Denny A. Carvajal

Comparative study of the optical and heat generation properties of IR820 and indocyanine green.

Near-infrared (NIR) fluorophores are the focus of extensive research for combined molecular imaging and hyperthermia. In this study, we showed that the cyanine dye IR820 has optical and thermal generation properties similar to those of indocyanine green (ICG) but with improved in vitro and in vivo stability. The fluorescent emission of IR820 has a lower quantum yield than ICG but less dependence of the emission peak location on concentration. IR820 demonstrated degradation half-times approximately double those of ICG under all temperature and light conditions in aqueous solution. In hyperthermia applications, IR820 generated lower peak temperatures than ICG (4–9%) after 3-minute laser exposure. However, there was no significant difference in hyperthermia cytotoxicity, with both dyes causing significant cell growth inhibition at concentrations ≥ 5 μM. Fluorescent images of cells with 10 μM IR820 were similar to ICG images. In rats, IR820 resulted in a significantly more intense fluorescence signal and significantly higher organ dye content than for ICG 24 hours after intravenous dye administration (p < .05). Our study shows that IR820 is a feasible agent in experimental models of imaging and hyperthermia and could be an alternative to ICG when greater stability, longer image collection times, or more predictable peak locations are desirable.

Covalent IR820-PEG-diamine nanoconjugates for theranostic applications in cancer.

Near-infrared dyes can be used as theranostic agents in cancer management, based on their optical imaging and localized hyperthermia capabilities. However, their clinical translatability is limited by issues such as photobleaching, short circulation times, and nonspecific biodistribution. Nanoconjugate formulations of cyanine dyes, such as IR820, may be able to overcome some of these limitations. We covalently conjugated IR820 with 6 kDa polyethylene glycol (PEG)-diamine to create a nanoconjugate (IRPDcov) with potential for in vivo applications. The conjugation process resulted in nearly spherical, uniformly distributed nanoparticles of approximately 150 nm diameter and zeta potential −0.4±0.3 mV. The IRPDcov formulation retained the ability to fluoresce and to cause hyperthermia-mediated cell-growth inhibition, with enhanced internalization and significantly enhanced cytotoxic hyperthermia effects in cancer cells compared with free dye. Additionally, IRPDcov demonstrated a significantly longer (P<0.05) plasma half-life, elimination half-life, and area under the curve (AUC) value compared with IR820, indicating larger overall exposure to the theranostic agent in mice. The IRPDcov conjugate had different organ localization than did free IR820, with potential reduced accumulation in the kidneys and significantly lower (P<0.05) accumulation in the lungs. Some potential advantages of IR820-PEG-diamine nanoconjugates may include passive targeting of tumor tissue through the enhanced permeability and retention effect, prolonged circulation times resulting in increased windows for combined diagnosis and therapy, and further opportunities for functionalization, targeting, and customization. The conjugation of PEG-diamine with a near-infrared dye provides a multifunctional delivery vector whose localization can be monitored with noninvasive techniques and that may also serve for guided hyperthermia cancer treatments.

Assessment of the Resistance to Uranium (VI) Exposure by Arthrobacter sp. Isolated from Hanford Site Soil

Production of nuclear fuel has resulted in hazardous waste streams that have contaminated the soil and groundwater. Arthrobacter strains, G975, G968, and G954 were used in the prescreening tests to evaluate their tolerance to UO2 2+ and investigate bacteria-U(VI) interactions under oxidizing pH-neutral conditions. Experiments have shown G975 is the fastest growing and the most uranium tolerant strain that removed about 90% of uranium from growth media. Atomic Force Microscopy images exhibited an irregular surface structure, which perhaps provided a larger surface area for uranium precipitation. The data indicate that aerobic heterotrophic bacteria may offer a solution to sequestering uranium in oxic conditions, which prevail in the vadose zone.

Lyophilized Kit for the Preparation of the PET Perfusion Agent [68Ga]-MAA

Rapid developments in the field of medical imaging have opened new avenues for the use of positron emitting labeled microparticles. The radioisotope used in our research was 68Ga, which is easy to obtain from a generator and has good nuclear properties for PET imaging. Methods. Commercially available macroaggregated albumin (MAA) microparticles were suspended in sterile saline, centrifuged to remove the free albumin and stannous chloride, relyophilized, and stored for later labeling with 68Ga. Labeling was performed at different temperatures and times. 68Ga purification settings were also tested and optimized. Labeling yield and purity of relyophilized MAA microparticles were compared with those that were not relyophilized. Results. MAA particles kept their original size distribution after relyophilization. Labeling yield was 98% at 75°C when a 68Ga purification system was used, compared to 80% with unpurified 68Ga. Radiochemical purity was over 97% up to 4 hours after the labeling. The relyophilized MAA and labeling method eliminate the need for centrifugation purification of the final product and simplify the labeling process. Animal experiments demonstrated the high in vivo stability of the obtained PET agent with more than 95% of the activity remaining in the lungs after 4 hours.

90Y-DOTA-CHS Microspheres for Live Radiomicrosphere Therapy: Preliminary In Vivo Lung Radiochemical Stability Studies

Chitosan (CHS) is used to prepare microspheres of 31 ± 8 µm size. Surface modification with p-SCN-Bn-DOTA was performed. A maximum 90Y capacity was found to be 12.1 ± 4.4 µCi/particle. The best obtained labeling yield was 87.7 ± 0.6%. More than 90% in vitro stability was found. Particle in vitro degradation half-life in PBS was found to be greater than 21 days. In vivo studies with 90Y-DOTA-CHS showed more than 95% of the injected activity (decay corrected) in the lungs 24 hours after tail vein administration. 90Y-DOTA-CHS in vivo label stability was superior to resin microspheres. The addition of p-SCN-Bn-DOTA served as a radioprotectant for bone marrow as the 5% 90Y released, during the first 24 hours, was quickly eliminated via urine.

Covalent IR820-PEG diamine conjugates: characterization and in vivo biodistribution.

Introduction: IR820 is a near-infrared probe with potential applications in optical imaging and hyperthermia. Its chlorosubstituted cyclohexene makes it amenable to forming conjugates as multifunctional probes. We prepared a novel covalent IR820/PEG-diamine (IRPDcov) nanoconjugate. Methods: IRPDcov was prepared using IR820 and 6kDa PEG-diamine, characterized by SEM, H-NMR, spectrophotometry, and spectrofluorometry; and studied in vitro and in vivo. Mice (n=36) were used to explore the biodistribution of IRPDcov compared to IR820 and indocyanine green (ICG) after i.v. injection of a 0.24 mg/kg dose of dye, with plasma samples collected at 15-30-60 minutes and 24 hours. The plasma concentrations were fit to a biexponential curve following a two compartment model. Organ samples were collected after 24 hours. Results and Discussion: IRPDcov retained the ability to fluoresce for in vivo optical imaging and also to generate heat, and was significantly more stable than IR820 in aqueous solution over a period of 72 hours. IRPDcov and IR820 demonstrated significantly longer (p<0.05) plasma half-lives, elimination half-lives, and area-under-the-curve values compared to ICG. This could pose an advantage in therapeutic probe applications such as hyperthermia or drug delivery. Both IR820 and IRPDcov showed a very strong signal in the liver and lower-intensity signal in the kidneys 24 hours after injection, whereas the predominant signal for ICG was weak and located in the intestines, demonstrating a much more rapid GI elimination. IR820 showed signal in the lungs, which was not present in IRPDcov subjects indicating that IRPDcov may have been able to escape detection by alveolar macrophages.

(68)Ga-NOTA-CHSg and (99m)Tc-CHSg Labeled Microspheres for Lung Perfusion and Liver Radiomicrospheres Therapy Planning.

Fast biodegradable (12 h < half-life < 48 h) radioactive labeled microspheres are needed for PET and SPECT lung perfusion and radiomicrosphere therapy planning. An emulsion method was used to create 30.1 ±4.8 μm size range microspheres with biodegradable Chitosan glycol (CHSg). Microspheres were characterized and labeled with 99mTc or 68Ga as an alternative to MAA in perfusion PET and SPECT studies. Surface decoration of CHSg microspheres with p-SCN-Bn-NOTA was performed to increase 68Ga  in vivo stability. 99mTc was labeled directly to the CHSg microspheres. Labeling yield and in vitro radiochemical stability were evaluated. In vitro CHSg microsphere degradation half-life was ~24 hours in porcine blood. Labeled microspheres were injected into Sprague Dawley rats and biodistribution was determined after 2 and 4 hours. Both 99mTc-CHSg and 68Ga-NOTA-CHSg were quickly allocated in the lungs after injection. 99mTc-CHSg showed 91.6 ± 6.5% and 83.2 ± 4.1% of the decay corrected injected activity remaining in the lungs after 2 and 4 hours, respectively. For the obtained 68Ga-NOTA-CHSg microspheres, lung allocation was very high with 98.9 ± 0.2% and 95.6 ± 0.9% after 2 and 4 hours, respectively. The addition of p-SCN-Bn-NOTA acts as a radioprotectant eliminating the released 68Ga activity from the lungs to the bladder protecting the other organs.

Development of Matlab Algorithm to Process Pressure Waveforms from Isolated Perfused Heart Experiments

The Langendorff isolated heart preparation allows for the collection of pressure waveforms in animal heart specimens. The data obtained provides information on physiological parameters such as left ventricular systolic pressure, rate of contractility, rate of relaxation, heart rate, and aortic pressure. Processing this data allows researchers to assess and monitor heart function in a close-to-physiological environment, but data processing may be time-consuming. Commercial software packages are available to process the data collected from the isolated heart experiment, but these packages are expensive and may not be adaptable to the exact application that researchers need. Our group has developed an easily customizable Matlab algorithm which includes a graphical user interface for intuitive processing. The algorithm reads text files containing raw aortic and left ventricular pressure data. From this information, it extracts and plots values for maximum dP/dt, minimum dP/dt, peak left ventricular end systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), left ventricular developed pressure (LVDP), mean time from LVEDP to LVSP, heart rate, and mean aortic pressure. The output information includes the standard deviation of the measurements. The input of the algorithm can vary from one to multiple files of data at a time, which allows for comparison of data collected at different time intervals in the experiment. This feature is very useful to monitor the progression of heart function during the experiment and the reproducibility of the experiments. The applicability of the algorithm is beyond the isolated heart experiment, because it could be modified for use in any application that requires processing of pressure waveforms.

Application of a Fluorescent Multiple Indicator Method to Study Changes in Cardiac Permeability with Chemotherapy

Classical measurements of tissue permeability use radioactive indicators, which are very accurate but also limited by radiation exposure hazards. We have developed a fluorescent multiple indicator dilution method using Texas Red-conjugated Dextran (TR, MW 70,000 Da) as the reference dye, and sodium fluorescein (NaFL, MW 376 Da) as the diffusible dye. The characteristic excitation/emission wavelengths are 485/515 nm for NaFL, and 590/630 nm for TR. By comparing the output profiles of the two dyes versus time using a spectrofluorometer, we can estimate the permeability-surface-area-product (PSP) of the capillary network. We have studied changes in cardiac capillary permeability after treatment with doxorubicin (DOX) using our method. Our hypothesis is that DOX treatment will cause endothelial damage and lead to increased cardiac capillary permeability. Sprague-Dawley rats were randomly assigned to a control (n= 4) or DOX group (n = 5). Rats were injected intraperitoneally with 3 mg/kg of either saline solution or DOX on days 1, 3, 5, 7, 9, and 11. On day 12, we performed the isolated heart experiment. In three replicates, we injected 25 μL of the fluorescent mixture above the aortic cannula (12.5 μL each of 1.56 μg/mL TR, 1.56 ng/mL NaFL), and collected output samples for 45 seconds. We measured the samples using a Fluorolog-3® spectrofluorometer. The results showed that the control group had a lower average PSP (0.04±0.01 cm3/s) than the DOX group (0.09±0.01 cm3/s), p<0.05. This indicates that DOX treatment causes an increase in cardiac capillary permeability related to its cardiotoxic effects. The results also show that our fluorescent indicator dilution method is able to sensitively detect changes in cardiac capillary permeability. The applicability of this method is not limited to the isolated heart setup, and the protocol can be adapted for use in other studies of organ or tissue permeability.