Human induced pluripotent stem cells are resistant to human cytomegalovirus infection primarily at the attachment level due to the reduced expression of cell-surface heparan sulfate.

Cytomegalovirus (CMV), a type of herpes virus, is the predominant cause of congenital anomalies due to intrauterine infections in humans. Adverse outcomes related to intrauterine infections with human cytomegalovirus (HCMV) vary widely, depending on factors such as fetal infection timing, infection route, and viral virulence. The precise mechanism underlying HCMV susceptibility remains unclear. In this study, we compared the susceptibility of neonatal human dermal fibroblast cells (NHDFCs) and human induced pluripotent stem cells (hiPSCs) derived from NHDFCs, which are genetically identical to HCMV, using immunostaining, microarray, in situ hybridization, quantitative PCR, and scanning electron microscopy. These cells were previously used to compare CMV susceptibility, but the underlying mechanisms were not fully elucidated. HCMV susceptibility of hiPSCs was significantly lower in the earliest phase. No shared gene ontologies were observed immediately post-infection between the two cell types using microarray analysis. Early-stage expression of HCMV antigens and the HCMV genome was minimal in immunostaining and in in situ hybridization in hiPSCs. This strongly suggests that HCMV does not readily bind to hiPSC surfaces. Scanning electron microscopy performed using the NanoSuit method confirmed the scarcity of HCMV particles on hiPSC surfaces. The zeta potential and charge mapping of the charged surface in NHDFCs and hiPSCs exhibited minimal differences when assessed using zeta potential analyzer and scanning ion conductance microscopy; however, the expression of heparan sulfate (HS) was significantly lower in hiPSCs compared with that in NHDFCs. Thus, HS expression could be a primary determinant of HCMV resistance in hiPSCs at the attachment level. IMPORTANCE: Numerous factors such as attachment, virus particle entry, transcription, and virus particle egress can affect viral susceptibility. Since 1984, pluripotent cells are known to be CMV resistant; however, the exact mechanism underlying this resistance remains elusive. Some researchers suggest inhibition in the initial phase of HCMV binding, while others have suggested the possibility of a sufficient amount of HCMV entering the cells to establish latency. This study demonstrates that HCMV particles rarely attach to the surfaces of hiPSCs. This is not due to limitations in the electrostatic interactions between the surface of hiPSCs and HCMV particles, but due to HS expression. Therefore, HS expression should be recognized as a key factor in determining the susceptibility of HCMV in congenital infection in vitro and in vivo. In the future, drugs targeting HS may become crucial for the treatment of congenital CMV infections. Thus, further research in this area is warranted.

A novel prognostic model of de novo metastatic hormone-sensitive prostate cancer to optimize treatment intensity.

BACKGROUND: The treatment and prognosis of de novo metastatic hormone-sensitive prostate cancer (mHSPC) vary. We established and validated a novel prognostic model for predicting cancer-specific survival (CSS) in patients with mHSPC using retrospective data from a contemporary cohort. METHODS: 1092 Japanese patients diagnosed with de novo mHSPC between 2014 and 2020 were registered. The patients treated with androgen deprivation therapy and first-generation anti-androgens (ADT/CAB) were assigned to the Discovery (N = 467) or Validation (N = 328) cohorts. Those treated with ADT and androgen-receptor signaling inhibitors (ARSIs) were assigned to the ARSI cohort (N = 81). RESULTS: Using the Discovery cohort, independent prognostic factors of CSS, the extent of disease score ≥ 2 or the presence of liver metastasis; lactate dehydrogenase levels > 250U/L; a primary Gleason pattern of 5, and serum albumin levels ≤ 3.7 g/dl, were identified. The prognostic model incorporating these factors showed high predictability and reproducibility in the Validation cohort. The 5-year CSS of the low-risk group was 86% and that of the high-risk group was 22%. Approximately 26.4%, 62.7%, and 10.9% of the patients in the Validation cohort defined as high-risk by the LATITUDE criteria were further grouped into high-, intermediate-, and low-risk groups by the new model with significant differences in CSS. In the ARSIs cohort, high-risk group had a significantly shorter time to castration resistance than the intermediate-risk group. CONCLUSIONS: The novel model based on prognostic factors can predict patient outcomes with high accuracy and reproducibility. The model may be used to optimize the treatment intensity of de novo mHSPC.

[Re-Administration of Pembrolizumab for Urothelial Carcinoma after immune-Related Myasthenia Gravis : A Case Report].

Immune checkpoint inhibitors (ICIs) provide excellent benefits to the treatment of various cancer types, including urothelial carcinoma. Conversely, they can cause immune-related adverse events (irAEs), and some of them are severe or fatal. Furthermore, evidence on the safety and effectiveness of the readministration of ICIs after the occurrence of irAEs is limited. In this case report, a 78-year-old man who suffered from metastatic right renal pelvic cancer was treated with pembrolizumab. He had a partial response to pembrolizumab, but he developed grade 3 myasthenia gravis. The myasthenia gravis symptoms were immediately relieved by corticosteroids and intravenous immunoglobulin therapy. When the disease rapidly progressed, he was treated again with pembrolizumab. After 5 days, a chest radiograph showed shrinkage of pulmonary metastases. Unfortunately, he died of multiple brain infarctions 7 days after the readministration. We report this case with a literature review on the efficacy and safety of the readministration of ICIs after the occurrence irAEs including myasthenia gravis.

Volatile profiling of fruits of 17 mango cultivars by HS-SPME-GC/MS combined with principal component analysis.

Headspace solid-phase microextraction combined with gas chromatography/mass spectrometry is one of the strongest tools for comprehensive analysis of volatile compounds and has been used to analyze aromatic components of mango and investigate its varietal characteristics. In this study, profiling of aroma compounds in 17 mango cultivars, grown in the same green house to exclude the effect of environmental factors, was conducted and the patterns were subjected to principal component analysis (PCA) to identify the relationship between the aroma components and cultivars. Fifty-nine different volatile constituents were detected from the blends of these 17 mango cultivars. The cultivars were divided into 4 clusters using PCA based on the volatile components determined in the study. Aiko was found to mainly contain δ-3-carene and showed a composition more similar to its pollen parent, Irwin, than to its seed parent, Chiin Hwang No. 1.

Impact of the objective response to and number of cycles of platinum-based first-line chemotherapy for metastatic urothelial carcinoma on overall survival of patients treated with pembrolizumab.

OBJECTIVES: To investigate the impact of the number of cycles and objective response to chemotherapy on overall survival in patients with metastatic urothelial carcinoma treated with pembrolizumab. METHODS: This multicenter, retrospective study included 755 patients from 59 institutions with advanced, chemoresistant urothelial carcinoma who received pembrolizumab. The associations of the overall survival with the number of cycles and best objective response were investigated using Cox multiple regression analysis. RESULTS: Overall, 391 patients received standard first-line chemotherapy and pembrolizumab as a second-line treatment, and were included in the final analysis. Of the 391 patients, 185 received less than four cycles, 134 received four to six cycles and 72 received more than six cycles of first-line chemotherapy. An objective response (complete or partial response) to chemotherapy was observed in 145 patients (37.1%). Univariate analysis showed that the overall survival of patients who received more than six cycles or responded to chemotherapy (complete or partial response) was significantly longer than that of patients who received less than four cycles or did not respond to chemotherapy (stable or progressive disease). At multivariate levels, no correlations were observed between overall survival and the number of cycles of or the response to chemotherapy. CONCLUSIONS: Therapeutic benefit of pembrolizumab can be expected irrespective of the objective response to and number of cycles of platinum-based first-line chemotherapy.

[A Case of Prostate Cancer with High Levels of Prostate-Specific Antigen Undetected by Prostate Biopsy].

A 79 year-old-man visited our hospital with right back pain. Computed tomography suggested external iliac and para-aortic lymphadenopathy. Serum prostate specific antigen (PSA) increased to 335 ng/ml and prostate cancer was highly suspected. We performed transperineal prostate biopsies two times, but could not detect prostate carcinoma cells. Multiparametric magnetic resonance imaging (MRI) indicated no suspicious malignant lesions in the prostate. Laparoscopic biopsy of the right obturator lymph nodes was performed and histological examination, including immunohistochemical staining with PSA, confirmed lymphnode metastasis from prostate cancer. After endocrine therapy was started, serum PSA levels declined and lymph nodes shrunk. In cases of negative prostate biopsies despite high serum PSA levels, aggressive indication for biopsy of metastatic lesion and histological inspection is highly recommended.

Combination therapy with liposomal neuroprotectants and tissue plasminogen activator for treatment of ischemic stroke.

For ischemic stroke treatment, extension of the therapeutic time window (TTW) of thrombolytic therapy with tissue plasminogen activator (tPA) and amelioration of secondary ischemia/reperfusion (I/R) injury are most desirable. Our previous studies have indicated that liposomal delivery of neuroprotectants into an ischemic region is effective for stroke treatment. In the present study, for solving the above problems in the clinical setting, the usefulness of combination therapy with tPA and liposomal fasudil (fasudil-Lip) was investigated in ischemic stroke model rats with photochemically induced thrombosis, with clots that were dissolved by tPA. Treatment with tPA 3 h after occlusion markedly increased blood-brain barrier permeability and activated matrix metalloproteinase (MMP)-2 and -9, which are involved in cerebral hemorrhage. However, an intravenous administration of fasudil-Lip before tPA markedly suppressed the increase in permeability and the MMP activation stemming from tPA. The combination treatment showed significantly larger neuroprotective effects, even in the case of delayed tPA administration compared with each treatment alone or the tPA/fasudil-treated group. These findings suggest that treatment with fasudil-Lip before tPA could decrease the risk of tPA-derived cerebral hemorrhage and extend the TTW of tPA and that the combination therapy could be a useful therapeutic option for ischemic stroke.-Fukuta, T., Asai, T., Yanagida, Y., Namba, M., Koide, H., Shimizu, K., Oku, N. Combination therapy with liposomal neuroprotectants and tissue plasminogen activator for treatment of ischemic stroke.

Systemic Administration of siRNA with Anti-HB-EGF Antibody-Modified Lipid Nanoparticles for the Treatment of Triple-Negative Breast Cancer.

Triple-negative breast cancer is one of the intractable cancers that are not sensitive to treatment with existing molecular-targeted drugs. Recently, there has been much interest in RNA interference-mediated treatment of triple-negative breast cancer. In the present study, we have developed lipid nanoparticles encapsulating siRNA (LNP-siRNA) decorated with an Fab' antibody against heparin-binding EGF-like growth factor (αHB-EGF LNP-siRNA). αHB-EGF LNP-siRNA targeting polo-like kinase 1 (PLK1) was prepared and evaluated for its anticancer effect using MDA-MB-231 human triple-negative breast cancer cells overexpressing HB-EGF on their cell surface. Biodistribution data of radioisotope-labeled LNP and fluorescence-labeled siRNA indicated that αHB-EGF LNP effectively delivered siRNA to tumor tissue in MDA-MB-231 carcinoma-bearing mice. Expression of PLK1 protein in the tumors was clearly suppressed after intravenous injection of αHB-EGF LNP-siPLK1. In addition, tumor growth was significantly inhibited by treatment with this formulation of siRNA and an antibody-modified carrier. These findings indicate that αHB-EGF LNP is a promising carrier for the treatment of HB-EGF-expressing cancers, including triple-negative breast cancer.

Nobiletin Reduces Intracellular and Extracellular ß-Amyloid in iPS Cell-Derived Alzheimer's Disease Model Neurons.

Alzheimer's disease (AD) is the most common cause of dementia, with progressive memory impairment. Recently, neprilysin, a ß-amyloid (Aß)-degrading enzyme has become featured as a drug target for AD. Previously, we identified nobiletin from citrus peels as a natural compound possessing anti-dementia activity. In addition, we demonstrated that nobiletin improved memory in memory-impaired animals and, further, that Aß levels were markedly decreased in the brains of these animals. We demonstrated in vitro that nobiletin up-regulates neprilysin expression and activity in human neuroblastoma cells. However, the action of nobiletin with regard to Aß degradation under in vitro AD pathological conditions remains unclear. In this study, we examined whether nobiletin could enhance the degradation of intra- and extracellular Aß using human induced pluripotent stem cell-derived AD model neurons, which generate an excess of Aß1-42 due to the familial AD presenilin-1 mutation. The neurons were treated in the presence or absence of nobiletin. The results of real-time quantitative RT-PCR indicated that neprilysin mRNA levels were significantly up-regulated by nobiletin. Furthermore, immunostaining with an anti-Aß antibody revealed that nobiletin substantially reduced the intraneuronal content of Aß. Interestingly, the results of Aß1-42 immunoassays confirmed that nobiletin also significantly decreased the levels of Aß1-42 released into the cellular medium. These results suggest that nobiletin enhanced the reduction of intra- and that extracellular Aß levels under AD pathologic conditions, and this is associated with the up-regulation of neprilysin expression. Collectively, nobiletin appears to be a promising novel prophylactic seed drug or functional food for AD.

Involvement of Tumor Lymphatic System in Translocation of Intratumorally Injected Liposomes.

The tumor microenvironment is one of the key factors contributing to the efficiency of drug delivery to a tumor. It has been reported that lymphangiogenesis is induced in certain tumors. Because the lymphatic system functions as a drainage one, it is possible that tumor lymphatic vessels alter not only the tumor microenvironment, but also the distribution of drug nanocarriers accumulated in the tumor tissue. Herein, we aimed to elucidate the involvement of the tumor lymphatic system in the translocation of intratumoral liposomes to regional lymph nodes by using vascular endothelial growth factor (VEGF)-C-overexpressing B16F10 tumor-bearing mice (B16/VEGF-C). When the amount of polyethylene glycol (PEG)-modified liposomes in lymph nodes (cervical, brachial, axillary, and inguinal lymph nodes) was measured after the radiolabeled liposomes had been intratumorally injected into B16/VEGF-C-bearing mice or wild-type B16-bearing mice, the accumulation of liposomes in the axillary and inguinal lymph nodes was significantly higher on the tumor-implanted side of B16/VEGF-C-bearing mice than on that of the B16-bearing ones. On the other hand, the accumulation of liposomes in these lymph nodes on the control side (no implantation) of either type of tumor-bearing mice was very low; and no difference could be observed between the 2 sides. Furthermore, the intratumoral distribution of liposomes was observed to be located near the lymphatic vessels. These results indicate that the tumor lymphatic system contributed to the extrusion of a portion of PEG-modified liposomes from the tumor tissue, suggesting that tumor lymphangiogenesis would be one of the key factors to determine the intratumoral distribution of liposomes and their subsequent fate.

Impact of divergent differentiation in urothelial carcinoma on oncological outcome in patients with T1 high-grade bladder cancer.

OBJECTIVE: T1 high-grade bladder cancer has a poor prognosis compared with other non-muscle-invasive bladder cancers. We investigated the clinical outcomes among patients with T1 high-grade bladder cancer to identify factors related to cancer recurrence and disease progression. METHODS: We retrospectively reviewed the data of 148 patients who were diagnosed with T1 high-grade bladder cancer by transurethral resection from January 2001 to February 2015 at our institution. Clinicopathological factors were analyzed using univariate and multivariate analyses. RESULTS: The median age and follow-up duration were 72 years and 45.4 months, respectively. Sixty-two patients (41.9%) had recurrence, 28 (18.9%) experienced progression and 13 (8.8%) died of bladder cancer. In the multivariate analysis, divergent differentiation was an independent factor related to recurrence (P = 0.0096, hazard ratio = 2.5), whereas a non-papillary tumor shape, divergent differentiation and presence of a residual tumor at the time of the second transurethral resection were independent factors related to progression (P = 0.0349, hazard ratio = 3.8; P = 0.0074, hazard ratio = 6.8 and P = 0.0449, hazard ratio = 4.1, respectively). There were no independent factors related to cancer-specific mortality. Divergent differentiation was the only independent factor associated with both recurrence and progression. In addition, patients with divergent differentiation had significantly worse recurrence-free survival and progression-free survival rates than did patients without divergent differentiation (log-rank P = 0.0009 and P = 0.0019, respectively). CONCLUSIONS: In this study, the presence of divergent differentiation was related to worse oncological outcomes in patients with T1 high-grade bladder cancer. Patients with divergent differentiation may require stringent follow-up and early cystectomy after recurrence to improve oncological outcomes.

The Efficacy of Trimodal Chemoradiotherapy with Cisplatin as a Bladder-Preserving Strategy for the Treatment of Muscle-Invasive Bladder Cancer.

INTRODUCTION: Open radical cystectomy (ORC) is currently the standard treatment for muscle-invasive bladder cancer (MIBC) without metastasis, while many patients with MIBC are not always appropriate candidates due to multiple comorbidities. To evaluate the bladder-preservation strategy, we compared the results with those obtained by ORC. PATIENTS AND METHODS: We retrospectively analyzed the data of 50 patients with MIBC treated by trimodal chemoradiotherapy with cisplatin (CDDP-radiation [CDDP-R]). Transurethral resection of the bladder tumor (TURBT) was performed before treatment to confirm pathological stage ≥T2. Extensive TURBT was performed after chemoradiotherapy to evaluate the pathological response to treatment. We compared the survival outcomes of our CDDP-R with those of ORC (retrospective cohort, n = 205) by propensity score matching analysis. RESULTS: The 2- and 5-year progression-free survival, bladder-intact survival, cancer-specific survival, and overall survival (OS) rates after treatment were 70.8 and 63.9%, 64.0 and 49.8%, 86.7 and 71.8%, and 84.3 and 64.8%, respectively. The 2- and 5-year OS rates after CDDP-R were 90.5 and 74.3%, respectively, and those after ORC were 71.8 and 59.9%, respectively, indicating a significant survival advantage conferred by CDDP-R over ORC (p < 0.05, HR 0.45, 95% CI 0.21-0.94). CONCLUSIONS: In selected patients, CDDP-R for MIBC may provide comparative oncological outcomes as ORC.

Bauhinia purprea agglutinin-modified liposomes for human prostate cancer treatment.

Bauhinia purprea agglutinin (BPA) is a well-known lectin that recognizes galactosyl glycoproteins and glycolipids. In the present study, we firstly found that BPA bound to human prostate cancer specimens but not to normal prostate ones. Therefore, we sought to develop BPA-PEG-modified liposomes (BPA-PEG-LP) encapsulating anticancer drugs for the treatment of prostate cancer. We examined the tumor targetability of BPA-PEG-LP with human prostate cancer DU145 cells, and observed that fluorescently labeled BPA-PEG-LP dominantly associated with the cells via the interaction between liposome-surface BPA and cell-surface galactosyl molecules. We also observed that BPA-PEG-LP accumulated in the prostate cancer tissue after the i.v. injection to DU145 solid cancer-bearing mice, and strongly bound to the cancer cells. In a therapeutic study, DU145 solid cancer-bearing mice were i.v. injected thrice with BPA-PEG-LP encapsulating doxorubicin (BPA-PEG-LPDOX, 2 mg/kg/day as the DOX dosage) or PEG-modified liposomes encapsulating DOX (PEG-LPDOX). As a result, BPA-PEG-LPDOX significantly suppressed the growth of the DU145 cancer cells, whereas PEG-LPDOX at the same dosage as DOX showed little anti-cancer effect. The present study suggested that BPA-PEG-LP could be a useful drug carrier for the treatment of human prostate cancers.

Clinical significance of a second-line chemotherapy regimen with paclitaxel, ifosfamide and nedaplatin for metastatic urothelial carcinoma after failure of cisplatin-based chemotherapy.

OBJECTIVE: Cisplatin-based chemotherapy has been commonly used as the first-line chemotherapy for metastatic urothelial carcinoma. However, after failure of the first-line cisplatin-based chemotherapy, there is no established standard second-line chemotherapy. Starting in 2006, paclitaxel, ifosfamide and nedaplatin chemotherapy has been performed as the second-line chemotherapy in our institution. Here, we report the treatment results of paclitaxel, ifosfamide and nedaplatin chemotherapy. METHODS: From 2006 to 2015, 33 patients with metastatic urothelial carcinoma were treated with paclitaxel, ifosfamide and nedaplatin chemotherapy after failure of first-line cisplatin-based chemotherapy in our institution. We retrospectively examined the treatment outcome and predictive factors for therapeutic effects of paclitaxel, ifosfamide and nedaplatin. The median age, treatment cycle and follow-up period were 62.5 years, 3 cycles and 10.4 months, respectively. RESULTS: The median overall survival and progression-free survival were 10.4 and 3.5 months, respectively. Complete and partial responses were found in 3 and 7 patients, respectively, with an overall response rate of 30%. All patients developed grade 3-4 neutropenia, but there was no treatment-related death. In multivariate analysis, the only prognostic factor for progression-free survival was 24-hour urinary creatinine clearance. CONCLUSIONS: A paclitaxel, ifosfamide and nedaplatin regimen as second-line chemotherapy for metastatic urothelial carcinoma was effective and tolerable. Moreover, paclitaxel, ifosfamide and nedaplatin chemotherapy may be more effective in patients with satisfactory renal function.

Risk stratification using Bmi-1 and Snail expression is a useful prognostic tool for patients with upper tract urothelial carcinoma.

OBJECTIVES: To investigate the expression levels of E-cadherin, Snail, Twist and Bmi-1 in the human upper tract urothelial carcinoma, and to assess whether these factors could be prognostic markers. METHODS: Immunohistochemistry was carried out to determine the expression of E-cadherin, Snail, Twist and Bmi-1 in upper tract urothelial carcinoma samples from 144 patients that underwent total nephroureterectomy between January 1995 and December 2010. The patient population had a median age of 71 years, and comprised 104 men and 40 women, with a median follow-up period of 40 months. The prognostic value of these markers was assessed by univariate and multivariate analysis. A risk stratification analysis was carried out. RESULTS: Snail and Bmi-1 expression predicted worse overall survival (P = 0.0075 and 0.0035), cancer-specific survival (P = 0.0919 and 0.0085) and recurrence-free survival (P = 0.0360 and 0.0817, respectively) compared with tumors that lacked Snail and Bmi-1 expression. Additionally, clinical parameters, grade, stage and lymphovascular invasion correlated with overall survival, cancer-specific survival and recurrence-free survival. Multivariate analysis showed that Bmi-1 expression was among the most significant factors in predicting cancer-specific survival (P = 0.0333). The combination of Snail, Bmi-1 and pathological stage was the most useful prognostic biomarker for upper tract urothelial carcinoma. CONCLUSION: Risk stratification by epithelial-mesenchymal transition and cancer stem cell-regulated genes, such as Snail and Bmi-1, might be useful prognostic markers for upper tract urothelial carcinoma.

Treatment of cerebral ischemia-reperfusion injury with PEGylated liposomes encapsulating FK506.

FK506 (Tacrolimus) has the potential to decrease cerebral ischemia-reperfusion injury. However, the clinical trial of FK506 as a neuroprotectant failed due to adverse side effects. This present study aimed to conduct the selective delivery of FK506 to damaged regions, while at the same time reducing the dosage of FK506, by using a liposomal drug delivery system. First, the cytoprotective effect of polyethylene glycol-modified liposomes encapsulating FK506 (FK506-liposomes) on neuron-like pheochromocytoma PC12 cells was examined. FK506-liposomes protected these cells from H2O2-induced toxicity in a dose-dependent manner. Next, we investigated the usefulness of FK506-liposomes in transient middle cerebral artery occlusion (t-MCAO) rats. FK506-liposomes accumulated in the brain parenchyma by passing through the disrupted blood-brain barrier at an early stage after reperfusion had been initiated. Histological analysis showed that FK506-liposomes strongly suppressed neutrophil invasion and apoptotic cell death, events that lead to a poor stroke outcome. Corresponding to these results, a single injection of FK506-liposomes at a low dosage significantly reduced cerebral cell death and ameliorated motor function deficits in t-MCAO rats. These results suggest that liposomalization of FK506 could reduce the administration dose by enhancing the therapeutic efficacy; hence, FK506-liposomes should be a promising neuroprotectant after cerebral stroke.

Upregulation of N-methyl-D-aspartate receptor subunits and c-Fos expressing genes in PC12D cells by nobiletin.

The N-methyl-D-aspartate (NMDA) receptor plays a key role in learning and memory. Our recent studies have shown that nobiletin from citrus peels activates the cAMP response element-binding protein (CREB) signaling pathway and ameliorates NMDA receptor antagonist-induced learning impairment by activating extracellular signal-regulated kinase. For the first time, we have shown that nobiletin significantly upregulated mRNA expression of the NMDA receptor subunits NR1, NR2A, and NR2B in PC12D cells. Furthermore, c-Fos mRNA expression also increased due to the action of nobiletin. Our results indicate that nobiletin modulates the expression of essential genes for learning and memory by activating the CREB signaling pathway, and suggest that this action mechanism of nobiletin plays a crucial role in improving NMDA receptor antagonist-induced learning impairment in model animals with dementia.

Real-time trafficking of PEGylated liposomes in the rodent focal brain ischemia analyzed by positron emission tomography.

Aliposomal drug delivery system was previously applied to ischemic brain model rats for the treatment of brain ischemia, and we observed that 100-nm-sized liposomes could extravasate and accumulate in the ischemic brain region even when cerebral blood flow was markedly reduced in permanent middle cerebral artery occlusion (p-MCAO) model rats. In the present study, we investigated the real-time cerebral distribution of polyethylene glycol (PEG)-modified liposomes (PEGliposomes) labeled with 1-[18F]fluoro-3,6-dioxatetracosane in p-MCAO rats by positron emission tomography (PET). [18F]-Labeled PEG-liposomes were intravenously injected into p-MCAO rats 1 h after the onset of occlusion, and then a PET scan was performed for 2 h. The PET scan showed that the signal intensity of [18F] gradually increased in the ischemic region despite the drastic reduction in cerebral perfusion, suggesting that PEG-liposomes had accumulated in and around the ischemic region. Therefore,drug delivery to the ischemic region by use of liposomes would be possible under ischemic conditions, and a liposomal drug delivery system could be a promising strategy for protecting the ischemic brain from damage before recovery from ischemia.

Development of a miR-92a delivery system for anti-angiogenesis-based cancer therapy.

BACKGROUND: RNA interference has received much attention as a novel therapeutic strategy. MicroRNA (miRNA) appears to be promising as a novel nucleic-acid medicine because it is able to suppress a series of protein expression that relates to a specific event such as angiogenesis. In the present study, we used dicetyl phosphate-tetraethylenepentamine-based polycation liposomes (TEPA-PCL) as a delivery system for miR-92a, one of the miRNAs regulating angiogenesis, and attempted to deliver miR-92a to angiogenic endothelial cells for the development of cancer therapy by anti-angiogenesis. METHODS: Cholesterol-grafted miR-92a (miR-92a-C) was bound to TEPA-PCL, and the ratio of nitrogen of TEPA-PCL to phosphorus of miR-92a-C (N/P ratio) was optimized. This complex was transfected into human umbilical vein endothelial cells (HUVECs), and the intracellular localization of miR-92a-C was observed under a confocal laser-scanning microscope by the use of fluorescein isothiocyanate-labeled miR-92a-C. After transfection of HUVECs with miR-92a-C/TEPA-PCL, the expression of miR-92a-target proteins (e.g. integrin α5, mitogen-activated protein kinase kinase 4, sphingosine-1-phosphate receptor 1) was examined by western blotting, and a tube formation assay was performed. RESULTS: The complex of miR-92a-C with TEPA-PCL was formed and miR-92a-C remained stable with TEPA-PCL at the N/P ratio of 10. After transfection of HUVECs with miR-92a-C complex, miR-92a-C spread into the whole cytoplasm of the cells without any change of cellular morphology, and the expression of several proteins encoded by miR-92a-target genes was suppressed. Furthermore, the capability of forming capillary tubes was impaired in complex-treated HUVECs. CONCLUSIONS: We have developed a miR-92a delivery system into angiogenic endothelial cells by the use of TEPA-PCL. These results suggest that miR-92a-C/TEPA-PCL is promising for the treatment of tumors via the suppression of angiogenesis.

Nanoparticles accumulate in ischemic core and penumbra region even when cerebral perfusion is reduced.

The use of a liposomal drug delivery system is a promising strategy for avoiding side effects and enhancing drug efficiency by changing the distribution of the intact drug. We have previously shown that liposomal agents quickly accumulated in an ischemia-reperfusion region and ameliorated cerebral ischemia-reperfusion injury when they were injected after reperfusion in transient middle cerebral artery occlusion (t-MCAO) rats. In the present study, we hypothesized that liposomes also act effectively as a drug carrier in the ischemic state, since the integrity of the blood brain barrier is disrupted at an early stage after an ischemic event. To test this hypothesis, the cerebral distribution of fluorescence-labeled liposomes was observed in permanent MCAO (p-MCAO) rats. The liposomes accumulated in the ischemic core and the penumbra region when injected at 1 or 2h after occlusion. The accumulation in the ischemic core region was clearly greater than that in the penumbra region, despite the cerebral blood perfusion of the core region being substantially reduced. This result suggests that drug delivery to an ischemic region using liposomes is possible even when cerebral blood circulation has not recovered. Because liposomal drug delivery systems have the potential to effectively employ a number of agents that have failed in clinical trials, they may offer an effective strategy for achieving neuroprotection in stroke patients.