In vitro and in vivo fluorescence monitoring of photosensitizers

The use of steady state fluorescence spectroscopy in the detection and monitoring of potential photochemotherapeutic agents is examined. Problems associated with both in vitro and in vivo fluorescence measurements are investigated, and typical data are presented. Recent results on the use of fluorescence in pharmacokinetic studies are discussed, and the relative merits of in vitro vs. in vivo methods are outlined.     

In vivo monitoring of amine neurotransmitters using microdialysis with on-line capillary electrophoresis.

Microdialysis sampling was coupled via a flow-gated interface on-line to capillary electrophoresis with laser-induced fluorescence (LIF) detection for in vivo monitoring of neuroactive amino acids and amines. In the instrument, analytes are derivatized precolumn with o-phthaldehyde and beta-mercaptoethanol to form fluorescent isoindole products. The instrument was improved over previous designs by incorporating a sheath-flow cuvette for reduced background in LIF detection which improved sensitivity by 15-fold. The methodology was improved by utilizing a voltage ramped injection which allowed generation of 500000 theoretical plates with 20 s separations. Resolution of the isoindole derivatives was further improved by addition of hydroxypropyl-modified beta-cyclodextrin to the electrophoresis buffer. The new instrumentation and methods allow resolution and detection of glutamate, gamma-aminobutyric acid, glycine, aspartate, serine, taurine, glutamine and dopamine (if levels are elevated) collected from in vivo sampling probes every 20 s. The technique is suited to continuous monitoring for dynamic measurements of these compounds in vivo.     

Evaluation of Zn and Fe in diets of patients with chronic renal failure

Having in mind the importance of Fe and Zn content in the diets of nondialyzed patients with chronic renal failure, diets of 39 patients with ages varying from 18 to 79 years under a conservative treatment were analyzed by INAA. The 24-hour recall method was used for sample collection. The content of proximate composition was also determined. The average daily dietary intake was compared to the new recommended values by the Food and Nutrition Board. It was observed that the diets were deficient for these elements and, therefore, there should be a nutritional follow-up to avoid possible negative effects. This revised version was published online in August 2006 with corrections to the Cover Date.     

In vivo monitoring of the distribution of a monoclonal antibody fragment

This study demonstrates the potential of in vivo, whole body fluorescence imaging for pharmacokinetic studies. The distribution of a novel human anti-tumour antibody fragment, NovoMab-G2-scFv, labelled with a fluorescent dye (Cy5.5.18) was monitored in vivo. The NovoMab-G2-scFv–Cy5 complex was injected via the tail vein into nude mice bearing subcutaneous human melanoma tumour cells. The distribution of fluorescence NovoMab-G2-scFv–Cy5 was imaged non-invasively using a charge-coupled device (CCD) camera. Whole body fluorescence images were acquired 2, 6, 12, 24, 48 and 72 h post-injection. Fluorescence was detected at the tumour site following injection of NovoMab-G2-scFv–Cy5 but not following injection of a labelled irrelevant antibody fragment, demonstrating specific binding of the antibody–dye complex to the tumour. Fluorescence from the tumour site peaked 2 h post-injection and gradually declined, reaching a minimum 72 h post-injection. Fitting an exponential decay to fluorescence data extracted from images allowed the half-time of the antibody at the tumour site to be calculated, and a value of 7.7 h was obtained.Fluorescence was also apparent in the kidneys, indicating clearance of the NovoMab-G2-scFv through the kidneys. Again, fluorescence intensity decreased with time, reaching a minimum 72 h post-injection. Imaging of isolated organs (ex vivo) confirmed the presence of the antibody–dye complex in the tumours, kidneys and liver. No fluorescence was observed in the brain, heart, lungs or spleen, suggesting that these organs do not accumulate the NovoMab-G2-scFv–dye complex.     

In vivo monitoring of the gastrooesophageal system using optical fibre sensors

In the present paper optical fibre sensors for the detection of foregut diseases are described, in particular, sensors for the detection of bile, carbon dioxide and pH. Bile-containing refluxes are measured by means of a sensor which uses bilirubin as natural marker. The sensor, which is already present on the market, has been clinically validated by various hospitals. The clinically relevant parameter is the exposure time of the stomach/oesophagus mucosa to the bile. When measured in the oesophagus, it has been shown to be closely correlated with the onset of Barrett's oesophagus or general oesophagitis. Recently, optical fibres have been proposed for the continuous monitoring of carbon dioxide in the stomach: an important parameter in critically ill patients. A clinically validated prototype has shown its superiority in comparison with the traditional method, that is based on gastric tonometry. For the sake of completeness, also gastric pH sensors are considered, although at the moment their development is stationary at the laboratory stage.     

In vivo monitoring of mercury ions using a rhodamine-based molecular probe

Exposure to mercury causes severe damage to various tissues and organs in humans. Concern over mercury toxicity has encouraged the development of efficient, sensitive, and selective methods for the in vivo detection of mercury. Although a variety of chemosensors have been exploited for this purpose, no in vivo monitoring systems have been described to date. In this report, we describe an irreversible rhodamine chemosensor-based, real-time monitoring system to detect mercury ions in living cells and, in particular, vertebrate organisms. The chemosensor responds rapidly, irreversibly, and stoichiometrically to mercury ions in aqueous media at room temperature. The results of experiments with mammalian cells and zebrafish show that the mercury chemosensor is cell and organism permeable and that it responds selectively to mercury ions over other metal ions. In addition, real-time monitoring of mercury-ion uptake by cells and zebrafish using this chemosensor shows that saturation of mercury-ion uptake occurs within 20-30 min in cells and organisms. Finally, accumulation of mercury ions in zebrafish tissue and organs is readily detected by using this rhodamine-based chemosensor.     

In vivo glucose monitoring: towards 'Sense and Act' feedback-loop individualized medical systems

Glucose biosensors are key components of closed-loop glycaemic control (insulin delivery) systems for effective management of diabetes. By providing a fast return of the analytical information in a timely fashion, such sensors offer direct and reliable assessment of rapid changes in the glucose level, as desired for making optimal and timely therapeutic interventions in cases of hypo- and hyperglycemia. The majority of sensors used for continuous glucose monitoring are amperometric enzyme electrodes. The successful realization of closed-loop glycaemic control requires innovative approaches for addressing major challenges of biofouling, inflammatory response, calibration, stability, selectivity, power, miniaturization, common to other remote sensor systems. The goal of this review article is to demonstrate how these challenges are being addressed towards achieving reliable continuous subcutaneous monitoring of glucose. While the concept is presented here in connection to the management of diabetes, such loop-based individualized integrated (sensing/release) medical systems should lead to a fine-tune drug therapy and should have an enormous impact upon the treatment and management of different diseases.     

In vivo NADH fluorescence monitoring as an assay for cellular damage in photodynamic therapy.

In this study the endogenous fluorescence signal attributed to reduced nicotinamide adenine dinucleotide (NADH) has been measured in response to photodynamic therapy (PDT)-induced damage. Measurements on cells in vitro have shown that NADH fluorescence decreased relative to that of controls after treatment with a toxic dose of PDT, as measured within 30 min after treatment. Similarly, assays of cell viability indicated that mitochondrial function was reduced immediately after treatment in proportion to the dose delivered, and the proportion of this dose response did not degrade further over 24 h. Measurements in vivo were used to monitor the fluorescence emission spectrum and the excited state lifetime of NADH in PDT-treated tissue. The NADH signal was defined as the ratio of the integrated fluorescence intensity of the 450 +/- 25 nm emission band relative to the fluorescence intensity integrated over the entire 400-600 nm range of collection. Measurements in murine muscle tissue indicated a 22% reduction in the fluorescence signal immediately after treatment with verteporfin-based PDT, using a dose of 2 mg/kg injected 15 min before a 48 J/cm2 light dose at 690 nm. Control animals without photosensitizer injection had no significant change in the fluorescence signal from laser irradiation at the same doses. This signal was monotonically correlated to the deposited dose used here and could provide a direct dosimetric measure of PDT-induced cellular death in the tissue being treated.     

In vivo monitoring of the monoamine neurotransmitters in the rat brain by microdialysis coupled with the liquid chromatography dual electrochemical detector

The carbon film based ring-disk dual electrodes in the thin-layer radial flow cell are used as the dual electrochemical detector (DECD) for liquid chromatography (LC) to determine the monoamine neurotransmitters. Cyclic voltammetric experiments show there has great difference in the reversibility of the oxidative reactions of dopamine and ascorbate. Therefore the ring-disk dual electrode arrangement in the radial flow cell can effectively remove the interference of ascorbate and determine dopamine in the LC-DECD. In order to obtain the better collection efficiency (CE) and better peak current of analytes in the LC-DECD, several operational parameters have been investigated: flow rate, pH and the working potentials. Under the optimum conditions, the method shows a good stability and reproducibility to determine dopamine (DA), norepinephrine (NE), 5-hydroxytryptamine (5-HT), epinephrine (E) and 3,4-dihydroxyphenylacetic acid (DOPAC). The limits of detection are 0.1 pmol for DA, 0.1 pmol for NE, 0.1 pmol for 5-HT, 1.0 pmol for E and 0.1 pmol for DOPAC. The application of this method, coupled with microdialysis sampling, for the in vivo determination of the monoamine neurotransmitters in the striatum of the rat brain is satisfactory.     

In vitro and in vivo imaging with quantum dots

Quantum dots (QDs), also named semiconductor nanocrystals, have initiated a new realm of bioscience by combining nanomaterials with biology, which will profoundly influence future biological and biomedical research. In this review, we describe the extraordinary optical properties of QDs and developments in methods for their synthesis. We focus on fluorescent imaging with QDs both in vitro and in vivo, and the cytotoxicity of QDs and potential barriers to their use in practical biomedical applications. Finally, we provide insights into improvements aimed at decreasing the cytotoxicity of QDs and the future outlook of QD applications in biomedical fields.

In vivo monitoring of tissue regeneration using a ratiometric lysosomal AIE probe

Tissue regeneration is a crucial self-renewal capability involving many complex biological processes. Although transgenic techniques and fluorescence immunohistochemical staining have promoted our understanding of tissue regeneration, simultaneous quantification and visualization of tissue regeneration processes is not easy to achieve. Herein, we developed a simple and quantitative method for the real-time and non-invasive observation of the process of tissue regeneration. The synthesized ratiometric aggregation-induced-emission (AIE) probe exhibits high selectivity and reversibility for pH responses, good ability to map lysosomal pH both in vitro and in vivo, good biocompatibility and excellent photostability. The caudal fin regeneration of a fish model (medaka larvae) was monitored by tracking the lysosomal pH change. It was found that the mean lysosomal pH is reduced during 24–48 hpa to promote the autophagic activity for cell debris degradation. Our research can quantify the changes in mean lysosomal pH and also exhibit its distribution during the caudal fin regeneration. We believe that the AIE-active lysosomal pH probe can also be potentially used for long-term tracking of various lysosome-involved biological processes, such as tracking the stress responses of tissue, tracking the inflammatory responses, and so on.

An AIE-active ratiometric probe for the first time achieved the long-term quantification of lysosomal pH during the medaka larva''s caudal fin regeneration.     

In vivo incorporation of radioactive 36Cl, a method for monitoring chloro compounds in biological material

A new method for fast and easy monitoring of the presence, isolation, and separation of natural chloro compounds in plants is described. The method relies on the in vivo incorporation of radioactive 36Cl and new enhancement methods in autoradiographic technology. The method allows the time of exposure to be limited to 4 days and is thus suitable for routine purposes.     

An orally administered microcapsule system for treating chronic renal failure patients

Ingestible adsorbents for the removal of uremic metabolites are being investigated as adjunctive therapy in the treatment of chronic uremia. In particular, a microcapsule product containing urease and zirconium phosphate (UZP) has been investigated for removing urea. A dog model, simulating chronic uremia, was developed to investigate: (1) the concentration of various nitrogenous metabolites (urea, creatinine, and uric acid) in the GI tract, (2) flux rates of H₂O and various nitrogenous metabolites in the GI tract, and (3) the efficacy of the microcapsule product. The results of these perfusion studies suggest that urea and creatinine can be removed from the GI tract via ingestible adsorbents. In addition, the model may be useful in investigating suspect uremic toxins, e.g., guanidinosuccinic acid (GSA). The reduction of blood urea nitrogen levels in the dog model when the animal was fed the microcapsule product was limited by the capacity of the zirconium phosphate to bind ammonium ion. Preliminary clinical studies with the microcapsule product indicate that it may be of potential adjunctive therapy in patients suffering from chronic renal failure.     

In vivo microdialysis/liquid chromatography/tandem mass spectrometry for the on-line monitoring of melatonin in rat

Liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been coupled to in vivo microdialysis for on-line monitoring of melatonin in a freely moving rat for a period of 15 hours. A microdialysis probe was surgically implanted into the jugular vein of the rat, and deionized water was used as the perfusion medium at a flow rate of 1.0 microL/min. Microdialysis samples were collected in an on-line injector with sample injection every 30 minutes. Melatonin was dosed by intraperitoneal (i.p.) injection and then monitored by microdialysis/LC/MS/MS. The whole experiment, including the microdialysis sampling and sample injection into the LC/MS system, was fully automated. Metabolites of melatonin were identified off-line by LC/MSn experiments. Two metabolites were identified as 6-hydroxymelatonin and cyclic 2-hydroxymelatonin, consistent with ones found previously in the literature.     

In vivo dosimetry with the use of silicon photodiodes

Miniature silicon photodiodes have been tested for utilization in radiotherapy. Their use in the treatment of ca laryngis is described. The actually given dose was monitored during irradiation. Treatment plan or the setting up of the entrance fields was modified according to the response of the diode. This method seems to be a useful tool for checking both the treatment plan and appropriate setting up of the patient in treatment position.     

Photodynamic activity of dibiotinylated aluminum sulfophthalocyanine in vitro and in vivo

The photodynamic activity of dibiotinylated aluminum sulfophthalocyanine was studied in vitro and in vivo. Dibiotinylated aluminum sulfophthalocyanine provided enhanced phototoxic action on OAT-75 cell monolayers as compared with the parent drug. Photodynamic therapy of mice with Ehrlich carcinoma using dibiotinylated aluminum sulfophthalocyanine (0.25 mg/kg) resulted in enhanced inhibition of tumor growth, pronounced vascular damage (thrombosis and destruction of vascular walls) and eventual tumor necrosis.