RESUMEN
MicroPET is a noninvasive imaging modality that can potentially track tumor development in nude rats using the radiotracer fluorine 18-fluorodeoxyglucose ((18)F-FDG). Our goal was to determine whether microPET, as opposed to more invasive techniques, could be used to noninvasively monitor the development of ovarian cancer in the peritoneal cavity of nude rats for monitoring treatment response in future studies. Female nude rats were inoculated intraperitoneally with 36 million NIH:OVCAR-3 cells. Imaging was carried out at 2, 4, 6, or 8 weeks postinoculation. Each rat was fasted overnight and intravenously injected with 11.1 MBq (300 microCi) of (18)F-FDG in 0.2 mL of saline. Thirty minutes following injection, the rats were placed in the microPET and scanned for 30 min. After imaging, rats were euthanized for ascites and tissue collection for biodistribution and histopathologic correlation. Standard uptake values (SUVs) of (18)F-FDG within the peritoneal cavity were also calculated from regions of interest analysis of the microPET images. MicroPET images showed diffuse increased uptake of (18)F-FDG throughout the peritoneal cavity of tumor rats (mean SUV=4.64) compared with control rats (mean SUV=1.03). Ascites gathered from tumor-bearing rats had increased (18)F-FDG uptake as opposed to the peritoneal fluid collected from control rats. Biodistribution data revealed that the percent injected dose per gram (% ID/g) was significantly higher in tumor-bearing rats (6.29%) than in control rats (0.59%) in the peritoneal lymph nodes. Pathology verified that these lymph nodes were more reactive in tumor-bearing rats. By 6 weeks, some rats developed solid masses within the peritoneum, which could be detected on microPET images and confirmed as tumor by histopathology. (18)F-FDG uptake in these tumors at necropsy was 2.83% ID/g. These results correlate with previous invasive laparoscopic studies of the same tumor model and demonstrate that microPET using (18)F-FDG is a promising noninvasive tool to localize and follow tumor growth in an intraperitoneal ovarian cancer model.
Asunto(s)
Carcinoma/diagnóstico , Carcinoma/patología , Neoplasias Ováricas/diagnóstico , Neoplasias Ováricas/patología , Tomografía de Emisión de Positrones/métodos , Ensayos Antitumor por Modelo de Xenoinjerto , Algoritmos , Animales , Progresión de la Enfermedad , Femenino , Fluorodesoxiglucosa F18/farmacocinética , Humanos , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Cavidad Peritoneal/patología , Ratas , Ratas Desnudas , Distribución Tisular , Células Tumorales CultivadasRESUMEN
Liposome-encapsulated hemoglobin (LEH) is being developed as an oxygen therapeutic. In this work, we evaluated a neutral formulation of PEGylated LEH for its circulation and distribution properties in rodent models of 25% hypovolemic exchange transfusion. About 25% of blood in rats and rabbits was exchanged with LEH that had been previously labeled with 99mTc radionuclide. The distribution of 99mTc-LEH was followed by gamma camera imaging and intermittent blood sampling during 48 h, and counting the tissue-associated radioactivity after necropsy at 48 h. On the basis of circulation kinetics, the half-life of 99mTc-LEH in blood was 30 and 39.8 h in rats and rabbits, respectively. Apart from blood, major organs of accumulation of LEH after 48 h included liver (rats, 10.3% and rabbits, 5.4% of injected dose) and spleen (rats, 2.4% and rabbits, 0.8% of injected dose). The results demonstrate that LEH circulates for a prolonged time after administration and that the animals tolerate at least 25% of blood exchange without any distress. Subsequent to the enhanced uptake in the RES, the rats clear LEH from the circulation faster than the rabbits.
Asunto(s)
Transfusión de Componentes Sanguíneos/métodos , Hemoglobinas/administración & dosificación , Hipovolemia/terapia , Animales , Semivida , Hemoglobinas/farmacocinética , Liposomas , Hígado/metabolismo , Masculino , Conejos , Ratas , Ratas Sprague-Dawley , Bazo/metabolismo , Distribución TisularRESUMEN
To prepare long-circulating liposomes, poly(ethylene glycol) (PEG)-lipid is usually mixed with other lipid components before vesicle formation. PEG-lipids can also be postinserted in the outer layer of liposomes after the preparation. In this study, PEG-distearoylphosphatidylethanolamine was incorporated by postinsertion technique into liposome-encapsulated hemoglobin (LEH) carrying neutral or negative charge. Postinsertion technique improved the encapsulation efficiency of hemoglobin from about 0.0017 to 0.017 (hemoglobin/phospholipid, molar ratio) for a similar lipid composition. Thus, neutral, anionic, PEG-neutral, and PEG-anionic LEHs were made and labeled with technetium-99m to follow their biodistribution. A small dose of LEH (approximately 15 mg of phospholipid) was injected intravenously in rabbits, and its distribution was monitored by blood sampling, gamma camera imaging, and tissue radioactivity counting on necropsy. The 24-h blood levels of neutral, PEG-neutral, anionic, and PEG-anionic LEHs were 14, 40.3, 13.1, and 35.7% of injected dose, respectively; calculated T(1/2) values of circulation were 8.9, 19.3, 9.6, and 16.5 h, respectively. PEGylation also influenced accumulation of LEH in the reticuloendothelial system. Liver uptake of neutral LEH dropped from 52.1 to 19.1%, whereas that of anionic LEH came down from 35.3 to 11.5% on PEGylation. In contrast, PEGylation increased the spleen uptake by 8.5- and 2.5-fold for neutral and anionic LEH, respectively. The results demonstrate that PEGylation by postinsertion not only improves the circulation t1/2 of LEH but also enhances hemoglobin content inside the vesicles for better oxygen-carrying capacity.
Asunto(s)
Hemoglobinas/administración & dosificación , Liposomas/química , Fosfatidiletanolaminas/química , Polietilenglicoles/química , Animales , Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Hemoglobinas/farmacocinética , Cinética , Conejos , Distribución TisularRESUMEN
To determine the largest size of liposomes that can retain stealth behavior conferred by poly(ethylene glycol)-DSPE, neutral liposomes were studied in rabbits for their circulation and distribution. Five sizes (136.2, 165.5, 209.2, 275 and 318 nm) of liposomes (DSPC, Cholesterol, PEG-DSPE and alpha-tocopherol, 90:80:4.5:3.9 molar ratio) were made by extrusion technique and radiolabeled with technetium-99m (Tc-99m) to follow their distribution through 24 h. Although all liposomes showed prolonged circulation in blood, the amount still in circulation at 24 h was dependent on their size. Radioactivity accumulation in spleen progressively increased with increase in size of the liposomes. In the size range of approximately 160-220 nm, liver uptake was minimum, spleen uptake was moderate while the amount of circulating liposomes was maximum. Gamma camera scintigraphy corroborated the distribution pattern of liposomes on necropsy. Images within 1h showed high blood pool activities for liposomes of all sizes. However, at 24h, the blood pool activity was diminished for 275 nm and negligible for 308 nm liposomes; the smaller sized liposomes (136.2-209.2 nm) continued to show high blood pool activity. The amounts of radioactivity still circulating at 24h were 46.4, 50.4, 46.8, 36.2 and 14.5% for 136.2, 165.5, 209.2, 275 and 318 nm liposomes, respectively. Corresponding circulation T(1/2)s were 21.7, 26.5, 24.9, 18.7 and 8.9h, respectively. Thus, the optimum size of PEG-liposomes for prolonged circulation in rabbits is 160-220 nm. Beyond this range, the stealth property of PEG-liposomes is significantly compromised and the distribution is characterized by high RES accumulation.
Asunto(s)
Liposomas/farmacocinética , Fosfatidiletanolaminas/farmacocinética , Polietilenglicoles/farmacocinética , Análisis de Varianza , Animales , Colesterol/sangre , Colesterol/química , Colesterol/farmacocinética , Semivida , Inyecciones Intravenosas , Marcaje Isotópico , Liposomas/sangre , Liposomas/química , Hígado/diagnóstico por imagen , Hígado/metabolismo , Masculino , Tamaño de la Partícula , Fosfatidilcolinas/sangre , Fosfatidilcolinas/química , Fosfatidilcolinas/farmacocinética , Fosfatidiletanolaminas/sangre , Fosfatidiletanolaminas/química , Polietilenglicoles/química , Conejos , Cintigrafía , Espectrometría gamma , Bazo/diagnóstico por imagen , Bazo/metabolismo , Pentetato de Tecnecio Tc 99m , Distribución TisularRESUMEN
Therapeutic intervention in inflammatory bowel diseases (IBDs) is often associated with severe toxicity related to the nonspecific and ubiquitous interaction of drugs with the organs and tissues. In order to prevent side effects from aggressive and prolonged treatment with glucocorticoids and immunosuppressive agents, preferential accumulation of these potent drugs in diseased tissue is desired. In this work, we report that liposomes show a remarkable tendency to accumulate in inflamed colon of rats with experimental colitis. The disposition of liposomes was monitored by labeling them with Tc-99m followed by gamma camera imaging, and determining biodistribution of radioactivity in various organs. The images showed distinct accumulation of radioactivity in the colon of rats with colitis, while the abdomen of normal rats was conspicuously free of any visible radioactivity. Although images acquired 4 h after Tc-99m-liposome injection were clear enough for diagnostic indication, the real potential of liposomes for drug delivery was evident in 24 h images where the major organs of liposome accumulation were dwarfed by intense colon activity in animals with colitis. On necropsy, 13.5% +/- 5.48 of the activity accumulated in the inflamed colon as compared to only 0.1% in the normal colon, giving a target-to-nontarget ratio of 135. The blood borne radioactivity was 9% +/- 2.12 (colitis) and 25.7% +/- 4.27 (normal), indicating that the decrease in circulating liposomes is associated with an increase in liposome accumulation in the inflammatory site. The other two major organs that accumulated liposomes were spleen (10.7% normal vs. 11% colitis) and liver (8% normal vs. 10.1% colitis). In conclusion, this study demonstrates the innate propensity of liposomes to accumulate in the sites of inflammation and potential of liposomes loaded with therapeutic drugs or diagnostic agents for targeting colitis.
Asunto(s)
Colon/metabolismo , Enfermedades Inflamatorias del Intestino/diagnóstico por imagen , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Polietilenglicoles , Animales , Colon/diagnóstico por imagen , Colon/patología , Modelos Animales de Enfermedad , Liposomas , Masculino , Cintigrafía , Ratas , Ratas Sprague-Dawley , Tecnecio , Distribución TisularRESUMEN
This study investigates the use of [(99m)Tc] liposomes for the detection of sentinel lymph nodes. A variety of [(99m)Tc] liposome formulations were compared with common lymphoscintigraphic agents including [(99m)Tc] regular-sulfur colloid (SC), [(99m)Tc] 0.22 microfiltered-SC, [(99m)Tc] reduced heating time 0.22 microfiltered-SC, and [(99m)Tc] human serum albumin (HSA) in rabbits. Images were acquired for the first 60 minutes and at 24 hours, followed by tissue biodistribution study. All agents except [(99m)Tc] regular SC demonstrated good migration from the injection site. Agents were retained in the popliteal node at 24 hours to varying degrees as follows: both [(99m)Tc] filtered SC preparations > [(99m)Tc] regular SC > [(99m)Tc] liposomes > [(99m)Tc] HSA. [(99m)Tc] liposome imaging can be used to develop novel liposome compositions with improved lymph node diagnostic and drug delivery characteristics.
Asunto(s)
Linfocintigrafia , Radiofármacos/administración & dosificación , Radiofármacos/farmacocinética , Agregado de Albúmina Marcado con Tecnecio Tc 99m/administración & dosificación , Agregado de Albúmina Marcado con Tecnecio Tc 99m/farmacocinética , Azufre Coloidal Tecnecio Tc 99m/administración & dosificación , Azufre Coloidal Tecnecio Tc 99m/farmacocinética , Animales , Portadores de Fármacos , Cámaras gamma , Humanos , Procesamiento de Imagen Asistido por Computador , Liposomas , Ganglios Linfáticos/diagnóstico por imagen , Masculino , Tamaño de la Partícula , ConejosRESUMEN
UNLABELLED: Colloidal radiopharmaceuticals are commonly used in combination with blue dye for localization of the sentinel node. Liposomes are colloidal particles composed of spontaneously forming lipid spheres that can carry a wide variety of diagnostic and therapeutic agents. Conventional liposomes are poorly retained in lymph nodes (<2% of the subcutaneously injected dose). We have previously described a system for increasing the retention of liposomes in the lymph nodes by approximately 7-fold. This system is comprised of subcutaneously injected biotin-coated liposomes, followed by an adjacent injection of avidin. When the avidin moves into the lymphatic vessels, it causes aggregation of the biotin-coated liposomes that are also in the process of migrating through the lymphatic vessels. These aggregated liposomes become entrapped in the next encountered lymph node. In this study, we use this novel lymph node delivery system with liposomes that encapsulate blue dye, resulting in intense blue staining of the sentinel node. These liposomes can also be labeled with (99m)Tc, permitting scintigraphic imaging and radioguided probe localization of the sentinel node. METHODS: Liposomes coated with biotin and coencapsulating blue dye and glutathione were labeled with (99m)Tc using hexamethylpropyleneamine oxime. Rabbits were subcutaneously administered 0.3 mL (99m)Tc-biotin-liposomes containing blue dye in both hind feet, followed by a subcutaneous injection (0.3 mL) of 5 mg avidin in only one hind foot (experimental). The other hind foot served as a control. RESULTS: Labeling efficiencies (mean +/- SEM) for liposomes encapsulating blue dye were 92.1% +/- 1.9%. Necropsy at 24 h revealed that the popliteal node on the experimental leg receiving the avidin was intensely blue stained compared with virtually no blue coloration of the control node. Tissue counts of these nodes were 12.2 +/- 1.5 percentage injected dose (%ID) in the experimental node compared with 1.2 +/- 0.1 %ID in the control nodes (P<0.0001). CONCLUSION: Biotin-liposomes encapsulating blue dye can be successfully labeled with (99m)Tc, providing a convenient option for the visualization and radiolocalization of the sentinel node. This biotin-liposome/avidin system may also have potential for the delivery of therapeutic drugs and radiopharmaceuticals to lymph nodes.
Asunto(s)
Ganglios Linfáticos/diagnóstico por imagen , Colorantes de Rosanilina , Biopsia del Ganglio Linfático Centinela , Exametazima de Tecnecio Tc 99m , Animales , Biotina , Liposomas , Masculino , Conejos , Cintigrafía , Radiofármacos/farmacocinética , Colorantes de Rosanilina/farmacocinética , Exametazima de Tecnecio Tc 99m/farmacocinética , Distribución TisularRESUMEN
Scintigraphic imaging is a valuable tool that can be used during the development of liposome-based therapeutic agents. It provides the ability to non-invasively track and quantitate the distribution of liposomes in the body. This review article provides a general overview of the methods involved in producing scintigraphic images as well as methods of radiolabeling liposomes. Liposomes labeled with technetium-99m ((99m)Tc) are particularly useful for scintigraphic imaging due to the physical characteristics of (99m)Tc, which provides a high quality image. Examples of how scintigraphic imaging studies have contributed to the development of a variety of liposome-based formulations are covered in this article. These liposome formulations include long-circulating liposome-based oxygen carriers, liposome-based anti-cancer drugs, liposomes encapsulating antibiotics and anti-fungals, and liposomes targeted to lymph nodes. Studies using scintigraphic imaging for the investigation of immune responses to liposomes are also discussed. These examples demonstrate the usefulness of scintigraphic imaging for the development of novel liposome formulations.
Asunto(s)
Quimioterapia , Procesamiento de Imagen Asistido por Computador , Liposomas , Tomografía Computarizada de Emisión de Fotón Único , Tomografía Computarizada de Emisión , Animales , Humanos , Marcaje Isotópico , Ganglios Linfáticos/diagnóstico por imagen , Neoplasias/diagnóstico por imagen , Compuestos de TecnecioRESUMEN
Intravenously administered liposomes are versatile carriers for drugs, contrast agents, biologics, and DNA. Liposomes and other colloidal particles are currently under investigation as lymph node delivery vehicles. After s.c. injection, conventional liposomes move into the lymphatic vessels, but are poorly retained in each draining lymph node (<2% injected dose). In this report, we describe a novel method for greatly enhancing the retention of liposomes in the lymph nodes. This system is comprised of a s.c. injection of biotin-coated liposomes in an area where lymph node targeting is desired, followed by an adjacent s.c. injection of avidin. As the avidin moves through the lymphatic vessels, it causes aggregation of biotin-coated liposomes that are also in the process of migrating through lymphatic vessels. These aggregated liposomes become trapped in the next encountered lymph node. In the present study, experimental rabbits were s.c. administered biotin-coated liposomes in both hind feet, followed by an adjacent injection of avidin, whereas control rabbits were administered biotin-coated liposomes in both hind feet without the avidin. At 24 h, rabbits receiving avidin retained 13.7% of the injected liposomes in popliteal nodes and 2.3% in iliac nodes, whereas control rabbits retained only 1.7% of the liposomes in popliteal nodes and 0.3% in iliac nodes. Blood and liver uptake of the biotin-coated liposomes was greatly decreased in the experimental rabbits receiving avidin. This novel liposome delivery system may prove useful for the delivery of chemotherapeutic drugs, vaccine antigens, and biologic agents to lymph nodes.
Asunto(s)
Liposomas/farmacocinética , Ganglios Linfáticos/metabolismo , Animales , Avidina/farmacocinética , Biotina/farmacocinética , Portadores de Fármacos , Masculino , ConejosRESUMEN
A major obstacle in the development of red cell substitutes has been overcoming their short circulation persistence. In this study, distearoyl phosphoethanolamine polyethylene glycol 5000 (PEG-PE) (10 mol%) was added to the formulation of liposome-encapsulated hemoglobin (LEH) to decrease reticuloendothelial system uptake and prolong LEH circulation persistence. PEG-LEH was radiolabeled with technetium-99m, infused into rabbits (25% of blood pool at 1 ml/min) (n = 5), and monitored by scintigraphic imaging at various times out to 48 h. At 48 h, animals were sacrificed, and tissue samples were collected for counting in a scintillation well counter. Tissue distribution data at 48 h revealed that 51.3 +/- 3.4% of the technetium-99m-PEG-LEH remained in circulation, a greater than 3-fold increase in the circulation half-life compared with circulation half-lives previously reported for non-PEG-containing LEH formulations. The liver had the greatest accumulation at 48 h (12.7 +/- 0.7%), followed by bone marrow (6.2 +/- 0.1%), whereas the spleen had only 1.4 +/- 0.2%. The addition of PEG-PE to the LEH formulation greatly prolongs the circulation persistence of LEH and represents a significant step in the development of red cell substitutes with prolonged oxygen delivery.
Asunto(s)
Sustitutos Sanguíneos/farmacocinética , Hemoglobinas/farmacocinética , Polietilenglicoles/farmacocinética , Animales , Hemoglobinas/administración & dosificación , Liposomas , Fosfatidilgliceroles/administración & dosificación , Fosfatidilgliceroles/sangre , Fosfatidilgliceroles/farmacocinética , Polietilenglicoles/administración & dosificación , Conejos , Tecnecio , Distribución TisularRESUMEN
UNLABELLED: We evaluated radiolabeled liposomes (liposomes labeled both with 99mTc and 111In) for the early detection of osteomyelitis in an experimental model. METHODS: Liposomes, containing 5% polyethylene glycol-distearoyl phosphatidylethanolamine with encapsulated glutathione and deferoxamine, were prepared and labeled with 99mTc and 111In by a previously described method. Acute osteomyelitis was induced in male New Zealand rabbits by intramedullary injection of sodium-morrhuate and Staphylococcus aureus in the tibial bone marrow. Serial imaging studies, consisting of radiolabeled liposome imaging (2-4 mCi 99mTc and 75-125 microCi 111In), 99mTc-methylene diphosphonate (MDP) (3-5 mCi) and 67Ga-citrate (500 microCi), were performed starting at the third day after injection. Each radionuclide study was separated by at least 2 days. The animals also underwent radiography of the lower extremities. The animals were then killed and the infected tibia was excised for histopathology. RESULTS: For interpreting relative efficacy of individual radiopharmaceuticals, only animals showing positive histopathological findings (n = 9) were considered. Radiographs (Days 12, 13) were conclusive for osteomyelitis in only 3 rabbits. Radiolabeled liposome imaging (Days 4-6) showed positivity in 8 cases and was equivocal in 1. Though the lesion could be delineated as early as 8 hr postinjection in the 99MTc window, the best target-to-nontarget ratio (T/NT) of 1.86 +/- 0.19 was obtained at 48 hr in the 111In window. Three-phase 99mTc-MDP scan (Day 7) was positive in only 5 rabbits with 3 hr T/NT of 1.6 +/- 0.23. Galium-67-citrate images (Days 9-11) were positive in 8 cases and equivocal in 1, the mean 48 hr T/NT being 1.74 +/- 0.24. These results show liposomes are better than 99mTc-MDP for imaging bone infection. Given the early localization and better quality of the images, radiolabeled liposomes also exhibited advantages over 67Ga-citrate for detection of acute osteomyelitis.
Asunto(s)
Radioisótopos de Indio , Osteomielitis/diagnóstico por imagen , Tecnecio , Enfermedad Aguda , Animales , Citratos , Galio , Liposomas , Masculino , Conejos , Cintigrafía , Radiofármacos , Sensibilidad y Especificidad , Exametazima de Tecnecio Tc 99m , Medronato de Tecnecio Tc 99mRESUMEN
Liposomes encapsulating both glutathione and deferoxamine were labeled with 99mTc-HMPAO and 111In-oxine at the same time. These dual radiolabeled liposomes were intravenously injected in rats with S. aureus infection in thigh. The target-to-background ratio (T/BG) increased from 2.9 at 2 h to 4.4 at 8 h in 99mTc images. In 111In images, T/BG of 5.5 at 8 h increased to 10.5 by 48 h. The 24-h spleen uptake of 111In- and 99mTc-liposomes was 24.14%ID and 8.91%ID. In femur, 99mTc-liposomes remained at approximately 10.5%ID, but 111In-liposomes increased from approximately 11%ID at 4 h to approximately 25.5%ID at 24 h. The simultaneous presence of 99mTc and 111In in the liposomes resulted in good early (2-8 h) as well as delayed (24-48 h) images delineating the infection site.
Asunto(s)
Infecciones/diagnóstico por imagen , Liposomas/farmacocinética , Radiofármacos/farmacocinética , Animales , Quelantes/farmacocinética , Deferoxamina/farmacocinética , Glutatión/farmacocinética , Procesamiento de Imagen Asistido por Computador , Radioisótopos de Indio , Inflamación/diagnóstico por imagen , Cintigrafía , Ratas , Ratas Sprague-Dawley , Infecciones Estafilocócicas/diagnóstico por imagen , Exametazima de Tecnecio Tc 99m/farmacocinética , Distribución TisularAsunto(s)
Sustitutos Sanguíneos , Hemoglobinas/metabolismo , Radioisótopos de Oxígeno/farmacocinética , Oxígeno/sangre , Animales , Bovinos , Eritrocitos/metabolismo , Recambio Total de Sangre , Humanos , Cinética , Liposomas , Masculino , Modelos Biológicos , Oxihemoglobinas/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Infusion of liposome-encapsulated hemoglobin (LEH) induces a transient thrombocytopenia in rats (Rabinovici R, Rudolph AS, Ligler FS, Smith EF, III, Feuerstein G [1992] Biological responses to exchange transfusion with liposome-encapsulated hemoglobin. Circ Shock 37:124). A specific mechanism such as a localization of platelets during this transient LEH-induced thrombocytopenia has not been reported previously in the literature. In this study, platelets were isolated and labeled with indium-111 (111In), then retransfused into the same animal. Fifteen minutes after the 111In platelets were retransfused and allowed to equilibrate with the blood pool, a 10% top load volume of either LEH (1.8 mL, 262 mg phospholipid/kg body weight, 92 mg hemoglobin (Hb)/kg body weight), liposome vehicle (LV) (1.8 mL, 262 mg phospholipid/kg body weight), or free bovine Hb (1.8 mL, 92 mg Hb/kg body weight) (n=6 per group) was infused. Serial blood samples were drawn to determine platelet counts by radioactivity and light scattering methods. LV and Hb demonstrated no significant changes from baseline levels in circulating platelet levels, whereas LEH caused a transient 50% decrease in 111In platelet activity 2-5 minutes postinfusion, which returned to baseline levels by 15 minutes. Platelet counts based on traditional light scattering methods were not significantly different among the three treatment groups over this same time course. Localization of these 111In platelets was monitored with a gamma scintigraphic camera. After infusion of LEH, 111In platelets rapidly moved out of the circulation and sequestered in the lungs and liver with subsequent return to the circulation by 15 minutes. In contrast, no significant changes in 111In platelet activity were noted in the lungs and liver after infusion of LV and Hb. 111In platelet activity in the spleen nearly doubled 30 minutes after Hb infusion and was significantly different than spleen 111In platelet activity in the LEH and LV groups. In vitro platelet aggregation studies were also performed to determine the direct effect of LEH, LV, and Hb on platelet aggregation. The rate of thrombin-induced aggregation did not differ among control samples and samples containing LEH, LV, or Hb; none of these agents induced platelet aggregation. We conclude that LEH-induced thrombocytopenia is associated with a transient (within minutes) sequestration of platelets in the lungs and liver, with subsequent release to the circulation within 15 minutes. Tetrameric bovine Hb is associated with increased platelet accumulation in the spleen. Light scattering methods for measuring platelet levels in blood samples containing LEH are unreliable because of particulate interference.
Asunto(s)
Plaquetas/fisiología , Sustitutos Sanguíneos/farmacología , Hemoglobinas/farmacología , Liposomas , Animales , Plaquetas/diagnóstico por imagen , Radioisótopos de Indio , Hígado/diagnóstico por imagen , Pulmón/diagnóstico por imagen , Masculino , Agregación Plaquetaria/efectos de la radiación , Recuento de Plaquetas/efectos de los fármacos , Cintigrafía , Ratas , Ratas Sprague-Dawley , Bazo/diagnóstico por imagen , Factores de TiempoRESUMEN
A method for determining oxygen-carrying capacity of blood substitutes has been developed using the short-lived cyclotron-produced positron-emitting isotope 15O. This method measures the oxygen-carrying capacity of the blood substitutes in vivo in the presence of red blood cells and allows determination of changes in the oxygen-carrying capacity over time after exchange transfusion. This method is applied to the blood substitutes of liposome-encapsulated hemoglobin (LEH) and cell-free hemoglobin (Hb). We have used 15O (half-life of 2 min) to quantitate the lung uptake and tissue delivery of [15O2]LEH. Lung uptake studies were performed in intubated, catheterized rats after a 40% exchange transfusion of bovine LEH (LEBH; 0.68 g Hb/kg body wt), human hemolysate LEH (LEHH; 1.0 g Hb/kg body wt), or free bovine hemoglobin (SFHS; 0.56 g Hb/kg body wt). A bolus inhalation of 15O2 (3-5 mCi) was given at 15 min, 3 h, and 24 h post-transfusion. Arterial blood samples were collected, spun, and separated into LEH, red blood cell, and plasma fractions. 15O activity and hemoglobin content were determined for each fraction. Oxygen-carrying capacity was calculated as a percentage of the original red blood cell fraction removed. For LEBH, the carrying capacity was 15% at 15 min, 13% at 3 h, and 1% at 24 h. For LEHH, the carrying capacity was 30% at 15 min, 26% at 3 h, and 19% at 24 h. The marked decrease in carrying capacity at 24 h for LEBH compared with LEHH was attributable to the increased formation of methemoglobin in the circulating LEBH rather than increased removal from circulation, because total hemoglobin concentrations measured for both LEH samples decreased at a similar rate during the 24 h. The presence of methemoglobin reductase and other naturally occurring antioxidants in the LEHH may be responsible for maintaining the higher levels of oxyhemoglobin. Oxygen-carrying capacity for SFHS also decreased over time but at a much sharper rate compared with both LEH formulations. The carrying capacity for SFHS of 8% measured at 15 min decreased to 0.3% at 3 h and undetectable levels at 24 h. This sharper decrease in carrying capacity for SFHS is attributable to the rapid removal of the hemoglobin from circulation.
Asunto(s)
Sustitutos Sanguíneos , Radioisótopos de Oxígeno , Animales , Volumen Sanguíneo , Hemoglobinas , Liposomas/química , Pulmón/irrigación sanguínea , Masculino , Oxígeno/sangre , Ratas , Ratas Sprague-DawleyRESUMEN
Previous studies have documented that liposome-encapsulated hemoglobin (LEH) can cause a rapid and transient thrombocytopenia following intravenous injection into small animals. The present study evaluated the role of complement during the LEH-induced thrombocytopenia in rats. We have compared changes in platelet levels in the blood, platelet organ distribution, and total hemolytic complement levels following intravenous administration of LEH in control and complement-depleted rats. Changes in platelet organ distribution at various times after LEH administration were monitored by labeling autologous platelets with indium-111 (111In)-oxine and imaging the 111In-platelets with a gamma camera after reinjection. Platelet counts were determined by light-scattering methods and by following 111In radioactivity at various times after LEH administration. Platelet levels did not significantly change for the complement-depleted rats during the 60 min following an injection of LEH, whereas thrombocytopenia (40% decrease) was noted within 4 min post-LEH-injection for control rats with a gradual return to baseline circulating platelet levels within 60 min. This drop in circulating platelets was correlated with a rapid redistribution of 111In-platelets from the circulation to the lungs and liver, whereas complement-depleted rats showed no transient movement of the 111In-platelets from the circulation. Baseline complement levels of 21.6 +/- 2.2 CH50/ml for control rats and 0.2 +/- 0.1 CH50/ml for complement-depleted rats did not significantly change during the 60 min following LEH administration. This study suggests that complement must be present during LEH-induced transient thrombocytopenia, as complement-depleted rats underwent no thrombocytopenia, and that the transient LEH-induced thrombocytopenia may be associated with complement activation.
Asunto(s)
Proteínas del Sistema Complemento/fisiología , Hemoglobinas/administración & dosificación , Hemoglobinas/efectos adversos , Liposomas , Trombocitopenia/etiología , Animales , Proteínas Inactivadoras de Complemento/farmacología , Venenos Elapídicos/farmacología , Radioisótopos de Indio , Inyecciones Intravenosas , Luz , Hígado/patología , Pulmón/patología , Miocardio/patología , Especificidad de Órganos , Recuento de Plaquetas , Ratas , Dispersión de Radiación , Bazo/patologíaRESUMEN
UNLABELLED: This study evaluated two 99mTc-liposome formulations as potential blood-pool agents in comparison with standard 99mTc-red cells and 99mTc-human serum albumin (HSA). METHODS: Liposomes with no surface modification or coated with polyethylene glycol (PEG) were labeled with 99mTc using the lipophilic chelator, HMPAO. Autologous red cells were labeled with 99mTc using in vitro or in vivo techniques. Technetium-99m-HSA was supplied commercially. Rabbits were injected intravenously with 99mTc-liposomes, 99mTc-red cells or 99mTc-HSA. Static images were acquired and blood samples collected. RESULTS: Technetium-99m-liposome images showed prominent blood-pool activity compared to lung and liver activities, which were similar to those acquired for 99mTc-red cells, but better than 99mTc-HSA. Heart-to-lung ratios were not significantly different between 99mTc-liposome formulations or for either formulation compared to 99mTc-red cells. The ratios were higher, however, than for 99mTc-HSA. Heart-to-liver ratios were higher for PEG 99mTc-liposomes than they were for neutral 99mTc-liposomes and 99mTc-HSA, but were not significantly different than 99mTc-red cells. Bladder activities for both 99mTc-liposome formulations were 3-6 times lower than for the other agents. PEG 99mTc-liposomes remained in circulation 1.6 times longer than any of the other agents. CONCLUSIONS: Technetium-99m-liposomes, independent of surface modification, had excellent circulation persistence and in vivo stability when compared to 99mTc-red cells and 99mTc-HSA. PEG 99mTc-liposomes performed better than neutral 99mTc-liposomes due to lower liver background activity. Advantages of PEG 99mTc-liposomes compared to 99mTc-red cells include: (a) only one venipuncture, (b) little exposure to patient's blood, (c) excellent in vitro and in vivo stability and (d) lack of drug interference.
Asunto(s)
Liposomas , Tecnecio , Animales , Eritrocitos , Imagen de Acumulación Sanguínea de Compuerta , Corazón/diagnóstico por imagen , Marcaje Isotópico , Liposomas/farmacocinética , Hígado/diagnóstico por imagen , Pulmón/diagnóstico por imagen , Masculino , Compuestos de Organotecnecio , Oximas , Polietilenglicoles , Conejos , Tecnecio/farmacocinética , Agregado de Albúmina Marcado con Tecnecio Tc 99m , Exametazima de Tecnecio Tc 99m , Distribución Tisular , Vejiga Urinaria/diagnóstico por imagenRESUMEN
Physiological responses and circulation properties of liposome-encapsulated hemoglobin (LEH) labeled with technetium-99m (99mTc) were measured in rats after a 10% (170 mg/kg hemoglobin; 430 mg/kg phospholipid) or a 50% (450 mg/kg hemoglobin, 2.3 g/kg phospholipid) hypovolemic exchange transfusion (n = 5 per exchange group). Mean arterial pressure returned to baseline values (105 +/- 8 mmHg) by 90 min post-infusion for both groups. By 20 h, mean arterial pressure remained at baseline values for the 10% group, but dropped to 30 +/- 14 mmHg for the 50% group. For both groups, bradycardia was seen after the exchange period, but heart rate recovered by 30 min for the 10% group and by 90 min for the 50% group. The 99mTc-LEH remained in circulation longer for the 50% group (18.2 h half-life) than for the 10% group (2.4 h half-life). Removal of 99mTc-LEH from the bloodstream was via the liver and spleen. At 20 h, 99mTc-LEH accumulation in these organs was greater for the 10% group (liver, 36.2 +/- 1.7%; spleen, 37.5 +/- 2.5%) than for the 50% group (liver, 17.0 +/- 1.4%; spleen, 17.1 +/- 1.4%). The data show that there is less clearance of 99mTc-LEH from the bloodstream by the reticuloendothelial system after a 50% hypovolemic exchange transfusion, thus supporting the possible use of LEH as an oxygen-carrying resuscitative fluid in situations of severe blood loss.
Asunto(s)
Circulación Sanguínea/fisiología , Sustitutos Sanguíneos/farmacocinética , Recambio Total de Sangre , Hemoglobinas/farmacocinética , Sistema Mononuclear Fagocítico/fisiopatología , Choque/terapia , Anestésicos , Animales , Sustitutos Sanguíneos/administración & dosificación , Portadores de Fármacos , Hemodinámica/efectos de los fármacos , Hemoglobinas/administración & dosificación , Liposomas , Masculino , Tasa de Depuración Metabólica , Ratas , Ratas Sprague-Dawley , Compuestos de Tecnecio , Distribución Tisular/fisiologíaRESUMEN
UNLABELLED: In this study, liposomes labeled with 99mTc have been evaluated as tumor imaging agents. METHODS: Liposomes containing reduced glutathione and carrying either a negative surface charge or no surface charge were labeled with 99mTc using the lipophilic chelator, hexamethylpropyleneamine oxime (HMPAO). The 99mTc-liposomes were intravenously injected into the tail vein of nuce mice which had been implanted intramuscularly in the thigh with nontransfected Chinese hamster ovary cells. Gamma camera images were acquired at 1, 4 and 22 hr and compared with tissue biodistribution studies at 24 hr postinjection. RESULTS: Tumors could be distinguished from normal thigh muscle at 4 hr postinjection for both formulations. Tumor-to-muscle ratios were not significantly different for the two formulations due to the increased normal muscle activity at 24 hr for the neutral liposomes. Liver-to-tumor, liver-to-blood, spleen-to-tumor and spleen-to-blood ratios were significantly lower for the neutral 99mTc-liposomes than for the negative 99mTc-liposomes. Neutral 99mTc-liposomes were cleared slower by the reticuloendothelial system, and therefore remained in the circulation for a longer period of time. CONCLUSION: The results of this study indicate that both formulations could be used as tumor imaging agents, but that neutral 99mTc-liposomes would be more suitable as a drug delivery agent due to their increased total uptake by the tumor and decreased nonspecific uptake by the reticuloendothelial system.
Asunto(s)
Liposomas , Neoplasias Experimentales/diagnóstico por imagen , Compuestos de Organotecnecio , Oximas , Animales , Células CHO/trasplante , Cricetinae , Cricetulus , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Compuestos de Organotecnecio/administración & dosificación , Compuestos de Organotecnecio/farmacocinética , Oximas/administración & dosificación , Oximas/farmacocinética , Cintigrafía , Exametazima de Tecnecio Tc 99m , Distribución TisularRESUMEN
OBJECTIVE: To evaluate the circulation persistence and organ biodistribution of a freeze-dried, oxygen carrying resuscitative fluid: liposome-encapsulated hemoglobin. DESIGN: Randomized, animal studies. SETTING: Accredited animal research facilities. SUBJECTS: Normal female Balb/c mice and male New Zealand rabbits. INTERVENTIONS: Two groups of normal female Balb/c mice were injected in the tail vein with either lyophilized liposome-encapsulated hemoglobin (n = 9) that was reconstituted just before administration, or with unlyophilized liposome-encapsulated hemoglobin (n = 9) as a comparison. Two groups of male New Zealand rabbits were injected in the ear vein with either lyophilized 99mTc-liposome-encapsulated hemoglobin (n = 6) or unlyophilized 99mTc-liposome-encapsulated hemoglobin as a comparison (n = 6). After injection, mice were anesthetized by brief inhalation of halothane followed by blood sampling through the retro-orbital sinus. Rabbits were anesthetized 30 mins before liposome-encapsulated hemoglobin administration with an intramuscular injection of a 5:1 mixture of ketamine (50 mg/kg) and xylazine (10 mg/kg). Rabbits were then dynamically imaged for 90 mins, housed, and at 20 hrs, imaging again followed by autopsy and tissue sampling to validate imaged organ biodistributions. MEASUREMENTS: Circulation persistence in the mouse was measured by removing a blood sample at various time points up to 24 hrs after injection. The blood sample was centrifuged in a hematocrit capillary tube and the disappearance of the sedimented liposome-encapsulated hemoglobin fraction was measured. The change in the sedimented fraction of the liposomes with time was used to generate circulation persistence profiles in mice. The circulation persistence and organ biodistribution of 99mTc-liposome-encapsulated hemoglobin was measured by circling regions of interest on computer-generated gamma camera images. These image intensities were then calculated as a function of total injected dose which was measured from a known volume and activity of 99mTc-liposome-encapsulated hemoglobin. Actual tissue uptake was estimated from images by subtracting blood pool contribution which was measured by injecting 99mTc-labeled rabbit red cells. Imaged organ biodistribution was validated at 20 hrs by measuring activity in weighed portions of tissue after autopsy. MAIN RESULTS: The mean circulation half-life of liposome-encapsulated hemoglobin in mice injected at a dose of 1.0 g phospholipid/kg mouse and 1.95 g hemoglobin/kg was approximately 10.4 +/- 0.5 (SD) hrs. The circulation half-life of lyophilized liposome-encapsulated hemoglobin was 10.7 +/- 0.7 hrs. The circulation profiles demonstrate a rapid removal phase over the first 4 hrs after injection, followed by a secondary slow removal measured up to 24 hrs. The rapid removal phase of liposome-encapsulated hemoglobin and lyophilized liposome-encapsulated hemoglobin in the rabbit (injected at the same dose) indicated that lyophilized liposome-encapsulated hemoglobin persists longer than the unlyophilized form in the first 4 hrs after injection. The organ biodistributions of unlyophilized 99mTc-liposome-encapsulated hemoglobin and lyophilized 99mTc-liposome-encapsulated hemoglobin in the rabbit demonstrate that the reticuloendothelial system is the primary site of removal, with significant uptake of lyophilized 99mTc-liposome-encapsulated hemoglobin by the liver (15.6 +/- 1.0%), bone marrow (12.6 +/- 1.6%), and spleen (9.7 +/- 1.1%). The kidneys showed little accumulation of unlyophilized 99mTc-liposome-encapsulated hemoglobin or lyophilized 99mTc-liposome-encapsulated hemoglobin (1.6 +/- 0.2% and 1.8 +/- 0.1%, respectively), an important result for the efficacy and safety of this hemoglobin-based blood substitute. (ABSTRACT TRUNCATED)