**3. Liposomes and membrane fusion mimicking**

A remote introduction to the processes of gene transfer is represented by a series of studies aiming at mimicking the process of cell fusion by using model membranes mainly composed of phosphatidylcholine (PC) and phosphatidylserine (PS). It was demonstrated that addition of liposomes containing these lipids fuse with plasma membranes in the presence of Ca2+ and Mg2+ (Papahadjopoulos et al., 1976). Considering that cellular fusion is one of the most fundamental processes in life and that its role has been recognized in the interaction of liposomes with endosomes within the processes of DNA transfection, such studies are highly significant for the knowledge of this particular step of the non viral GT and in designing lipids with the highest characteristics of fusion. The study of the effect of divalent metal cations in the interaction and mixing of membrane components in vesicles prepared from phospholipids led to find that low amounts of Ca2+ and Mg2+ induce extensive mixing of vesicle membrane components and important structural rearrangements to form new membrane structures. The result is a true fusion rather than a simple mixing of vesicles that occurs in the absence of cations. Some evidence was found that fusion of vesicle membranes by Ca2+ and Mg2+ is not simply due to electrostatic charge neutralization, but rather to changes in molecular packing. It is possible to see here an anticipation of the phase transitions that many years after was demonstrated to occur in DNA transfection with co-lipid added lipoplexes, as reported in the previous section. These results have been confirmed by finding that Ca2+ and Mg2+ produce structurally different complexes with PE (Newton et al., 1978). A different behaviour in fusion induced by these two cations was found in mixture of PS/PC, PS/PE and PS/PC/PE (Düzgünes et al. 1981). The extent of fusion by Ca2+ in mixed PE/PC was lower compared to that of pure PS vesicles and was completely inhibited when PC reached 50% in the mixture; rapid fusion was instead obtained in mixtures PS/PE. Mg2+ can fuse PS only in the presence of PE. The fusogenic capacity of Mg2+ was instead completely absent in mixtures PS/PC/PE with 10% of PC. These results show clearly a marked difference between Ca2+ and Mg2+ against fusion: as we will see later, this difference will appear also in some processes concerning the formation of complexes of DNA with neutral liposomes.

A rational mechanism to interpret these results was tried out: kinetics of the interaction between PS vesicles in the presence of Ca2+ (Portis et al., 1979) show the formation of two different complexes: the former develops when the cations bind only to individual vesicles, the latter, which seems correlated with the beginning of membrane fusion, when the vesicles come to close apposition; the former complex is obtained also with Mg2+. Its characteristics led to suggest that this complex is formed when the divalent cations bind to PS head groups on one bilayer only (cis complex). The latter, obtained only with Ca2+, shows different characteristics and seems to involve a polydentate chelation of Ca2+ with

liposomes with endosomes and structural changes that favour the release of DNA in the cytosol. Three phosphoramidates with a cationic polar head derived from natural aminoesters or a methylimidazolium salt were also synthesized and these cationic lipids were formulated with each one of the two new helpers and with cholesterol or DOPE for a comparison of transfections; it is worth noticing that the new helper lipids can improve the

A remote introduction to the processes of gene transfer is represented by a series of studies aiming at mimicking the process of cell fusion by using model membranes mainly composed of phosphatidylcholine (PC) and phosphatidylserine (PS). It was demonstrated that addition of liposomes containing these lipids fuse with plasma membranes in the presence of Ca2+ and Mg2+ (Papahadjopoulos et al., 1976). Considering that cellular fusion is one of the most fundamental processes in life and that its role has been recognized in the interaction of liposomes with endosomes within the processes of DNA transfection, such studies are highly significant for the knowledge of this particular step of the non viral GT and in designing lipids with the highest characteristics of fusion. The study of the effect of divalent metal cations in the interaction and mixing of membrane components in vesicles prepared from phospholipids led to find that low amounts of Ca2+ and Mg2+ induce extensive mixing of vesicle membrane components and important structural rearrangements to form new membrane structures. The result is a true fusion rather than a simple mixing of vesicles that occurs in the absence of cations. Some evidence was found that fusion of vesicle membranes by Ca2+ and Mg2+ is not simply due to electrostatic charge neutralization, but rather to changes in molecular packing. It is possible to see here an anticipation of the phase transitions that many years after was demonstrated to occur in DNA transfection with co-lipid added lipoplexes, as reported in the previous section. These results have been confirmed by finding that Ca2+ and Mg2+ produce structurally different complexes with PE (Newton et al., 1978). A different behaviour in fusion induced by these two cations was found in mixture of PS/PC, PS/PE and PS/PC/PE (Düzgünes et al. 1981). The extent of fusion by Ca2+ in mixed PE/PC was lower compared to that of pure PS vesicles and was completely inhibited when PC reached 50% in the mixture; rapid fusion was instead obtained in mixtures PS/PE. Mg2+ can fuse PS only in the presence of PE. The fusogenic capacity of Mg2+ was instead completely absent in mixtures PS/PC/PE with 10% of PC. These results show clearly a marked difference between Ca2+ and Mg2+ against fusion: as we will see later, this difference will appear also in some processes concerning the formation of complexes of

A rational mechanism to interpret these results was tried out: kinetics of the interaction between PS vesicles in the presence of Ca2+ (Portis et al., 1979) show the formation of two different complexes: the former develops when the cations bind only to individual vesicles, the latter, which seems correlated with the beginning of membrane fusion, when the vesicles come to close apposition; the former complex is obtained also with Mg2+. Its characteristics led to suggest that this complex is formed when the divalent cations bind to PS head groups on one bilayer only (cis complex). The latter, obtained only with Ca2+, shows different characteristics and seems to involve a polydentate chelation of Ca2+ with

transfection by a factor of 100 compared with DOPE.

DNA with neutral liposomes.

**3. Liposomes and membrane fusion mimicking** 

the head groups of PS from apposed membranes (trans complex). The formation of this PS/Ca2+ complex is of crucial importance for the fusion of the vesicles. Apart from the names used to identify these complexes, it is worth noting that the structure suggested for the latter complex agrees with the one found in the complex between the neutral lipid DPPC and DNA, promoted by divalent metal cations, showing a CL phase. A more <sup>α</sup> detailed study (Wilschut et al., 1980) allowed to obtain further information about the process: it was demonstrated that fusion is one of the earliest events during the Ca2+ induced aggregation of SUVs (small unilamellar vesicles) of PS and occurs at a similar time scale, which means that fusion doesn't require initial rupture of the vesicles. The close contact between the vesicles induced by Ca2+ is sufficient to trigger the immediate fusion of the two membranes and the mixing of the internal volumes with a relative low leakiness of their content: which makes the Ca2+/PS system an almost ideal model for membrane fusion. This model has been later deeply developed and is the basis to explain the processes which occur in the cytosol when the complexes liposomes/DNA encounter the endosomes and release the DNA. With these last findings the route to the DNA delivery to cells by means of liposomes was opened.
