**Neutral Liposomes and DNA Transfection**

Michela Pisani, Giovanna Mobbili and Paolo Bruni

*Marche Polytechnic University Italy* 

#### **1. Introduction**

318 Non-Viral Gene Therapy

Zuhorn, I.S.; Bakowsky, U.; Polushkin, E.; Visser, W.H.; Stuart, M.C.; Engberts, J.B. &

Zhang, Z.; Yang, C.; Duan, Y.; Wang, Y.; Liu, J.; Wang, L. & Kong, D. (2010). Poly(ethylene

Zhang, S.B.; Zhao, Y.N.; Zhao, B.D. & Wang, B. (2010). Hybrids of Nonviral Vectors for Gene Delivery. *Bioconjugate Chem.*, Vol.21, No.6, pp. 1003-1009, ISSN 1043-1082 Zhdanov, R.I.; Podobed, O.V. & Vlassov, V.V. (2002). Cationic Lipid–DNA Complexes-

Zhi, D.F.; Zhang, S.B.; Wang, B., Zhao, Y.N., Yang B.L. & Yu S.J. (2010). Transfection

Delivery. *Bioconjugate Chem.*, Vol.21, No.4, pp. 563-577, ISSN 1043-1082

No.5, pp. 801-810, ISSN 1525-0016

No.1, pp. 1-10, ISSN 0168-3659

64, ISSN 1567-5394

Hoekstra, D. (2005). Nonbilayer Phase of Lipoplex-membrane Mixture Determines Endosomal Escape of Genetic Cargo and Transfection Efficiency. *Mol. Ther.*, Vol.11,

glycol) Analogs Grafted with Low Molecular Weight Poly(ethylene imine) As Nonviral Gene Vectors. *Acta Biomater.*, Vol.6, No.7, pp. 2650-2657, ISSN 1742-7061 Zhang, S.B.; Zhao, B.D.; Jiang, H.M.; Wang, B. & Ma, B.C. (2007). Cationic Lipids and

Polymers Mediated Vectors for Delivery of siRNA. *J. Controlled Release*, Vol.123,

lipoplexes-for Gene Transfer and Therapy. *Bioelectrochemistry.* Vol.58, No.1, pp. 53-

Efficiency of Cationic Lipids with Different Hydrophobic Domains in Gene

Non viral gene transfer vectors for human gene therapy (HGT) applications represent today one of the widest fields of chemical, biological and medical research. Some authors (Kostaleros & Miller, 2005) have expressed the opinion that the future of a safe and efficient gene therapy will depend on suitable synthetic vectors of genetic material, rather than on viruses. The reason for this preference is based on the consideration that viruses, although characterized by high transfection efficiency of genetic material, may suffer from some serious disadvantages such as immune response (Marshall, 2000) and potential oncogenic activity (Hacein-Bey-Abina et al., 2003), as well as a high cost of preparation of the transferring system. On the contrary, synthetic vectors have many potential advantages, such as lack of immunogenicity and oncogenicity, no limits to the size of nucleic acids to be carried inside the cells (Harrington et al., 1997; Roush, 1997; Willard, 2000) and finally preparation procedures cheap and easy to perform. Nevertheless an awkward problem that accompanies their use is the low efficiency of the transfections *in vivo*. Among the synthetic vectors, the most explored by the researchers are the cationic liposomes (CLs), after Felgner and collaborators (Felgner et al., 1997) described the synthesis of the first cation lipid, *N*-[1- (2,3-dioleoyloxy)propyl]-*N*,*N*,*N*-trimethylammonium chloride (DOTMA) and demonstrated that it was able to bind DNA and transfect it both *in vitro* and *in vivo* experiments. Other cationic lipids followed and became popular, such as the commercially widely used N-[1- (2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), dimethyldioctadecylammonium bromide (DDAB) and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol); many others were synthesized during the past years and are still being synthesized. A few years after the Felgner's discovery scientists' interest was focused also on cationic polymers (Wu, G.Y. & Wu; C.H., 1987), that became popular after the discovery of polyethylenimines (Boussif, 1995) and are still the object of great interest. Both classes of cationic compounds owe their interest to the formation of stable complexes with DNA, called lipoplexes and polyplexes respectively, formed through an electrostatic interaction between the cationic head of lipids and the negative phosphates of DNA. The great amount of data and experiments reported in the literature with cationic vectors and some encouraging results in *in vivo* transfection experiments have led to the significant milestone of 20% of the ongoing clinical trials run with synthetic vectors (Edelstein et al., 2004): however the goal of a higher efficiency is still a problem to be solved. As recently stated (Safinya et al., 2006), the long-term target of research in this area is to develop a general

Neutral Liposomes and DNA Transfection 321

the amount of the toxic cationic component and, more important, to alter the physical properties of the delivery vehicle, in a way that favours some of the most complex steps of the whole mechanism of transfection: the consequence is that both actions affect the quality of the transfection. The most widely used neutral helpers in DNA transfection experiments are the 1,2-dioleoyl-*sn*-glycero-3-phosphocholine (DOPC) and the 1,2-dioleoyl-*sn*-glycero-3 phosphoethanolamine (DOPE), sometimes in combination with cholesterol: the main

lipoplexes, while DOPE induces an inverted hexagonal <sup>C</sup> HII phase. Earlier studies led to propose that DOPE is responsible for more efficient transfections because its hexagonal phase is able to fuse readily with anionic vesicles (Koltover et al., 1998) and destabilize the bilayer membranes, making easier the DNA escape from the endosomes, once the lipoplex has entered the cells. Therefore it was believed that DOPE was more useful than DOPC to realize the most efficient transfections. In support of this claim it was also suggested (Mui et al., 2000) that the inverted hexagonal phase promoted by DOPE has a higher ability to disrupt the membrane integrity than the lamellar one induced by DOPC. It turned soon evident that the situation was not so simple and the interpretation was not unambiguous: indeed, if it is verified that the DNA complex with cationic DOTAP, in which DOPE is present as helper in an amount of 70%, transfects better than the corresponding complex

showing similar transfection efficiency, were also synthesized ( Lin et al., 2003). More generally, many literature data show the difficulty in finding a general correlation between vector formulations and transfection efficiency. Trying to explain these apparently contradictory results, some researchers have recently suggested the opportunity to consider the possibility that complexes can bear a structural modification within the phase of the

phase being an example among others (Safinya, 2001). Starting from the observation that a

(Caracciolo & Caminiti, 2005) that perhaps a compelling correlation between the structure of a complex and its transfection efficiency does not simply exist and that the lower transfection efficiency of the synthetic carriers of DNA with respect to virus depends on a poor understanding of the supramolecular structures of the complexes, on the mechanism of their interaction with cells and of the release of DNA within the nucleus. If these statements are true, and there are reasons to confirm them, a more exhaustive knowledge of the subject is particularly important also with reference to the use of NLs as independent carriers of DNA, because it is likely that similar problems will arise and the need to overcome them will be even more important. Of course, understanding the mechanism is crucial for any successful design of a non viral gene delivery, whatever path has been chosen. Besides some early studies, that assumed a fusion between liposomes and cell membranes as the initial step of the process, it is today recognized that the uptake into an endocytic pathway is required for fusion to occur **(**Wrobel **&** Collins, 1995) and the whole aspect has been the object of deep attention (Liu & Huang, 2002) leading to suggest some main steps, namely non specific interaction with the cell surface, endocytosis into endocytic vesicles, trafficking and release of the DNA from endosomal compartment, nuclear uptake

Special attention was also devoted to the intracellular trafficking of cationic vectors and the role of neutral helpers **(**Elouahabi & Ruysschaert, 2005)**:** six steps of the whole transfection

α

α

phase, it was suggested

α

phase in the

complexes,

αto <sup>C</sup> HII

difference between the two phospholipids is that DOPC induces a lamellar CL

including the same amount of DOPC (Koltover et al., 1998), lamellar CL

interaction of the systems DNA-carrier with each individual cell, the transition CL

large number of efficient complexes are assembled in the CL

and transgene expression.

fundamental theory, that may help to design and implement the synthesis of specialized vectors able to offer the highest efficiency in the various *in vivo* applications. Accordingly, the prevailing opinion is that more exhaustive research and development will be required before such efficiency becomes competitive with viral vectors. As a matter of fact the experience shows that also the cationic carriers suffer from some serious drawbacks, affecting more or less negatively their efficiency of transfection: namely some inherent cytotoxicity (Filion & Phillips, 1998; Lv et al., 2006) that causes negative effects on cells, such as shrinking and inhibition of the protein kinase C (PKC) and a limited stability of their complexes with plasmid DNA in serum (Foradada et al., 2000), responsible for the current restriction of a generalized and extensive use. In this situation the idea of using neutral liposomes as carriers of DNA seems to be interesting and can offer good prospects. It is well known that neutral liposomes are generally non toxic (Koiv et al., 1995) and relatively stable in serum (Tardi et al., 1996), which makes them potentially interesting gene transfer vectors. Despite these strategic features, neutral liposomes (NLs) have not yet received wide attention in the context of the HGT, even though the researchers' initial interest for DNA entrapment was turned to neutral liposomes (Budker et al., 1978). Likely, there are two causes for this situation: the first, and more important, is the lack of positive charge that makes virtually impossible to realize an interaction stable enough between NLs and DNA; the second is the great leading role assumed by the cationic carriers, which have polarized the researchers' interest, leaving other alternatives aside.

In this chapter we will deal with NLs following two separate paths, according to different functions they exert in HGT applications. The former will deal with their role of helpers of DNA transfection when used in mixture with cationic liposomes; the latter with their achievements and perspectives as autonomous and independent carriers of DNA. A survey of the literature published so far, though not too extensive, enables to foresee interesting prospects for NLs as promising synthetic vectors of genetic material. Perhaps they will be considered in a near future as an alternative to cationic vectors, rather than a provocative challenge. This forecast is supported by the results of several studies: of course deeper investigation is necessary in order to define a frame appropriate to treat correctly the many aspects of the transfection process and find better experimental conditions to warrant high transfection efficiency, particularly *in vivo*. The large number of studies carried on so far and the very large number of data collected in the field of cationic liposomes will help in building this frame and exploring the different aspects of the specific transfection with NLs.

#### **2. Neutral lipids as helpers of cationic liposomes in DNA transfection**

Since the discovery of the cationic liposomes as DNA carriers for gene transfer applications it has been clear that higher efficiencies could be obtained by adding a neutral lipid to the lipoplexes with the role of transfection helper. The approach followed in this section is not to evaluate all the issues related to DNA transfection with lipoplexes mediated by neutral lipids, but rather discuss the specific role of neutral lipids in determining the lipoplex transfection efficiency. The ultimate purpose is to highlight the factors that cause such increase of efficiency and check how they may help in designing the best experimental conditions for the transfections procedures with independent NLs.

Cationic systems for gene therapy are generally prepared by mixing a cationic lipid with DNA and a neutral helper co-lipid; such non toxic helper induces the dual result to reduce

fundamental theory, that may help to design and implement the synthesis of specialized vectors able to offer the highest efficiency in the various *in vivo* applications. Accordingly, the prevailing opinion is that more exhaustive research and development will be required before such efficiency becomes competitive with viral vectors. As a matter of fact the experience shows that also the cationic carriers suffer from some serious drawbacks, affecting more or less negatively their efficiency of transfection: namely some inherent cytotoxicity (Filion & Phillips, 1998; Lv et al., 2006) that causes negative effects on cells, such as shrinking and inhibition of the protein kinase C (PKC) and a limited stability of their complexes with plasmid DNA in serum (Foradada et al., 2000), responsible for the current restriction of a generalized and extensive use. In this situation the idea of using neutral liposomes as carriers of DNA seems to be interesting and can offer good prospects. It is well known that neutral liposomes are generally non toxic (Koiv et al., 1995) and relatively stable in serum (Tardi et al., 1996), which makes them potentially interesting gene transfer vectors. Despite these strategic features, neutral liposomes (NLs) have not yet received wide attention in the context of the HGT, even though the researchers' initial interest for DNA entrapment was turned to neutral liposomes (Budker et al., 1978). Likely, there are two causes for this situation: the first, and more important, is the lack of positive charge that makes virtually impossible to realize an interaction stable enough between NLs and DNA; the second is the great leading role assumed by the cationic carriers, which have polarized

In this chapter we will deal with NLs following two separate paths, according to different functions they exert in HGT applications. The former will deal with their role of helpers of DNA transfection when used in mixture with cationic liposomes; the latter with their achievements and perspectives as autonomous and independent carriers of DNA. A survey of the literature published so far, though not too extensive, enables to foresee interesting prospects for NLs as promising synthetic vectors of genetic material. Perhaps they will be considered in a near future as an alternative to cationic vectors, rather than a provocative challenge. This forecast is supported by the results of several studies: of course deeper investigation is necessary in order to define a frame appropriate to treat correctly the many aspects of the transfection process and find better experimental conditions to warrant high transfection efficiency, particularly *in vivo*. The large number of studies carried on so far and the very large number of data collected in the field of cationic liposomes will help in building this frame and exploring the different aspects of the specific transfection with NLs.

**2. Neutral lipids as helpers of cationic liposomes in DNA transfection** 

conditions for the transfections procedures with independent NLs.

Since the discovery of the cationic liposomes as DNA carriers for gene transfer applications it has been clear that higher efficiencies could be obtained by adding a neutral lipid to the lipoplexes with the role of transfection helper. The approach followed in this section is not to evaluate all the issues related to DNA transfection with lipoplexes mediated by neutral lipids, but rather discuss the specific role of neutral lipids in determining the lipoplex transfection efficiency. The ultimate purpose is to highlight the factors that cause such increase of efficiency and check how they may help in designing the best experimental

Cationic systems for gene therapy are generally prepared by mixing a cationic lipid with DNA and a neutral helper co-lipid; such non toxic helper induces the dual result to reduce

the researchers' interest, leaving other alternatives aside.

the amount of the toxic cationic component and, more important, to alter the physical properties of the delivery vehicle, in a way that favours some of the most complex steps of the whole mechanism of transfection: the consequence is that both actions affect the quality of the transfection. The most widely used neutral helpers in DNA transfection experiments are the 1,2-dioleoyl-*sn*-glycero-3-phosphocholine (DOPC) and the 1,2-dioleoyl-*sn*-glycero-3 phosphoethanolamine (DOPE), sometimes in combination with cholesterol: the main difference between the two phospholipids is that DOPC induces a lamellar CLα phase in the lipoplexes, while DOPE induces an inverted hexagonal <sup>C</sup> HII phase. Earlier studies led to propose that DOPE is responsible for more efficient transfections because its hexagonal phase is able to fuse readily with anionic vesicles (Koltover et al., 1998) and destabilize the bilayer membranes, making easier the DNA escape from the endosomes, once the lipoplex has entered the cells. Therefore it was believed that DOPE was more useful than DOPC to realize the most efficient transfections. In support of this claim it was also suggested (Mui et al., 2000) that the inverted hexagonal phase promoted by DOPE has a higher ability to disrupt the membrane integrity than the lamellar one induced by DOPC. It turned soon evident that the situation was not so simple and the interpretation was not unambiguous: indeed, if it is verified that the DNA complex with cationic DOTAP, in which DOPE is present as helper in an amount of 70%, transfects better than the corresponding complex including the same amount of DOPC (Koltover et al., 1998), lamellar CLα complexes, showing similar transfection efficiency, were also synthesized ( Lin et al., 2003). More generally, many literature data show the difficulty in finding a general correlation between vector formulations and transfection efficiency. Trying to explain these apparently contradictory results, some researchers have recently suggested the opportunity to consider the possibility that complexes can bear a structural modification within the phase of the interaction of the systems DNA-carrier with each individual cell, the transition CLα to <sup>C</sup> HII phase being an example among others (Safinya, 2001). Starting from the observation that a large number of efficient complexes are assembled in the CLα phase, it was suggested (Caracciolo & Caminiti, 2005) that perhaps a compelling correlation between the structure of a complex and its transfection efficiency does not simply exist and that the lower transfection efficiency of the synthetic carriers of DNA with respect to virus depends on a poor understanding of the supramolecular structures of the complexes, on the mechanism of their interaction with cells and of the release of DNA within the nucleus. If these statements are true, and there are reasons to confirm them, a more exhaustive knowledge of the subject is particularly important also with reference to the use of NLs as independent carriers of DNA, because it is likely that similar problems will arise and the need to overcome them will be even more important. Of course, understanding the mechanism is crucial for any successful design of a non viral gene delivery, whatever path has been chosen. Besides some early studies, that assumed a fusion between liposomes and cell membranes as the initial step of the process, it is today recognized that the uptake into an endocytic pathway is required for fusion to occur **(**Wrobel **&** Collins, 1995) and the whole aspect has been the object of deep attention (Liu & Huang, 2002) leading to suggest some main steps, namely non specific interaction with the cell surface, endocytosis into endocytic vesicles, trafficking and release of the DNA from endosomal compartment, nuclear uptake and transgene expression.

Special attention was also devoted to the intracellular trafficking of cationic vectors and the role of neutral helpers **(**Elouahabi & Ruysschaert, 2005)**:** six steps of the whole transfection

Neutral Liposomes and DNA Transfection 323

CATIONIC LIPIDS

O N<sup>+</sup> <sup>O</sup> <sup>H</sup> Cl-

> N+ Br-

O NH2 +

> NH3 +

+H2N

+H3N

O

O H N

DOGS

O O O

O

H

<sup>P</sup> <sup>O</sup> O

<sup>O</sup> N+

Cl-

O

EDOPC

DOTAP O

DDAB

O N<sup>+</sup> O H Cl-

O

DODAP <sup>O</sup>

H

O N O H

N+ CH3 Cl-

N+ CH3 Cl-

actions depending on the cationic lipid and the target cells (Fasbender et al., 1997).

The escape of DNA from the endosomes is strictly depending on the nature of the neutral co-lipid: it was found that the fusion with endosomes is likely the way for the release of DNA into the cytoplasm. This mechanism was supported by the finding that efficient transfections require the fusogenic lipid DOPE, which is able to promote a transition from bilayers to hexagonal structures, the latter being known to catalyze the fusion process (Koltover et al., 1998; Mok & Cullis, 1997). The ability of liposomes to fuse with endosomal membranes was also proved by several studies (Koltover et al., 1998; Farhood et al., 1995; Mok & Cullis, 1997) and some evidence was found that the helper lipids can adapt their

DC-chol N

In order to better clear up the situation, an interesting study was made a few years ago (Zuhorn et al., 2005). It provides deeper insight into the involvement of helper lipids in the liposomes mediated gene delivery. Two different helpers, DOPE, which has a propensity to adopt an inverted hexagonal phase, and the lamellar phase forming dipalmitoylphosphatidylethanolamine (DPPE), have been compared as neutral co-lipids in lipoplexes formed with SAINT-2 and plasmid DNA, with the specific aim of studying the endosomal escape of the genetic cargo in the cytosol for transport to the nucleus. As usual, it was found that the helper determines the *in vitro* transfection efficiency (COS-7 cells were used), DPPE inducing a significantly lower efficiency (≅ 25% of cell transfected) than DOPE

SAINT-2

H H H

SAINT-5

DOTMA

N+ H N

Cl-

O O H H

process were identified and analyzed, namely (i) interaction between vectors and nucleic acids with formation of complexes, (ii) binding of a complex to the cell, (iii) effect of serum, (iv) uptake of the complex by the cell, (v) escape from endosomes and dissociation of the complex and finally (vi) nuclear entry of DNA.

Fig. 1. A schematic representation of the steps involved in the liposome mediated DNA transfection.

We will consider here only the aspects likely to have significant involvements on the transfection process operated by NLs as unique carriers of genetic material. Neutral helpers play a not marginal role in the formation of the complexes: meaningful, though in some way contradictory, is the role of the helpers in determining the effect of serum on the uptake of lipoplexes, an issue that has implications in the *in vivo* transfections. No significant inhibition of serum was observed in transfecting COS-7 cells with cationic pyridinium– derived lipids (SAINT) in DOPE/DNA lipoplexes (Zuhorn et al., 2002), while a strong inhibitory effect operates in lipoplexes obtained by polycationic lipids like DOGS. However this negative effect may be avoided if one operates at slight alkaline pH that favours a lamellar organisation of the lipoplexes (Boukhnikachvili et al., 1997). Likewise DOTAP/DOPE and DC-Chol/DOPE prepared at high +/- charge ratio are not sensitive to the inhibitory effect of serum and indeed, at some ratios, are even more efficient (Yang & Huang, 1997). A different perspective on the role of helpers is offered by other researchers: it was observed (Fasbender & al., 1997) that when DOPE is incorporated into the complexes of DNA with three different cationics, its effect on the gene expression in COS-1 cells is different, depending on each cationic lipid. When the cationic lipids and DOPE were formulated separately and then complexed with DNA, no difference in activity was observed over that obtained with cationic lipids alone. Finally (Felgner et al., 1994) unsaturated PE co-lipids enhance lipoplexes activity while saturated PE and PC have no enhancing effect or even have an inhibitory effect.

process were identified and analyzed, namely (i) interaction between vectors and nucleic acids with formation of complexes, (ii) binding of a complex to the cell, (iii) effect of serum, (iv) uptake of the complex by the cell, (v) escape from endosomes and dissociation of the

Fig. 1. A schematic representation of the steps involved in the liposome mediated DNA

We will consider here only the aspects likely to have significant involvements on the transfection process operated by NLs as unique carriers of genetic material. Neutral helpers play a not marginal role in the formation of the complexes: meaningful, though in some way contradictory, is the role of the helpers in determining the effect of serum on the uptake of lipoplexes, an issue that has implications in the *in vivo* transfections. No significant inhibition of serum was observed in transfecting COS-7 cells with cationic pyridinium– derived lipids (SAINT) in DOPE/DNA lipoplexes (Zuhorn et al., 2002), while a strong inhibitory effect operates in lipoplexes obtained by polycationic lipids like DOGS. However this negative effect may be avoided if one operates at slight alkaline pH that favours a lamellar organisation of the lipoplexes (Boukhnikachvili et al., 1997). Likewise DOTAP/DOPE and DC-Chol/DOPE prepared at high +/- charge ratio are not sensitive to the inhibitory effect of serum and indeed, at some ratios, are even more efficient (Yang & Huang, 1997). A different perspective on the role of helpers is offered by other researchers: it was observed (Fasbender & al., 1997) that when DOPE is incorporated into the complexes of DNA with three different cationics, its effect on the gene expression in COS-1 cells is different, depending on each cationic lipid. When the cationic lipids and DOPE were formulated separately and then complexed with DNA, no difference in activity was observed over that obtained with cationic lipids alone. Finally (Felgner et al., 1994) unsaturated PE co-lipids enhance lipoplexes activity while saturated PE and PC have no

complex and finally (vi) nuclear entry of DNA.

enhancing effect or even have an inhibitory effect.

transfection.

The escape of DNA from the endosomes is strictly depending on the nature of the neutral co-lipid: it was found that the fusion with endosomes is likely the way for the release of DNA into the cytoplasm. This mechanism was supported by the finding that efficient transfections require the fusogenic lipid DOPE, which is able to promote a transition from bilayers to hexagonal structures, the latter being known to catalyze the fusion process (Koltover et al., 1998; Mok & Cullis, 1997). The ability of liposomes to fuse with endosomal membranes was also proved by several studies (Koltover et al., 1998; Farhood et al., 1995; Mok & Cullis, 1997) and some evidence was found that the helper lipids can adapt their actions depending on the cationic lipid and the target cells (Fasbender et al., 1997).

In order to better clear up the situation, an interesting study was made a few years ago (Zuhorn et al., 2005). It provides deeper insight into the involvement of helper lipids in the liposomes mediated gene delivery. Two different helpers, DOPE, which has a propensity to adopt an inverted hexagonal phase, and the lamellar phase forming dipalmitoylphosphatidylethanolamine (DPPE), have been compared as neutral co-lipids in lipoplexes formed with SAINT-2 and plasmid DNA, with the specific aim of studying the endosomal escape of the genetic cargo in the cytosol for transport to the nucleus. As usual, it was found that the helper determines the *in vitro* transfection efficiency (COS-7 cells were used), DPPE inducing a significantly lower efficiency (≅ 25% of cell transfected) than DOPE

CATIONIC LIPIDS

Neutral Liposomes and DNA Transfection 325

the C18:0 did not. All these new perspectives must be seriously considered by all aiming at studying the DNA transfection with NLs, since an analogous behaviour will be

Before ending these considerations on the role of the helper co-lipids in the processes of non viral DNA transfection, it is advisable to say something about a neglected aspect of the topic. As a matter of fact it is surprising that the continuous growing of number and features of cationic lipids, in the search for most suitable vectors of genetic material, no analogous interest has been reserved, for many years, for new co-lipids. The idea that improved transfections could be realized also with the aid of new and more appropriate helpers has developed only in the last ten years. After the discovery that high transfection efficiency could be obtained with fluorinated double chain lipospermines, forming fluorinated lipoplexes (Gaucheron et al., 2001a, 2001b), a partially fluorinated analogue of DOPE, identified as [F8E11][C16]OPE from the number of fluorine atoms, was synthesized and compared with DOPE as helper of fluorinated lipoplexes (Boussif et al., 2001): this compound, inactive itself in promoting transfection, increased the *in vitro* and *in vivo* gene transfer of the lipoplex obtained from the pentacationic pcTG90 to a larger extent than DOPE. The synthesis was then extended to more fluorinated glycerophosphoethanolamines (Gaucheron et al., 2001) confirming that lipoplexes formulated with fluorinated helper lipids are attractive candidates for gene delivery both *in vitro* and *in vivo*. Several reasons were identified to explain these results: fluorinated colipids have a larger ability to preserve the integrity of complexed DNA in a biological environment and a larger propensity to promote fusion with endosomes and subsequent destabilisation, allowing more efficient DNA release in the cytosol; their high hydrophobic and lipophobic character can preserve the lipoplexes from the effect of the interactions with lipophilic and hydrophilic biocompounds; finally fluorinated DOPE compounds are expected to have a greater tendency to promote a lamellar to a an inverted hexagonal phase transition with the consequence of a higher effectiveness in disrupting

It is commonly accepted that one of the main features of DOPE as helper depends on its polymorphism under various concentration and temperature conditions. Its ability to enhance transfection efficiency is related to its preference for the fusogenic HII phase, which can promote fusion with cellular membranes, especially the endosomal ones, thereby facilitating the escape of the genetic material; however, the low Lα/HII phase transition temperature (Th = 10 °C) makes cationic liposomes too unstable in the *in vivo* environment. An approach to solve the problem might be to synthesize analogues of DOPE in which the phase transition is near the physiological temperature. Some molecules having these characteristics have been synthesized (Fletcher et al., 2006) and correspond to a series of dialkynoyl analogues of DOPE where the cis-double bond in the two oleoyl fatty acid chains is replaced by a triple bond located in different positions of the hydrocarbon tails. With this modified geometry a new intermolecular packing is realized, able to induce an increase of

The achievements just shown were based on the concept to modify DOPE: a different approach to the search for more efficient helpers has been realized by synthesizing completely new lipids characterized by the presence of an imidazole polar head (Mével et al., 2008). These lipophosphoramides are neutral at physiological pH: the protonation occurring in the acidic compartments of the cell, namely the endosomes, induces fusion of

probably characterize those experimental setups.

membranes than DOPE.

the phase transition at the physiological conditions.

(≅ 75%), despite an equal interaction of both SAINT-2/DNA/DOPE and SAINT-2/DNA/DPPE with cells. Assuming that the translocation of the nucleic acids through the endosomal membrane is the crucial step of the overall process, a mimic membrane consisting of phosphatidylserine (PS): phosphatidylcholine (PC): phosphatidylethanolamine (PE) anionic vesicle was used to simulate this step. Without helper lipids, a limited fraction of DNA was released from SAINT-2 lipoplex and no effect was promoted by the inclusion of DOPC. On the contrary, inclusion of DOPE significantly enhanced the amount of DNA released and, more interesting, a comparable effect was induced by DPPE (40% release for DPPE versus 50% for DOPE). This result can find an explanation since the *x*-ray diffraction analysis after incubation of SAINT-2/DPPE with the anionic PS:PC:PE (1:1:2) revealed the presence of a mixed lamellar-hexagonal phase. The lower efficiency of the DPPE containing complex is consistent with this partial transition from the lamellar to the hexagonal phase. These results confirm that the limiting step in the overall transfection pathway depends on the level of DNA translocation through the endosomal membrane. The results obtained in the transfections with two different lipoplexes containing SAINT with different tails, namely SAINT-2 (C18:1) and SAINT-5 (C18:0) lead (Zuhorn et al., 2002) to analogous conclusions. Both amphiphiles may make transfection and DOPE strongly promotes the SAINT-2 mediated one, but not the SAINT-5. The relatively rigid SAINT-5 membrane forms structurally deformed lipoplexes hampering the plasmid translocation through endosomal and/or nuclear membranes.

What it has been said so far makes clear that there are still many aspects concerning the DNA transfection process that require a better investigation: among them, the need of a comprehensive knowledge of what really happens when a lipid-DNA complex interacts with a cell, an issue extremely important also in the neutral lipid mediated DNA transfections. In this connection a new perspective has gained recently ground: it identifies as one of the most critical factors of the transfection process the evolution of the structure of the lipoplexes that occurs when they interact with cells. That means to introduce the idea that the differences in the transfection efficiency, often observed and not always unambiguously interpreted, may depend on each particular cellular variety and emphasize the importance to consider both lipid composition of lipoplexes and target membranes (Koynova et al., 2005). Ancestors of this new perspective are some studies that demonstrated the ability of anionic lipids to promote the release of DNA from lipoplexes, by neutralizing their positive charge (Szoka et al. 1996; Zelphati & Szoka, 1996; McDonald et al., 1999). It was observed that the DNA release from complexes with the cationic lipids o-ethyldioleoylphosphatidylcholinium (EDOPC) or DOTAP, after mixing them with some different negatively charged lipids, depends on both lipoplexes and negative lipids. Most significant, the transfection efficiencies of DNA complexes with two very similar cationic phospholipids, bearing only a minimal structural difference in one of the two hydrocarbon tails, the carbon-carbon double bond bearing oleoyldecanoyl-ethylphosphatidylcholine (C18:1/C10-EPC) and the completely saturated stearoyldecanoyl-ethylphosphatidylcholine (C18:0/C10-EPC) were compared (Koynova et al., 2006). The former complex shows a 50-fold higher transfection efficiency than the latter in human umbilical artery endothelial cells. A reasonable explanation of this different behaviour lies in the great difference of these lipids in the phase evolution found in mixing with biomembrane mimicking lipid mixtures (DOPC/DOPE/DOPS/chol). The C18:1 lipoplex underwent a transition to the fusogenic non lamellar cubic phase, whereas

(≅ 75%), despite an equal interaction of both SAINT-2/DNA/DOPE and SAINT-2/DNA/DPPE with cells. Assuming that the translocation of the nucleic acids through the endosomal membrane is the crucial step of the overall process, a mimic membrane consisting of phosphatidylserine (PS): phosphatidylcholine (PC): phosphatidylethanolamine (PE) anionic vesicle was used to simulate this step. Without helper lipids, a limited fraction of DNA was released from SAINT-2 lipoplex and no effect was promoted by the inclusion of DOPC. On the contrary, inclusion of DOPE significantly enhanced the amount of DNA released and, more interesting, a comparable effect was induced by DPPE (40% release for DPPE versus 50% for DOPE). This result can find an explanation since the *x*-ray diffraction analysis after incubation of SAINT-2/DPPE with the anionic PS:PC:PE (1:1:2) revealed the presence of a mixed lamellar-hexagonal phase. The lower efficiency of the DPPE containing complex is consistent with this partial transition from the lamellar to the hexagonal phase. These results confirm that the limiting step in the overall transfection pathway depends on the level of DNA translocation through the endosomal membrane. The results obtained in the transfections with two different lipoplexes containing SAINT with different tails, namely SAINT-2 (C18:1) and SAINT-5 (C18:0) lead (Zuhorn et al., 2002) to analogous conclusions. Both amphiphiles may make transfection and DOPE strongly promotes the SAINT-2 mediated one, but not the SAINT-5. The relatively rigid SAINT-5 membrane forms structurally deformed lipoplexes hampering the plasmid translocation through endosomal

What it has been said so far makes clear that there are still many aspects concerning the DNA transfection process that require a better investigation: among them, the need of a comprehensive knowledge of what really happens when a lipid-DNA complex interacts with a cell, an issue extremely important also in the neutral lipid mediated DNA transfections. In this connection a new perspective has gained recently ground: it identifies as one of the most critical factors of the transfection process the evolution of the structure of the lipoplexes that occurs when they interact with cells. That means to introduce the idea that the differences in the transfection efficiency, often observed and not always unambiguously interpreted, may depend on each particular cellular variety and emphasize the importance to consider both lipid composition of lipoplexes and target membranes (Koynova et al., 2005). Ancestors of this new perspective are some studies that demonstrated the ability of anionic lipids to promote the release of DNA from lipoplexes, by neutralizing their positive charge (Szoka et al. 1996; Zelphati & Szoka, 1996; McDonald et al., 1999). It was observed that the DNA release from complexes with the cationic lipids o-ethyldioleoylphosphatidylcholinium (EDOPC) or DOTAP, after mixing them with some different negatively charged lipids, depends on both lipoplexes and negative lipids. Most significant, the transfection efficiencies of DNA complexes with two very similar cationic phospholipids, bearing only a minimal structural difference in one of the two hydrocarbon tails, the carbon-carbon double bond bearing oleoyldecanoyl-ethylphosphatidylcholine (C18:1/C10-EPC) and the completely saturated stearoyldecanoyl-ethylphosphatidylcholine (C18:0/C10-EPC) were compared (Koynova et al., 2006). The former complex shows a 50-fold higher transfection efficiency than the latter in human umbilical artery endothelial cells. A reasonable explanation of this different behaviour lies in the great difference of these lipids in the phase evolution found in mixing with biomembrane mimicking lipid mixtures (DOPC/DOPE/DOPS/chol). The C18:1 lipoplex underwent a transition to the fusogenic non lamellar cubic phase, whereas

and/or nuclear membranes.

the C18:0 did not. All these new perspectives must be seriously considered by all aiming at studying the DNA transfection with NLs, since an analogous behaviour will be probably characterize those experimental setups.

Before ending these considerations on the role of the helper co-lipids in the processes of non viral DNA transfection, it is advisable to say something about a neglected aspect of the topic. As a matter of fact it is surprising that the continuous growing of number and features of cationic lipids, in the search for most suitable vectors of genetic material, no analogous interest has been reserved, for many years, for new co-lipids. The idea that improved transfections could be realized also with the aid of new and more appropriate helpers has developed only in the last ten years. After the discovery that high transfection efficiency could be obtained with fluorinated double chain lipospermines, forming fluorinated lipoplexes (Gaucheron et al., 2001a, 2001b), a partially fluorinated analogue of DOPE, identified as [F8E11][C16]OPE from the number of fluorine atoms, was synthesized and compared with DOPE as helper of fluorinated lipoplexes (Boussif et al., 2001): this compound, inactive itself in promoting transfection, increased the *in vitro* and *in vivo* gene transfer of the lipoplex obtained from the pentacationic pcTG90 to a larger extent than DOPE. The synthesis was then extended to more fluorinated glycerophosphoethanolamines (Gaucheron et al., 2001) confirming that lipoplexes formulated with fluorinated helper lipids are attractive candidates for gene delivery both *in vitro* and *in vivo*. Several reasons were identified to explain these results: fluorinated colipids have a larger ability to preserve the integrity of complexed DNA in a biological environment and a larger propensity to promote fusion with endosomes and subsequent destabilisation, allowing more efficient DNA release in the cytosol; their high hydrophobic and lipophobic character can preserve the lipoplexes from the effect of the interactions with lipophilic and hydrophilic biocompounds; finally fluorinated DOPE compounds are expected to have a greater tendency to promote a lamellar to a an inverted hexagonal phase transition with the consequence of a higher effectiveness in disrupting membranes than DOPE.

It is commonly accepted that one of the main features of DOPE as helper depends on its polymorphism under various concentration and temperature conditions. Its ability to enhance transfection efficiency is related to its preference for the fusogenic HII phase, which can promote fusion with cellular membranes, especially the endosomal ones, thereby facilitating the escape of the genetic material; however, the low Lα/HII phase transition temperature (Th = 10 °C) makes cationic liposomes too unstable in the *in vivo* environment. An approach to solve the problem might be to synthesize analogues of DOPE in which the phase transition is near the physiological temperature. Some molecules having these characteristics have been synthesized (Fletcher et al., 2006) and correspond to a series of dialkynoyl analogues of DOPE where the cis-double bond in the two oleoyl fatty acid chains is replaced by a triple bond located in different positions of the hydrocarbon tails. With this modified geometry a new intermolecular packing is realized, able to induce an increase of the phase transition at the physiological conditions.

The achievements just shown were based on the concept to modify DOPE: a different approach to the search for more efficient helpers has been realized by synthesizing completely new lipids characterized by the presence of an imidazole polar head (Mével et al., 2008). These lipophosphoramides are neutral at physiological pH: the protonation occurring in the acidic compartments of the cell, namely the endosomes, induces fusion of

Neutral Liposomes and DNA Transfection 327

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.

membranes of living systems would be obtained.

**4.1 Liposomes and polynucleotide entrapment** 

**4. The neutral liposomes as independent DNA transfection agents** 

After the Bangham's work (Bangham et al., 1965) liposomes were extensively used as models of biological membranes (Sessa & Weissmann, 1968) on the basis of their lamellar structure. It was seen that they are able to discriminate ions as natural membranes do, and that it is easy to vary their surface charge, in order to modulate the diffusion of a large amount of cations and anions. It was proved that it is possible to incorporate proteins in their lamellar structure and that their composition can be modified to mimic the properties of a large variety of natural membranes. Basically, it was recognized that liposomes are a valuable instrument to study many problems concerning natural membrane structure and function. What's more, it was assumed that, if liposomes were able to incorporate proteins, enzymes, drugs or nucleic acids, an important step towards a true *in vitro* replica of the

Soon these foreseen opportunities began to turn into actual tasks: liposomes started being applied as carriers of different molecules into target cells (Dimitriadis, 1979; Tyrrell et al., 1976; Finkelstein & Weissmann, 1978) or of enzymes in enzyme replaced therapy (Gregoriadis & Buckland, 1973). It was in those years that the entrapment of synthetic polynucleotides (Magee et al., 1976), as well as natural ones (Hoffman et al., 1978; Lurquin, 1979), was undertaken. Large unilamellar liposomes were obtained (Dimitriadis, 1978) by adding ribonucleic acid (globin mRNA) to PS and it was demonstrated that mRNA is really entrapped and not simply adhering to the surface. A different experiment was realized with the aim of clearing up the mechanism of crossing the hydrophobic barriers formed by protein-lipid membranes and the nature of bonds, providing adsorption of polynucleotides in the membranes (a mechanism unknown at that time). It was demonstrated (Budker et al., 1978) that polynucleotides are adsorbed by liposomes of PC forming stable complexes in the presence of Mg2+ or Ca2+ ions, but not in the absence of these ions. This result suggested that this interaction is due to the action of bivalent cations, which crosslink phosphate groups of polynucleotides with the ones of PC. It was also found that the complexes

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 transfection by a factor of 100 compared with DOPE.
