Peritoneal retention of liposomes: Effects of lipid composition, PEG coating and liposome charge.

In the treatment of peritoneal carcinomatosis, systemic chemotherapy is not quite effective due to the poor penetration of cytotoxic agents into the peritoneal cavity, whereas intraperitoneal administration of chemotherapeutic agents is generally accompanied by quick absorption of the free drug from the peritoneum. Local delivery of drugs with controlled-release delivery systems like liposomes could provide sustained, elevated drug levels and reduce local and systemic toxicity. In order to achieve an ameliorated liposomal formulation that results in higher peritoneal levels of the drug and retention, vesicles composed of different phospholipid compositions (distearoyl [DSPC]; dipalmitoyl [DPPC]; or dimiristoylphosphatidylcholine [DMPC]) and various charges (neutral; negative, containing distearoylphosphatidylglycerol [DSPG]; or positive, containing dioleyloxy trimethylammonium propane [DOTAP]) were prepared at two sizes of 100 and 1000nm. The effect of surface hydrophilicity was also investigated by incorporating PEG into the DSPC-containing neutral and charged liposomes. Liposomes were labeled with (99m)Tc and injected into mouse peritoneum. Mice were then sacrificed at eight different time points, and the percentage of injected radiolabel in the peritoneal cavity and the tissue distribution in terms of the percent of the injected dose/gram of tissue (%ID/g) were obtained. The ratio of the peritoneal AUC to the free label ranged from a minimum of 4.95 for DMPC/CHOL (cholesterol) 100nm vesicles to a maximum of 24.99 for DSPC/CHOL/DOTAP 1000nm (DOTAP 1000) vesicles. These last positively charged vesicles had the greatest peritoneal level; moreover, their level remained constant at approximately 25% of the injected dose from 2 to 48h. Among the conventional (i.e., without PEG) 100nm liposomes, the positively charged vesicles again showed the greatest retention. Incorporation of PEG at this size into the lipid structures augmented the peritoneal level, particularly for negatively charged liposomes. The positively charged PEGylated vesicles (DOTAP/PEG 100) had the second-greatest peritoneal level after DOTAP 1000; however, their peritoneal-to-blood AUC ratio was low (3.05). Overall, among the different liposomal formulations, the positively charged conventional liposomes (100 and 1000nm) provided greater peritoneal levels and retention. DOTAP/PEG100 may also be a more efficient formulation because this formulation can provide a high level of anticancer drug into the peritoneal cavity and also can passively target the primary tumor.

Polyethylene glycol (PEG) modified 99mTc-HMPAO-liposome for improving blood circulation and biodistribution: the effect of the extent of PEGylation.

Modification of liposomes using polyethylene glycol (PEG) results in steric hindrance to the phagocyte system and prolongation of blood circulation time. However, PEGylation can reduce radiolabeling efficiency (RE) when using the glutathione method for radiolabeling the liposomes. Therefore, we investigated the effect of the extent of PEGylation (PEG extent (PEGExt): 0, 5, 9.6, and 13.7 mol%) on the in vivo biodistribution of liposomes in Wistar rats, and RE with technetium-(99m) ((99m)Tc). PEGylated liposomes were prepared with egg phosphatidylcholine (egg PC, 1.85 mol%), cholesterol (1.0 mol%), and distearoylphosphatidylethanolamine-N-[polyethylene glycol] (DSPE-PEG; 0, 5, 9.6, and 13.7 mol%, respectively). The size distribution of the PEGylated liposomes was analyzed by a dynamic light scattering. The (99m)Tc-hexamethylpropylene-amine oxime ((99m)Tc-HMPAO) complexes were used for radiolabeling of preformed liposomes. The labeling efficiency and stability was analyzed with Sephadex G-15 column, and the biodistribution studies of (99m)Tc-liposomes after intravenous (i.v.) injection were also investigated with Wistar rats. The sizes of PEGylated liposomes decreased by increasing the PEGExt to 9.6 mol%, whereas sizes increased at 13.7 mol%. RE of (99m)Tc were greater than 90% for all PEGExt tested, and radiolabeling stability in human plasma was enhanced as a function of PEGExt. Liposomes without PEG were cleared rapidly from the blood and accumulated preferentially in the liver and the spleen. When PEGExt was increased, the accumulation in the organs decreased. This accumulation of PEG was maximized at 9.6 mol%. Accumulation of the liposomes in the spleen was increased again when PEGExt increased to 13.7 mol%. The splenic uptake of liposomes seemed to be dependent not only on PEGExt but also on the size of the liposomes. In conclusion, the PEG chains on the surface of liposome have no influence on the labeling efficiency, and the prolongation of circulation time was maximized at the 9.6 mol% of PEGylation.

Localization and diagnosis of septic endoprosthesis infection by using 99mTc-HMPAO labelled leucocytes.

The aim of this study was to evaluate, retrospectively, the diagnostic value of Tc hexamethylpropylene amine oxime (99mTc-HMPAO) labelled autologous leucocytes for the preferred septic localizations of the infection of the endoprosthesis. We retrospectively reviewed 67 patients with implanted endoprostheses. Diagnosis was found in 42/67 patients. In 25/67 patients we were able to negate an acute pathological process of infection of the endoprosthesis. Our patients were divided into three groups according to the type of endoprosthesis (hip joint, knee joint, shoulder joint). The localizations of the endoprosthesis disorders are shown. The preferred localizations of the acute infection of the hip endoprosthesis are the regio intertrochanterica and the middle part of the shaft of the prosthesis. The preferred localization of the acute infection of the knee endoprosthesis is the proximal shaft of the tibia. The preferred localization of the acute infection of the shoulder endoprosthesis is the distal end of the prosthesis in the proximal humerus. It is hoped that the knowledge of these preferred localizations of infection of endoprosthesis will help patients and doctors in diagnosis and treatment in the future.

Noninvasive measurement of cerebral blood flow with (99m)Tc-hexamethylpropyleneamine oxime single-photon emission computed tomography and 1-point venous blood sampling.

BACKGROUND AND PURPOSE: The arterial and venous blood concentration of technetium 99m-labeled hexamethylpropyleneamine oxime ((99m)Tc-HMPAO) reaches an equilibration more rapidly than other CBF tracers. We hypothesized that (99m)Tc radioactivity of a venous sample at equilibrium, which is similar to that of an arterial sample, would allow estimation of the integrated input function for the clinical measurement of CBF by use of single-photon emission CT. METHODS: In 53 patients with stable cerebrovascular disease, the radioactivity of a venous sample 5 minutes after injection of (99m)Tc-HMPAO was correlated with 5-minute arterial blood radioactivity and the first 5 minutes of the integrated arterial curves of the lipophilic tracer. The measured CBF values were compared with those of xenon 133. RESULTS: The radioactivity of 5-minute venous blood was almost equivalent to that of 5-minute arterial blood (r(2) = 0.987; y = 0.993x + 1.63; P : <0.0001). The correlation between the venous blood radioactivity and the integrated arterial lipophilic fraction was excellent (r(2) = 0.935, P : <0.0001). A strong correlation was obtained between (99m)Tc-HMPAO and (133)Xe CBF values (r(2) = 0.825, P : <0.0001). CBF values were reproducible (coefficient of variation, 8.6%). CONCLUSIONS: This approach is fast, simple, and an alternative to continuous blood sampling in clinical quantitative (99m)Tc-HMPAO CBF studies.

Planar scintigraphy and SPET with 99Tcm-HMPAO-labelled leukocytes in patients with median sternotomy: normal patterns.

The aim of this study was to determine the normal planar and SPET patterns of the thoracic distribution of 99Tcm-hexamethylpropylene amine oxime (99Tcm-HMPAO) in 20 patients who had undergone a previous median sternotomy and without infectious complications at follow-up. The study included anterior and oblique anterior planar views at 4 and 20 h. SPET of the chest was also carried out at 4 and 20 h. At 4 h, the planar views showed low background vascular activity in the lungs and cardiac region in addition to the sternal uptake, which showed two patterns: homogeneous in five patients and heterogeneous in 15. A long and narrow defect of uptake along the sternal midline was the most characteristic finding. At 4 h, in addition to the background vascular activity in the lungs and cardiac region, the greatest uptake on SPET was in the sternum anteriorly and the marrow spine posteriorly without any focal uptake, allowing visualization of the mediastinum free of focal activity. At 20 h, both the planar and SPET images showed a higher organ-to-background ratio. Knowledge of these post-surgical patterns will make it easier to interpret planar and SPET images when 99Tcm-HMPAO-labelled leukocytes are used in the diagnosis of mediastinitis and sternal infections in patients who had previously undergone median sternotomy. Planar views were better for the assessment of sternal uptake, but SPET views were better for the direct visualization of the mediastinum by eliminating overlapping sternal uptake.

A trial study of leukocyte labeling with stabilized Tc-99m D,L-HMPAO by methylene blue and sodium phosphate buffer.

UNLABELLED: We attempted to label leukocytes with stabilized Tc-99m D,L-HMPAO by methylene blue and sodium phosphate buffer (S-HMPAO). METHODS: The results were compared with unstabilized Tc-99m D,L-HMPAO (U-HMPAO). U-HMPAO was obtained by reconstituting a commercial vial of D,L-HMPAO. Stabilization of the kit was performed by the addition of methylene blue. The leukocytes were labeled using a modified published method. The test samples of S-HMPAO and U-HMPAO were prepared immediately, and stood for 0.5, 1, 2, 4, and 6 h, respectively, at room temperature before analysis. RESULTS: In comparison with U-HMPAO: (1) the radiochemical purity of S-HMPAO was higher; (2) the labeling efficiencies of S-HMPAO labeled leukocytes were higher and consistent; (3) the viability of S-HMPAO labeled leukocytes was as high as the viability of U-HMPAO labeled leukocytes at any time; and (4) the percentages of disintegrated from S-HMPAO labeled leukocytes in plasma were lower. CONCLUSION: S-HMPAO is more stable than U-HMPAO and can provide higher leukocyte labeling efficiency. S-HMPAO, therefore, has the potential to replace U-HMPAO as a leukocyte-labeling agent.

Stabilization of Tc-99m D,L-HMPAO preparations as a leucocyte labelling agent.

An attempt was made to use stabilized Tc-99m D,L-HMPAO (S-HMPAO) to label leucocytes. The radiochemical purity of Tc-99m D,L-HMPAO, labelling efficiency of leucocytes, cell viability of labelled leucocytes, and stability of S-HMPAO labelled leucocytes were calculated. In comparison with commercial Tc-99m D,L-HMPAO (C-HMPAO) without stabilization, immediately, at 0.5, 1, 2, 4, and 6 h after HMPAO preparation, the radiochemical purity of S-HMPAO and the labelling efficiencies of S-HMPAO labelled leucocytes were higher. S-HMPAO is more stable than C-HMPAO and can provide higher leucocyte labelling efficiency. S-HMPAO, therefore, has the potential to replace C-HMPAO as a leucocyte-labelling agent.

Labelling lymphocytes with technetium99m-hexamethyl propyleneamine oxime for scintigraphy: an improved labelling procedure.

Leukocyte scintigraphy has been used as a standard diagnostic procedure for the detection of inflammation in vivo. In this study, we developed a method of labelling purified lymphocytes with technetium99m-hexamethyl propyleneamine oxime (Tc99m-HMPAO) without significantly impairing their function. This was confirmed by measurements of in vitro lymphocyte adhesion and migration and of both necrotic and apoptotic cell death. The results of the in vitro control studies indicate that the dysfunction of leukocytes caused by Tc99m-HMPAO labelling can be minimized by using a gentle labelling method and low Tc99m activity. Because lymphocytes have been thought to participate specifically in the pathogenesis of inflammatory bowel disease (IBD), we compared scintigraphies obtained with Tc99m-HMPAO-labelled purified lymphocytes and mixed leukocytes in colitis patients. We found that a lower number of Tc99m-HMPAO-labelled peripheral blood lymphocytes accumulated in the inflamed colon during the first 4 h than labelled mixed leukocytes. The results are likely to reflect the dissimilar kinetics of lymphocyte traffic compared with granulocytes in IBD. We do not recommend the use of Tc99m-HMPAO-labelled purified lymphocytes as a diagnostic tool in chronic colitis. However, the in vitro data indicate that Tc99m-HMPAO-labelled lymphocytes may be suitable for studying short term lymphocyte recirculation and lymphocyte kinetics in other types of inflammation.

Interference of patient medication in the radiolabelling of white blood cells: an update.

Evidence is now accumulating that patient medication can adversely affect the radiolabelling of white cells. We have undertaken a survey for the years 1981-97 to examine instances of unusually low labelling efficiencies of 111In-tropolone and 99Tcm-HMPAO labelled white cells. Respondents were asked to ascertain which drugs were being taken on the day of the test. Fifty adverse reports were received during that period. Many patients were taking drugs which are known to affect white cell function, including cephalosporins, azathioprine, prednisolone, cyclophosphamide, nifedipine, suphasalazine, iron salts and heparin. Using Bradford-Hill's criteria to assess whether an association between two variables is also one of causation, it was found that there was a high probability that the above drugs caused the low labelling efficiencies.

Radiolabelled mixed leukocytes and pure granulocytes with stabilized 99Tcm-exametazime.

Although the methylene blue stabilizer extends the shelf life of 99Tcm-exametazime to 4-6 h after reconstitution, the dark blue appearance of the mixture of stabilized 99Tcm-exametazime and blood components makes it impossible to separate out the leukocyte button. The aim of this study was to assess the feasibility of using stabilized 99Tcm-exametazime to radiolabel mixed leukocytes separated by Volex sedimentation with hypotonic lysis (VL) and pure granulocytes isolated by a single-density Ficoll-Hypaque gradient with hypotonic lysis (FL). Isolated cells from 40-ml and 80-ml donor blood samples were mixed with 0.5 ml stabilized 99Tcm-exametazime (approximately 925 MBq 99Tcm and 62.5 micrograms exametazime) and incubated at room temperature for 15 min. After incubation, two dilution steps with 3 ml and 9 ml of 12.6% ACD/NS (anticoagulant citrate dextrose, solution A, USP, mixed with 0.9% NaCl, v/v) were conducted to dilute the dark blue mixture and to remove any unbound 99Tcm activity. With the addition of 9 ml of 12.6% ACD/NS solution to the 1-ml bottom portion from the first dilution, the supernatant of the centrifuged preparation was clear enough to be withdrawn. The overall labelling efficiency (LE) of labelled leukocytes and granulocytes was 87.1 +/- 4.9% and 87.7 +/- 6.2%, respectively (n = 12 each). Overall, radiolabelled cells (n = 12) from the 80-ml blood samples (LE = 90.3 +/- 2.8%) had an approximately 6% higher labelling efficiency than from the 40-ml blood samples (LE = 84.5 +/- 6.0%) and also had a slightly better in vitro stability compared to the 40-ml samples. The in vitro stability studies showed that only approximately 2% (n = 48) 99Tcm activity was eluted each hour from the radiolabelled leukocytes or granulocytes for the 40-ml or 80-ml blood samples during the 6-h evaluation period. Cell viability of all labelled leukocyte samples was confirmed by the trypan blue staining technique. In conclusion, mixed leukocytes separated by the VL method and pure granulocytes isolated by the FL method can be effectively labelled with stabilized 99Tcm-exametazime with the use of the 'double dilution' technique.