Anu Bali

Carbamyl analogs of phosphatidylcholines: Synthesis, interaction with phospholipases and permeability behavior of their liposomes

A novel class of phospholipase-resisting phosphatidylcholine analogs, in which the C-2 ester group or both C-1 and C-2 ester groups have been replaced by carbomyloxy functions (Formula--see text), have been synthesized. These lipids were not degraded by phospholipase A2, while complete hydrolysis occurred with phospholipase C. Ultrasonic irradiation of the aqueous dispersions of the phospholipids in the presence as well as in the absence of cholesterol resulted in the formation of closed bilayer structures as evidenced by negative staining electron microscopy and also by their ability to entrap [14C]glucose. The leakage rates of glucose at 37 degrees C from liposomes of these compounds have also been measured. Liposomes consisting of 1,2-dipentadecanylcarbamyloxy-sn-glycero-3-phosphorylcholine were found to be more leaky (2.1%/h) as compared to the liposomes of 1-palmitoyl-2-pentadecanylcarbamyloxy-sn-glycero-3-phosphorylcholine (0.5%/h). Moreover, inclusion of cholesterol (33 mol%) into the bilayers of the former phospholipid had no effect on the leakage rate (2.4%/h) while it effectively reduced permeability of the latter (0.22%/h). These phosphatidylcholines are useful for studying the possible role of phospholipases in the capture and lysis of liposomes in vivo.

Specific interactions of liposomes with PMN leukocytes upon incorporating tuftsin in their bilayers

Incorporation of tuftsin derivatives, Thr‐Lys‐Pro‐Arg‐NH‐C18H37 or Thr‐Lys‐Pro‐Arg‐NH‐(CH2)2‐NH‐COC15H31, into an egg phosphatidylcholine/cholesterol liposome bilayer led to significantly enhanced binding of the liposomes to PMN leukocytes at 37°C but not at 0°C. Under identical conditions, no such enhanced binding of the liposomes was observed with erythrocytes and lymphocytes. These results demon‐strate that grafting of tuftsin on the liposome bilayer enables the liposome to recognize specifically the PMN leukocytes and to deliver its contents to these cells.

Protection of mice against Plasmodium berghei infection by a tuftsin derivative

In Plasmodium berghei infections, the mortality rate and parasitaemias were significantly reduced and the mean survival time was considerably enhanced by pretreating the animals with a tuftsin derivative, Thr‐Lys‐Pro‐Arg‐NH‐(CH2)2‐NHCOC15H31. This effect of the modified tuftsin was further increased upon its incorporation in the liposome bilayer. These results indicate that tuftsin and its derivatives may prove useful in enhancing nonspecific host resistance against protozoan infections.

Influence of the phospholipid structure on the stability of liposomes in serum

The effect of serum on the structural integrity of liposomes consisting of ether and/or carbamyl analogs of 1,2-diester phosphatidylcholine (PC) has been evaluated by measuring both the efflux of the entrapped 6-carboxyfluorescein and the lipid transfer to serum proteins, and the results have been compared with the egg PC liposomes. Replacement of the C-1 ester bond in PC by an ether linkage did not significantly enhance the liposome stability, but it was markedly increased upon introducing further structural changes in the C-2 ester region of the resulting 1-ether-2-ester PC. However, the stability was not influenced by altering the steric configuration of the latter phospholipid. These results strongly suggest that lysis of liposomes in serum can be prevented by structurally modifying the ester bond(s) in the phospholipid component of liposomes.

Modification of phospholipid structure results in greater stability of liposomes in serum

Previous studies have revealed that the replacement of the C-2 ester group in phosphatidylcholine by the carbamyloxy function renders the resulting lipids, without affecting the properties of the liposomes, resistant to hydrolysis by phospholipase A2 (Gupta, C.M. Bali, A. (1981) Biochim. Biophys, Acta 663, 506-515). As an extension of this work, the effect of serum on the stability of liposomes, prepared from 1-palmitoyl-2-heptadec-10-cis-enylcarbamyloxyphosphatidylcholine (carbamylphosphatidylcholine), has been examined. The stability has been measured in terms of (a) bilayer permeability to solutes, and (b) the lipid transfer to serum proteins. Replacement of egg phosphatidylcholine in liposomes by the carbamyl analog prevented serum-induced leakage of the entrapped solutes and also inhibited the lipid (phospholipid and cholesterol) transfer. Manipulation of the cholesterol content of the liposomes had no effect on the stability. These observations indicate that the interaction of serum proteins with liposomes probably involves a highly specific binding of the proteins to the liposome surface.

Metastable phase behavior of a sphingolipid analogue.

The phase behavior of the sphingolipid analogue 1-palmitoyl-2-tridecanylcarbamyloxy-sn-glycero-3-phosphocholine (CM-PC) has been studied by differential scanning calorimetry. When CM-PC is cooled at rates greater than 5 K/min, subsequent heating runs exhibit metastable behavior: a low enthalpy exotherm is observed at about 9 degrees C (delta H = -(1-2)kcal/mol), followed by a high enthalpy endotherm at 38 degrees C (delta H = 13 kcal/mol). Systematic variation of cooling/heating protocols indicates that CM-PC exhibits two low temperature states, one metastable and the other stable. Cooling from the liquid crystalline state results in formation of the metastable low-temperature polymorph I, which must transform into the stable low-temperature polymorph II before the liquid crystalline state can be reached again. This metastable thermal behavior is virtually identical to that recently reported for synthetic palmitoyl cerebroside (Ruocco, M.J., Atkinson, D., Small, D.M., Skarjune, R.P., Oldfield, E. and Shipley, G.G. (1981) Biochemistry 20, 5957-5966) and for bovine brain n-acylcerebrosides (Curatolo, W. (1982) Biochemistry, 21, 1761-1764). The observation of the metastable phase behavior of CM-PC indicates that the sphingosine backbone is not a prerequisite for such metastable behavior. Furthermore, the carbamyl group in CM-PC is reversed in orientation compared with the amide of sphingolipids (-NH-CO- vs. -CO-NH-), suggesting that the intermolecular hydrogen bonding potential, rather than some highly specific steric or conformational constraint, is responsible for the observed metastability of sphingolipids.

Effect of phospholipid structure on stability and survival times of liposomes in circulation.

The phosphatidylcholine (PC) component of liposomes was structurally modified by replacing its C-1, or both C-1 and C-2, ester linkage(s) with an ether and/or carbamyl bond(s) or by changing its steric configuration. Small unilamellar liposomes were formed from PC, traces of the corresponding 14C-labeled PC and cholesterol in the presence of 6-carboxyfluorescein (02.M) by sonication, and purified by centrifugation. These liposomes were administered intravenously to rats, and their stability in blood as well as the rate of their clearance from the circulation were determined. Stability and survival times of liposomes were markedly increased by modifying both the C-1 and the C-2 ester linkages in PC. A similar but quantitatively smaller effect was observed when only the C-1 ester linkage was modified. However, the stability remained unaffected by changing the steric configuration of PC, but this modification influenced the clearance rate of liposomes from the circulation. These results demonstrate that both stability in blood and the clearance rate from circulation can be modulated by structurally modifying the ester linkages in the phospholipid component of liposomes.

Stability of liposomes in circulation is markedly enhanced by structural modification of their phospholipid component

Replacement of the C‐2 ester group in phosphatidylcholine by the carbamyloxy function rendered its liposomes completely stable and longer living in the circulation of rats.

Synthesis of carbamyl and ether analogs of phosphatidylcholines

A complete synthesis of 1-O-hexadecyl-2-O-N-(heptadec-8-cis-enyl)carbamyl-sn-glycero-3-Phosphocholine, a novel analog of phosphatidylcholine, has been described. Each step is simple to perform and gives the desired products in high yield. Also, some of the intermediates formed during the synthesis have been efficiently utilized to prepare 1-O-hexadecyl-2-O-oleyl-sn-glycero-3-phosphocholine, 1-O-hexadecyl-2-oleoyl-sn-glycero-3-phosphochloine and 3-O-hexadecyl-2-oeloyl-sn-glycero-1-phosphocholine. These phosphatidylcholine (PC) analogs are useful for studying the possible role of phospholipases in the capture and lyses of liposomes in vivo.