Ex-vivo Fluorescence Research Articles

In-vivo toxicological study of cysteamine modified carbon dots derived from Ruellia simplex on fruit fly for potential bioimaging

Modern medicine must shift its paradigm from conventional treatment strategies to theranostics to meet individuals' needs. Carbon dots, as a class of fluorescent materials, provide biocompatible and multifunctional solutions for a wide range of applications including clinical sensing, imaging, and drug delivery. Various studies have focused on their synthesis, photophysical properties, and innoxious nature but their potential biomedical applications for the in-vivo toxicity are yet limited. In this work, previously synthesized C-Dots (CDs) were utilized to determine their effect on the development of Drosophila melanogaster. Simultaneously, the genotoxic potential of CDs was evaluated on specific larval cell types that play important roles in immunological defence as well as growth and development. The gut organ toxicity of both CDs was studied using DAPI and DCFH-DA dyes wherein RS-CDs didn't show significant toxicity to the concentration 500 μg/mL whereas RS-Cys-CDs showed nuclear fragmentation and modest ROS (reactive oxygen species) production. Subsequently, trypan-blue assay, larvae crawling assay, touch sensitivity, adult phenotype, and survivability assay were performed. The trypan-blue assay shows the non-toxic nature of both CDs even at the concentration of 500 μg/mL. The high concentrations of RS-Cys-CD (500 μg/mL) were further associated with the alteration in touch behaviour and decrease in pupa hatching. In-vivo and ex-vivo fluorescence assessment of both the CDs exhibit bright fluorescence in green and red channels upon excitation at 485 and 577 nm respectively. The prominent imaging results from RS-Cys-CDs highlight the positive impact of surface modification.

Pharmaceutical Nanoplatforms Based on Self-nanoemulsifying Drug Delivery Systems for Optimal Transport and Co-delivery of siRNAs and Anticancer Drugs

Small interfering RNAs (siRNAs) have the ability to induce selective gene silencing, although siRNAs are vulnerable to degradation in vivo. Various active pharmaceutical ingredients (APIs) are currently used as effective therapeutics in the treatment of cancer. However, routes of administration are limited due to their physicochemical and biopharmaceutical properties. This research aimed to develop oral pharmaceutical formulations based on self-nanoemulsifying drug delivery systems (SNEDDS) for optimal transport and co-delivery of siRNAs related to cancer and APIs. Formulations were developed using optimal mixing design (Design-Expert 11 software) for SNEDDS loading with siRNA (water/oil emulsion), API (oil/water emulsion), and siRNA-API (multiphase water/oil/water emulsion). The final formulations were characterized physicochemically and biologically. The nanosystems less than 50 nm in size had a drug loading above 48 %. The highest drug release occurred at intestinal pH, allowing drug protection in physiological fluids. SNEDDS-siRNA-APIs showed a twofold toxicity effect than APIs in solution and higher transfection and internalization of siRNA in cancer cells with respect to free siRNAs. In the duodenum, higher permeability was observed with SNEDDS-API than with the API solution, as determined by ex-vivo fluorescence microscopy. The multifunctional formulation based on SNEDDS was successfully prepared, siRNA, hydrophobic chemotherapeutics (doxorubicin, valrubicin and methotrexate) and photosensitizers (rhodamine b and protoporphyrin IX) agents were loaded, using a chitosan-RNA core, and Labrafil® M 1944 CS, Cremophor® RH40, phosphatidylcholine shell, forming stable hybrid SNEDDS as multiphasic emulsion, suitable as co-delivery system with a potent anticancer activity.

Neonatal stress disrupts the glymphatic system development and increases the susceptibility to Parkinson's disease in later life.

Neonatal stress disrupts brain development and increases the risk of neurological disorders later in life. However, the impact of neonatal stress on the development of the glymphatic system and susceptibility to Parkinson's disease (PD) remains largely unknown. Neonatal maternal deprivation (NMD) was performed on mice for 14 consecutive days to model chronic neonatal stress. Adeno-associated virus expressing A53T-α-synuclein (α-syn) was injected into the substantia nigra to establish PD model mice. Glymphatic activity was determined using invivo magnetic resonance imaging, exvivo fluorescence imaging and microplate assay. The transcription and expression of aquaporin-4 (AQP4) and other molecules were evaluated by qPCR, western blotting, and immunofluorescence. Animal's responses to NMD and α-syn overexpression were observed using behavioral tests. Glymphatic activity was impaired in adult NMD mice. AQP4 polarization and platelet-derived growth factor B (PDGF-B) signaling were reduced in the frontal cortex and hippocampus of both young and adult NMD mice. Furthermore, exogenous α-syn accumulation was increased and PD-like symptoms were aggravated in adult NMD mice. The results demonstrated that NMD could disrupt the development of the glymphatic system through PDGF-B signaling and increase the risk of PD later in life, indicating that alleviating neonatal stress could be beneficial in protecting the glymphatic system and reducing susceptibility to neurodegeneration.

MP80-07 MICROSCOPICAL ANALYSIS OF FRESH PROSTATE SPECIMENS: PRELIMINARY FINDINGS ABOUT NERVE DISTRIBUTION ON PROSTATE APEX

MP80-07 MICROSCOPICAL ANALYSIS OF FRESH PROSTATE SPECIMENS: PRELIMINARY FINDINGS ABOUT NERVE DISTRIBUTION ON PROSTATE APEX

Ex-vivo fluorescence confocal microscopy with digital staining for characterizing basal cell carcinoma on frozen sections: A comparison with histology.

Ex-vivo fluorescence confocal microscopy (FCM) has been used on fresh tissue, but there is little experience on frozen sections. We evaluated the applicability of FCM on frozen sections of basal cell carcinomas (BCCs), stained with acridine orange and digitally colored to simulate hematoxylin and eosin (H&E) dyes. We compared our diagnostic accuracy in detecting and subtyping BCCs with FCM to our gold standard (H&E stained frozen sections used in 3D horizontal micrographic surgery). Fourty-six primary BCCs were analyzed for free margins as well as histological subtype with all FCM modes and conventional H&E staining. Adnexa, artifacts and diagnostic confidence were evaluated. Free margins were identified with a sensitivity and specificity of 92% and 91%. Concordance for tumor subtype was 88%. FCM may be used on both fresh tissue and frozen samples, although with reduced performance and different artifacts. The device is useful for the intraoperative diagnosis, subtyping and margin-mapping of BCCs.

Assessment of a liposomal CT/optical contrast agent for image-guided head and neck surgery

This study evaluates a long-acting liposomal fluorescence / CT dual-modality contrast agent (CF800) in head and neck cancer to enhance intraoperative tumor demarcation with fluorescence imaging and cone-beam computed tomography (CBCT). CF800 was administered to 12 buccal cancer-bearing rabbits. Imaging was acquired at regular time points to quantify time-dependent contrast enhancement. Surgery was performed 5–7 days after, with intraoperative near-infrared fluorescence endoscopy and CBCT, followed by histological and ex-vivo fluorescence assessment. Tumor enhancement on CT was significant at 24, 96 and 120 hours. Volumetric analysis of tumor segmentation showed high correlation between CBCT and micro-CT. Fluorescence signal was apparent in both ex-vivo and in-vivo imaging. Histological correlation showed [100%] specificity for primary tumor. Sensitivity and specificity of CF800 in detecting nodal involvement require further investigation.CF800 is long acting and has dual function for CT and fluorescence contrast, making it an excellent candidate for image-guided surgery.

PD54-12 EX-VIVO FLUORESCENCE CONFOCAL MICROSCOPY: EVALUATION OF THE LEARNING CURVE FOR INTERPRETATION OF PROSTATIC BENIGN AND MALIGNANT TISSUES

INTRODUCTION AND OBJECTIVE:Ex-vivo fluorescence confocal microscopy (FCM) is an optical technology that provides fast H&E like images of freshly excised tissues and it has been mainly used for “rea...

UP42 - Ex-vivo fluorescence confocal microscopy: Evaluation of the learning curve for interpretation of prostatic benign and malignant tissues

UP42 - Ex-vivo fluorescence confocal microscopy: Evaluation of the learning curve for interpretation of prostatic benign and malignant tissues

Diagnostic accuracy of exvivo fluorescence confocal microscopy in Mohs surgery of basal cell carcinomas: aprospective study on 753 margins.

Frozen histological sections are used for intraoperative margin assessment during Mohs surgery. Fluorescence confocal microscopy (FCM) is a new tool that offers a promising and faster alternative to frozen histology. To evaluate prospectively in a clinical setting the accuracy of FCM vs. frozen sections in margin assessment of basal cell carcinoma (BCC). Patients with BCC scheduled for Mohs surgery were prospectively enrolled. Freshly excised surgical specimens were examined by FCM and then frozen sections were evaluated. Permanent sections were obtained, in order to validate the sample technique. A blind re-evaluation was also performed for discordant cases. Sensitivity and specificity levels, as well as positive and negative predictive values (PPV and NPV, respectively), were calculated and receiver-operating characteristic curves generated. We enrolled 127 BCCs in as many patients (40·2% females). Seven hundred and fifty-three sections were examined. All BCCs were located in the head and neck area. In evaluating the performance of FCM vs. frozen sections, sensitivity was 79·8%, specificity was 95·8%, PPV was 80·5% and NPV was 95·7% [area under the curve 0·88, 95% confidence interval 0·84-0·92 (P < 0·001)]. Forty-nine discordant cases were re-evaluated; 24 were false positive and 25 false negative. The performance of FCM and frozen sections was also evaluated according to the final histopathological assessment. We found high levels of accuracy for FCM vs. frozen section evaluation in intraoperative BCC margin assessment during Mohs surgery. Some technical issues prevent the wide use of this technique, but new devices promise to overcome these limitations.

Near-infrared mediated quantum dots and paclitaxel co-loaded nanostructured lipid carriers for cancer theragnostic

Timing is an important factor in cancer management. Theragnostic systems have benefit of improving patients' life-quality by expediting therapeutic decisions. The objective of this study was to explore the potential of co-loaded [quantum dots (CdTe/CdS/ZnS) and paclitaxel] NLC (nanostructured lipid carriers) as a parenteral multifunctional delivery system. The co-loaded NLC was prepared by emulsion-evaporation and low temperature-solidification method utilising glyceryl monostearate, oleic acid, and soya phosphatidylcholine as lipid matrix. In characterising the co-loaded NLC, physicochemical properties of particle size, polydispersity index (PDI), zeta potential (ZP), morphology, encapsulation efficacy (EE) and drug loading (DL) were investigated. Moreover, in-vitro paclitaxel release profile, cytotoxicity, histopathological, in-vivo anti-tumour efficacy, and in-vivo and ex-vivo fluorescence optical imaging abilities of the co-loaded NLC were assessed. The mean particle size, PDI and ZP were reported to be 115.93±1.61nm, 0.17±0.04 and −0.22±0.03mV, respectively. The particles were spheroid-like in shape with relatively smooth surface. A higher EE (80.70±2.11%) and DL (4.68±0.04%) were recorded. The coloaded NLC exhibited a biphasic pattern of drug release. IC50 value was found to be 1.05±0.58μM. The tumour growth inhibition rate of 77.85% was registered. The in-vivo and ex-vivo imaging results indicated capability of the co-loaded NLC to specifically target and detect the H22 tumour. Tissues showed no significant cytoarchitectural differences. We can satisfactorily conclude that co-loaded NLC formulation can be qualified as a splendid parenteral drug delivery system foundation for cancer theragnostic.

MP79-18 EX-VIVO EVALUATION OF INTRAPROSTATIC TRACER DISTRIBUTION OF TARGETED VERSUS TEMPLATE PERINEAL INJECTION OF FREE INDOCYANINE GREEN FOR FLUORESCENCE TARGETED PELVIC LYMPH NODE DISSECTION (F-T PLND) IN PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Staging II1 Apr 2016MP79-18 EX-VIVO EVALUATION OF INTRAPROSTATIC TRACER DISTRIBUTION OF TARGETED VERSUS TEMPLATE PERINEAL INJECTION OF FREE INDOCYANINE GREEN FOR FLUORESCENCE TARGETED PELVIC LYMPH NODE DISSECTION (F-T PLND) IN PROSTATE CANCER Christine Englberger, Tobias Schätz, Thomas Kunit, Martin Drerup, Matthias Meissnitzer, Rosemarie Forstner, Esra Foditsch, Peter Törzsök, Martina Hager, Günter Janetschek, Karl-Dietrich Sievert, and Stephan Hruby Christine EnglbergerChristine Englberger More articles by this author , Tobias SchätzTobias Schätz More articles by this author , Thomas KunitThomas Kunit More articles by this author , Martin DrerupMartin Drerup More articles by this author , Matthias MeissnitzerMatthias Meissnitzer More articles by this author , Rosemarie ForstnerRosemarie Forstner More articles by this author , Esra FoditschEsra Foditsch More articles by this author , Peter TörzsökPeter Törzsök More articles by this author , Martina HagerMartina Hager More articles by this author , Günter JanetschekGünter Janetschek More articles by this author , Karl-Dietrich SievertKarl-Dietrich Sievert More articles by this author , and Stephan HrubyStephan Hruby More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2016AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES In recent works, we described the evolution and outcome of ft-PLND utilizing Indocyan Green (ICG) in Prostate cancer. The purpose of this study was to evaluate how free ICG dissolves throughout the prostate and if a TRUS/MRI-targeted injection could add more precision. METHODS We evaluated 8 targeted and 7 template- injected prostate specimens of patients in whom a high or intermediate risk prostate cancer was detected. All patients underwent transperitoneal laparoscopic prostatectomy and f-t PLND using a Storz camera system. In patients, where a distinct index tumor was detected by TRUS/MRI Fusion biopsy 1cc free ICG/ 1 cc cancer volume was injected perineally into and around the tumor by the same means at the beginning of the operation. In the other cases ICG was injected TRUS-guided perineally into the peripheral zone into three definded templates (2.5 cc/ 3 portions (Base/Mid/Apex )/ each lobe). After removal of all ICG visualized lymph node templates, a routine super-extended PLND was carried out as control. From all lymph nodes 250 µm paraffin sections were prepared and analyzed by immunohistochemistry. In addition, we analyzed the intraprostatic tracer distribution and concentration of the locally injected ICG to by using a fluorescence imaging system ex vivo. RESULTS In total, 125 sections of 15 prostates have been analyzed. A fluorescence signal in the different sections of the prostate could be visualized in every patient. Free ICG stayed at the site of injection and did not dissolve completely throughout the prostate. The average diameter of fluorescence activity was 8 mm in the targeted vs. 11 mm in the template injected group. F-activity in the index tumor was found in all patients in the targeted group vs. 4 patients (57 %) in the template group. Additional tumor was not covered in 53 % (4 targeted vs 3 template). The average number of F-visualized Lymphnodes was 15 in the template group vs 8,2 LN in the targeted group. CONCLUSIONS Ex-vivo fluorescence imaging of the prostate could show that free ICG stays at the injection side and does not dissolve throughout the prostate. Although a targeted injection into and around the area of the tumor allows for accurate studying the drainage pattern of the index lesion, it could also miss some tumor, which is maybe not visualized by MRI. Therefore a combined approach seems to be the most promising method and should be advised to identify the complete drainage pattern of the all tumor tissue of the prostate. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1041 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Christine Englberger More articles by this author Tobias Schätz More articles by this author Thomas Kunit More articles by this author Martin Drerup More articles by this author Matthias Meissnitzer More articles by this author Rosemarie Forstner More articles by this author Esra Foditsch More articles by this author Peter Törzsök More articles by this author Martina Hager More articles by this author Günter Janetschek More articles by this author Karl-Dietrich Sievert More articles by this author Stephan Hruby More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Targeting Triple Negative Breast Cancer with a Small-sized Paramagnetic Nanoparticle.

There is no available targeted therapy or imaging agent for triple negative breast cancer (TNBC). We developed a small-sized dendrimer-based nanoparticle containing a clinical relevant MRI contrast agent, GdDOTA and a NIR fluorescent dye, DL680. Systemic delivery of dual-modal nanoparticles led to accumulation of the agents in a flank mouse model of TNBC that were detected by both optical and MR imaging. In-vivo fluorescence images, as well as ex-vivo fluorescence images of individual organs, demonstrated that nanoparticles accumulated into tumor selectively. A dual modal strategy resulted in a selective delivery of a small-sized (GdDOTA)42-G4-DL680 dendrimeric agent to TNBC tumors, avoiding other major organs.

FRI0038 Fluorescent fusion protein for molecular imaging of TNF in mouse autoimmune disease models

BackgroundTumor necrosis factor (TNF) plays an essential function in host defense. On the other hand, its overexpression may be deleterious and lead to autoimmune disorders. Therapeutic inhibition of TNF (mostly...

Anti-tumor activity of liposomal glucocorticoids: The relevance of liposome-mediated drug delivery, intratumoral localization and systemic activity

Tumor-associated inflammation has been recognized as an important tumor growth propagator and, therefore, represents an attractive target for anti-cancer therapy. In the current study, inspired by recent findings on the anti-tumor activity of liposomal glucocorticoids, we introduce paramagnetic and fluorescent liposomes, encapsulating prednisolone phosphate (PLP), to evaluate the local delivery of liposomal glucocorticoids to the tumor and its importance for the therapeutic response. The new multifunctional liposomes (Gd–PLP–L) (120 nm diameter, 5.8 mg PLP/60 μmol lipid, bioexponential blood-clearance kinetics (T 1/2α = 2.4 ± 0.5 h, T 1/2β = 42.0 ± 12.4 h), drug leakage of 15%/72 h ( in vitro)), containing 25 mol% Gd–DTPA–lipid and 0.1 mol% of rhodamine–lipid, were tested in B16F10 melanoma subcutaneously inoculated in C57BL/6 mice, and compared to the original PLP formulation (PLP–L). A single dose of Gd–PLP–L (20 mg PLP/kg/week, i.v.) was found to significantly inhibit tumor growth compared to non-treated mice (P < 0.05), similarly to PLP–L. The accumulation efficacy of the liposomal agent in the tumor was assessed with MRI, using the increase in the longitudinal relaxation rate (ΔR 1) as a marker. Interestingly, large inter-tumor differences in ΔR 1 (0.009–0.063 s − 1 , 24 h post-administration), corresponding to highly variable intratumoral Gd–PLP–L levels, did not correlate to the effectiveness of tumor growth inhibition. Uptake of liposomes by tumor-associated macrophages (TAM), determined by ex-vivo fluorescence microscopy, was limited to only 5% of the TAM population. Furthermore, the therapy did not lead to TAM depletion. Importantly, a 90% drop in white blood cell count both after Gd–PLP–L and PLP–L administration was observed. This depletion may reduce tumor infiltration of monocytes, which stimulate angiogenesis, and, thus, possibly co-contributes to the anti-tumor effects. In conclusion, MRI provides a powerful instrument to monitor the delivery of liposomal therapeutics to tumors and guided us to reveal that the activity of liposomal glucocorticoids is not limited to the tumor site only.

Hochauflösende Fluoreszenzmikroskopie des retinalen Pigmentepithels mittels strukturierter Beleuchtung

The accumulation of autofluorescent bodies in retinal pigment epithelium (RPE) cells has an impact on the pathogenesis of retinal diseases, including age-related macular degeneration. While current invivo fluorescence microscopy allows a lateral resolution of fluorophores in a micrometer range, with ex vivo microscopy a lateral resolution down to 200nm is possible. For the first time, we used structured illumination microscopy for exvivo high-resolution fluorescence microscopy of RPE cells. Histological sections were prepared from a 68-year-old patient. With epifluorescence microscopy, fluorescent RPE cells were detectable. Structured illumination uses inhomogeneous illumination for resolution of previously nonresolvable structures, similar to the Moiré effect. Images were taken from RPE cells at different excitation wavelengths (488, 568, and 647nm) and were reconstructed with special software. The different excitation patterns of the fluorescent granules in the RPE cells were colour-coded and analysed. With structured illumination microscopy, autofluorescence signals of RPE cells were detectable, and a lateral resolution of 110nm could be achieved. Using varying wavelengths, different pigments were excitable. Lipofuscin gave the highest signals, at 488 and 568nm. The improved resolution showed inhomogeneous intragranular fluorophore patterns. Structured illumination microscopy enabled us to generate images of fluorescent structures in RPE cells exvivo with a lateral resolution of 110nm. With the use of different excitation wavelengths, intracellular fluorescence patterns in single cell compartments are visible and allow further differentiation.