William T. Phillips

Circulation and biodistribution profiles of long-circulating PEG-liposomes of various sizes in rabbits

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.

A simple method for producing a technetium-99m-labeled liposome which is stable In Vivo

A new method for labelling preformed liposomes with technetium-99m (99mTc) has been developed which is simple to perform and stable in vivo. Previous 99mTc-liposome labels have had variable labeling efficiencies and stability. This method consistently achieves high labeling efficiencies (greater than 90%) with excellent stability. A commercially available radiopharmaceutical kit--hexamethylpropyleneamine oxime (HM-PAO)--is reconstituted with 99mTcO4- and then incubated with preformed liposomes that encapsulate glutathione. The incubation takes only 30 min at room temperature. Liposomes that co-encapsulate other proteins such as hemoglobin or albumin, in addition to glutathione, also label with high efficiency. Both in vitro and in vivo studies indicate good stability of this label. Rabbit images show significant spleen and liver uptake at 2 and 20 h after liposome infusion without visualization of thyroid, stomach or bladder activity. This labeling method can be used to study the biodistribution of a wide variety of liposome preparations that are being tested as novel drug delivery systems. This method of labeling liposomes with 99mTc may also have applications in diagnostic imaging.

Delivery of gamma-imaging agents by liposomes.

Liposomes are spherical bilayers which spontaneously form when water is added to a dried lipid mixture. Much progress has been made in the use of liposomes as vehicles for the delivery of gamma imaging agents. These radiolabeled liposomes have a wide variety of potential diagnostic uses including the detection of sites of infection, inflammation, gastrointestinal bleeding, tumor, cardiac blood pool imaging and lymphoscintigraphy. The ability to modify the surface of the liposomes permits the customization of liposome formulations for each particular diagnostic use.

Rapid Gastric Emptying of a Solid Pancake Meal in Type II Diabetic Patients

OBJECTIVE To estimate the rate of gastric emptying of a solid pancake carbohydrate meal in recently diagnosed asymptomatic type II diabetic patients compared with nondiabetic control subjects. RESEARCH DESIGN AND METHODS Gastric emptying studies using radiolabeled meals were performed on eight recently diagnosed asymptomatic diabetic patients and on eight sex-, BMI- and age-matched nondiabetic control subjects. Although a liquid protein drink was administered along with the pancake meal, the radioactivity was adherent to only the pancake portion of the meal. Plasma glucose and serum insulin levels were measured in fasting and postprandial blood samples collected at 15-min intervals up to 120 min after ingestion of the mixed nutrient meal. RESULTS The average gastric half-emptying time (time it takes for one-half of the meal to empty) was significantly more rapid for the diabetic patients (45.3 ± 4.8 min) when compared with the nondiabetic control subjects (60.4 ± 5.1 min; P = 0.05). The serum insulin concentrations were not statistically different between the two groups. Plasma glucose values were significantly higher in the diabetic patients compared with the nondiabetic control subjects. CONCLUSIONS Type II diabetic patients with no clinical evidence of neuronal dysfunction have a significantly more rapid rate of gastric emptying of a solid high-carbohydrate meal when compared with nondiabetic control subjects.

In vivo PET imaging and biodistribution of radiolabeled gold nanoshells in rats with tumor xenografts.

Here we report the radiolabeling of gold nanoshells (NSs) for PET imaging in rat tumor model. A conjugation method was developed to attach NSs with the radionuclide, (64)Cu. The resulting conjugates showed good labeling efficiency and stability in PBS and serum. The pharmacokinetics of (64)Cu-NS and the controls ((64)Cu-DOTA and (64)Cu-DOTA-PEG2K) were determined in nude rats with a head and neck squamous cell carcinoma xenograft by radioactive counting. Using PET/CT imaging, we monitored the in vivo distribution of (64)Cu-NS and the controls in the tumor-bearing rats at various time points after their intravenous injection. PET images of the rats showed accumulation of (64)Cu-NSs in the tumors and other organs with significant difference from the controls. The organ biodistribution of rats at 46h post-injection was analyzed by radioactive counting and compared between the (64)Cu-NS and the controls. Different clearance kinetics was indicated. Neutron activation analysis (NAA) of gold concentration was performed to quantify the amount of NSs in major tissues of the dosed rats and the results showed similar distribution. Overall, PET images with (64)Cu had good resolution and therefore can be further applied to guide photothermal treatment of cancer.

Radioactive liposomes: Radioactive liposomes

Many methods of labeling liposomes with both diagnostic and therapeutic radionuclides have been developed since the initial discovery of liposomes 40 years ago. Diagnostic radiolabels can be used to track nanometer-sized liposomes in the body in a quantitative fashion. This article reviews the basic methods of single photon emission computed tomography (SPECT) and positron emission tomography (PET) imaging and labeling of liposomes with single photon and dual photon positron emission radionuclides. Examples of the use of these diagnostic imaging agents will be shown. The ability to track the uptake of liposomes in humans and research animals on a whole body basis is providing researchers with an excellent tool for developing liposome-based drug delivery agents. The attachment of therapeutic radionuclides to liposomes also has great promise in cancer therapy. Recent developments in the use of liposomes carrying therapeutic radionuclides for cancer therapy will also be reviewed. Many of the radiolabeling and tracking technologies developed for nanosized liposomes will also be useful for the imaging and tracking of other nanoparticles.

Circulation Kinetics and Organ Distribution of Hb-Vesicles Developed as a Red Blood Cell Substitute

Phospholipid vesicles encapsulating concentrated human hemoglobin (Hb-vesicles, HbV), also known as liposomes, have a membrane structure similar to that of red blood cells (RBCs). These vesicles circulate in the bloodstream as an oxygen carrier, and their circulatory half-life times (t1/2) and biodistribution are fundamental characteristics required for representation of their efficacy and safety as a RBC substitute. Herein, we report the pharmacokinetics of HbV and empty vesicles (EV) that do not contain Hb, in rats and rabbits to evaluate the potential of HbV as a RBC substitute. The samples were labeled with technetium-99m and then intravenously infused into animals at 14 ml/kg to measure the kinetics of HbV elimination from blood and distribution to the organs. The t1/2 values were 34.8 and 62.6 h for HbV and 29.3 and 57.3 h for EV in rats and rabbits, respectively. At 48 h after infusion, the liver, bone marrow, and spleen of both rats and rabbits had significant concentrations of HbV and EV, and the percentages of the infused dose in these three organs were closely correlated to the circulatory half-life times in elimination phase (t1/2β). Furthermore, the milligrams of HbV per gram of tissue correlated well between rats and rabbits, suggesting that the balance between organ weight and body weight is a fundamental factor determining the pharmacokinetics of HbV. This factor could be used to estimate the biodistribution and the circulation time of HbV in humans, which is estimated to be equal to that in rabbit.

Treatment With an Oral Proteinase Inhibitor Slows Gastric Emptying and Acutely Reduces Glucose and Insulin Levels After a Liquid Meal in Type II Diabetic Patients

OBJECTIVE To determine whether an oral trypsin/chymotrypsin inhibitor, POT II, will delay the rate of gastric emptying in recently diagnosed type II diabetic patients and improve their postprandial metabolic parameters. RESEARCH DESIGN AND METHODS Two gastric emptying studies were performedon each of six type II diabetic patients. During one study, the patient ingested a glucose/protein solution, and during the other study, the patientingested the same glucose/protein solution with the addition of 1.5 g ofPOT II, a putative stimulant of cholecystokinin (CCK) release. Each patient served as their own control subject. Each of the two oral solutions were administered to the patients in a counterbalanced order separated by at least 1 week. RESULTS Serum insulin, plasma glucose, plasma gastric inhibitory polypeptide (GIP) values, and the rate of gastric emptying were all significantly (P <0.05) decreased over the 2-h testing period when POT II was added tothe oral glucose/protein meal. The area under the curve above baseline for glucose with POT II was 75% of the glucose value without POT II. The areaunder the curve above baseline for insulin with POT II was 68% of the value without POT II. Plasma CCK was significantly increased by POT II 15 min postprandially. CONCLUSIONS A trypsin/chymotrypsin inhibitor, POT II, can delay therate of gastric emptying, and decrease postprandial plasma glucose levels, GIP levels, and serum insulin levels in type II diabetic patients diagnosed recently. Delay of gastric emptying in diabetic patients may provide a uniqueor adjunctive approach to the treatment of type II diabetes.

Integrin αvβ3-targeted gold nanoshells augment tumor vasculature-specific imaging and therapy

Purpose Gold nanoshells (NSs) have already shown great promise as photothermal actuators for cancer therapy. Integrin αvβ3 is a marker that is specifically and preferentially overexpressed on multiple tumor types and on angiogenic tumor neovasculature. Active targeting of NSs to integrin αvβ3 offers the potential to increase accumulation preferentially in tumors and thereby enhance therapy efficacy. Methods Enzyme-linked immunosorbent assay (ELISA) and cell binding assay were used to study the in vitro binding affinities of the targeted nanoconjugate NS–RGDfK. In vivo biodistribution and tumor specificity were analyzed using 64Cu-radiolabeled untargeted and targeted NSs in live nude rats bearing head and neck squamous cell carcinoma (HNSCC) xenografts. The potential thermal therapy applications of NS–RGDfK were evaluated by subablative thermal therapy of tumor xenografts using untargeted and targeted NSs. Results ELISA and cell binding assay confirmed the binding affinity of NS–RGDfK to integrin αvβ3. Positron emission tomography/computed tomography imaging suggested that tumor targeting is improved by conjugation of NSs to cyclo(RGDfK) and peaks at ~20 hours postinjection. In the subablative thermal therapy study, greater biological effectiveness of targeted NSs was implied by the greater degree of tumor necrosis. Conclusion The results presented in this paper set the stage for the advancement of integrin αvβ3-targeted NSs as therapeutic nanoconstructs for effective cancer therapy.

The use of scintigraphic imaging as a tool in the development of liposome formulations.

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.