Near-infrared-fluorescence imaging of lymph nodes by using liposomally formulated indocyanine green derivatives.

Liposomally formulated indocyanine green (LP-ICG) has drawn much attention as a highly sensitive near-infrared (NIR)-fluorescence probe for tumors or lymph nodes in vivo. We synthesized ICG derivatives tagged with alkyl chains (ICG-Cn), and we examined NIR-fluorescence imaging for lymph nodes in the lower extremities of mice by using liposomally formulated ICG-Cn (LP-ICG-Cn) as well as conventional liposomally formulated ICG (LP-ICG) and ICG. Analysis with a noninvasive preclinical NIR-fluorescence imaging system revealed that LP-ICG-Cn accumulates in only the popliteal lymph node 1h after injection into the footpad, whereas LP-ICG and ICG accumulate in the popliteal lymph node and other organs like the liver. This result indicates that LP-ICG-Cn is a useful NIR-fluorescence probe for noninvasive in vivo bioimaging, especially for the sentinel lymph node.

Preparation and characterization of phospholipid-conjugated indocyanine green as a near-infrared probe.

We have rationally designed and synthesized a novel near-infrared (NIR) photoactivating probe, designated by iDOPE, in which an indocyanine green (ICG) fluorophore is covalently conjugated with a phospholipid moiety, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), to incorporate into liposome bilayers. NIR irradiation showed that iDOPE retained the optical and fluorescence properties of ICG and demonstrated photoactivator characteristics: fluorescence emission at around 820 nm in a solvent, singlet oxygen production, and concentration-dependent heat generation. Additionally, iDOPE was incorporated into liposome bilayers and maintained stable liposomally formulated iDOPE (LP-iDOPE) over 1week under physiological conditions. We also observed the tumor-specific biodistribution of LP-iDOPE of in vivo xenografts. These findings suggest that LP-iDOPE might be a promising tool for NIR optical imaging, photodynamic therapy, and photothermal therapy.

Modulation of the activity of cytosolic phospholipase A2alpha (cPLA2alpha) by cellular sphingolipids and inhibition of cPLA2alpha by sphingomyelin.

We examined the effect of the cellular sphingolipid level on the release of arachidonic acid (AA) and activity of cytosolic phospholipase A2alpha (cPLA2alpha) using two Chinese hamster ovary (CHO)-K1-derived mutants deficient in sphingolipid synthesis: LY-B cells defective in the LCB1 subunit of serine palmitoyltransferase for de novo synthesis of sphingolipid species, and LY-A cells defective in the ceramide transfer protein CERT for SM synthesis. When LY-B and LY-A cells were cultured in Nutridoma medium and the sphingolipid level was reduced, the release of AA stimulated by the Ca(2+) ionophore A23187 increased 2-fold and 1.7-fold, respectively, compared with that from control cells. The enhancement in LY-B cells was decreased by adding sphingosine and treatment with the cPLA2alpha inhibitor. When CHO cells were treated with an acid sphingomyelinase inhibitor to increase the cellular SM level, the release of AA induced by A23187 or PAF was decreased. In vitro studies were then conducted to test whether SM interacts directly with cPLA2alpha. Phosphatidylcholine vesicles containing SM reduced cPLA2alpha activity. Furthermore, SM disturbed the binding of cPLA2alpha to glycerophospholipids. These results suggest that SM at the biomembrane plays important roles in regulating the cPLA2alpha-dependent release of AA by inhibiting the binding of cPLA2alpha to glycerophospholipids.

Medical and Dental Visits of Chronic Kidney Disease-Diagnosed Participants Analyzed From the Specific Health Checkups Results in Japan: TAMA MED Project-CKD.

BACKGROUND: Since 2012, Tama City has promoted the early detection of chronic kidney disease (CKD), through an initiative that measures serum creatinine as part of the specific health checkups. We examined preventive measures against CKD deterioration based on the outcomes of this initiative. METHODS: The complications, medication status, body mass index, smoking status and other determining factors were surveyed among CKD-diagnosed participants over 3 years between 2013 and 2015. Moreover, factors aggravating CKD were investigated via a survey of medical and dental visits based on health insurance claim data over the same period. RESULTS: There was an increased rate of comorbid hypertension with each increase in the CKD stage. Comorbidity rates of diabetes mellitus, dyslipidemia, obesity, and smoking increased until CKD stage G4, and then decreased from stage G5. A substantial number of participants with CKD stage G3b and above were not medicated despite comorbidities like hypertension, diabetes mellitus and dyslipidemia. While the rate of regular visits at medical institutions was seen to increase significantly in accordance with the worsening degree of CKD, there were also individuals who, despite having severe CKD, did not visit medical institutions specializing in internal medicine. The rate of dental visits decreased as the CKD stage increased, and further decreased as the diabetic control status worsened. CONCLUSIONS: CKD patients should become aware of the importance of the dental visit because only a limited number of patients with advanced CKD received dental care.

Use of Aeromonas spp. as General Indicators of Antimicrobial Susceptibility among Bacteria in Aquatic Environments in Thailand.

Antimicrobials are widely used, not only for treating human infections, but also for treatment of livestock and in fish farms. Human habitats in Southeastern Asian countries are located in close proximity to aquatic environments. As such, the human populations within these regions are at risk of exposure to antimicrobial resistant bacteria, and thereby disseminating antimicrobial resistance genes (ARGs). In this study, we collected water samples from 15 sites (5 sites in Chao Phraya River, 2 sites at the mouth of Chao Phraya River, 3 sites in Ta Chin River, and 5 sites at city canals) and 12 sites (6 sites at city canals; 2 sites at chicken farms; 2 sites at pig farms; and 2 samples from sites at pig farms, which were subsequently treated at a biogas plant) in Thailand in 2013 and 2014, respectively. In total, 117 Aeromonas spp. were isolated from the water samples, and these organisms exhibited various antimicrobial susceptibility profiles. Notably, there was a significant correlation between the environmental concentration of tetracyclines and the rates of tetracycline resistance in the isolated Aeromonas spp.; however, both the concentration and rates of tetracycline resistance in samples derived from pig farms were higher than those of samples harvested from other aquatic environments. These findings suggest that the high concentrations of antimicrobials observed in these aquatic environments likely select for ARGs. Furthermore, they indicate that Aeromonas spp. comprise an effective marker for monitoring antimicrobial resistance in aquatic environments.

Liposomally formulated phospholipid-conjugated indocyanine green for intra-operative brain tumor detection and resection.

Some tumor-specific near-infrared (NIR) fluorescent dyes such as indocyanine green (ICG), IDRye800CW, and 5-aminolevulinic acid have been used clinically for detecting tumor margins or micro-cancer lesions. In this study, we evaluated the physicochemical properties of liposomally formulated phospholipid-conjugated ICG, denoted by LP-iDOPE, as a clinically translatable NIR imaging nanoparticle for brain tumors. We also confirmed its brain-tumor-specific biodistribution and its characteristics as the intra-operative NIR imaging nanoparticles for brain tumor surgery. These properties of LP-iDOPE may enable neurosurgeons to achieve more accurate identification and more complete resection of brain tumor.

Detection of peritoneal dissemination with near-infrared fluorescence laparoscopic imaging using a liposomal formulation of a synthesized indocyanine green liposomal derivative.

BACKGROUND/AIM: Although conventional staging laparoscopy (SL) has improved the diagnostic accuracy of peritoneal dissemination, novel technology is needed to increase the sensitivity of SL. We herein describe a new imaging method employing near-infrared (NIR) fluorescence imaging using a liposomal synthesized indocyanine green (ICG) liposomal derivative, LP-ICG-C18. METHODS AND RESULTS: LP-ICG-C18 is a NIR-photoactivating probe in which an ICG fluorophore is covalently conjugated with a phospholipid moiety. Nude mice were intraperitoneally injected with gastric cancer cells. Twelve days later, the mice were given intravenous injections of LP-ICG-C18 at a dose of 0.15 mg/kg. A NIR imaging system was used to identify the disseminated tumors. The disseminated nodules in mice were detected without any difficulties. Disseminated tumor nodules were collected from mice with or without injections of liposomal formulation and were transferred into the swine peritoneal cavity. The nodules in the swine peritoneal cavity were clearly and promptly defined by the NIR imaging system. CONCLUSION: NIR-fluorescing liposomal probes can effectively target peritoneal disseminated tumors and can be easily detected by a NIR imaging system. These results warrant future clinical trials of our imaging system and may contribute to a more precise diagnosis and therapeutic approach for gastric cancer patients.

Treatment of near-infrared photodynamic therapy using a liposomally formulated indocyanine green derivative for squamous cell carcinoma.

INTRODUCTION: Photodynamic therapy (PDT) is a less invasive option for cancer treatment that has evolved through recent developments in nanotechnology. We have designed and synthesized a novel liposome system that includes an indocyanine green (ICG) derivative, ICG-C18, in its bilayer. In addition to its use as an optical imager to visualize blood, lymphatic, and bile flow, ICG has also been used as an optical sensitizer. In the present report, we evaluate the use of our novel liposome system, LP-ICG-C18, in PDT for squamous cell carcinoma in an autologous murine model. MATERIALS AND METHODS: An excitation pulse beam (300 µJ/pulse) of a single band (800 nm) was used for sensitization. The cytotoxicity of the photodynamic therapy was evaluated in terms of cellular morphology changes, methyl thiazolyl tetrazolium (MTT) assay results, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining. We tested the enhanced permeability and retention effect of LP-ICG-C18 in tumor-bearing C3H/He mice using a near-infrared fluorescence imaging system and fluorescence microscopy. We also examined the antitumor effect of PDT by measuring tumor volume in tumor-bearing mice. RESULTS: Cell death and apoptosis were only observed in the PDT group receiving LP-ICG-C18. LP-ICG-C18 itself had no cytotoxic activity and showed good biocompatibility. LP-ICG-C18 accumulated on the tumor 24 hours after injection and was retained for approximately 3 weeks. Tumor cell apoptosis following PDT with LP-ICG-C18 was also observed under optical microscopy, MTT assay, and TUNEL staining. CONCLUSION: These findings suggest that LP-ICG-C18 may be an effective intervening material in PDT for malignant disease.

Longitudinal evaluation of local muscle conditions in a rat model of gastrocnemius muscle injury using an in vivo imaging system.

This study aimed to evaluate the time course of local changes during the acute phase of gastrocnemius muscle strain, in a rat model, using an in vivo imaging system. Thirty-eight, 8-week-old Sprague-Dawley male rats were used in our study. Experimental injury of the right gastrocnemius muscle was achieved using the drop-mass method. After inducing muscle injury, a liposomally formulated indocyanine green derivative (LP-iDOPE, 7 mg/kg) was injected intraperitoneally. We evaluated the muscle injuries using in vivo imaging, histological examinations, and enzyme-linked immunosorbent assays. The fluorescence peaked approximately 18 h after the injury, and decreased thereafter. Histological examinations revealed that repair of the injured tissue occurred between 18 and 24 h after injury. Quantitative analyses for various cytokines demonstrated significant elevations of interleukin-6 and tumor necrosis factor-α at 3 and 18 h post-injury, respectively. The time course of fluorescence intensity, measured using in vivo imaging, demonstrated that the changes in cytokine levels and histopathologic characteristics were consistent. Specifically, these changes reached peaked 18 h post-injury, followed by trends toward recovery.

Surface properties and biocompatibility of nitrided titanium for abrasion resistant implant materials.

Corrosion, other related properties and biocompatibility of surface nitrided titanium were investigated to examine its possible use as an abrasion resistant implant material. The nitrided layer about 2 microm thick composed of TiN and Ti2N was formed on titanium by a gas nitriding method. The dissolved amount of titanium ion in SBF was as low as the detection limit of ICP, and that in the 1% lactic acid showed no significant difference from titanium. The tissue reaction of the cylindrical implant in soft tissue of rats showed no inflammation, and fine particles of 1 microm induced phagocytosis, which was similar to titanium. The implantation in the femor showed the new bone formed in direct contact with implants. All the results suggested that the wettability, corrosion resistance, S. mutans adhesion and biocompatibility were nearly equivalent to those of titanium. The surface of nitrided titanium was promising, with biocompatibility comparable with titanium, as an implant material such as for an abutment part of a dental implant, which requires high abrasion resistance.

Inhibition of hemoglobin S polymerization in vitro by a novel 15-mer EF-helix beta73 histidine-containing peptide.

Our mutational studies on Hb S showed that the Hb S beta73His variant (beta6Val and beta73His) promoted polymerization, while Hb S beta73Leu (beta6Val and beta73Leu) inhibited polymerization. On the basis of these results, we speculated that EF-helix peptides containing beta73His interact with beta4Thr in Hb S and compete with Hb S, resulting in inhibition of Hb S polymerization. We, therefore, studied inhibitory effects of 15-, 11-, 7-, and 3-mer EF-helix peptides containing beta73His on Hb S polymerization. The delay time prior to Hb S polymerization increased only in the presence of the 15-mer His peptide; the higher the amount, the longer the delay time. DIC image analysis also showed that the fiber elongation rate for Hb S polymers decreased with increasing concentration of the 15-mer His peptide. In contrast, the same 15-mer peptide containing beta73Leu instead of His and peptides shorter than 11 amino acids containing beta73His including His alone showed little effect on the kinetics of polymerization and elongation of polymers. Analysis by protein-chip arrays showed that only the 15-mer beta73His peptide interacted with Hb S. CD spectra of the 15-mer beta73His peptide did not show a specific helical structure; however, computer docking analysis suggested a lower energy for interaction of Hb S with the 15-mer beta73His peptide compared to peptides containing other amino acids at this position. These results suggest that the 15-mer beta73His peptide interacts with Hb S via the beta4Thr in the betaS-globin chain in Hb S. This interaction may influence hydrogen bond interaction between beta73Asp and beta4Thr in Hb S polymers and interfere in hydrophobic interactions of beta6Val, leading to inhibition of Hb S polymerization.