no lymphatic therapy system exists that can deliver drugs to LNs located outside the dissection area. Here, we demonstrate proof of concept for a drug delivery system using MXH10/Mo-lpr/lpr mice that exhibit systemic lymphadenopathy, with some peripheral LNs being as large as 10 mm in diameter. We report that a fluorescent solution injected into the subiliac LN (defined as the upstream LN within the dissection area) was delivered successfully to the proper axillary LN (defined as the downstream LN outside the dissection area) through the lymphatic vessels. Our results suggest that this approach could be used before surgical resection to deliver drugs to downstream LNs outside the dissection area. We think that our methodology could be applied clinically, before surgical resection, to cure/prevent micrometastasis in LNs outside the dissection area.

Cancer death is mainly caused by metastasis via lymphatic or cardiovasucular system. Normally, solid tumor has a high interstitial fluid pressures (IFP) and it makes difficult to deliver drugs into the tumor region. It has been reported that IFP increases in tumor or pre-tumor regions because of tumor vessels and impaired lymphatic vessels, and high IFP may have correlation with lymph node metastasis. Although it is reported that drugs are delivered to metastatic lymph node via lymphogenous, there is no reliable methods to evaluate chemotherapy-induced treatment effect. Here we show the correlation between intermodal pressure (INP) and tumor-bearing lymph node progression / degression caused by chemotherapy by CDDP. We found that tumor-bearing lymph node was progressed with increasing INP, and chemotherapy decreased. There was no significant change in of blood vessel density in lymph node. Our results demonstrate an importance of INP for assessing tumor-bearing lymph node. We anticipate that this study contributes to develop an evaluation method of diagnosis and treatment for lymph node metastasis.

Sonoporation has the potential to deliver extraneous molecules into a target tissue non-invasively. There have been numerous investigations of cell membrane permeabilization induced by microbubbles, but very few studies have been carried out to investigate sonoporation by inertial cavitation, especially from a temporal perspective. In the present paper, we show the temporal variations in nano/micro-pit formations following the collapse of inertial cavitation bubbles, with and without Sonazoid microbubbles. Using agarose S gel as a target material, erosion experiments were conducted in the presence of 1-MHz focused ultrasound applied for various exposure times, T-ex (0.002-60 s). Conventional microscopy was used to measure temporal variations in micrometer-scale pit numbers, and atomic force microscopy utilized to detect surface roughness on a nanometer scale. The results demonstrated that nanometer-scale erosion was predominantly caused by Sonazoid (R) microbubbles and C4F10 gas bubbles for 0.002 s &lt; T-ex &lt; 1 s, while the number of micrometer-scale pits, caused mainly by inertial cavitation bubbles such as C4F10 gas bubbles and vapor bubbles, increased exponentially with increasing T-ex in the range 0.1 s &lt; T-ex &lt; 10 s. The results of the present study suggest that cavitation-induced sonoporation can produce various pore sizes in membranes, enabling the delivery of external molecules of differing sizes into cells or tissues. (C) 2014 Elsevier B.V. All rights reserved.

Curing/preventing micrometastasis to lymph nodes (LNs) located outside the surgically resected area is essential for improving the morbidity and mortality associated with breast cancer and head and neck cancer. However, no lymphatic therapy system exists that can deliver drugs to LNs located outside the dissection area. Here, we demonstrate proof of concept for a drug delivery system using MXH10/Mo-lpr/lpr mice that exhibit systemic lymphadenopathy, with some peripheral LNs being as large as 10 mm in diameter. We report that a fluorescent solution injected into the subiliac LN (defined as the upstream LN within the dissection area) was delivered successfully to the proper axillary LN (defined as the downstream LN outside the dissection area) through the lymphatic vessels. Our results suggest that this approach could be used before surgical resection to deliver drugs to downstream LNs outside the dissection area. We anticipate that our methodology could be applied clinically, before surgical resection, to cure/prevent micrometastasis in LNs outside the dissection area, using techniques such as ultrasound-guided internal jugular vein catheterization. (C) 2014 Optical Society of America

Chemotherapy based on hematogenous administration of drugs to lymph nodes (LNs) located outside the surgically resected area shows limited tissue selectivity and inadequate response rates, resulting in poor prognosis. Here, we demonstrate proof of concept for a lymphatic drug delivery system using nano/microbubbles (NMBs) and ultrasound (US) to achieve sonoporation in LNs located outside the dissection area. First, we demonstrated the in vitro effectiveness of doxorubicin (Dox) delivered into three different tumor cell lines by sonoporation. Sonoporation increased the Dox autofluorescence signal and resulted in a subsequent decrease in cell viability. Next, we verified the antitumor effects of Dox in vivo using MXH10/Mo-lpr/lpr mice that exhibit systemic lymphadenopathy, with some peripheral LNs reaching 10 mm in diameter. We defined the subiliac LN (SiLN) as the upstream LN within the dissection area, and the proper axillary LN (PALN) as the downstream LN outside the dissection area. Dox and NMBs were injected into the SiLN and delivered to the PALN via lymphatic vessels; the PALN was then exposed to US when it had filled with solution. We found that sonoporation enhanced the intracellular uptake of Dox leading to high cytotoxicity. We also found that sonoporation induced extravasation of Dox from lymphatic endothelia and penetration of Dox into tumor tissues within the PALN. Furthermore, our method inhibited tumor growth and diminished blood vessels in the PALN while avoiding systemic toxic effects of Dox. Our findings indicate that a lymphatic drug delivery system with sonoporation represents a promising method for treating metastatic LNs located outside the dissection area.

Lymph node (LN) metastasis is thought to be an important factor to determine the treatment regimen or prognosis of patients. Chemotherapy of antitumor agents is one of the candidates for LN metastasis. However, systemic administration has shown poor efficacy in LN metastasis because only a small fraction of drugs can reach the target tissues. Here we show that local administration of antitumor agent, Doxorubicin (Dox), through lymphatic vessel (LV) with nano/micro bubbles (NMBs) and ultrasound (US) exposure enhances antitumor efficacy. We found that Dox and NMBs could reach the tumor-bearing LN through LV, and that US exposure to the LN enhanced the delivery of Dox into the tumor cells. Furthermore, we found that administration into the LV reduced the side effects compared to systemic administration. Our results demonstrate that lymphatic administration through LV and US exposure to the LN with tumor is a more valid treatment method than systemic administration.

Intravenous chemotherapy is a therapeutic option for the treatment of lymph node metastasis, but the drugs often have difficulty accessing the lymphatic system. The aim of this study was to determine whether the combination of intralymphatic chemotherapy with ultrasound and nano-/microbubbles is active against tumors in mouse lymph nodes. Intralymphatic chemotherapy in mice with lymph nodes containing tumors was found to have a marked anti-tumor effect, compared with intravenous administration, and the addition of ultrasound combined with nano-/microbubbles enhanced the effect of the anti-cancer drug, but only when the drug was administered intralymphatically. Furthermore, decreases in the volumes and blood vessel densities of tumor-bearing lymph nodes are reliable measures of therapeutic effect, confirmed by histopathological evaluation. The main conclusion is that combining ultrasound with nano-/microbubbles and intralymphatic chemotherapy improves drug delivery to the lymphatic system and has a more potent anti-tumor effect. (E-mail: kodama@bme.tohoku.ac.jp) (C) 2014 World Federation for Ultrasound in Medicine & Biology.

This paper proposes a method for detecting contrast agents in ultrasound image sequences to develop diagnostic ultrasound imaging systems for tumor diagnosis. The conventional methods are based on simple subtraction of ultrasound images to detect ultrasound contrast agents, where the conventional methods need ultrasound image sequences with and without contrast agents. Even if the subject slightly moves, the detection result of the conventional methods includes significant errors. On the other hand, the proposed method employs the spatio-temporal analysis of the pixel intensity variation over several frames. The proposed method also employs motion estimation to select optimal image frames for detecting contrast agents. Through a set of experiments using mice, we demonstrate that the proposed method exhibits efficient performance compared with the conventional methods.

Lymph node dissection for regional nodal metastasis is a primary option, but is invasive and associated with adverse effects. The development of non-invasive therapeutic methods in preclinical experiments using mice has been restricted by the small lymph node size and the limited techniques available for non-invasive monitoring of lymph node metastasis. Here, we show that photothermal therapy (PTT) using gold nanorods (GNRs) and near-infrared (NIR) laser light shows potential as a non-invasive treatment for tumors in the proper axillary lymph nodes (proper-ALNs) of MXH10/Mo-lpr/lpr mice, which develop systemic swelling of lymph nodes (up to 13 mm in diameter, similar in size to human lymph nodes). Tumor cells were inoculated into the proper-ALNs to develop a model of metastatic lesions, and any anti-tumor effects of therapy were assessed. We found that GNRs accumulated in the tumor in the proper-ALNs 24 h after tail vein injection, and that irradiation with NIR laser light elevated tumor temperature. Furthermore, combining local or systemic delivery of GNRs with NIR irradiation suppressed tumor growth more than irradiation alone. We propose that PTT with GNRs and NIR laser light can serve as a new therapeutic method for lymph node metastasis, as an alternative to lymph node dissection. (C) 2013 Elsevier B.V. All rights reserved.

Rationale: Appropriate ischemia models are required for successful studies of therapeutic angiogenesis. While collateral routes are known to be present within the innate vasculature, there are no reports describing the detailed vascular anatomy of the murine hindlimb. In addition, differences in the descriptions of anatomical names and locations in the literature impede understanding of the circulation and the design of hindlimb ischemia models. To understand better the collateral circulation in the whole hindlimb, clarification of all the feeding arteries of the hindlimb is required. Objective: The aim of this study is to reveal the detailed arterial anatomy and collateral routes in murine hindlimb to enable the appropriate design of therapeutic angiogenesis studies and to facilitate understanding of the circulation in ischemia models. Methods and Results: Arterial anatomy in the murine hindlimb was investigated by contrast-enhanced X-ray imaging and surgical dissection. The observed anatomy is shown in photographic images and in a schema. Previously unnoticed but relatively large arteries were observed in deep, cranial and lateral parts of the thigh. The data indicates that there are three collateral routes through the medial thigh, quadriceps femoris, and the biceps femoris muscles. Furthermore, anatomical variations were found at the origins of the three feeding arteries. Conclusions: The detailed arterial anatomy of murine hindlimb and collateral routes deduced from the anatomy are described. Limitations on designs of ischemia models in view of anatomical variations are proposed. These observations will contribute to the development of animal studies of therapeutic angiogenesis using murine hindlimb ischemia models.

Monitoring angiogenesis is potentially an effective strategy for the early detection of cancer. In this study, early detection was achieved by evaluating blood vessel density in the liver using a three-dimensional contrast-enhanced high-frequency ultrasound (CE-HFUS) system and Sonazoid microbubbles. Three-dimensional CE-HFUS detected an increase in blood vessel density in the liver after intrasplenic injection of breast tumor cells into mice. The results were in agreement with immunohistochemical analysis of blood vessel density. Three-dimensional CE-HFUS using microbubbles is an attractive, novel approach for the early detection of liver metastases through quantification of new, pathological vascular growth (i.e. tumor angiogenesis). (C) 2013 Elsevier Ireland Ltd. All rights reserved.

While islet transplantation is considered a useful therapeutic option for severe diabetes mellitus (DM), the outcome of this treatment remains unsatisfactory. This is largely due to the damage and loss of islets in the early transplant stage. Thus, it is important to monitor the condition of the transplanted islets, so that a treatment can be selected to rescue the islets from damage if needed. Recently, numerous trials have been performed to investigate the efficacy of different imaging modalities for visualizing transplanted islets. Positron emission tomography (PET) and magnetic resonance imaging (MRI) are the most commonly used imaging modalities for this purpose. Some groups, including ours, have also tried to visualize transplanted islets by ultrasonography (US). In this review article, we discuss the recent progress in islet imaging. © 2013 Landes Bioscience.

Reactive oxygen species (ROS) act as intracellular signaling molecules in the regulation of receptor activator of nuclear factor-kappa B ligand (RANKL)-dependent osteoclast differentiation, but they also have cytotoxic effects that include peroxidation of lipids and oxidative damage to proteins and DNA. Cellular protective mechanisms against oxidative stress include transcriptional control of cytoprotective enzymes by the transcription factor, nuclear factor E2-related factor 2 (Nrf2). This study investigated the relationship between Nrf2 and osteoclastogenesis. Stimulation of osteoclast precursors (mouse primary peritoneal macrophages and RAW 264.7 cells) with RANKL resulted in the up-regulation of kelch-like ECH-associated protein 1 (Keap1), a negative regulator of Nrf2. It also decreased the Nrf2/Keap1 ratio, and it down-regulated cytoprotective enzymes (heme oxygenase-1, gamma-glutamylcysteine synthetase, and glucose-6-phosphate dehydrogenase). Nrf2 overexpression up-regulated the expression of cytoprotective enzymes, decreased ROS levels, decreased the number of tartrate-resistant acid phosphatase-positive multinucleated cells, reduced marker genes for osteoclast differentiation, and attenuated bone destruction in both in vitro and in vivo models. Overexpression of Keap or RNAi knockdown of Nrf2 exerted the opposite actions. In addition, in vivo local Nrf2 overexpression attenuated lipopolysaccharide-mediated RANKL-dependent cranial bone destruction in vivo. This is the first study to show that the Keap1/Nrf2 axis regulates RANKL-dependent osteoclastogenesis through modulation of intracellular ROS signaling via expression of cytoprotective enzymes. This raises the exciting possibility that the Keap1-Nrf2 axis may be a therapeutic target for the treatment of bone destructive disease.

Lymph node size is an important variable in ultrasound diagnosis of lymph node metastasis. However, the size criterion often leads to oversight of tumor-positive lymph nodes within the range of "normal" size, such that more accurate diagnostic criteria for lymph node metastasis are required. In this study, we show how diagnosis of lymph node metastasis can be improved by evaluating changes in blood vessel volume and density using a novel contrast-enhanced high-frequency ultrasound (CE-HFUS) system with Sonazoid. An MRL/MpJ-lpr/lpr (MRL/lpr) mouse model of lymph node metastasis was used in which lymph nodes are similar in size to humans. Metastasis via lymphatic vessels to proper axillary lymph nodes (proper ALN) was induced by injection of tumor cells into the subiliac lymph nodes. Within 21 days of injection, significant increases in blood vessel volume and density, but no increases in the size of the proper ALNs, were observed. The increase in blood vessel density was confirmed with immunohistochemical analysis and was positively related to tumor cell proliferation as measured using bioluminescence imaging. Together, our results showed that alterations in blood vessel volume and density precede alterations in lymph node size in the early stages of lymph node metastasis. Detection of these changes by ultrasonography may offer new criteria for early diagnosis of lymph node metastasis. © 2012 American Association for Cancer Research.

Preclinical models of lymph node (LN) metastasis are fundamental to the study and design of new techniques for the diagnosis and treatment of LN metastasis. However, the identification of LNs and lymphatic vessels (LVs) in mice is challenging with conventional imaging modalities, since the LN diameter in normal mice is 1-2 mm. Here, we describe MXH10/ Mo-lpr/lpr (MXH10/Mo/lpr) inbred mice, which develop systemic swelling of LNs up to 10 mm in diameter, allowing investigation of the topography of LNs and LVs. Using a gross anatomy dissection approach, we identified 22 different LNs situated in the head and neck, limbs, thoracic and abdominal regions. Furthermore, four peripheral inter-LN vessels were found: from the subiliac LN (SiLN) to the proper axillary LN (PALN); from the parotid LN to the caudal deep cervical LN; and from the popliteal LN to both the sciatic LN and the SiLN. Metastasis to the PALN via LVs was induced by inoculating FM3A/Luc mouse mammary carcinoma cells into the SiLN. Our results demonstrate that the MXH10/Mo/lpr mouse strain is an excellent model in which to investigate lymphatic drainage and inter-LN metastasis of cancer. This paper unveils the anatomy of murine lymphatics to give new insights into the investigation of inter-LN metastasis of cancer, especially the mechanisms involved in the trafficking of cancer cells through inter-LN vessels. The results provide data that may prove very useful in the quest to develop better lymph drainage-based drug delivery systems. (C) 2013 Elsevier B.V. All rights reserved.

Animal studies of lymph node metastasis are constrained by limitations in the techniques available for noninvasive monitoring of the progression of lymph node metastasis, as well as difficulties in the establishment of appropriate animal models. To overcome these challenges, this study has developed a mouse model of inter-lymph-node metastasis via afferent lymphatic vessels for use in the development of imaging modalities. We used 14- to 18-week-old MRL/MpJ-/lpr/lpr (MRL/lpr) mice exhibiting remarkable systemic lymphadenopathy, with proper axillary lymph nodes (proper-ALNs) and subiliac lymph nodes (SiLNs) that are 6 to 12 mm in diameter (similar in size to human lymph nodes). When KM-Luc/GFP malignant fibrous histiocytoma-like cells stably expressing the firefly luciferase gene were injected into the SiLN, metastasis could be detected in the proper-ALN within 3 to 9 days, using in vivo bioluminescence imaging. The metastasis route was found to be via the efferent lymphatic vessels of the SiLN, and metastasis incidence depended on the number of cells injected, the injection duration and the SiLN volume. Three-dimensional contrast-enhanced high-frequency ultrasound imaging showed that the blood vessel volume and density in the metastasized proper-ALN significantly increased at 14 days after tumor cell inoculation into the SiLN. The present metastasis model, with lymph nodes similar in size to those of humans, has potential use in the development of ultrasound imaging with high-precision and high-sensitivity as well as other imaging modalities for the detection of blood vessels in lymph nodes during the progression of metastasis. © 2013 Li et al.

The present study was to understand the true power irradiated to the cell line cultured on a culture well, in relation to the nominal power from ultrasonic transducer, and to characterize the temporal variations of the acoustic pressure exerted on the cell. Numerical simulation was carried out for a typical culture well exposed to 1 MHz continuous ultrasound generated by a circular transducer contact underneath the well. The results showed that the ultrasonic pressure exposed to the cell layer in the well was 6.7 times larger than the nominal pressure of the ultrasonic transducer. The ultrasonic pressure in the transient period rose rapidly and was widely variable, and the temporal peak was even greater than that of the steady state period. This suggests that the cells undergo characteristically different ultrasonic exposure between the transient and the steady state period. © 2013 IEEE.

Ultrasound contrast agents (UCAs) are nano/microbubbles that contain air or a high-molecular-weight, low-solubility gas encapsulated in a lipid or albumin shell. Previous studies have developed acoustic liposomes (ALs), liposomes that encapsulate perfluoropropane (C3F8) gas. These ALs can be used as just UCAs, for early diagnostic or observation of angiogenesis. They can also be used for drug delivery, through their ultrasound-induced destruction leading to permeabilization of the neighboring cells. However, the echogenicity of ALs decreases within minutes, raising the need for more stable preparations. Here we show that the in vitro stability of ALs is affected by fluidity changes in the bilayer, the presence of anionic phospholipids and the density of the PEG coating layer. These results allowed the preparation of "optimized" ALs displaying a 50% enhanced detection time in vitro. We anticipate their stability to be enhanced in a similar manner, in vivo. Further research aims at further improvement of the stability of gas encapsulation by surface modification and coating of the liposomes, and in vivo characterization of the optimized ALs.

The antitumor effects of tumor necrosis factor (TNF-alpha) were evaluated following transfection of TNF-alpha plasmid DNA into solid mouse tumors using the nanobubbles (NBs) and ultrasound (US) gene delivery system. Murine breast carcinoma (EMT6) cells expressing luciferase (1 x 10(6) cells) were injected intradermally into the flanks of 6-7-week-old male SCID mice on day 0. Ten microliters of TNF-alpha (5 mu g/mu L) or TNF-alpha mock plasmid DNA (5 mu g/mu L) with/without NBs (15 mu L) and saline was injected intratumorally in a total volume of 30 mu L, and tumors were exposed to US (frequency, 1 MHz; intensity, 3.0 W/cm(2); duty cycle, 20%; number of pulses, 200; and exposure time, 60 s) on days 2, 4, 7, and 9. Changes in tumor size were measured with an in vivo bioluminescent imaging system and a mechanical caliper. Changes in tumor vessel area were quantified using contrast-enhanced US imaging with Sonazoid and a high frequency US imaging system (40 MHz) and immunohistochemistry (CD31). At the mRNA level, expression of TNF-alpha, caspase-3, and p53 were quantified using real-time quantitative RT-PCR. At the protein level, expression of caspase-3 and p53 were confirmed by immunohistochemistry. We show that repeated TNF-a gene delivery using NBs and US can lead to the local production of TNF-alpha. This results in antitumor effects, including activation of p53-dependent apoptosis, decrease in tumor vessel density, and suppression of tumor size. In this study, we showed the effectiveness of using NBs and US for TNF-alpha gene delivery into tumor cells. (Cancer Sci 2011; 102: 2082-2089)

Acoustic liposomes (AL) have their inherent echogenicity and can add functionality in serving as drug carriers with tissue specificity. Nonuniform vascular structures and vascular branches/bends are evaluated by imaging the intravascular movement locus of ALs with high-frequency ultrasound (HF-US) imaging. However, the evaluation of antitumor effects on angiogenesis by ALs and HF-US imaging has not been reported. Here, we show that the combination of ALs and an HF-US imaging system is capable of noninvasively evaluating antitumor volumetric and angiogenic effects in preclinical mouse models of various cancers. In this study, the antitumor effects of cisplatin on tumor growth and angiogenesis in mice bearing two different types of tumor cells were assessed. By tracking each AL flowing in the vessel and transferring the images to personal computers, microvessel structures were mapped and reconstructed using the color difference based on SD method. The antitumor effects were confirmed with an in vivo bioluminescence imaging system and immunohistochemical analysis. Our results show that cisplatin inhibits tumor growth by decreasing intratumoral vessel area but does not affect the angiogenesis ratio in the tumor. The vascular occupancy in the outer region of the tumor was larger than that in the inner region; however, both occupancies were similar to those of the control tumor. We propose that this method of mapping microvessels with ALs and an HF-US system can serve as a new molecular imaging method for the assessment of angiogenesis and can be applied to evaluate the antitumor effects by various therapeutic agents. Cancer Res; 71(22); 6957-64. (C)2011 AACR.

In the case of extracorporeal shock wave lithotripsy (ESWL), a shock wave-bubble interaction inevitably occurs near the focusing point of stones, resulting in stone fragmentation and subsequent tissue damage. Because shock wave-bubble interactions are high-speed phenomena occurring in tissue consisting of various media with different acoustic impedance values, numerical analysis is an effective method for elucidating the mechanism of these interactions. However, the mechanism has not been examined in detail because, at present, numerical simulations capable of incorporating the acoustic impedance of various tissues do not exist. Here, we show that the improved ghost fluid method (IGFM) can treat shock wave-bubble interactions in various media. Nonspherical bubble collapse near a rigid or soft tissue boundary (stone, liver, gelatin and fat) was analyzed. The reflection wave of an incident shock wave at a tissue boundary was the primary cause for the acceleration or deceleration of bubble collapse. The impulse that was obtained from the temporal evolution of pressure created by the bubble collapse increased the downward velocity of the boundary and caused subsequent boundary deformation. Results of this study showed that the IGFM is a useful method for analyzing the shock wave-bubble interaction near various tissues with different acoustic impedance.

Islet transplantation is a cell replacement therapy to improve glycometabolic control in type 1 diabetic patients. Establishing methods to monitor engrafted islets, as well as the islet preparation, is important when performing islet transplantation. Since current imaging techniques are still not available to directly detect transplanted islets, we propose a novel method to visualize transplanted islets using high-frequency ultrasound (HF-US), and to evaluate the correlation between these US findings and metabolic parameters. We transplanted syngeneic (BALB/c mice) and xenogeneic (SD rats) islets into the renal subcapsular space of diabetic mice. The recipient mice were examined by HF-US until post-operative day (POD) 28 and, while syngeneic islets could be detected by HF-US throughout the observational period, the xenogeneic islets had vanished by POD 28. The islet volume calculated by HF-US was correlated with the number of transplanted islets (R-2 = 0.31, p = 0.0003) and the metabolic function of islets (blood glucose: R-2 = 0.15, p &lt; 0.0001, serum insulin: R-2 = 0.22, p &lt; 0.0001). In conclusion, HF-US may be a useful imaging modality for visualizing the islet mass in renal subcapsular transplantation models. It may also be an available modality for clinical settings in the future.

A challenge in the field of nanobubbles, including lipobubbles and polymeric nanobubbles, is identification of formulation approaches to enhance circulation time or "bubble life" in the specific organ to allow for organ visualization. The aim of this study was to investigate the potential of two specific preparation approaches, polymeric surface modification to lipobubbles and a one-step approach for the preparation of ionotropically originated polymeric hydrogel nanobubbles for the production of biocompatible, biodegradable, and sufficiently echogenic (&apos;flexible&apos;) bubbles, preferably within the nanometer range, that possess an enhanced in vivo lifetime compared to an unmodified lipobubble to allow visualization of the lymph node vasculature. In the first approach, formed liposomes (basic and polymerically enhanced) were sequentially layered with appropriate cationic and anionic polyelectrolytes followed by transformation into polymer-coated nanobubbles. In addition, a one-step approach was employed for the fabrication of ionotropically originated polymeric hydrogel bubbles. Bubble lifetime was marginally enhanced by self-deposition of polyelectrolytes onto the normal lipobubble, however, not significantly (P = 0.0634). In general, formulations possessing a higher ratio of anionic:cationic coats and highly anionic overall surface charge (-20.62 mV to -17.54 mV) possessed an enhanced lifetime. The improvement in bubble lifetime was significant when a purely polymeric polyionic hydrogel bubble shell was instituted compared to a normal unmodified lipobubble (P = 0.004). There was enhanced persistence of these systems compared to lipobubbles, attributed to the highly flexible, interconnected hydrogel shell which minimized gas leakage. The prolonged contrast signal may also be attributed to a degree of polymeric deposition/endothelial attachment. This study identified the relevance of polymeric modifications to nanobubbles for an improved circulating lifetime, which would be essential for application of these systems in passive drug or gene targeting via the enhanced permeability and retention effect.

A dual-intensity ultrasound system (DIUS) using nanobubbles offers opportunities for localized gene delivery. This system consists of low-/high-ultrasound intensities. The bladder is a balloon-shaped closed organ in which the behavior of nanobubbles can be controlled spatially and temporally by ultrasound exposure. We hypothesized that when a DIUS with nanobubbles was used, low-intensity ultrasound would direct nanobubbles to targeted cells in the bladder, whereas high-intensity ultrasound intensity would collapse nanobubbles and increase cell membrane permeability, facilitating entry of exogenous molecules into proximate cells. A high-frequency ultrasound imaging system characterized movement and fragmentation of nanobubbles in the bladder. Confocal microscopy revealed that fluorescent molecules were delivered in the localized bladder wall, whereas histochemical examination indicated that the molecular transfer efficiency depended on the acoustic energy. A bioluminescence imaging system showed luciferase plasmid DNA was actually transfected in the bladder wall and subsequent transfection depended on acoustic energy. These findings indicate that delivery of exogenous molecules in the bladder using this approach results in high localization of molecular delivery, facilitating gene therapy for bladder cancer. (E-mail: kodama@bme.tohoku.ac.jp) (C) 2010 World Federation for Ultrasound in Medicine & Biology.

Background: We investigated the effectiveness of ultrasound-mediated destruction of bubble liposome (UBL) for siRNA transfer by observing reduction in the luciferase activity of human bladder tumor RT-112 cells transfected with the luciferase gene (RT-112Luc) following luciferase siRNA transfer into the cells. Results: siRNA was transferred to 26% of RT-112Luc cells by UBL and the luciferase activity of RT-112Luc cells was significantly suppressed by UBL using the luciferase siRNA, compared with that using nonspecific siRNA in vitro (p = 0.036). The luciferase activity of RT-112Luc tumor was suppressed by UBL using luciferase siRNA compared with that using nonspecific siRNA 2 days after the in vivo treatment. Conclusion: This study showed that UBL is suitable for siRNA transfer to mammalian cells. © 2010 Future Science Ltd.

We demonstrate the self-organization process of a stable pore structure in a phospholipid bilayer by unsteady and nonequilibrium molecular dynamics simulations. The simulation is started from an initial state including some amount of water molecules in its hydrophobic region, which is a model of a cell membrane stimulated by ultrasound radiation for the membrane permeabilization (sonoporation). We show that, in several nanoseconds, the bilayer-water system can spontaneously develop into a water-filled pore structure without any mechanical and electrical forcing from outside, when the initial number of water molecules in the hydrophobic region exceeds a critical value. The increase in the initial number of water molecules enhances the probability of pore formation, and sometimes induces the formation of transient micellelike structures of phospholipid molecules.

Recent studies have revealed that ultrasound contrast agents with low-intensity ultrasound, namely, sonoporation, can noninvasively deliver therapeutic molecules into target sites. However, the efficiency of molecular delivery is relatively low and the methodology requires optimization. Here, we investigated three types of nano/microbubbles (NMBs)-human albumin shell bubbles, lipid bubbles and acoustic liposomes-to evaluate the efficiency of gene expression in skeletal muscle as a function of their physicochemical properties and the number of bubbles in solution. We found that acoustic liposomes showed the highest transfection and gene expression efficiency among the three types of NMBs under ultrasound-optimized conditions. Liposome transfection efficiency increased with bubble volume concentration; however, neither bubble volume concentration nor their physicochemical properties were related to the tissue damage detected in the skeletal muscle, which was primarily caused by needle injection. (E-mail: kodama@bme.tohoku.ac.jp) (C) 2010 World Federation for Ultrasound in Medicine & Biology.

Sonoporation is achieved by ultrasound-mediated destruction of ultrasound contrast agents (UCA) microbubbles. For this, UCAs must be tissue specific and have good echogenicity and also function as drug carriers. Previous studies have developed acoustic liposomes (ALs), liposomes that encapsulate phosphate buffer solution and perfluoropropane (C(3)F(8)) gas and function as both UCAs and drug carriers. Few studies have examined the co-existence of gas and liquid in ALs. This study aims to elucidate AL structure using TEM. The size, zeta potential and structure of ALs were compared with those of two other UCAs, human albumin shell bubbles (ABs; Optison) and lipid bubbles (LBs). ABs and LBs encapsulate the C(3)F(8) gas. Particle size was measured by dynamic light scattering. The zeta potential was determined by the Smoluchowski equation. UCA structure was investigated by TEM. ALs were similar to 200 nm in size, smaller than LBs and ABs. ALs and LBs had almost neutral zeta potentials whereas AB values were strongly negative. The negative or double staining TEM images revealed that similar to 20% of ALs contained both liquid and gas, while similar to 80% contained liquid alone (i.e. nonacoustic). Negative staining AB images indicated electron beam scattering near the shell surface, and albumin was detected in filament form. These findings suggest that AL is capable of carrying drugs and high-molecular-weight, low-solubility gases.

Microbubble destruction and the induced cavitation bubble behavior are regarded as key phenomena affecting the enhancement of sonoporation. In the present study an experiment was carried out for investigating the interaction of ultrasound waves with microbubbles. Test liquid was a suspension of Sonazoid ultrasound contrast agent diluted with distilled water with the volumetric concentration of 10 % [v/v], which was set in a cylindrical vessel, i.e. one of a 24 well-plate, conventionally used in <i>in vitro</i> experiments and irradiated by ultrasound waves with the frequency of 1 MHz. It was found that microbubbles were rapidly destroyed and the survival number was reduced to half the original number within 100ms for all the liquid depths examined here. A maximum number of cavitation bubbles was achieved at a certain exposure time less than one second where more than sixty cavitation bubbles were generated, subsequently behaving in the observation volume (= 14 mm³). Although almost the microbubbles were crushed within one second, we occasionally observed a special situation where plenty of cavitation bubbles happened at the exposure time of five seconds due to the circulation flow generated by the acoustic radiation pressure coupled with the flow induced by the free surface fluctuation.

For destroying Sonazoid microbubbles 1 MHz - ultrasound waves was irradiated by employing a concave ultrasound probe with the vibrating diameter of 40 mm and the arithmetic focal length of 80 mm. The probe was set in distilled water at temperature of 25 ℃. A suspension of Sonazoid ultrasound contrast agent diluted with distilled water with the volumetric concentration of 5 % [v/v] was shut in a cuvette which was positioned at x_<c,b> measured from the bottom of the ultrasound probe. It was found that Sonazoid microbubbles were rapidly destroyed and the survival number was reduced to 40 % of the original number at the exposure time of 100 ms. When a cuvette containing suspension of Sonazoid microbubbles was set at the arithmetic focal point, a limited volume of Sonazoid suspension was exposed to the ultrasound waves, resulting in a relatively larger survival number of Sonazoid microbubbles. Sonazoid destruction was observed by taking snapshots using a nano-pulse light with the pulse duration of 180 ns as a light source.

Three-dimensional high-frequency ultrasound (HF-US) combined with nanobubbles (NBs) permit detection of vessels in tumor. Description of the vessel density in tumors has recently been used for differentiating benign from malignant disease, but it was rarely reported in detection of liver metastases. In this study, we aimed to evaluate the possibility of detecting liver metastases at a minimum size, by evaluation the vessel density in the preclinical liver metastatic models with three-dimensional HF-US and NBs longitudinally. Tumor cells with luciferase-expressing were injected into the spleen slowly to establish the liver metastases models. Liver metastases were confirmed by luciferase activity located in the mouse liver area which was measured by in vivo bioluminescence system (IVIS). Ultrasound imaging on mice livers was performed by a high-frequency ultrasound system after NBs were administered from the tail vein. Vessels in the liver were constructed and the vessel density in the whole liver was calculated by the contrast mode soft. Contrast ultrasound images showed the change of vessels in normal and metastatic parts of liver with time. Vessel density could used to compare the angiogenesis in the metastatic mouse liver with the normal livers. These results demonstrated a system of HF-US combined with NBs could be useful for detection of liver metastases at a minimum size by evaluation the vessel density.

Newly formed tumor vasculature is usually abnormally leaky due to the extensive angiogenesis induced by the growing tumor, which enables extravasation of particles with a diameter of less than 200 nm: this phenomenon is recognized as the enhanced permeability and retention (EPR) effect. Due to their versatility, liposomes are potential candidates to take advantage of the EPR effect and passively target tumor cells. In this study, liposomes encapsulating fluorescent dye or bearing covalently-bound fluorophores on their surface were prepared and characterized in vitro. DLS and fluorescence measurements respectively showed that they currently display a diameter of less than 200nm and a fluorescence intensity similar to that of commercially available agents. Further research aims at an in vivo observation of the EPR effect through real-time visualization of the accumulation of such liposomes.

A non-viral gene delivery approach with nano/microbubbles and ultrasound offers opportunities for targeting soft tissues for gene therapy. The periodontium is a complex structure comprised of hard (cementum, alveolar bone) and soft tissues (periodontal ligament, gingivae). We hypothesized that our established gene delivery method would allow the periodontal tissue to be targeted for transfection for gene therapy. Expression kinetics and sites of transfection sites with this approach were investigated in rat periodontal tissue. Bioluminescence imaging revealed that transient gene expression was induced at day 1 post-transfection, while confocal microscopy showed that gene expression was localized in the muscle cells of gingival tissues. These findings indicate that regular transfection with this approach results in high gene expression, facilitating gene therapy for periodontal disease involving alveolar bone resorption.

In dendritic cell (DC)-based cancer immunotherapy, it is important that DCs present peptides derived from tumor-associated antigens on MHC class I. and activate tumor-specific cytotoxic T lymphocytes (CTLs). However, MHC class I generally present endogenous antigens expressed in the cytosol. We therefore developed an innovative approach capable of directly delivering exogenous antigens into the cytosol of DCs: i.e., a MHC class I-presenting pathway. In this study, we investigated the effect of antigen delivery using perfluoropropane gas-entrapping liposomes (Bubble liposomes, BLs) and ultrasound (US) exposure on MHC class I presentation levels in DCs, as well as the feasibility of using this antigen delivery system in DC-based cancer immunotherapy. DCs were treated with ovalbumin (OVA) as a model antigen, BLs and US exposure. OVA was directly delivered into the cytosol but not via the endocytosis pathway, and OVA-derived peptides were presented on MHC class I. This result indicates that exogenous antigens can be recognized as endogenous antigens when delivered into the cytosol. Immunization with DCs treated with OVA, BLs and US exposure efficiently induced OVA-specific CTLs and resulted in the complete rejection of E.G7-OVA tumors. These data indicate that the combination of BLs and US exposure is a promising antigen delivery system in DC-based cancer immunotherapy. (C) 2008 Elsevier B.V. All rights reserved.

Preclinical mouse models are essential to the study of liver metastasis, yet their utility has been limited by difficulty in tracking the progression of metastases through time. In this study, we attempted to applicate some non-invasive methods to study the murine liver metastasis. Livers of mice were observed by camera system termed photo dynamic eye (PDE) with the indocianine green (ICG) injected into the tail vein. Mice received and intrasplenic injection of luciferase-expressing tumor cells. Luciferase activity was measured by in vivo bioluminescence system (IVIS). Ultrasound imaging was performed by a high-frequency ultrasound system (VEVO), after nanobubbles were administered from the tail vein. As the result, murine liver metastases were identified using PDE and IVIS. Two-dimensional and three dimensional images by ultrasound and nanobubbles showed vessels in the liver. These results could be benefit for early diagnosis of liver metastases with a minimum detection size in preclinical model.

Seventy percent of patients with bladder cancer present superficial tumors with high recurrence rate. In order to develop effective superficial bladder cancer gene therapy, effective gene delivery using nanobubbles (NBs) and ultrasound (US) is our ultimate goal. To achieve this goal, we propose localized gene delivery in bladder using NBs and low-high-intensity US (LHUS) while low-intensity US direct NBs proximate to targeted cells in the bladder and high-intensity US collapse NBs to increase cell membrane permeability resulting the entry of exogenouse molecules into proximate cells. In the present study, to demonstrate the effectiveness of NBs and LHUS to localize the gene delivery to a designated bladder wall, we have assessed: i) in vivo local delivery and fragmentation of NBs with high frequency US imaging system; ii) localized molecular delivery of fluorescent molecules and quantification of the transfection efficacy; and iii) transfection of plasmid DNA and quantification of the transfection efficacy.

Molecular delivery using ultrasound (US) and nano/microbubbles (NBs), i.e., sonoporation, has applications in gene therapy and anticancer drug delivery. When NBs are destructed by ultrasound, the surrounding cells are exposed to mechanical impulsive forces generated by collapse of either the NBs or the cavitation bubbles created by the collapse of NBs. In the present study, experimental, theoretical and numerical analyses were performed to investigate cavitation bubbles mediated molecular delivery during sonoporation. Experimental observation using lipid NBs indicated that increasing US pressure increased uptake of fluorescent molecules, calcein (molecular weight: 622), into 293T human, and decreased survival fraction. Confocal microscopy revealed that calcein molecules were uniformly distributed throughout the some treated cells. Next, the cavitation bubble behavior was analyzed theoretically based on a spherical gas bubble dynamics. The impulse of the shock wave (i.e., the pressure integrated over time) generated by the collapse of a cavitation bubble was a dominant factor for exogenous molecules to enter into the cell membrane rather than bubble expansion. Molecular dynamics simulation revealed that the number of exogenous molecules delivered into the cell membrane increased with increasing the shock wave impulse. We concluded that the impulse of the shock wave generated by cavitation bubbles was one of important parameters for causing exogenous molecular uptake into living cells during sonoporation.

Cell permeabilization using microbubbles (MB) and low-intensity ultrasound (US) have the potential for delivering molecules into the cytoplasm. The collapsing MB and cavitation bubbles created by this collapse generate impulsive pressures that cause transient membrane permeability, allowing exogenous molecules to enter the cells. To evaluate this methodology in vitro and in vivo, we investigated the effects of low-intensity 1-MHz pulsed US and MB combined with cis-diamminedichloroplatinum (II) (CDDP) on two cell lines (Colon 26 murine colon carcinoma and EMT6 murine mammary carcinoma) in vitro and in vivo on severe combined immunodeficient mice inoculated with HT29-luc human colon carcinoma. To investigate in vitro the efficiency of molecular delivery by the US and MB method, calcein molecules with a molecular weight in the same range as that of CDDP were used as fluorescent markers. Fluorescence measurement revealed that approximately 10(6)-10(7) calcein molecules per cell were internalized. US-MB-mediated delivery of CDDP in Colon 26 and EMT6 cells increased cytotoxicity in a dose-dependent manner and induced apoptosis (nuclear condensation and fragmentation, and increase in caspase-3 activity). In vivo experiments with xenografts (HT29-luc) revealed a very significant reduction in tumor volume in mice treated with CDDP + US + MB compared with those in the US + CDDP groups for two different concentrations of CDDP. This finding suggests that the US-MB method combined with chemotherapy has clinical potential in cancer therapy. (Cancer Sci 2008; 99: 2525-2531).

A non-invasive molecular delivery method using nano/microbubbles (NB) and ultrasound (US) is an attractive technique for gene therapy. This method is able to deliver macromolecules such as plasmid DNA to a tissue specific site non-invasively. In addition, a non-invasive quantification method of gene expression has been studied by monitoring ^<124> accumulation caused by the of Na/I symporter (NIS) gene expression with positron emission tomography (PET). In the present study, we delivered NIS gene into the skeletal muscle of vasculitis and muscular dystrophy mice by using the US/NB method, and succeed in visualizing the ^<124>I accumulation caused by NIS gene expression in the muscle with PET. The combination of the US/NB delivery with NIS/PET visualization will be a valuable methodology in gene therapy.

Unsteady and nonequilibrium molecular dynamics simulations of the response of dipalmitoylphosphatidylcholine (DPPC) bilayers; to the shock waves of various incident angles are presented. The action of an incident shock wave is modeled by adding a momentum in an oblique direction to water molecules adjacent to a bilayer. We thereby elucidate the effects of incident shock angles on (i) collapse and rebound of the bilayer, (ii) lateral displacement of headgroups, (iii) tilts of lipid molecules, (iv) water penetration into the hydrophobic region of the bilayer, and (v) momentum transfer across the bilayer. The number of water molecules delivered into the hydrophobic region is found to be insensitive to incident shock angles. The most important structural changes are the lateral displacement of headgroups and tilts of lipid molecules, which are observed only in the half of the bilayer directly exposed to a shock wave for all incident shock angles studied here. As a result, only the normal component of the added oblique momentum is substantially transferred across the bilayer. This also suggests that the irradiation by shock waves may induce a jet-like streaming of the cytoplasm toward the nucleus. (C) 2008 Elsevier B.V. All rights reserved.

A physical method using ultrasound (US) and nano/microbubbles (NBs) can deliver exogenous molecules noninvasively into a specific target site. In this study, we evaluated the application of this technology to cancer gene therapy using prodrug activation therapy. Low-intensity pulsed ultrasound (1 MHz; 1.3 W/cm(2)) and NBs were used to transduce the herpes simplex thymidine kinase (HSVtk) gene in vitro, leading to gene transfer. The addition of ganciclovir (GCV) to the transduced cells led to HSVtk/GCV-dependent cell death mediated by apoptosis. This technology was then assessed in vivo, using mice bearing subcutaneous tumors (1 MHz; 3.0 W/cm(2)). Gene transfer to the tumor, measured by luciferase activity, was transient, with a peak of expression 24 h after transduction, and decreased at 48 h, demonstrating the transient nature of US/NB-mediated gene transfer. The therapeutic potential of this approach was evaluated through repeated intratumoral gene delivery using US/NB-mediated transfer of the HSVtk gene, followed by recurrent administration of GCV, using two different experimental treatment protocols. In both cases, dramatic reductions of the tumor size by a factor of four were observed. Altogether, these data demonstrate the potential of US/NB as a new physical gene delivery method for cancer gene therapy.

Recently, we reported an animal semiconductor PET with the spatial resolution of 0.8mm FWHM within the central 20mm-diameter of FOV for the purpose of biomedical study using rats and mice. This ultra high spatial resolution was obtained by the use of small CdTe elements of 1.1mm x 1.0mm x 5mm. The FOV of this PET is 64mm in diameter and 26 mm in axis. We applied to observe small tumors in mouse and succeeded to obtain [18F]FDG images of mouse mammary carcinoma of ∼1mm size. ©2008 IEEE.

Preclinical mouse models are essential to the study of liver metastasis, yet their utility has been limited by difficulty in tracking the progression of metastases through time. In this study, liver metastases were identified by in vivo bioluminescence system and a high-frequency ultrasound system with nanobubbles. Tumor grows and vessel densities were calculated. Liver metastases were observed on the second days after inoculation and confirmed by the HE staining. Vessels density in the liver metastasis group was increased compared to the control group. This study show that three-dimensional high-frequency ultrasound imaging combined with nanobubbles may be suited for early detection, quantitative assessment of metastatic progression in liver metastasis models.

It is important of diagnosing not only primary tumors but also metastases accurately. Recently, we have established murine lymph node metastasis model and developed a new method to reconstruct the two- and three-dimensional (2D/3D) vessel structures in the model by using nanobubbles (NBs) and high-frequency ultrasound imaging system. In the present study, we investigated the characteristics of 2D/3D vessel structures in the metastasis model, and compared to normal lymph node. This technique may be useful for early diagnosis of lymph node micrometastasis.

In the present study, the kinetics of gene transfer mediated by ultrasound contrast agents (albumin, lipid micelle, and acoustic liposome nano/microbubbles) and ultrasound in the skeletal muscle was investigated with varying bubble concentrations. The gene activity depended on the bubble concentration, therefore, gene transfer efficiency would be improved with increasing bubble concentrations. However, the transient nature of transgene expression combined with the damage to the muscle observed at the peak of expression restricts the potential therapeutic application of this methodology.

Spinal gene therapy is a promising option for treating various spinal-related disorders. Several previous studies using viral vectors reported successful transfer of therapeutic genes into the spinal nerve system. However, because of the considerable immunogenicity related to the use of viruses, non-viral gene transfer still needs to be developed. One possible approach is the combined use of ultrasound and echo-contrast microbubbles. The present study shows that this method can be applied for targeted intrathecal gene delivery. We intrathecally injected a mixture of plasmid-DNA encoded with luciferase and commercially available albumin microbubbles by needle puncture at the lower lumbar intervertebral space in mice. Subsequent percutaneous ultrasonication on the lumbar vertebrae significantly enhanced the luciferase expression, analyzed by imaging luciferin bioluminescence, in the dorsal meningeal cells at the insonated region. No apparent neurological damages were induced by the present spinal interventions. In addition to the general benefits of the combined use of ultrasound and microbubbles, our approach can offer some advantages specific to spinal gene transfection including minimal invasiveness of simple percutancous dural puncture, targetability due to the limited access of ultrasound waves through anatomical apertures of the vertebrae, and possible paracrine delivery of therapeutic molecules to the spinal nerve system. (c) 2006 Elsevier B.V. All rights reserved.

Cell membrane permeabilization technique utilizing ultrasound and micro- nano- bubbles (i.e. sonoporation) is a promising way of drug and gene delivery. The mechanisms of the permeabilization and subsequent molecular transport across the membrane are underlying the development of the technique for clinical applications. We thereby have studied the mechanisms in the molecular level by molecular dynamics (MD) simulations of lipid bilayers, which are fundamental of all biological cell membranes. Firstly, we performed MD simulations of structural changes of the bilayer induced by shock wave and revealed the resulting collapse and rebound of the bilayer followed by water penetration into the hydrophobic region of the bilayer. Next, we studied the water pore formation resulted from the water penetration into the hydrophobic region. It was found that the water pore is formed within several nanoseconds when large amount of water molecules are existed in the hydrophobic region. Finally, we analyzed the diffusion of anti-cancer drug in the water pore and deduced that the water pore induced by the action of shock wave may be one of the processes of drug delivery in the presence of shock waves.

In the present study, we quantified antitumor effect by cisplatin with thee-dimensional tumor diagnostic technique using high-frequency ultrasound and nanobubbles. Solid tumors were developed in mice and the antitumor efficacy was compared to a conventional caliper method. It was found that high-frequency ultrasound method was able to evaluate intratumor vascular structure and density as well as tumor volume. The caliper method overestimated tumor volume by a factor of two compared to this technique.

The structural change of a phospholipid bilayer in water under the action of a shock wave is numerically studied with unsteady nonequilibrium molecular dynamics simulations. The action of shock waves is modeled by the momentum change of water molecules, and thereby we demonstrate that the resulting collapse and rebound of the bilayer are followed by the penetration of water molecules into the hydrophobic region of the bilayer. The high-speed phenomenon that occurs during the collapse and rebound of the bilayer is analyzed in detail, particularly focusing on the change of bilayer thickness, the acyl chain bend angles, the lateral fluidity of lipid molecules, and the penetration rate of water molecules. The result shows that the high-speed phenomenon can be divided into two stages: in the first stage the thickness of bilayer and the order parameter are rapidly reduced, and then in the second stage they are recovered relatively slowly. It is in the second stage that water molecules are steadily introduced into the hydrophobic region. The penetration of water molecules is enhanced by the shock wave impulse and this qualitatively agrees with a recent experimental result.

A Japanese word, monozukuri ( literally translated "making things'') is the philosophy of first having the idea and then the faith in the technical expertise and experience to accomplish the result. We believe that the concept of engineering is monozukuri. Through the process of monozukuri, engineered natural science based on mathematics and physics has been developed. Medicine is the field of study which has been developed for maintaining daily healthy life with diagnosis, treatment, examination, and protection. Biomedical engineering is the interdisciplinary study of engineering and medicine, and should be developed based on monozukuri. In this particular research, we have developed a physical molecular delivery method for cancer gene therapy using nano/microbubbles and ultrasound. First, the behavior of cavitation bubbles and subsequent shock wave phenomena involved in the mechanism of molecular delivery were analyzed, combining theory and computer simulation. In a second step, the methodology was optimized in vitro and in vivo. Finally, the therapeutic potential of the method in pre-clinical models was evaluated using transgenes relevant to cancer gene therapy instead of reporter genes, and whole body, non-invasive imaging using single photon emission computed tomography (SPECT/CT) was used to evaluate the selectivity of gene delivery in vivo.

The combination of ultrasound and ultrasound contrast agents (UCAs) is able to induce transient membrane permeability leading to direct delivery of exogenous molecules into cells. Cavitation bubbles are believed to be involved in the membrane permeability; however, the detailed mechanism is still unknown. In the present study, the effects of ultrasound and the UCAs, Optison((TM)) on transfection in vitro for different medium heights and the related dynamic behaviors of cavitation bubbles were investigated. Cultured CHO-E cells mixed with reporter genes (luciferase or beta-gal plasmid DNA) and UCAs were exposed to 1MHz ultrasound in 24-well plates. Ultrasound was applied from the bottom of the well and reflected at the free surface of the medium, resulting in the superposition of ultrasound waves within the well. Cells cultured on the bottom of 24-well plates were located near the first node (displacement node) of the incident ultrasound downstream. Transfection activity was a function determined with the height of the medium (wave traveling distance), as well as the concentration of UCAs and the exposure time was also determined with the concentration of UCAs and the exposure duration. Survival fraction was determined by MTT assay, also changes with these values in the reverse pattern compared with luciferase activity. With shallow medium height, high transfection efficacy and high survival fraction were obtained at a low concentration of UCAs. In addition, capillary waves and subsequent atomized particles became significant as the medium height decreased. These phenomena suggested cavitation bubbles were being generated in the medium. To determine the effect of UCAs on bubble generation, we repeated the experiments using crushed heat-treated Optison (TM) solution instead of the standard microbubble preparation. The transfection ratio and survival fraction showed no additional benefit when ultrasound was used. These results suggested that cavitation bubbles created by the collapse of UCAs were a key factor for transfection, and their intensities were enhanced by the interaction of the superpose ultrasound with the decreasing the height of the medium. Hypothesizing that free cavitation bubbles were generated from cavitation nuclei created by fragmented UCA shells, we carried out numerical analysis of a free spherical bubble motion in the field of ultrasound. Analyzing the interaction of the shock wave generated by a cavitation bubble and a cell membrane, we estimated the shock wave propagation distance that would induce cell membrane damage from the center of the cavitation bubble. (E-mail: kodama@tubero.tohoku.ac.jp) (c) 2006 World Federation for Ultrasound in Medicine & Biology.

The diffusion process of a 5-fuluorouracil (5FU) in the water layer confined by two leaflets of lipid monolayer is elucidated by molecular dynamics simulation. The calculated diffusion constant of 5FU is 1.1×10^<-5>cm^2/s in bulk water with SPC/E water model at 25℃. This is in rather good agreement with that obtained in a recent NMR experiment (1.0×10^<-5>cm^2/s). It is found that the diffusion constant of 5FU in the confined water layer decreases as it moves from the center of the water layer toward the lipid monolayer surface. This decrease may be caused by the effect of the charges of the hydrophilic head groups of the lipid molecules.

Water molecule penetration into a bilayer hydrophobic region with a shock wave impulse has been investigated using molecular dynamics simulations [Koshiyama et al., AIP Conference Proceedings, 754, 104-106, (2005)]. Here we report results of simulation of spontaneous water pore formation in a bilayer that contains water molecules in the hydrophobic region in an initial state. The bilayers of 128 DPPC lipid and 3655 water molecules with insertion of 392, 784, and 1176 water molecules in the hydrophobic region are simulated. A water pore is spontaneously formed when 1176 water molecules exist in the hydrophobic region. The water pore diameter is estimated to be c.a. 1.9 nm, which is three times larger than that of 5-fluorouracil (5FU) used in cancer treatment.

Ultrasound contrast agents (UCAs), are capable of enhancing non-invasive cytoplasmic molecular delivery in the presence of ultrasound. Collapse of UCAs may generate nano-scale cavitation bubbles, resulting in the transient permeabilization of the cell membrane. In the present study, we investigated the interaction of a cavitation bubble-induced shock wave with a cell membrane using acoustic theory and molecular dynamics (MD) simulation. From the theory, we obtained the shock wave propagation distance from the center of a cavitation bubble that would induce membrane damage. The MD simulation determined the relationship between the uptake of water molecules into the lipid bilayer and the shock wave. The interaction of the shock wave induced a structural change of the bilayer and subsequently increased the fluidity of each molecule. These changes in the bilayer due to shock waves may be an important factor in the use of UCAs to produce the transient membrane permeability during sonoporation.

Therapy with naked oligodeoxynucleotides (ODNs, molecular weight: 3000 to 7500) provides an elegant means of modulating gene expression without the problems associated with conventional gene therapy, but the relatively low transfer efficiency on intravascular administration is a limitation to clinical application. Ultrasound, which can be potentiated by microbubbles, shows promise as a method of delivering macromolecules such as plasmid DNA and other transgenes into cells. Since uptake of molecules into cells depends on their molecular weight, it might be expected that the delivery of ODNs, which are relatively small, will be facilitated by ultrasound and microbubbles. In the present study, we delivered ODNs into veins using ultrasound and microbubbles. First, we quantified the uptake of fluorescent-labeled ODNs into intact ex vivo human saphenous veins and isolated smooth muscle cells from the veins, evaluating the effect of ultrasound and microbubbles on uptake. Ultrasound potentiated the delivery of ODN in cells, except at high concentrations. When intact veins were studied, we achieved nuclear localization of fluorescent-labeled ODNs in cells. This increased with increasing concentration and incubation time and was not potentiated by ultrasound, even when microbubbles were used. We then applied a therapeutic ODN (antisense to intercellular adhesion molecule 4, ICAM-1) to vein samples and documented a functional inhibition of gene expression in a sequence-specific manner at the protein level with immunohistochemistry and western blot analysis. Again, no significant difference was seen with adjunct ultrasound. These observations suggest high diffusion of ODNs into human saphenous veins in this ex vivo model, indicating potential applications to inhibition of vascular bypass graft occlusion and other vasculopathies. Although microbubble-ultrasound was of value with cells in culture, it was not beneficial with intact veins. (c) 2005 World Federation for Ultrasound in Medicine & Biology.

Nano/micro-bubbles are capable of enhancing non-invasive cytoplasmic molecular delivery in the presence of ultrasound. Collapse of the bubbles might generate nano-scale free cavitation bubbles, resulting in transient membrane permeabilization. In the present study, we used two different nano bubbles to investigate the mechanism of molecular delivery into the cytoplasm with theory and experiment.

Molecular dynamics (MD) simulations of molecular delivery into a lipid bilayer utilizing shock-wave-based techniques were carried out for 5Fuluorouracil (5FU). Although water penetration into the bilayer hydrophobic region was observed for a shock wave impulse range of 9.5〜57mPa.s, 5FU penetration was not observed. Therefore larger structural changes (e.g. water pore formation) than those observed in current simulations are thought to be required for 5FU penetration.

Cell permeabilization by shock waves may have application in gene therapy and anticancer drug delivery. In the present study we performed direct molecular dynamic (MD) simulation of the interaction of a single shock wave with a cell membrane to investigate the mechanism of the cell permeabilization. The shock wave was characterized by an impulse that was expressed with a velocity determined by the change in the momentum. The cell membrane was designed as a dipalmitoylphosphatidylcholine (DPPC) lipid bilayer placed between two layers of water molecules. The MD simulation determined the relationship between water penetration into the bilayer, the order parameter, the fluidity of each lipid molecule, and the intensity of impulse. These structural changes in the bilayer may be an important factor in the use of shock waves to produce transient membrane permeability.

Cell permeabilization by shock waves may have application in gene therapy and anticancer drug delivery. In the present study we performed direct molecular dynamic (MD) simulation of the interaction of a single shock wave with a cell membrane to investigate the mechanism of the cell permeabilization. The shock wave was characterized by an impulse that was expressed with a velocity determined by the change in the momentum. The cell membrane was designed as a dipalmitoylphosphatidylcholine (DPPC) lipid bilayer placed between two layers of water molecules. The MD simulation determined the relationship between water penetration into the bilayer, the order parameter, the fluidity of each lipid molecule, and the intensity of impulse. These structural changes in the bilayer may be an important factor in the use of shock waves to produce transient membrane permeability.

Ultrasound (US) is a promising tool for facilitating direct gene transfer to skeletal muscle, but no systematic optimisation study has been performed. We exposed H2K myoblast cells to US with varying intensity of exposure and duration to evaluate its effect on cell viability and transfection efficiency using as endpoints transfection rate, average fluorescence intensity (fluorescence normalised by the number of transfected cells) and overall expression (the product of transfection rate and average fluorescence intensity) as indices. Cell viability decreased with exposure time and intensity, consistent with previous findings. Optimal setting of US was observed at the range of 0.5 to 1 W cm(-2) with duration of 20 s, producing maximum efficiency (transfection = 4.5%) in gene transfection with minimum cell toxicity (cell viability = 83 %). Higher intensity alone or in combination with low intensity and long duration did not improve cell viability and transfection. The increase of eGFP (enhanced green fluorescence protein) plasmid concentration up to 200 mug per mL was related to an increase in average fluorescence intensity and overall expression. However, transfection rate saturated when DNA concentration reached 50 jig per mL despite initial increase with DNA concentration. The average fluorescence intensity was linearly proportional to the logarithm of DNA concentration, suggesting a diffusion-based model for DNA uptake under sonoporation. We conclude that low-intensity US irradiation provides a safe and effective alternative for gene delivery. (C) 2004 World Federation for Ultrasound in Medicine Biology.

Cell permeabilization using shock waves may be a promising way of introducing macromolecules and small polar molecules into the cytoplasm and may have applications in gene therapy and anticancer drug delivery. The detailed mechanism of the cell permeabilization however is still unclear. To investigate the mechanism, we have performed molecular dynamics (MD) simulations of the interaction of the shock wave impulse with a lipid-bilayer. The MD simulation determined the relationship between the uptake of water molecules into the lipid bilayer and the impulse. The interaction of a shock wave with a lipid bilayer induced a structural change of the bilayer and subsequently increased the fluidity of each molecule. These changes in the bilayer may be an important factor for the mechanism of cell permeabilization using shock waves.

The dynamics of a laser-induced cavitation bubble near a composite surface, consisting of either a thin rigid plate glued on a foam rubber (composite surface A) or a deformable rubber plate glued on a foam rubber (composite surface B), was investigated experimentally with high-speed photography. To understand the interaction between a cavitation bubble and a composite surface, the dynamic properties of the composite surfaces were measured with a modal analysis by providing a maximum frequency to 5 kHz, since the period of the bubble motion with the radius of 1 mm is about 200 mus. It was found that bubble migration was significantly influenced by the dynamic property of composite surfaces, showing a range of response between the free surface and rigid boundary cases. For one of the composite surfaces with a deformable rubber, bubble splitting was generated for gamma&lt;1.1, where gamma=L/R-max with L being the initial bubble location and R-max the maximum bubble radius. A neutral bubble collapse occurred at a specified bubble location defined as gamma(N) that was fairly correlated with the compliance, G(n), of the composite surfaces. The value of gamma(N) decreased with increasing G(n). (C) 2003 American Institute of Physics.

The motion of single- and two-cavitation bubbles generated by laser beams directly beneath a free surface is studied experimentally, using high-speed photography, and theoretically using the highly accurate boundary integral method. Favorable comparisons of bubble shape history and centroid motion are observed while the numerical calculations provide information on the pressure field surrounding the bubbles. A range of responses, including the null impulse state, is obtained for the two bubbles depending on the bubble size ratio and the interbubble and bubble-free surface distances, although in all cases reported in this article, the bubble nearest the free surface yields a high-speed liquid jet directed away from the free surface. It is also found that when the free-surface-bubble interaction is strong, a fast free-surface spike is formed for both the single- and two-bubble cases. © 2001 American Institute of Physics.

A prior study has reported that a rapid recanalization therapy of cerebral embolism, using liquid jet impacts generated by the interaction of gas bubbles with shock waves, can potentially penetrate through thrombi in as little as a few μs with very efficient ablation (Kodama et al. 1997). The present study was undertaken to examine the liquid jet impact effect on fibrinolysis in a tube model of an internal carotid artery. First, the conditions for generating the maximum penetration depth of liquid jets in the tube were investigated. Gelatin was used to mimic thrombi. The shock wave was generated by detonating a silver azide pellet weighing about a few μg located in a balloon catheter. The collapse of the inserted gas bubbles and the subsequent liquid jet formation were recorded with high-speed photography. Second, thrombi were formed using fresh human blood from healthy volunteers. The fibrinolysis induced by the liquid jet impact with urokinase was explored. This was conducted under selected conditions based on the experiment using the gelatin. Fibrinolysis was calculated as the percentage of the weight loss of the thrombus. Fibrinolysis with urokinase alone and with a single liquid jet impact with urokinase was 1.9 ± 3.7% (n = 16) and 20.0 ± 9.0% (n = 35), respectively, for an incubation time of 60 min. Statistical differences were obtained between all groups (ANOVA). These results suggest that liquid jet impact thrombolysis has the potential to be a rapid and effective therapeutic modality in recanalization therapy for patients with cerebral embolism and other clinical conditions of intra- arterial thrombosis.

We have developed a novel, less invasive, shock wave source that can be introduced into an arbitrary position in a human body percutaneously. Using this technique we can disrupt cells locally. The shock wave source consists of an explosive, an optical fiber, a balloon catheter, and a Nd:YAG laser, which generates a spherical explosive shock wave. The destructive potential of the present source for injuring tissue was confirmed and the subsequent cell elongation and split in the direction of the shock wave has been observed.

The interaction of air bubbles attached to gelatin surfaces, extirpated livers or abdominal aortas of rats with underwater shock waves was investigated to help clarify the tissue-damage mechanism associated with cavitation bubbles induced during extracorporeal shock-wave lithotripsy. The overpressure of the shock wave was 10.2 ± 0.5 MPa. The initial bubble radii varied from 0.12 to 3.06 min. The subsequent collapse of the bubbles was recorded by a high-speed camera. The liver-cell damage was histochemically evaluated. The bubble attached to gelatin or rat's liver surface migrates away from the surface with an oscillatory growth/collapse behavior after the shock-wave interaction. The penetration depth of the liquid jet into the gelatin and the radius of the subsequent damage pit on the surface depend on the initial bubble radius. The elongation and split of the nuclei in the liver parenchymal cells in the direction of the liquid jet and the increase in the cell density within the circumference of the injured region are revealed histologically.

Transurethral prostatectomy is performed using Ho: YAG laser in this study. Prostatic tissue is cut by underwater shockwave induced by Ho: YAG laser irradiation. The advantage of this method compared with transurethral prostatectomy using high frequency electrical current is that bleeding is little and there is no possibility of having hyponatremia. Another advantage of this method compared with transurethral prostatectomy using Nd: YAG laser is that the force of urinary stream greatly improves just after the operation.

The shock-induced collapse and jet formation of a single air bubble inside a cylinder filled with gelatin and an artificial thrombus were investigated using high-speed photography. A thrombus was prepared <I>in vitro</I> and inserted in a cylinder modeling an artery to establish a treatment for brain embolism by the interaction of an underwater shock wave with an air bubble. When the shock wave was applied to an air bubble of mm in diameter, the liquid jet that formed within the bubble penetrated the thrombus. Liquid jet impact ablation of the for thrombus was found to be very effective, and provided valuable information for the treatment of brain embolism.

Revascularization time is the dominant factor in the treatment of acute cerebral embolism. In this paper we describe a rapid revascularization therapy using liquid jets generated by the interaction of gas bubbles with shock waves, which impact on the thrombi. The interaction of a shock wave with a gas bubble attached to an artificial thrombus which was inserted into a tube model of a cerebral artery was investigated. The shock wave was generated by detonating a microexplosive pellet. The overpressure of the shock wave was 3.0 ± 0.6 MPa (n = 7) and 12.7 ± 0.4 MPa (n = 3). The initial air bubble radii were varied from 0.87 mm to 2.18 min. The subsequent collapse of the bubble was photographed using a high-speed framing camera, and the liquid jet penetrating into the artificial thrombus was visualized using x-ray photography. The penetration depth of the liquid jet increased with increasing bubble size. There was an optimal separation distance between the bubble and the shock wave source to obtain the maximum penetration depth. Liquid jets have the potential to penetrate through thrombi in as little as a few microseconds, and with very efficient ablation.

An experimental study has been made on the interaction of an underwater shock wave with two air bubbles attached to a gelatin surface. The shock wave was generated by detonating a microexplosive pellet, and the subsequent behavior of bubble collapse was visualized by high-speed photography. By measuring the directivity and the maximum depth of the liquid jets of the two bubbles penetrating into the gelatin, it was found that when the bubbles are beyond a critical separation distance, the depth of the penetration was no longer affected by the presence of the other bubble, and that the penetration depth for a given bubble in a two-bubble arrangement is a function of the size of the other bubble. © 1996 American Institute of Physics.

This is an experimental study visualizing the interaction of air bubbles with underwater shock waves and is related to the investigation of tissue damage induced during extracorporeal shockwave lithotripsy treatment. Two optical visualization techniques, such as holographic interferometry and conventional photography were intensively used. The result of visualization revealed that various collapse patterns exist depending on properties of surrounding materials and the tissue damage could be generated mainly due to liquid jets formed inside the bubbles.

This experimental study has revealed damage to red blood cells that is quantitatively related to the acoustic pressure during irradiation with 0.75-MHz continuous-wave ultrasound, using a range of intensities comparable to those employed by ultrasonic physiotherapy equipment (0.25 to 7 W cm(-2) spatial average temporal average). Damage to the red blood cells was investigated by measuring the extent of haemolysis using a UV/VIS spectrophotometer. There was a clear correlation between the amount of haemoglobin released and the intensity of harmonic emissions recorded from the sample during irradiation. The observed degree of haemolysis could not be produced by temperature rises in the absence of the sound field. This suggests that the damage was a direct result of acoustic cavitation.

Evidence of acoustic cavitation was identified in the form of transient echoes in ultrasound B-scan images of patients receiving extracorporeal shock-wave lithotripsy treatment on a Storz Modulith SL20. This lithotripter generates 10-mu s duration pulses with a centre frequency of 0.2 MHz at a pulse repetition frequency of 1 Hz. The visual appearance of B-scan images was examined in a total of 30 patients and a quantitative analysis of echogenicity changes was carried out in six cases involving lithotripsy treatment of stones in the renal pelvis. In these patients new echoes were identified in images unaffected by movement artefacts and were found to occur in perinephric fat and adjacent muscle and kidney tissue at positions close to the axis of the shock-wave field between 1 and 2 cm in advance of the indicated beam focus of the lithotripter. The echogenicity within each region increased significantly above the background level when the output of the lithotripter was increased above a threshold value. The acoustic pressures corresponding to this threshold were measured in water using a calibrated PVDF membrane hydrophone. After correction for attenuation in tissue the cavitation thresholds, in terms of the temporal peak negative pressure, are found to lie between 1.5 MPa and 3.5 MPa in all six cases. Interpretation of the measured values in terms of the likely threshold at the higher frequencies used in diagnostic ultrasound is considered using a theoretical model.

An investigation was made of the motion of a cavitation bubble in the vicinity of a free surface experimentally as well as theoretically. A bubble was produced by focusing a ruby laser into water, and subsequent behavior was observed with a high speed camera. The deformable nature of both a bubble and a free surface becomes significant when a bubble behaves relatively close to the surface. Immediately after the bubble rebound a secondary cavitation occurs, at around zero dynamic pressure region, which was developed in water between a rising free surface and a re-expanding bubble due to lower pressure caused by the interaction of reflected expansion waves from the surface. Some aspects of the bubble motion were compared with image theory with good agreement.

An experimental investigation was made of the motion of a cavitation bubble in the vicinity of a free surface in order to study an induced secondary cavitation during the bubble rebound. A bubble was produced by focusing a ruby laser into water, and its subsequent behavior was observed with a high-speed camera. The deformable nature of both a bubble and a free surface becomes significant as the mutual distance between them is decreased. Immediately after bubble rebound, a secondary cavitation occurs at around zero dynamic pressure region which is developed in water between a rising free surface and a collapsing bubble, due to the local pressure reduction mainly caused by the interaction of expansion waves originated from the surface.