Paul L. Beaumier

Encapsulation, with high efficiency, of radioactive metal ions in liposomes

The encapsulation of radioactive metalic cations, such as 111In3+ or 67Ga3+, in the internal aqueous compartment of liposomes can be achieved with an efficiency of about 90%. The efficient loading of a high specific activity of cations into liposomes involves the transport of 111In3+ or 67Ga3+ through the lipid bilayer to an encapsulated strong chelate, such as nitrilotriacetic acid, by 8-hydroxyquinoline, in conjunction with an efficient anion-exchange resin technique for the removal of the external cations. The efficiency of loading cations to liposomes is affected markedly by the concentration of 8-hydroxyquinoline-metal, and the presence of the chelating agents in the loading incubation mixture. However, the loading efficiency is not affected by the pH of the internal aqueous compartment of liposomes over a range of pH 5-9, the concentration of the liposomes, the method of liposomal preparation, the lamellar structure of the liposomes, and the composition of liposomes. Furthermore, the loading procedures do not appear to affect the size and the permeability of liposomes. There is a good agreement in the tissue distributions of the liposomes prepared by the present loading methods and those by the conventional method of encapsulation by sonication. Liposomes entrapping high specific activity of 67Ga3+ or 111In3+ will be useful for future studies of the in vivo kinetics of liposomes by the combined techniques of scintigraphic imaging and the gamma-ray perturbed angular correlation.

Uptake of small liposomes by non-reticuloendothelial tissues☆

The distribution of liposomes within the intravascular space and the extent to which they escape into extravascular space strongly impact on the application of lipid vesicles as a carrier for pharmacologically active agents. The present study investigates how intact small unilamellar vesicles (SUV) may be taken up by different tissues after intravenous injection into mice, using various types of SUV with different entrapped markers, lipid composition, size, doses of liposomal lipids and stability in the blood. Our focus was specifically on sphingomyelin (or distearoyl phosphatidylcholine)/cholesterol (2:1, mol/mol) SUV, which are known to be stable in the blood circulation. Our results indicated that, in addition to the reticuloendothelial tissues, intact SUV were taken up in several other parts of the body, including intestine, skin, carcass and legs. It appears that the accumulation of SUV in the intestine and the skin increases with time post-injection. Furthermore, from the kinetic data, the process of uptake of SUV by the skin and intestine is compatible with a non-saturable pathway, which follows first-order kinetics. This suggests that the cells involved in the uptake of SUV in the intestine and skin are not phagocytic cells, which are normally saturable.

Effects of liposome size on the degradation of bovine brain sphingomyelin/cholesterol liposomes in the mouse liver

The relative rates of degradation of the outer lipid bilayer of large multilamellar and small unilamellar bovine brain sphingomyelin/cholesterol (2:1; mol/mol) liposomes in the livers of Balb/c mice were compared. The rate of the release of entrapped In-111 ions from the aqueous reservoir of small unilamellar liposomes or from the outermost aqueous compartment of multilamellar liposomes was used to monitor the rate of degradation of the exterior lipid bilayer surface of these liposomes. The technique of gamma-ray perturbed angular correlation and a method for loading In-111 ions into the outermost aqueous compartment of liposomes were used in this investigation. It was found that in the liver the exterior lipid bilayer of large multilamellar liposomes was degraded more rapidly than the bilayer of small unilamellar liposomes in vivo. In contrast to the situation for small unilamellar liposomes, the degradative process for large multilamellar liposomes in the liver was not maintained under ischemic conditions. Our results suggest that multiple pathways operate in the degradation of liposomes in the liver. The rate of degradation of liposomes in the liver may depend on accessibility of liposomes to degradative sites.

Autoradiolysis of iodinated monoclonal antibody preparations

We investigated the effects of ionizing radiation on the immunointegrity of antibody fragments (Fab) because large amounts of high specific activity 131I may damage the proteins. We found that 1000 Gy of external 137Cs gamma radiation was sufficient to destroy 80-90% of the immunointegrity of the initial preparation. This effect was also produced by internally added [131I]NaI in a quantity sufficient to provide the same radiation absorbed dose. Since radioiodinated monoclonal antibodies labeled to high specific activity are being evaluated for radioimmunotherapy, the above observation is significant since high levels of internal radiation occur with therapeutic doses of 131I-labeled antibody. Human serum albumin in low concentration (2%) added to the iodinated antibody solutions was successful in preventing loss of immunoreactivity and can be used to protect and stabilize therapeutic quantities of radiolabeled monoclonal antibody preparations.

Volume of distribution and transcapillary passage of small unilamellar vesicles

This study investigated the biodistribution of bovine brain sphingomyelin (SM)/cholesterol (CH) (2/1; M/M) small unilamellar vesicles (SUV) in mice, addressing specifically the volume of distribution and transcapillary passage of the SUV. The complex of nitrilotriacetic acid with In-111 or Ga-67 ions was encapsulated in the SUV as the radioactive marker for various studies. The structural integrity of liposomes in vitro and in vivo was monitored by the technique of gamma ray perturbed angular correlation. Our data suggested that initially the SM/CH SUV remained within the vascular system and occupied a volume of distribution approximately 1.28 times larger than that of erythrocytes in the vascular system of mice. However, our data also indicated that with time the SM/CH SUV could get out of the vascular system of mice and were taken up by surrounding tissues over a period of 24 hours.

Approaches to improved antibody- and peptide-mediated targeting for imaging and therapy of cancer

Antibodies with their ability to selectivity bind antigens have been of great interest in targeting radiation, drugs and toxins to tumors. Limited success in delivery of radioactivity has been enjoyed with conventional attachment to antibodies. This is due to slow tumor targeting processes and slow disappearance from blood and variable uptake and disappearance from the excretory organs, liver and kidney. Preliminary studies in animal models and patients have shown promise in increasing the tumor to blood exposure ratio by pretargeting antibody followed by small molecule delivery of radioactivity using a molecular capture system. Efficient capture of the small molecule radioactivity carrier by tumor localized antibody and rapid clearance and excretion of the untargeted radioactivity decreases the background problem for imaging and lowers marrow toxicity for radioimmunotherapy. Small peptide ligands that bind to receptor bearing tumors offer similar advantages.