**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 membranes of living systems would be obtained.

#### **4.1 Liposomes and polynucleotide entrapment**

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

Neutral Liposomes and DNA Transfection 329

(Wilson et al., 1979). A comparison between large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs) of PS indicates that LUVs deliver their content to cell cytoplasm much more efficiently than MLVs and that LUV-entrapped poliovirus RNA produces infection titers 10 to 100 fold higher than when delivered with other techniques. Likewise, DNA isolated from simian virus 40 (SV40) was encapsulated in LUVs of PS and delivered to a monkey cell line (Fraley et al., 1980). The infectivity realized with this method was enhanced at least 100 fold over that of free naked DNA. This process was then used as a probe to study liposome-cell interactions and determine conditions favouring the intracellular delivery of liposome content to cells (Fraley et al., 1981). The efficiency of DNA delivery was found dependent both on size of vesicles and the resistance of liposomes to cell induced leakage of content. Acidic phospholipids are much more effective in both binding and delivery, and PS was found to be the best in both events. Inclusion of cholesterol in liposomes reduces the cell-induced leakage of vesicle content and enhances the delivery of DNA to cells. A brief exposure of cells to glycerol solutions enhances infectivity of the SV40 DNA when encapsulated into the negatively charged liposome of PS, but not in neutral and positively charged liposomes. Morphological studies indicate that the glycerol treatment stimulates membrane vacuolisation and suggest that the enhanced uptake of liposomes occurs by an endocytic-like process. As it was said in a previous paragraph, endocytosis is the mechanism followed in the phase of the internalisation of the complexes liposome/DNA into cells. Additional attempts to transfect DNA to cells by means of bivalent cations mediated complexes of neutral liposomes are reported by the

**4.3 The problem of the uptake of liposomes by the reticuloendothelial system** 

It is known that many liposomes are removed in liver and spleen from the blood circulation within minutes: this property, beneficial when they are employed to carry drugs for treating intracellular infections of the reticuloendothelial system (RES), has limited their use as delivery carriers of material to sites beyond the RES. Designing liposomes with prolonged circulation time requires a reduction of the rate of their clearance by the RES and of the leakage of liposome cargo in blood stream. The search for a solution of the problem has led to the discovery of the so called stealth liposomes, sterically stabilized by the presence of bulky groups: the so-called PEG-liposomes, namely polyethyleneglycol functionalized liposomes, are the most important tools in ensuring a prolonged circulation time in blood. The incorporation of PEG into conventional liposomes provides a steric barrier at the liposome surface that inhibits opsonisation, therefore extending the persistence time of liposomes in the blood. Positive effects are a prolonged circulation lifetime of lipoplexes and a reduced formation of aggregates (Klibanov et al., 1990; Papahadjopoulos et al., 1991). The incorporation of 1,2-dioleoyl-N- (methoxy-polyethyleneglycol-succinyl-)phosphatidylethanolamine (PEG-PE) in liposomes composed of egg phosphatidylcholine-cholesterol, exposed to human serum at 37 °C increases the blood circulation half-life ten times the one of simple phosphatidylcholinecholesterol liposome (Klibanov et al., 1990). While PEG-lipids play an active role in limiting excessive inhibition and fusion during the self-assembling phase when cationic lipids associate with anionic DNA protecting DNA from nuclease degradation in plasma, the steric barrier introduced by PEG is expected to inhibit also the process of fusion with the endosomes and by consequence reduce the transfection activity. Conflicting results have been obtained so far. It has been found (Song et al., 2002) that, owing to the presence

literature (Kovalenko et al., 1996).

obtained are stable, but that the addition of monovalent cations reduces the extent of complexation.

It has been said already that one of the main reasons of the success of cationic liposomes resides in their positive charge that enables attractive interactions both with the negative phosphate groups of the polynucleotides and the negatively charged cell wall. Encapsulation of DNA into a vector is of course the first and irreplaceable step to realize a synthetic vector driven gene therapy and must be solved within neutral liposomes, where the absence of charge does not allow the formation of a stable aggregation with negative DNA. However gene transfer and gene therapy need an efficient encapsulation of plasmid DNA into neutral liposomes and an attractive interaction with the negative cell wall which is the necessary step for the endocytic internalisation of the construct. The methods generally used to realize a stable entrapment can be schematically indicated in three main classes: reverse phase evaporation, dehydration-rehydration and freeze-thawing. According to the first method the nucleic acids are dissolved in water and the solution is added to lipids dispersed in an organic solvent, then evaporated to induce vesiculation (Szoka & Papahadjopoulos, 1978). In the second procedure the nucleic acids are added to a dispersion of SULs (small unilamellar liposomes) and the mixture is dehydrated until almost dryness; afterwards the material is rehydrated and vortexed to induce the formation of liposomal aggregates (Deamer & Barchfeld, 1982). The third method involves the addition of nucleic acids to a dispersion of SULs followed by numerous freeze-thawing operations and by a final extrusion to obtain homogeneously sized vesicles (Chapman et al., 1990). The last method was applied to encapsulate a 3368 base pair DNA (Monnard et al., 1997), using liposomes prepared with 1-palmitoyl-2-oleoyl-*sn*-glycero-3-phosphocholine (POPC) mixed with a little amount of the negative PS or the cationic didodecyl-methylammonium bromide (DDAB) as cosurfatants. The yields of entrapment calculated over the amount of the initial material were 27% in pure POPC, 26% in POPC/PS 9:1 and 50% in POPC/DDAB 99:1. While the addition of PS has evidently no influence on the entrapment, the one of traces of the positive DDAB, doubles the percentage of entrapped DNA. An important contribute to the entrapment of nucleic acids in neutral liposomes was done some years later (Bayley & Sullivan, 2000). Plasmid DNAs (pDNA) were trapped into pure DOPC, DOPC/DOPE 1:1 and DOPC/DOPE/Chol 1:1:1 by simply adding CaCl2 and ethanol to the initial mixture DNA/lipids. With optimized amounts of ethanol and calcium the entrapment percentages were 65-70% for DOPC, 70-80% for DOPC/DOPE 1:1 and only 35-40% for DOPC/DOPE/Chol 1:1:1. Most important the neutral liposome complexes obtained from DOPC and DOPC/DOPE are stable for at least two weeks in PBS (phosphate buffered saline) at 4 °C.

#### **4.2 Some early experiments of DNA delivery to cells by means of liposomes**

The need of modifying the expression of the eukaryotic genome to study the protein synthesis led to encapsulate a functional rabbit globin mRNA in lecithin liposomes, made by neutral PC and PE, to realize its selective insertion into differentiated eukaryotic cells in vitro and express a globin-like protein (Ostro et al., 1978). The authors claimed this result as the first successful attempt to entrap and deliver high molecular weight RNA with a liposome. New applications followed and led to improve the technique. Poliovirus RNA was encapsulated in liposomes of PS and delivered efficiently to cells in an infectious form

obtained are stable, but that the addition of monovalent cations reduces the extent of

It has been said already that one of the main reasons of the success of cationic liposomes resides in their positive charge that enables attractive interactions both with the negative phosphate groups of the polynucleotides and the negatively charged cell wall. Encapsulation of DNA into a vector is of course the first and irreplaceable step to realize a synthetic vector driven gene therapy and must be solved within neutral liposomes, where the absence of charge does not allow the formation of a stable aggregation with negative DNA. However gene transfer and gene therapy need an efficient encapsulation of plasmid DNA into neutral liposomes and an attractive interaction with the negative cell wall which is the necessary step for the endocytic internalisation of the construct. The methods generally used to realize a stable entrapment can be schematically indicated in three main classes: reverse phase evaporation, dehydration-rehydration and freeze-thawing. According to the first method the nucleic acids are dissolved in water and the solution is added to lipids dispersed in an organic solvent, then evaporated to induce vesiculation (Szoka & Papahadjopoulos, 1978). In the second procedure the nucleic acids are added to a dispersion of SULs (small unilamellar liposomes) and the mixture is dehydrated until almost dryness; afterwards the material is rehydrated and vortexed to induce the formation of liposomal aggregates (Deamer & Barchfeld, 1982). The third method involves the addition of nucleic acids to a dispersion of SULs followed by numerous freeze-thawing operations and by a final extrusion to obtain homogeneously sized vesicles (Chapman et al., 1990). The last method was applied to encapsulate a 3368 base pair DNA (Monnard et al., 1997), using liposomes prepared with 1-palmitoyl-2-oleoyl-*sn*-glycero-3-phosphocholine (POPC) mixed with a little amount of the negative PS or the cationic didodecyl-methylammonium bromide (DDAB) as cosurfatants. The yields of entrapment calculated over the amount of the initial material were 27% in pure POPC, 26% in POPC/PS 9:1 and 50% in POPC/DDAB 99:1. While the addition of PS has evidently no influence on the entrapment, the one of traces of the positive DDAB, doubles the percentage of entrapped DNA. An important contribute to the entrapment of nucleic acids in neutral liposomes was done some years later (Bayley & Sullivan, 2000). Plasmid DNAs (pDNA) were trapped into pure DOPC, DOPC/DOPE 1:1 and DOPC/DOPE/Chol 1:1:1 by simply adding CaCl2 and ethanol to the initial mixture DNA/lipids. With optimized amounts of ethanol and calcium the entrapment percentages were 65-70% for DOPC, 70-80% for DOPC/DOPE 1:1 and only 35-40% for DOPC/DOPE/Chol 1:1:1. Most important the neutral liposome complexes obtained from DOPC and DOPC/DOPE are stable for at least two weeks in PBS (phosphate buffered

**4.2 Some early experiments of DNA delivery to cells by means of liposomes** 

The need of modifying the expression of the eukaryotic genome to study the protein synthesis led to encapsulate a functional rabbit globin mRNA in lecithin liposomes, made by neutral PC and PE, to realize its selective insertion into differentiated eukaryotic cells in vitro and express a globin-like protein (Ostro et al., 1978). The authors claimed this result as the first successful attempt to entrap and deliver high molecular weight RNA with a liposome. New applications followed and led to improve the technique. Poliovirus RNA was encapsulated in liposomes of PS and delivered efficiently to cells in an infectious form

complexation.

saline) at 4 °C.

(Wilson et al., 1979). A comparison between large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs) of PS indicates that LUVs deliver their content to cell cytoplasm much more efficiently than MLVs and that LUV-entrapped poliovirus RNA produces infection titers 10 to 100 fold higher than when delivered with other techniques. Likewise, DNA isolated from simian virus 40 (SV40) was encapsulated in LUVs of PS and delivered to a monkey cell line (Fraley et al., 1980). The infectivity realized with this method was enhanced at least 100 fold over that of free naked DNA. This process was then used as a probe to study liposome-cell interactions and determine conditions favouring the intracellular delivery of liposome content to cells (Fraley et al., 1981). The efficiency of DNA delivery was found dependent both on size of vesicles and the resistance of liposomes to cell induced leakage of content. Acidic phospholipids are much more effective in both binding and delivery, and PS was found to be the best in both events. Inclusion of cholesterol in liposomes reduces the cell-induced leakage of vesicle content and enhances the delivery of DNA to cells. A brief exposure of cells to glycerol solutions enhances infectivity of the SV40 DNA when encapsulated into the negatively charged liposome of PS, but not in neutral and positively charged liposomes. Morphological studies indicate that the glycerol treatment stimulates membrane vacuolisation and suggest that the enhanced uptake of liposomes occurs by an endocytic-like process. As it was said in a previous paragraph, endocytosis is the mechanism followed in the phase of the internalisation of the complexes liposome/DNA into cells. Additional attempts to transfect DNA to cells by means of bivalent cations mediated complexes of neutral liposomes are reported by the literature (Kovalenko et al., 1996).

#### **4.3 The problem of the uptake of liposomes by the reticuloendothelial system**

It is known that many liposomes are removed in liver and spleen from the blood circulation within minutes: this property, beneficial when they are employed to carry drugs for treating intracellular infections of the reticuloendothelial system (RES), has limited their use as delivery carriers of material to sites beyond the RES. Designing liposomes with prolonged circulation time requires a reduction of the rate of their clearance by the RES and of the leakage of liposome cargo in blood stream. The search for a solution of the problem has led to the discovery of the so called stealth liposomes, sterically stabilized by the presence of bulky groups: the so-called PEG-liposomes, namely polyethyleneglycol functionalized liposomes, are the most important tools in ensuring a prolonged circulation time in blood. The incorporation of PEG into conventional liposomes provides a steric barrier at the liposome surface that inhibits opsonisation, therefore extending the persistence time of liposomes in the blood. Positive effects are a prolonged circulation lifetime of lipoplexes and a reduced formation of aggregates (Klibanov et al., 1990; Papahadjopoulos et al., 1991). The incorporation of 1,2-dioleoyl-N- (methoxy-polyethyleneglycol-succinyl-)phosphatidylethanolamine (PEG-PE) in liposomes composed of egg phosphatidylcholine-cholesterol, exposed to human serum at 37 °C increases the blood circulation half-life ten times the one of simple phosphatidylcholinecholesterol liposome (Klibanov et al., 1990). While PEG-lipids play an active role in limiting excessive inhibition and fusion during the self-assembling phase when cationic lipids associate with anionic DNA protecting DNA from nuclease degradation in plasma, the steric barrier introduced by PEG is expected to inhibit also the process of fusion with the endosomes and by consequence reduce the transfection activity. Conflicting results have been obtained so far. It has been found (Song et al., 2002) that, owing to the presence

Neutral Liposomes and DNA Transfection 331

NEUTRAL LIPIDS

O

DOPE O

DMPE O

O

O O O

O O O

O

DPPC O

O

n=2 18:1 6-PE n=5 18:1 12-PE H

O O O

n

NH3 +

H

H

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

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

<sup>O</sup> NH3

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

<sup>O</sup> N+

<sup>O</sup> NH3

+

+

O

O

O

DPPE O

O O O

O O O

H

O O O

undertaken mainly by means of x-ray diffraction technique.

H POPC <sup>O</sup>

H DOPC <sup>O</sup>

<sup>P</sup> <sup>O</sup> O - <sup>O</sup> N+

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

> <sup>P</sup> <sup>O</sup> O - <sup>O</sup> N+

<sup>O</sup> NH3

+

O

This new class of complexes consists of ternary systems NLs/DNA/M2+ where M refers mostly to Ca, Mg, a choice consistent with the previously reported experiments of membrane fusion, and sometimes Mn. The formation of the ternary complexes is the result of a self assembling process in which the driving force is represented by the release of the counter-ion entropy upon neutralization of DNA phosphate groups by metal cations (Cl— in the examples discussed). Studies on the structure of these ternary complexes were

In all the experiments performed by the authors of this review, XRD measurements were carried out at the high brilliance beamline ID02 of the European Synchrotron Radiation Facility (Grenoble, France). The energy of the incident beam was 12.5 keV (λ= 0.995 Å), the beam size 100x100 μm2, and the sample-to-detector distance 1.2 m. The 2D diffraction patterns were collected by a CCD detector. The small angle q range from qmin = 0.1 nm-1 to qmax = 4 nm-1 with a resolution of 5 x 10-3 nm-1 (fwhm) was investigated: the samples were held in a 1 mm-sized glass capillary. To avoid radiation damage, each sample was exposed to radiation for 3 sec/frame. To calculate the electron density maps, the integrated intensities of the diffraction peaks were determined by fitting the data with series of Lorentz functions, using a nonlinear baseline. The Lorentz correction was performed multiplying each integrated intensity by sin *θ* and the intensities were then calibrated dividing by the multiplicity of the reflection (Harper et al., 2001; Francescangeli et al., 1996). The square root of the corrected peak was finally used to determine the modulus of the form factor *F* of each

O O O

O

H

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

<sup>O</sup> <sup>N</sup> H


of PEG lipids with long acyl chains (< 14 carbons), the contact between complexes and endosomal membranes doesn't allow membrane disruption. In general it seems that the biological and physicochemical characteristics of the DNA/copolymer complexes, including PEG, are influenced by the copolymer architecture (Deshpande et al., 2004) and that the transfection efficiency is strongly correlated with the level of cellular association and uptake of the DNA/copolymer complexes.

#### **4.4 The structure of the complexes of DNA with zwitterionic liposomes**

In previous paragraphs we pointed out that the structure of the lipoplexes represents a fundamental tool in understanding and planning DNA transfection systems. The same remarks are valid for complexes of DNA with neutral (zwitterionic) liposomes in order to evaluate correctly their behaviour and, in case, design the necessary developments to achieve better results in DNA transfection experiments both *in vitro* and *in vivo*. These complexes were initially studied in order to understand the influence of DNA structural transition of neutral lipids: DSC thermograms of the DPPC/DNA/Ca2+ complex (Tarahovsky et al., 1996) reveal a distinct maximum at the temperature of 316.3 K in addition to the main maximum at 314.6 K. Since a direct relationship was observed between the molar proportion of DNA in samples and the value of the height of the second peak, it was hence assumed that the higher temperature transition corresponds to the formation of the complex. In another work (Kharakoz et al., 1999) it was demonstrated that DPPC/DNA complexes could be obtained by simply mixing the DNA solutions with an aqueous lipid dispersion in the presence of Ca2+ and that their formation could be obtained with both MLVs and ULVs. The stoichiometry was determined in 4.5 to 5 strongly bound lipid molecules per molecule of nucleotide, depending on the method used in a temperature-scanning ultrasonic study. From this result and the ones obtained in a small angle x-ray scattering experiment (SAXS), a model was proposed (McManus et al., 2003) for the interaction of DNA and DPPC in the presence of CaCl2. The lamellar repeat distance in complexes with MLVs at 298 K increases slightly as Ca2+ concentration increases, but it drops to a minimum at a Ca2+ concentration equal to 5 mM. At this concentration a special compact structural arrangement is observed, indicative of increased order. Combining this finding with the above result on the ratio of 4.5 to 5 lipid molecules per molecule of nucleotide, it was inferred that roughly one CaCl2 binds two DPPC molecules and a model was proposed where every Ca2+ bridges two adjacent DPPC molecules through their phosphate groups. A different possibility was formerly considered (Bruni et al., 1997) in a study on the interactions of bivalent metal cations with double-stranded polynucleotides or DNA and egg yolk PC. Scatchard plots of PC/DNA/Mn2+ and DNA/Mn2+ complexes, combined with data of elemental analysis, support an arrangement where each Mn2+ bridges two DNA phosphates with three PC molecules. One more schematic model for interpreting the DNA-lipid interaction mediated by Ca2+ and Mg2+ has been working on the zwitterionic 1,2-dimyristoylphopsphoetyhanolamine (DMPE). Following this suggestion (Gromelski & Brezesinski, 2006)thedivalent cations bridge the negative part of the zwitterionic phospholipid headgroups, thereby making the lipid monolayers positive. Divalent cations also interact with the negative DNA phosphate moieties, condensing the DNA and leading to an ordered alignment of the DNA strands. If not all charges are screened by the divalent cations, the DNA aggregate remains partially negative and can interact either via divalent cations with the lipid phosphate groups or directly with the positively charged ethanolamine groups of DMPE when the lipid phosphate groups are bridged by divalent cations.

of PEG lipids with long acyl chains (< 14 carbons), the contact between complexes and endosomal membranes doesn't allow membrane disruption. In general it seems that the biological and physicochemical characteristics of the DNA/copolymer complexes, including PEG, are influenced by the copolymer architecture (Deshpande et al., 2004) and that the transfection efficiency is strongly correlated with the level of cellular association

In previous paragraphs we pointed out that the structure of the lipoplexes represents a fundamental tool in understanding and planning DNA transfection systems. The same remarks are valid for complexes of DNA with neutral (zwitterionic) liposomes in order to evaluate correctly their behaviour and, in case, design the necessary developments to achieve better results in DNA transfection experiments both *in vitro* and *in vivo*. These complexes were initially studied in order to understand the influence of DNA structural transition of neutral lipids: DSC thermograms of the DPPC/DNA/Ca2+ complex (Tarahovsky et al., 1996) reveal a distinct maximum at the temperature of 316.3 K in addition to the main maximum at 314.6 K. Since a direct relationship was observed between the molar proportion of DNA in samples and the value of the height of the second peak, it was hence assumed that the higher temperature transition corresponds to the formation of the complex. In another work (Kharakoz et al., 1999) it was demonstrated that DPPC/DNA complexes could be obtained by simply mixing the DNA solutions with an aqueous lipid dispersion in the presence of Ca2+ and that their formation could be obtained with both MLVs and ULVs. The stoichiometry was determined in 4.5 to 5 strongly bound lipid molecules per molecule of nucleotide, depending on the method used in a temperature-scanning ultrasonic study. From this result and the ones obtained in a small angle x-ray scattering experiment (SAXS), a model was proposed (McManus et al., 2003) for the interaction of DNA and DPPC in the presence of CaCl2. The lamellar repeat distance in complexes with MLVs at 298 K increases slightly as Ca2+ concentration increases, but it drops to a minimum at a Ca2+ concentration equal to 5 mM. At this concentration a special compact structural arrangement is observed, indicative of increased order. Combining this finding with the above result on the ratio of 4.5 to 5 lipid molecules per molecule of nucleotide, it was inferred that roughly one CaCl2 binds two DPPC molecules and a model was proposed where every Ca2+ bridges two adjacent DPPC molecules through their phosphate groups. A different possibility was formerly considered (Bruni et al., 1997) in a study on the interactions of bivalent metal cations with double-stranded polynucleotides or DNA and egg yolk PC. Scatchard plots of PC/DNA/Mn2+ and DNA/Mn2+ complexes, combined with data of elemental analysis, support an arrangement where each Mn2+ bridges two DNA phosphates with three PC molecules. One more schematic model for interpreting the DNA-lipid interaction mediated by Ca2+ and Mg2+ has been working on the zwitterionic 1,2-dimyristoylphopsphoetyhanolamine (DMPE). Following this suggestion (Gromelski & Brezesinski, 2006)thedivalent cations bridge the negative part of the zwitterionic phospholipid headgroups, thereby making the lipid monolayers positive. Divalent cations also interact with the negative DNA phosphate moieties, condensing the DNA and leading to an ordered alignment of the DNA strands. If not all charges are screened by the divalent cations, the DNA aggregate remains partially negative and can interact either via divalent cations with the lipid phosphate groups or directly with the positively charged ethanolamine groups of

**4.4 The structure of the complexes of DNA with zwitterionic liposomes** 

DMPE when the lipid phosphate groups are bridged by divalent cations.

and uptake of the DNA/copolymer complexes.

#### NEUTRAL LIPIDS

This new class of complexes consists of ternary systems NLs/DNA/M2+ where M refers mostly to Ca, Mg, a choice consistent with the previously reported experiments of membrane fusion, and sometimes Mn. The formation of the ternary complexes is the result of a self assembling process in which the driving force is represented by the release of the counter-ion entropy upon neutralization of DNA phosphate groups by metal cations (Cl— in the examples discussed). Studies on the structure of these ternary complexes were undertaken mainly by means of x-ray diffraction technique.

In all the experiments performed by the authors of this review, XRD measurements were carried out at the high brilliance beamline ID02 of the European Synchrotron Radiation Facility (Grenoble, France). The energy of the incident beam was 12.5 keV (λ= 0.995 Å), the beam size 100x100 μm2, and the sample-to-detector distance 1.2 m. The 2D diffraction patterns were collected by a CCD detector. The small angle q range from qmin = 0.1 nm-1 to qmax = 4 nm-1 with a resolution of 5 x 10-3 nm-1 (fwhm) was investigated: the samples were held in a 1 mm-sized glass capillary. To avoid radiation damage, each sample was exposed to radiation for 3 sec/frame. To calculate the electron density maps, the integrated intensities of the diffraction peaks were determined by fitting the data with series of Lorentz functions, using a nonlinear baseline. The Lorentz correction was performed multiplying each integrated intensity by sin *θ* and the intensities were then calibrated dividing by the multiplicity of the reflection (Harper et al., 2001; Francescangeli et al., 1996). The square root of the corrected peak was finally used to determine the modulus of the form factor *F* of each

Neutral Liposomes and DNA Transfection 333

the DNA strands are sandwiched between the lipid bilayers and bound together through the hydrated metal ions: the value of 2.8 nm between two lipid bilayers is sufficient to

Fig. 3. A pictorial image of the DOPC/DNA/Mn2+ (left) and of the DOPE/DNA/M2+ (right)

The simultaneous presence of two lamellar structures, confirmed by an analogous XRD study (Uhrikova et al., 2005), was interpreted by plotting (Figure 4, left) the integrated intensities of the first order diffraction peaks of the DNA complex and of the DOPC liposome as a function of the ratio of the metal ion concentration versus the one of the DNA

Fig. 4. Left: integrated intensities of the first order diffraction peak of the ternary complex (▲) and of DOPC liposome (⌂) as a function of the ratio of the metal ion concentration to the concentration of DNA phosphate groups. (Reprinted from Phys. Rev. E, 2003, 67, 11904).

177 nm

Right: freeze-fracture EM micrograph of the DOPC/DNA/Mn2+ complex.

012345678

[Mn2+]/[PO2- 4 ] DNA

accommodate a hydrated double strand of DNA (Figure 2, right).

ternary complexes.

phosphate groups.

0.0

0.2

0.4

Intensity (arb. units)

0.6

0.8

1.0

respective reflection. The electron density profile Δρ along the normal to the bilayers was calculated by Fourier sum,

$$\Delta \rho = \frac{\rho \left( z \right) - \left\langle \rho \right\rangle}{\left[ \left\langle \rho^2 \left( z \right) \right\rangle - \left\langle \rho \right\rangle^2 \right]^{1/2}} = \sum\_{l=1}^{N} F\_l \cos \left( 2\pi \left\langle l \frac{z}{d} \right\rangle \right)$$

where ρ*(z)* is the electron density, ρ its average value, *N* the highest order of fundamental reflection observed in the SAXS pattern; *Fl* is the form factor of the (*00l*) reflection, *d* the thickness of the repeating unit and the origin of the *z* axis is chosen in the middle of the lipid bilayers. The phase problem was solved by means of a pattern recognition approach based on the histogram of the electron density map (Tristram-Nagle et al., 1998) and the results were found to be in agreement with those obtained with different approaches.

In a first example (Francescangeli et al., 2003), a DOPC liposome was mixed with calf thymus DNA in hepes buffered aqueous solutions of divalent cations and simultaneous small (SAXS) and wide (WAXS) angle x-ray scattering measurements were carried out.

Fig. 2. Left: synchrotron SAXS pattern of DOPC/DNA/Mn2+ complex at molar ratio 3:4:12. (Reprinted from MROC, 2011, 8, 38) Right: the model proposed for the CL phase of the <sup>α</sup> ternary complex, reporting the main structural parameters.

In a typical experiment the mole ratio DOPC:DNA:Mn2+ was 3:4:12 and the corresponding synchrotron *x*-ray diffraction (XRD) pattern (Figure 2, left) at 298 K is reported*.* Two series of spacings are present in the x-ray pattern: the one indicated with the symbol CL (d= 7.34 <sup>α</sup> nm), independent of the concentration of the cation, has been attributed to the ternary complex and the one indicated with the symbol Lα (d= 5.88 nm) to the complex DOPC/Mn2+. The ternary complex is characterized by the lamellar symmetry of the CL <sup>α</sup> (Luzzati, 1968), consisting of an ordered multilamellar assembly where the hydrated DNA helices are sandwiched between the liposome bilayers. This structure is similar to that found in CLs/DNA complexes (Rädler et al., 1997; Podgornik et al. 1989). A pictorial representation (Figure 3, left) of the ternary complex DOPC/DNA/Mn2+ has been proposed: 332 Non-Viral Gene Therapy

( ) 1/2 <sup>2</sup> <sup>2</sup> <sup>1</sup>

reflection observed in the SAXS pattern; *Fl* is the form factor of the (*00l*) reflection, *d* the thickness of the repeating unit and the origin of the *z* axis is chosen in the middle of the lipid bilayers. The phase problem was solved by means of a pattern recognition approach based on the histogram of the electron density map (Tristram-Nagle et al., 1998) and the results

In a first example (Francescangeli et al., 2003), a DOPC liposome was mixed with calf thymus DNA in hepes buffered aqueous solutions of divalent cations and simultaneous small (SAXS) and wide (WAXS) angle x-ray scattering measurements were carried out.

Fig. 2. Left: synchrotron SAXS pattern of DOPC/DNA/Mn2+ complex at molar ratio 3:4:12. (Reprinted from MROC, 2011, 8, 38) Right: the model proposed for the CL phase of the <sup>α</sup>

In a typical experiment the mole ratio DOPC:DNA:Mn2+ was 3:4:12 and the corresponding synchrotron *x*-ray diffraction (XRD) pattern (Figure 2, left) at 298 K is reported*.* Two series of spacings are present in the x-ray pattern: the one indicated with the symbol CL (d= 7.34 <sup>α</sup> nm), independent of the concentration of the cation, has been attributed to the ternary

DOPC/Mn2+. The ternary complex is characterized by the lamellar symmetry of the CL <sup>α</sup> (Luzzati, 1968), consisting of an ordered multilamellar assembly where the hydrated DNA helices are sandwiched between the liposome bilayers. This structure is similar to that found in CLs/DNA complexes (Rädler et al., 1997; Podgornik et al. 1989). A pictorial representation (Figure 3, left) of the ternary complex DOPC/DNA/Mn2+ has been proposed:

α

(d= 5.88 nm) to the complex

ternary complex, reporting the main structural parameters.

*L*α(003)

(003)

*L C* α

*L C* α

(004)

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

q(nm-1 )

*L*α(002)

(002)

*L C* α

*L*α(001)

*L C* α(001)

Intensity (arb.units)

complex and the one indicated with the symbol L

 = <sup>−</sup> Δ = <sup>=</sup> <sup>−</sup>

 ρ

 ρ

( )

were found to be in agreement with those obtained with different approaches.

*z* ρ

ρ

ρ

Δρ

*N l l <sup>z</sup> <sup>z</sup> F l*

cos 2

 π

*d*

2.8 nm 7.3 nm

its average value, *N* the highest order of fundamental

along the normal to the bilayers was

respective reflection. The electron density profile

*(z)* is the electron density,

ρ

calculated by Fourier sum,

where ρ the DNA strands are sandwiched between the lipid bilayers and bound together through the hydrated metal ions: the value of 2.8 nm between two lipid bilayers is sufficient to accommodate a hydrated double strand of DNA (Figure 2, right).

Fig. 3. A pictorial image of the DOPC/DNA/Mn2+ (left) and of the DOPE/DNA/M2+ (right) ternary complexes.

The simultaneous presence of two lamellar structures, confirmed by an analogous XRD study (Uhrikova et al., 2005), was interpreted by plotting (Figure 4, left) the integrated intensities of the first order diffraction peaks of the DNA complex and of the DOPC liposome as a function of the ratio of the metal ion concentration versus the one of the DNA phosphate groups.

Fig. 4. Left: integrated intensities of the first order diffraction peak of the ternary complex (▲) and of DOPC liposome (⌂) as a function of the ratio of the metal ion concentration to the concentration of DNA phosphate groups. (Reprinted from Phys. Rev. E, 2003, 67, 11904). Right: freeze-fracture EM micrograph of the DOPC/DNA/Mn2+ complex.

DNA

−*P*β*'* −*L*α

the Lα

5.5

5.5

6.0

6.5

7.0

unit cell (nm)

7.5

8.0

6.0

6.5

unit cell (nm)

7.0

7.5

8.0

goes directly from '

*c L*β

phase of DOPC, respectively.

0 5 10 15 20 25

n

Delivery. Mol. Cryst. Liq. Cryst., 2005, 434, 643).

0 5 10 15 20 25

n

Neutral Liposomes and DNA Transfection 335

organization of the DNA chains between the liposome layers. The thermotropic phase behaviour in a temperature range between 303 K and 328 K, well above the main transition temperature of the pure lipid (Tm = 314 K) was studied (Figure 7, right), leading to the important conclusion that coexistence of complexed and uncomplexed phases persists over the whole explored thermal range. A further relevant effect is observed: while the uncomplexed lipid exhibits the same thermotropic phase behaviour as pure DPPC, i.e. *L*

This is highlighted by the disappearance of the ripple phase and the remarkable increase of the main transition temperature: the observed thermotropic phase sequence of the complex

the formation of the DOPC/DNA/Mn2+ complex has been determined (Francescangeli et al., 2003): figure 8 shows the temperature evolution of the XRD patterns of this complex in the range 290 to 320 K (left) and the temperature dependence (right) of the integrated intensities of the two small-angle reflections for the CL phase for the ternary complex and <sup>α</sup>

**Mn2+**

**Fe2+**

Fig. 5. Lamellar d-spacings of the CL of ternary complex DOPC/DNA <sup>α</sup> ct/M2+ (•) and of the L

 phase of DOPC (∀) as a function of the metal mole number n in the DOPC/DNAct/M2+ complexes at molar ratios 3:4:n. (Reprinted from: M. Pisani, P. Bruni, C. Conti, E. Giorgini, O. Francescangeli. Self-Assembled Liposome-DNA-Metal Complexes Related to DNA

, the mesomorphic behaviour of the bound lipid in the complex is partially altered.

5.5

5.5

6.0

6.5

7.0

unit cell (nm)

7.5

8.0

6.0

6.5

7.0

unit cell (nm)

7.5

8.0

to *<sup>c</sup> La* . (Pisani et al. 2006). In addition the effect of the temperature on

β*'* 

**Co2+**

**Mg2+**

0 5 10 15 20 25

n

0 5 10 15 20 25

n

α

An increase of the Mn2+ concentration favours the formation of the ternary complex accompanied to a complementary reduction of the DOPC: the saturation is reached at a ratio [Mn2+]:[PO4 2-] ≅ 6, corresponding to a constant volume fraction of the two structures (~ 70% to ~ 30% respectively). A freeze-fracture EM micrograph of the ternary complex has also been made and reported in figure 4 (right). An analogous study was made with the neutral liposome 1,2-dipalmitoylphosphatidylcholine (DPPC), bearing completely saturated hydrocarbon tails (McManus et al., 2003) : as for DOPC two coexisting phases are present and the ternary complex shows a lamellar structure, the DNA layers being embedded in the DPPC layers. The repeat distance is 7.84 nm at 298 K. At this temperature the DPPC/DNA/Ca2+ complex is in the gel thermotropic phase (Lβ').

These works have been followed by an extended approach to ternary complexes based on NLs, bearing unsaturated (DOPC, DLPC and DOPE) or saturated (DPPC) hydrocarbon tails. A twofold goal has been pursued when investigating the microscopic structures of lipids and their corresponding ternary complexes: to test whether different metal cations are equally active in promoting the DNA condensation with different lipids and ascertain to what extent structure and phase symmetry of the lipids affect the structure of the complexes; two aspects that have fundamental implications in view of an approach to gene delivery based application of these complexes. Different varieties of DNA (calf-thymus, salmon sperm and plasmid) complexed with DOPC and DLPC (Pisani et al., 2005) or DPPC (Pisani et al. 2006), in the presence of different cations (Ca2+, Mn2+, Co2+, Fe2+, Mg2+), exhibit the already discussed multilamellar liquid-crystalline CL phase, consisting of ordered <sup>α</sup> assemblies, where hydrated DNA helices are sandwiched between the lipid bilayers, and the metal cations mediate the binding of the phosphate groups of DNA with the lipid polar heads. Also within these assemblies the CL phase coexists with the uncomplexed L <sup>α</sup> α phase of the parent lipid. A systematic series of SAXS measurements in DOPC/DNAct/M2+ complexes, prepared with different metal cations, was performed as a function of the number of metal ion moles (n).

The results obtained from these spectra are reported in figure 5: a remarkable constancy of the lamellar spacings of the CL accompanied by a slight decrease of the lamellar repeat <sup>α</sup> distance of the uncomplexed Lα , reported in the figure agrees with the model proposed in figure 3. As an example we report (Figure 6) the analysis of the ternary complex with Mg2+: again two sets of peaks (each one including fundamental and high-order harmonics) related to distinct lamellar structures CL and L <sup>α</sup> α , with layer spacings d1= 7.52 nm and d2= 5.9 nm respectively, are present. The SAXS pattern (A), and the relative electron density profile (B) are shown: in the latter, the two peaks with the maximum of electron density correspond to phospholipids' polar headgroups, while the minimum correlates with the terminal hydrocarbon chain region.

The distance between the centres of the density maxima gives a good approximation of the bilayer thickness (dPP = 4.51 nm): it follows that the water-layer thickness can be calculated as dW = d1 – dPP = 7.52 – 4.51 = 3.01 nm sufficient to accommodate a double stranded DNA helix surrounded by one water hydration layer plus two thin layers of hydrated metal ions. Likewise, it was calculated a water layer thicknesses dW in the range of 2.8-3.0 nm in the complexes with DLPC and in the range of 2.9-3.2 nm in the ones with DPPC, depending on metal cation.

The SAXS pattern of the DPPC/DNA/Ca2+ (Figure **7,** left) shows a correlation peak, marked as DNA in the figure, corresponding to the DNA-DNA interaction, indicative of a higher

An increase of the Mn2+ concentration favours the formation of the ternary complex accompanied to a complementary reduction of the DOPC: the saturation is reached at a ratio

to ~ 30% respectively). A freeze-fracture EM micrograph of the ternary complex has also been made and reported in figure 4 (right). An analogous study was made with the neutral liposome 1,2-dipalmitoylphosphatidylcholine (DPPC), bearing completely saturated hydrocarbon tails (McManus et al., 2003) : as for DOPC two coexisting phases are present and the ternary complex shows a lamellar structure, the DNA layers being embedded in the DPPC layers. The repeat distance is 7.84 nm at 298 K. At this temperature the

These works have been followed by an extended approach to ternary complexes based on NLs, bearing unsaturated (DOPC, DLPC and DOPE) or saturated (DPPC) hydrocarbon tails. A twofold goal has been pursued when investigating the microscopic structures of lipids and their corresponding ternary complexes: to test whether different metal cations are equally active in promoting the DNA condensation with different lipids and ascertain to what extent structure and phase symmetry of the lipids affect the structure of the complexes; two aspects that have fundamental implications in view of an approach to gene delivery based application of these complexes. Different varieties of DNA (calf-thymus, salmon sperm and plasmid) complexed with DOPC and DLPC (Pisani et al., 2005) or DPPC (Pisani et al. 2006), in the presence of different cations (Ca2+, Mn2+, Co2+, Fe2+, Mg2+), exhibit the already discussed multilamellar liquid-crystalline CL phase, consisting of ordered <sup>α</sup> assemblies, where hydrated DNA helices are sandwiched between the lipid bilayers, and the metal cations mediate the binding of the phosphate groups of DNA with the lipid polar

heads. Also within these assemblies the CL phase coexists with the uncomplexed L <sup>α</sup>

α

of the parent lipid. A systematic series of SAXS measurements in DOPC/DNAct/M2+ complexes, prepared with different metal cations, was performed as a function of the

The results obtained from these spectra are reported in figure 5: a remarkable constancy of the lamellar spacings of the CL accompanied by a slight decrease of the lamellar repeat <sup>α</sup>

figure 3. As an example we report (Figure 6) the analysis of the ternary complex with Mg2+: again two sets of peaks (each one including fundamental and high-order harmonics) related

respectively, are present. The SAXS pattern (A), and the relative electron density profile (B) are shown: in the latter, the two peaks with the maximum of electron density correspond to phospholipids' polar headgroups, while the minimum correlates with the terminal

The distance between the centres of the density maxima gives a good approximation of the bilayer thickness (dPP = 4.51 nm): it follows that the water-layer thickness can be calculated as dW = d1 – dPP = 7.52 – 4.51 = 3.01 nm sufficient to accommodate a double stranded DNA helix surrounded by one water hydration layer plus two thin layers of hydrated metal ions. Likewise, it was calculated a water layer thicknesses dW in the range of 2.8-3.0 nm in the complexes with DLPC and in the range of 2.9-3.2 nm in the ones with DPPC, depending on

The SAXS pattern of the DPPC/DNA/Ca2+ (Figure **7,** left) shows a correlation peak, marked as DNA in the figure, corresponding to the DNA-DNA interaction, indicative of a higher

, reported in the figure agrees with the model proposed in

, with layer spacings d1= 7.52 nm and d2= 5.9 nm

αphase

DPPC/DNA/Ca2+ complex is in the gel thermotropic phase (Lβ').

α

2-] ≅ 6, corresponding to a constant volume fraction of the two structures (~ 70%

[Mn2+]:[PO4

number of metal ion moles (n).

distance of the uncomplexed L

hydrocarbon chain region.

metal cation.

to distinct lamellar structures CL and L <sup>α</sup>

organization of the DNA chains between the liposome layers. The thermotropic phase behaviour in a temperature range between 303 K and 328 K, well above the main transition temperature of the pure lipid (Tm = 314 K) was studied (Figure 7, right), leading to the important conclusion that coexistence of complexed and uncomplexed phases persists over the whole explored thermal range. A further relevant effect is observed: while the uncomplexed lipid exhibits the same thermotropic phase behaviour as pure DPPC, i.e. *L*β*'*  −*P*β*'* −*L*α , the mesomorphic behaviour of the bound lipid in the complex is partially altered. This is highlighted by the disappearance of the ripple phase and the remarkable increase of the main transition temperature: the observed thermotropic phase sequence of the complex goes directly from ' *c L*β to *<sup>c</sup> La* . (Pisani et al. 2006). In addition the effect of the temperature on the formation of the DOPC/DNA/Mn2+ complex has been determined (Francescangeli et al., 2003): figure 8 shows the temperature evolution of the XRD patterns of this complex in the range 290 to 320 K (left) and the temperature dependence (right) of the integrated intensities of the two small-angle reflections for the CL phase for the ternary complex and <sup>α</sup> the Lαphase of DOPC, respectively.

Fig. 5. Lamellar d-spacings of the CL of ternary complex DOPC/DNA <sup>α</sup> ct/M2+ (•) and of the Lα phase of DOPC (∀) as a function of the metal mole number n in the DOPC/DNAct/M2+ complexes at molar ratios 3:4:n. (Reprinted from: M. Pisani, P. Bruni, C. Conti, E. Giorgini, O. Francescangeli. Self-Assembled Liposome-DNA-Metal Complexes Related to DNA Delivery. Mol. Cryst. Liq. Cryst., 2005, 434, 643).

Neutral Liposomes and DNA Transfection 337

The evolution of the equilibrium concentrations of the two phases clearly shows that the increase of the temperature favours the formation of the complex, the relative concentrations of the lamellar phases of pure lipids lowering in favour of the one of the ternary complex.

Fig. 8. Left: temperature evolution of the XRD patterns of DOPC/DNA/Mn2+ in the range 290 to 320 K. Right: temperature dependence of the integrated intensities of the two small-

0

1

2

3

intensity (arb.units)

4

5

The complex DOPC/DNA/Mn2+ has also been studied in a solid supported phase (Caracciolo et al., 2004) by Energy Dispersion *X*-ray Diffraction (Caminiti & Rossi Albertini, 1999; Caracciolo et al., 2002) and it has been found that its structure is essentially identical to that in aqueous solution. The effect of hydration on the structural features of these multilamellar systems has also been explored (Caminiti et al., 2005), considering that adsorbed water plays a major role in the effectiveness of lipid drug delivery systems where lipid-cell interactions are involved. The hydration kinetics of oriented DOPC shows that the long-range order in a multilamellar lipid system strictly depends on the hydration level: adsorbed water molecules first promote a spatial coherence along the normal to the lipid bilayers, then penetrate the interbilayer region and behave as bulk water, producing disorder. The existence of a correlation between the degree of hydration of lipid bilayers and the structure of interbilayer water (Ge & Freed, 2003; Zhou et al., 1999) has been confirmed. We have already reported that DOPE induces a structural transformation of the lipoplexes when added as a co-lipid: the equilibrium phase of pure DOPE in excess water consists of an inverted hexagonal HII lattice (Turner & Gruner, 1992)*,* whose structure elements are infinitely long rigid rods, all identical and cristallographically equivalent, regularly packed in a 2D hexagonal lattice. The cylinders are filled by water and dispersed in the continuous medium of the hydrocarbon chains, whereas the polar groups are located at the waterhydrocarbon interface. The SAXS pattern of pure DOPE allows to calculate a unit cell

phases. (Reprinted from Rec. Res. Devel. in

290 295 300 305 310 315 320 325

T (K)

α

290 K

295 K

300 K

305 K

310 K

320 K

angle reflections for the CL and the L <sup>α</sup>

0.8 1.2 1.6 2 q (nm-1)

Macromol., 2003, 7, 247).

intensity (arb.units)

Fig. 6. Left: synchrotron SAXS pattern of the ternary complex DLPC/DNAct/Mn2+ at 3:4:12 molar ratio. Right: electron density profile along the normal to the bilayers in the CLα phase. (Reprinted from: M. Pisani, P. Bruni, C. Conti, E. Giorgini, O. Francescangeli. Self-Assembled Liposome-DNA-Metal Complexes Related to DNA Delivery. Mol. Cryst. Liq. Cryst., 2005, 434, 643).

Fig. 7. Left: SAXS pattern of DPPC/DNA/Ca2+ complex at molar ratio 3:4:24. Right: synchrotron XRD patterns as a function of temperature.

Fig. 6. Left: synchrotron SAXS pattern of the ternary complex DLPC/DNAct/Mn2+ at 3:4:12 molar ratio. Right: electron density profile along the normal to the bilayers in the CLα phase.




Δρ(z) (arb.units)

0

0.5

1


z/d

1234

q (nm-1 )

β

*L c* β

DNA

*L* β

*'*(001)

*L c* β

Intensity (arb. units)

*'*(001)

> *'*(002)

> > *'*(002)

> > > *L* β

*'*(003) *<sup>L</sup>* 328 K 323 K

318 K 313 K

308 K

303 K

*L c* α(003)

*L* α(002)

*L* α(001)

*L c* α(001)

> *L c* α(002)

Assembled Liposome-DNA-Metal Complexes Related to DNA Delivery. Mol. Cryst. Liq.

(Reprinted from: M. Pisani, P. Bruni, C. Conti, E. Giorgini, O. Francescangeli. Self-

2 2.5 3 3.5 4

*L*c α (004)

*L*c α (003)

0.5 1 1.5 2 2.5 3 3.5 4 q (nm-1 )

q (nm-1)

Fig. 7. Left: SAXS pattern of DPPC/DNA/Ca2+ complex at molar ratio 3:4:24. Right:

synchrotron XRD patterns as a function of temperature.

1234

q (nm-1 )

α

 (003) *<sup>L</sup>*α

(002)

*L c* α

(001)

DNA

*L*α

(001)

*L c* α

Intensity (arb. units)

(002)

*L*α

(004) *L*

(004)

*L c* α

Cryst., 2005, 434, 643).

*L*α (001) *L*<sup>c</sup> α (002)

Intensity (arb. units)

*L*c α (001)

Intensity (arb. units)

*L*α (002) The evolution of the equilibrium concentrations of the two phases clearly shows that the increase of the temperature favours the formation of the complex, the relative concentrations of the lamellar phases of pure lipids lowering in favour of the one of the ternary complex.

Fig. 8. Left: temperature evolution of the XRD patterns of DOPC/DNA/Mn2+ in the range 290 to 320 K. Right: temperature dependence of the integrated intensities of the two smallangle reflections for the CL and the L <sup>α</sup> α phases. (Reprinted from Rec. Res. Devel. in Macromol., 2003, 7, 247).

The complex DOPC/DNA/Mn2+ has also been studied in a solid supported phase (Caracciolo et al., 2004) by Energy Dispersion *X*-ray Diffraction (Caminiti & Rossi Albertini, 1999; Caracciolo et al., 2002) and it has been found that its structure is essentially identical to that in aqueous solution. The effect of hydration on the structural features of these multilamellar systems has also been explored (Caminiti et al., 2005), considering that adsorbed water plays a major role in the effectiveness of lipid drug delivery systems where lipid-cell interactions are involved. The hydration kinetics of oriented DOPC shows that the long-range order in a multilamellar lipid system strictly depends on the hydration level: adsorbed water molecules first promote a spatial coherence along the normal to the lipid bilayers, then penetrate the interbilayer region and behave as bulk water, producing disorder. The existence of a correlation between the degree of hydration of lipid bilayers and the structure of interbilayer water (Ge & Freed, 2003; Zhou et al., 1999) has been confirmed. We have already reported that DOPE induces a structural transformation of the lipoplexes when added as a co-lipid: the equilibrium phase of pure DOPE in excess water consists of an inverted hexagonal HII lattice (Turner & Gruner, 1992)*,* whose structure elements are infinitely long rigid rods, all identical and cristallographically equivalent, regularly packed in a 2D hexagonal lattice. The cylinders are filled by water and dispersed in the continuous medium of the hydrocarbon chains, whereas the polar groups are located at the waterhydrocarbon interface. The SAXS pattern of pure DOPE allows to calculate a unit cell

Neutral Liposomes and DNA Transfection 339

in different contexts (Koynova et al., 1997) : this ability, together with the well known fusogenic property of DOPE and its destabilizing effect on targeted endosomal membranes makes the complexes DOPE/DOPE-PEG(350)/DNA/Mn2+ extremely

HII (20)

HII HII (11) (10)

Fig. 10. Synchrotron XRD patterns of the DOPE/DOPE-PEG(350)/DNA/Mn2+ complex as a function of different concentrations of the DOPE/PEG component in the lipid mixture. The pattern of the cubic phase is clearly visible at 6%, 9% and 15% concentration of the pegilated

q (nm-1)

1234

HC II (20)

HC II (11)

HC II (10)

> II H (30) <sup>C</sup> II (21)

15%

HC II (31)

HC II (22) HC

9%

6%

3%

HII (31) HII (22) HII (30) HII (21)

A first attempt of *in vitro* transfection was made with a couple of complexes DOPC/pDNA/M2+, with M = Ca or Mn, on a mouse fibroplast NIH 3T3 cell line (Bruni et al., 2006): using standard methods the green fluorescent protein was expressed by both complexes. Figure 11 reports an improved result obtained later, using a 15 mM

Other attempts of *in vitro* transfections made in our laboratories are compared in a series of histograms (Figure 12) which show that the low efficiency of pure DOPC can be increased by addition of both 1, 2-dioleoyl-*sn*-glycero-3-phosphoethanolamine-N-hexanoylamine (6PE) or 1,2-dioleoyl-*sn*-glycero-3-phosphoethanolamine-N-dodecanoylamine (12PE) to DOPC/DNA/M2+. This result confirms that the transfection efficiency is strongly dependent

interesting for application in HGT.

DOPE.

**4.5 DNA transfection experiments** *in vitro* 

on an appropriate mixutures of liposomes as DNA carriers.

Q Q

Q

Intensity (arb. units)

concentration of Ca2+ in the complex.

spacing a = 7.44 nm (Francescangeli et al., 2004) and its electron density profile calculated along the [10] direction (Figure 9, left) shows an average diameter of the water core dW = 3.02 nm. DOPE and divalent metal cations Mn2+, Mg2+, Co2+ and Fe2+ in water solution condense DNA into ternary complexes DOPE/DNA/M2+ characterized by an invertedhexagonal phase CHII . Also in this case two different sets of peaks with different unit cell spacings, namely a = 7.45 nm and aC*=* 6.87 nm respectively, have been observed. The former corresponds to the phase HII of pure DOPE, the latter is instead consistent with the 2D columnar inverted hexagonal phase CHII of the DOPE/DNA/Fe2+ complexes.

Fig. 9. Left: electron density profile of the pure DOPE along the [10] direction of the unit cell: the origin corresponds to the centre of water core. Right: electron density profile of the complex DOPE/DNA/Fe2+.

In this structure DNA strands are supposed to fill the water gap inside the cylinders of pure DOPE, as it is supported by the electron density profiles (Figure 9 right) calculated along the [10] direction. The two shoulders, at z/d ~ 0.26 and 0.73 respectively, correspond to phosphate groups and are used to localize the centres of the polar head. From the structural data, values of dPP = 3.26 nm and dL = 4.36 nm were calculated, leading to a water layer thickness dW = 2.51 nm, large enough to accommodate a double-stranded DNA molecule surrounded by a hydration layer (Podgornik et al., 1989). A pictorial representation of this structure is reported in Figure 3 (right). Unlike complexes organized in the CL the ratio <sup>α</sup> between HII and CHII depends also on the incubation time: after 48 hours, phase HII disappears completely and is transformed into the CHII of ternary complex.

The effect of pegylation on NLs has also been studied (Pisani at al., 2008, 2009) in mixed complexes DOPE/DOPE-PEG(350)/DNA/M2+ (M = Ca, Mg, Mn). XRD investigation on the complex with Mn2+ shows that with 3% of DOPE-PEG, the two phases CHII and HII coexist as usual: the former being attributed to DOPE/DOPE-PEG(350)/DNA/Mn2+, the latter to DOPE/DOPE-PEG(350)/Mn2+. Interestingly a new phase, indexed in the SAXS pattern as Q (Figure 10), appears at higher concentrations of DOPE-PEG (6, 9 and 15%): the corresponding peaks are spaced in the ratios 2; 3 ; 4 ; 6 ; 8 ; 9; 10 consistent with a cubic Q224 phase with the space group *Pn3m.* A transition HII → QII has been found

spacing a = 7.44 nm (Francescangeli et al., 2004) and its electron density profile calculated along the [10] direction (Figure 9, left) shows an average diameter of the water core dW = 3.02 nm. DOPE and divalent metal cations Mn2+, Mg2+, Co2+ and Fe2+ in water solution condense DNA into ternary complexes DOPE/DNA/M2+ characterized by an invertedhexagonal phase CHII . Also in this case two different sets of peaks with different unit cell spacings, namely a = 7.45 nm and aC*=* 6.87 nm respectively, have been observed. The former corresponds to the phase HII of pure DOPE, the latter is instead consistent with the 2D

Fig. 9. Left: electron density profile of the pure DOPE along the [10] direction of the unit cell: the origin corresponds to the centre of water core. Right: electron density profile of the




0

Δρ (x) (arb. units)

2

4

6

0.0 0.2 0.4 0.6 0.8 1.0

x/a

In this structure DNA strands are supposed to fill the water gap inside the cylinders of pure DOPE, as it is supported by the electron density profiles (Figure 9 right) calculated along the [10] direction. The two shoulders, at z/d ~ 0.26 and 0.73 respectively, correspond to phosphate groups and are used to localize the centres of the polar head. From the structural data, values of dPP = 3.26 nm and dL = 4.36 nm were calculated, leading to a water layer thickness dW = 2.51 nm, large enough to accommodate a double-stranded DNA molecule surrounded by a hydration layer (Podgornik et al., 1989). A pictorial representation of this structure is reported in Figure 3 (right). Unlike complexes organized in the CL the ratio <sup>α</sup> between HII and CHII depends also on the incubation time: after 48 hours, phase HII

The effect of pegylation on NLs has also been studied (Pisani at al., 2008, 2009) in mixed complexes DOPE/DOPE-PEG(350)/DNA/M2+ (M = Ca, Mg, Mn). XRD investigation on the complex with Mn2+ shows that with 3% of DOPE-PEG, the two phases CHII and HII coexist as usual: the former being attributed to DOPE/DOPE-PEG(350)/DNA/Mn2+, the latter to DOPE/DOPE-PEG(350)/Mn2+. Interestingly a new phase, indexed in the SAXS pattern as Q (Figure 10), appears at higher concentrations of DOPE-PEG (6, 9 and 15%): the corresponding peaks are spaced in the ratios 2; 3 ; 4 ; 6 ; 8 ; 9; 10 consistent with a cubic Q224 phase with the space group *Pn3m.* A transition HII → QII has been found

disappears completely and is transformed into the CHII of ternary complex.

complex DOPE/DNA/Fe2+.




0

Δρ (x) (arb. units)

2

4

6

0.0 0.2 0.4 0.6 0.8 1.0

x/a

columnar inverted hexagonal phase CHII of the DOPE/DNA/Fe2+ complexes.

in different contexts (Koynova et al., 1997) : this ability, together with the well known fusogenic property of DOPE and its destabilizing effect on targeted endosomal membranes makes the complexes DOPE/DOPE-PEG(350)/DNA/Mn2+ extremely interesting for application in HGT.

Fig. 10. Synchrotron XRD patterns of the DOPE/DOPE-PEG(350)/DNA/Mn2+ complex as a function of different concentrations of the DOPE/PEG component in the lipid mixture. The pattern of the cubic phase is clearly visible at 6%, 9% and 15% concentration of the pegilated DOPE.

#### **4.5 DNA transfection experiments** *in vitro*

A first attempt of *in vitro* transfection was made with a couple of complexes DOPC/pDNA/M2+, with M = Ca or Mn, on a mouse fibroplast NIH 3T3 cell line (Bruni et al., 2006): using standard methods the green fluorescent protein was expressed by both complexes. Figure 11 reports an improved result obtained later, using a 15 mM concentration of Ca2+ in the complex.

Other attempts of *in vitro* transfections made in our laboratories are compared in a series of histograms (Figure 12) which show that the low efficiency of pure DOPC can be increased by addition of both 1, 2-dioleoyl-*sn*-glycero-3-phosphoethanolamine-N-hexanoylamine (6PE) or 1,2-dioleoyl-*sn*-glycero-3-phosphoethanolamine-N-dodecanoylamine (12PE) to DOPC/DNA/M2+. This result confirms that the transfection efficiency is strongly dependent on an appropriate mixutures of liposomes as DNA carriers.

Neutral Liposomes and DNA Transfection 341

by K+, as revealed by x-rays, no transfection has been observed in this case. Instead the efficiency increases in the order Mg2+ < Ca2+ < La3+, the last being 2.6-fold higher than the lipoplex DNA/DOTAP/DOPE. It is also of great importance that the highest efficiency measured with La3+ complex has been obtained with ion concentration of three orders of magnitude lower than that of Ca2+: a result extremely favourable in relation to toxicity, as it

At present the use of NLs as autonomous carriers of genetic material in human gene therapy can be considered an opportunity that needs extensive exploration to become a real alternative to CLs. Considering the many limits the latter still meet, particularly in the *in vivo* applications, and their slow progress, it seems important to take also the parallel way of NLs as possible autonomous carriers: lack of toxicity and high stability in serum are important characteristics in their favour. Some of the results outlined here are worth interesting developments. It has been found that complexes reflect the structure and symmetry of the parent lipids and that the different bivalent metal cations are equally active in promoting the DNA condensation into the ternary complexes; these achievements will provide structure-composition correlation, that may be used in designing at the best these materials as non-viral DNA carriers in HGT. Additional developments of the research in this field, currently investigated in our laboratories, concern the use of pegylated NLs in the management of brain related diseases, where CLs have started being experimented (Zhang et al., 2002; Pardridge, 2007; Boardo, 2007). Better results could be perhaps obtained with NLs, thanks to their ability to reduce opsonisation. The recent interest in the so-called intelligent carriers which is developing on CLs (Voinea & Simionescu, 2002; Shi et al., 2002; Alvarez-Lorenzo et al., 2009) could also represent an interesting opportunity for NLs. The structural knowledge of complexes of DNA with NLs is only one of the aspects which will presumably affect the transfection: many other aspects, such as Z-potential values, complex size, and efficient DNA entrapment are all very important acquisitions to be obtained. The entry of NLs in the world of HGT and the consequent opportunity to compare properties and activity with the ones of cationic and anionic liposomes will lead to a better understanding of that processes. In this connection is encouraging to quote the opinion of Rädler, one of the most outstanding experts in cationic lipids: "the resources devoted to creating less toxic cationic-DNA complexes, may perhaps, in the future be balanced by research exploiting the possibility of creating comparable complexes from entirely non toxic

Alvarez-Lorenzo, C.; Bromberg, L. & Concheiro, A. (2009). Light-sensitive intelligent drug

Bailay, A.L. & Sullivan, S.M. (2000). Efficient encapsulation of DNA plasmids in small

Bangham, A.D., Standish, M.M. & Weissmann, G. (1965). The action of steroids and

neutral liposomes induced by ethanol and calcium. *Biochim. Biophys. Acta*, 1468,

streptolysin S on the permeability of phospholipids structures to cations. *J. Mol.* 

delivery systems. *Photochem. Photobiol*., 85, 848–860, 0031-8655.

has been proved.

**6. References** 

239-252, 005-2736.

*Biol.*, 13, 253-259, 0022-2836.

**5. Conclusions and perspectives** 

components such as the NLs/DNA/M2+ complexes".

Fig. 11. Fluorescence micrograph of mouse fibroplast NIH 3T3 cells transfected with pGreen Lantern complexed with DOPC liposome in presence of Ca2+.

Fig. 12. Luciferase expression following 6hrs transfection with different complexes in NIH3T3 cell line. Expression efficiency is expressed as Relative Luminometric Units per cell (RLU/cell).

An interesting comparison among in vitro transfection efficiencies by DOPE/DNA complexes mediated by cations bearing different charges such as K+, Mg2+, Ca2+, La3+ has been proposed (Tresset et al., 2007). At physiological pH pure DOPE has a slightly negative charge which is not altered by K+ owing to its low density of binding sites. On the contrary high charge density has been measured for Mg2+ and Ca*2+* and particularly for La3+ (100-fold higher than the two bivalent cations). SAXS of the corresponding ternary complexes show the absence of any ordered structure induced by K+, whereas the usual presence of the two HII and CHII phases has been confirmed with the other cations. Transfection efficiency has been measured on the two cell lines U87 and hepG2: due to the absence of a complexation

Fig. 11. Fluorescence micrograph of mouse fibroplast NIH 3T3 cells transfected with pGreen

Fig. 12. Luciferase expression following 6hrs transfection with different complexes in NIH3T3 cell line. Expression efficiency is expressed as Relative Luminometric Units per cell

6PE(15)-DOPC

An interesting comparison among in vitro transfection efficiencies by DOPE/DNA complexes mediated by cations bearing different charges such as K+, Mg2+, Ca2+, La3+ has been proposed (Tresset et al., 2007). At physiological pH pure DOPE has a slightly negative charge which is not altered by K+ owing to its low density of binding sites. On the contrary high charge density has been measured for Mg2+ and Ca*2+* and particularly for La3+ (100-fold higher than the two bivalent cations). SAXS of the corresponding ternary complexes show the absence of any ordered structure induced by K+, whereas the usual presence of the two HII and CHII phases has been confirmed with the other cations. Transfection efficiency has been measured on the two cell lines U87 and hepG2: due to the absence of a complexation

12PE(15)-DOPC

DOPC-DOTAP

(RLU/cell).

Lantern complexed with DOPC liposome in presence of Ca2+.

DOPC

RLU/Cell

DOPE

by K+, as revealed by x-rays, no transfection has been observed in this case. Instead the efficiency increases in the order Mg2+ < Ca2+ < La3+, the last being 2.6-fold higher than the lipoplex DNA/DOTAP/DOPE. It is also of great importance that the highest efficiency measured with La3+ complex has been obtained with ion concentration of three orders of magnitude lower than that of Ca2+: a result extremely favourable in relation to toxicity, as it has been proved.
