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.

Effect of lipid composition on insulin-mediated fusion of small unilamellar liposomes: a kinetic study.

Liposome-entrapped insulin could be used to prolong the hypoglycemic action of insulin. Also, the conjugation of insulin to the surface of liposomes allows the potential application of using insulin as a transporting molecule to deliver liposome-entrapped drugs to insulin-receptor rich tissues. The success of these two approaches of drug delivery depends on how insulin may interact with liposomes. The present study describes the application of the principle of kinetics to investigate the effect of insulin on the stability of various preparations of liposomal drug carriers. The technique of fluorescence resonance energy transfer was employed to study the destabilization of liposomal formulations through the process of insulin-mediated fusion of liposomes. The kinetics of insulin-mediated fusion appeared to be compatible with a model whereby the initial rate of fusion is governed by the mechanism of fusion of two small unilamellar, unfused liposomal particles. The rate constants of insulin-mediated fusion of various liposomal formulations were estimated from the initial rate of fusion, using the model of two-particle fusion. Arrhenius analysis of the rate constants of fusion at different temperatures suggests that the mechanism of insulin-mediated fusion of small unilamellar vesicles is not governed merely by the energy and frequency of collision between liposomal particles. Other factors, such as the binding of insulin with the surface of liposomes and the temperature effect on the dynamics of the liposomal membrane, as well as conformation of insulin, could potentially be important.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and purification of NB1-palmitoyl insulin.

A procedure for synthesizing NB1-palmitoyl insulin for incorporation into liposomes for targeting to hepatocytes was developed. The amino group of the first amino acid phenylalanine on the B chain (B1) of insulin was selected for conjugation with palmitic acid in anticipation that its binding to the insulin receptor would be preserved. Two other free amino groups present in insulin, the first amino acid glycine on the A chain (A1) and the 29th amino acid lysine on the B chain (B29), were first protected with a t-butoxycarbonyloxy (t-Boc) group to yield NA1, B29-di-(t-Boc) insulin. The identity of this di-(t-Boc) insulin was confirmed by amino acid analysis as well as by enzyme hydrolysis coupled with matrix-assisted laser-desorption time of flight mass spectrometry (MALDI-TOF MS). NA1,B29-Di-(t-Boc) insulin was then reacted with the N-hydroxysuccinimide ester of palmitic acid, followed by deblocking the t-Boc groups, to yield NB1-palmitoyl insulin, the structure of which was further confirmed by MALDI-TOF MS analysis. NB1-palmitoyl insulin was found to interact with the insulin receptor on fat cells, thereby catalyzing the conversion of [14C]glucose into lipids, at reduced efficiency (30-40%).

Targeting small unilamellar liposomes to hepatic parenchymal cells by dose effect.

A major research goal of liposome pharmacology is the selective delivery of drugs to target cell populations while minimizing extraction by phagocytic macrophages and blood monocytes of the reticuloendothelial system. The liver is an ideal organ for studying targeting strategies using a variety of liposomes, inasmuch as its discontinuous capillaries have fenestrae through which liposomes less than 0.2 microns in diameter may escape into the extravascular space. In a previous kinetic study, we proposed that the hepatic uptake of small unilamellar vesicles (SUV) in mice was compatible with a model of uptake involving dual, parallel pathways. One is a saturable, phagocytic pathway of uptake mediated by Kupffer cells, the other is a nonsaturable, pinocytotic pathway of uptake mediated by parenchymal cells, favoring the latter pathway at high liposomal dose (Beaumier et al., 1983). In the present study, we demonstrated by the techniques of liver cells fractionation that the uptake of either the bovine brain sphingomyelin/cholesterol (2:1; mole/mole) SUV or distearoyl phosphatidylcholine/cholesterol (2:1; mole/mole) SUV by hepatic parenchymal cells was enhanced markedly by increasing the amount of injected dose of SUV. As high as 85 to 90% of the total liver dose can be attributed to the uptake of SUV by the hepatic parenchymal cells alone, when the injected dose reaches at or above 7.5 to 10 micrograms of lipid per g b.wt. The dose effect on the uptake of liposomes by hepatocytes appears to be a general phenomenon of neutral SUV. Our data suggested that blockade by dose permits a feasible approach to target SUV to hepatic parenchymal cells.