**2.2 Monocationic lipids**

352 Non-Viral Gene Therapy

Commercially available, DC-Chol is widely used for the development of novel approaches for DNA delivery, i.e. as a component of cationic solid lipid nanoparticles (SLNs) (Choi et al., 2008). The SLNs for gene delivery composed of DC-Chol, DOPE, and Tween 80 with TC (tricaprin) as a core in various ratios were produced by the melt homogenization method. The SLNs were bound to the cellular membrane 10 min after transfection, and translocated to the cytosol 60 min later. After 24 h, the SLNs were detectable in the nucleus and cytosol. SLNs showed high transfection efficiency in comparison with commercially available Lipofectin. In fact, PCR analysis indicated that SLNs prolonged the mRNA expression of the plasmid in various organs for up to 5 days. The SLNs-mediated transfection of the p53 gene resulted in the efficient reestablishment of wild-type p53 function in lung cancer cells and

The use of the most thoroughly investigated cationic lipids DC-Chol, DOTAP, and dimethyldioctadecylammonium (DDA) as vaccine delivery systems to form an antigen depot at the site of injection (SOI) and to induce immunological responses *in vivo* was reported (Henriksen-Lacey et al., 2011). DC-Chol, DOTAP, and DDA liposomes incorporating immunomodulating trehalose dibehenate (TDB) were prepared. DC-Chol/TDB liposomes were stable under storage and were retained at a significantly better degree at the SOI, with nearly 40% of the original dose still detectable 14 days p.i, in comparison with DOTAP/TDB liposomes. With regards to the depot effect at the SOI, the formulations were able to cause antigen retention between the range of 59 and 79% of the

The key step in lipid-mediated DNA delivery may be the structural changes of lipid carriers resulting in DNA release (Tarahovsky et al., 2004; Koynova et al., 2006; Hoekstra et al., 2007). In recent times, it was shown that multicomponent lipoplexes are superior in transfection with respect to the binary ones usually employed for gene delivery (Caracciolo et al., 2005a; 2005b; 2006). For instance, the four-component lipid system incorporating cationic lipids DOTAP, DC-Chol and neutral helper lipids dioleoylphosphocholine (DOPC) and DOPE transfer DNA into mouse fibroblast (NIH 3T3) and tumoral myofibroblast-like (A17) cell lines more efficiently than the thoroughly studied DOTAP/DOPC and DC-Chol/DOPE cationic liposomes separately. To answer the question concerning how TE will change with an increasing number of lipid components, the multicomponent lipoplexes were studied incorporating three to six lipid species simultaneously and the TE was then evaluated with respect to mouse fibroblast (NIH 3T3), ovarian (CHO) and tumoral myofibroblast-like (A17) cell lines (Caracciolo et al., 2007). These multicomponent lipoplexes exhibited a much higher TE (about two orders of magnitude) than binary lipoplexes that are more commonly employed for gene delivery. Furthermore, a trend was discovered that the TE increases in correlation with the number of lipid components (with some exceptions as a result of lipid composition). This discovery may be related to the higher fusogenicity and compatibility of vesicles composed of several lipid components with respect to single lipids (Caracciolo et al., 2007). The existence of different regimes of stability was demonstrated for these multicomponent lipoplexes: the most efficient lipoplexes exhibited intermediate 'optimal stability'. To this end, lipoplexes DOTAP/DOPC-DNA were the least resistant mixture to disintegration; DC-Chol/DOPE/DOPC-DNA, the most resistant mixture to disintegration; and DOTAP/DC-Chol/DOPC/DOPE-DNA, the mixture exhibiting an intermediated behavior and characterized by a high TE. The extent of DNA release estimated by electrophoresis was in total concurrence with the structural stability of

restored the apoptotic pathway (Choi et al., 2008).

lipoplexes revealed by SAXS and TE (Caracciolo et al., 2007).

antigen dose recovered one day p.i.

The basic structure of the cholesterol-based cationic lipids used in gene therapy includes four functional domains: 1) Appositively charged headgroup capable to bind with the negatively charged phosphate group of nucleic acid; 2) a hydrophobic cholesterol anchor, which interacts with the cellular membrane; 3) a spacer group; 4) a linker group, which connects the positively charged head and the hydrophobic domain.

In order to estimate the contribution of each functional domain into the efficacy of DNA delivery and cytotoxicity, various types of cholesterol cationic lipids were synthesized. In the first investigation of the structure-activity relationship, it was revealed that in order to achieve an efficient transfection, the tertiary ammonium group must be connected to the cholesterol by a short spacer *via* the ester or urethane bond. (Farhood et al., 1992).

In further studies the 3-deoxycholesterol cationic derivatives **2a-с** were synthesized, which are shown to be more efficient than DC-Chol (**1a**) or lipid **1b** (Takeuchi et al. 1996). It was observed that introduction of the ethyl or propyl groups into the cationic head results in the decrease of the transfection efficiency (TE). The value of the surface charge (ζ-potential) for the liposomes based on lipids **1a,b, 2a-с, 3а** and their analogs was in positive correlation with TE of the HeLa, COS-7, and NIH 3T3 cells. Cationic liposomes **2а**/DOPE, having the highest ζ-potential, demonstrated the highest TE in all cell lines tested. Furthermore, the activity of the liposomes derived from the compounds **2с** and **3а** having the lowest ζ-potential, was less than 20% of the activity of lipid **2а** (Takeuchi et al., 1996). Moreover, it was found that to achieve an efficient transfection, the size of the complexes should be neither smaller (<400 nm) nor larger (>1.4 μm) (Kawaura et al., 1998).

Lipid **2d,** containing the 2-hydroxyethyl group was found to be more active than the compound **2a** with dimethylamine group, both in the presence and absence of serum (Okayama et al., 1997). Further modifications of the lipid structure by introduction of additional 2-hydroxyethyl groups (compounds **2e-i**) into the cationic head resulted in the collapse of transfection activity. Similarly, the derivative **2f** containing the primary amino group was almost not active. The TE of the lipid **2d** was 2-fold higher in comparison with the activity of lipids **2a** and **2h** (Hasegawa et al, 2002). Based on fluorescence resonance energy transfer (FRET) it was observed that DNA is released differently from the lipoplexes by means of anionic liposomes. Furthermore, both the release rate and the amount of unbound DNA have a positive correlation with the TE. The transfection efficiency of nanoparticles composed of the lipid **2d** was 11.5-fold higher than the TE of DC-Chol (**1а**) and was comparable with Lipofectamine 2000, DMRIE-C and Tfx-20 (Hattori et al., 2007). The size of the nonoplex was 290 nm, and the highest TE *in vitro* was observed for the nitrogen/phosphate (N/P) ratio of 3, when the nanoplex was positively charged. The *in vivo* delivery directly into the tumor demonstrated that the optimum TE corresponds to the N/P ratio of 1; meanwhile the size of nanocomplexes was around 145 nm, and ξ-potential was negative (-16.9 mV). After the intravenous, intramuscular and peroral administration, there was no transgene expression detected in any organ.

Lipids containing ester (**3b,c**), ether (**3d-f**) and urethane (**1b**) bonds were synthesized (Ghosh et al., 2000; Ghosh et al., 2002) in order to study the influence of linker type on the TE. It was subsequently demonstrated, that the presence of the urethane-based linkage in the compound **1b** led to the 6-fold decrease of the TE, in comparison to the ether lipid **3d**. However, in our opinion, this kind of comparison is fallacious due to presence of the different type of cationic head in the structure of the lipids **1b** and **3d**. Cholesterol lipids

Non-Viral Gene Delivery Systems Based on

**1c** <sup>O</sup> <sup>N</sup>

**1j** 

**1k** 

al., 2009).

O

H

NH2

**1h 2h**

**1d 2d** <sup>N</sup>

Cholesterol Cationic Lipids: Structure-Activity Relationships 355

**2с 3c**

H N

OH **3d**

Me

Me

 **R R R** 

H

**1e 2e 3e** Br- <sup>O</sup> <sup>N</sup> OH

It was found that heterocyclic cationic lipids containing imidazolinium (Solodin et al., 1995) or pyridinium polar heads (Ilies et al., 2006) reveal a higher TE and a reduced level of cytotoxicity in comparison with the classical transfectants. To study the influence of this type of heterocyclic polar head on the TE, the cholesterol-based lipids containing heterocyclic amine connected to the cholesterol residue *via* urethane (**4a-f**, **6a**), ether (**5a-5h**) and ester linkers (**6b-f**) were synthesized (Gao & Hui, 2001; Bajaj et al., 2008c; Medvedeva et

The study of the transfection activity for the lipids **4a-f** revealed that liposomes **4c**/DOPE and **4f**/DOPE displayed the highest TE, which from 3 to 6-fold exceeded the TE of DC-Chol (**1а**)/DOPE and Lipofectamine 2000 (Gao & Hui, 2001). The serum (from 1 to 10%) have no effect on cells transfection mediated by these cationic liposomes at different N/P ratios. It was observed that these liposomes gained a negative charge in the presence of serum. *In* 

O

**1a 2a 3а**

**1b 2b 3b**

**1f 2f 3f**

**1g 2g 3g**

with the ether linkage **3d,e** were significantly more efficient than their analogues **3b,c** with ester bond. The increase of the spacer length by one oxyethylene unit (lipid **3f**) resulted in the decrease of the TE. The efficiency of the **3d**-based liposomes was comparable with the Lipofectamine. Lipid **3e** with 2-hydroxyethyl substituent efficiently transected cells without DOPE. A comparison of the liposome mediated siRNA delivery in the presence of serum was carried out for the lipids with ether (**3g**) and urethane (**1c**) linkage. (Han et al., 2008). The cellular accumulation of the fluorescein-labeled siRNA mediated by liposome **1с**/DOPE was inhibited by serum, while liposomes **3g/**DOPE had the ability to efficiently deliver siRNA into the cell in the presence of serum. Liposomes **3g/**DOPE were successfully applied for silencing survivin and green fluorescent protein (GFP).

A comparative study of the TE of DC-Chol (**1a**), and lipids **1c-e** was performed in order to estimate the effect of the cationic headgroup in the cholesterol lipids containing the urethane linker group (Kearns et al., 2008). The lipids **1с** and **1d,** containing both primary and secondary amino groups, were able to transport DNA into the melanoma B16-F10 cells. These data differ from the results received for the 3-deoxycholesterol derivatives **2af**, where the lipid **2f** with primary amino group display extremely low TE in comparison of the analogue **2a** with the tertiary amino group. It was observed that lipids **1с** and **1d** containing primary and secondary amino groups are less toxic, in comparison to the lipids with tertiary (**1a**) and quaternary (**1e**) amino groups. The highest TE achieved for liposomes with lipids **1c** and **1d** could be a result of the ability of these liposomes to penetrate into the cells, to interact with the endosomal membrane and to release the nucleic acid into the cytoplasm.

Cationic liposomes formed by lipid **1f** in combination with DOPE were able to deliver DNA into the cells (Reynier et al., 2002**;** Lesage, 2002**)**. The TE of these liposomes was 2-fold higher in comparison with DC-Chol (**1a)**/DOPE liposomes. What is more, the TE increased in the presence of 4% PEG 8000. Using the liposomes **1e**/DOPE and **1f**/DOPE (1:1) the relocation of DNA within the lipoplexes upon the transfection was monitored in addition to the localization of the plasmid DNA inside the cell nucleus was visualized using immunogold labeling (Briane, et al., 2002).

As mentioned above, the introduction of the 2-hydroxyethyl substituent into the cationic headgroup can increase the TE (Okayama et al., 1997; Hasegawa et al, 2002). The cationic lipid **1h** was synthesized and formed stable liposomes with DOPE with the monomodal size distribution (Percot et al, 2004). *In vivo* direct injection into the tumor of the liposomes **1h/**DOPE and **1g/**DOPE demonstrated that the TE of the 2-hydroxyethyl-containing lipid **1h** was slightly lower than the TE of the lipid **1g** containing trimethylammonium headgroup. In order to study the effect of 2-hydroxyethyl group on the TE more thoroughly, the activity the lipids **1b** and **1h-k** was tested and compared with the activity of DC-Chol (**1a**) and 3 deoxycholesterol derivative **2d** (Ding, et al., 2008). It was discovered that lipids without ОНgroups (DC-Chol and **1b**) had the higher TE in comparison with the 2-hydroxyethylcontaining lipids **1h-k**, excluding lipid **2d**. Liposomes **2d**/DOPE possessed the highest TE *in vitro* (comparable with Lipofectamine2000) when N/P ratio was equal to 3, and were characterized by the big sizes of both liposomes itself (400 nm) and lipoplexes. The maximal level of the luciferase gene expression *in vivo* after intratracheal administration of the lipoplexes **1k/**DOPE-pDNA was 2-fold higher than the levels for the **1h/**DOPE, **1j/**DOPE and 4-fold higher than for DC-Chol (**1a**)/DOPE. The liposomes containing lipid **2d** were the least active.

with the ether linkage **3d,e** were significantly more efficient than their analogues **3b,c** with ester bond. The increase of the spacer length by one oxyethylene unit (lipid **3f**) resulted in the decrease of the TE. The efficiency of the **3d**-based liposomes was comparable with the Lipofectamine. Lipid **3e** with 2-hydroxyethyl substituent efficiently transected cells without DOPE. A comparison of the liposome mediated siRNA delivery in the presence of serum was carried out for the lipids with ether (**3g**) and urethane (**1c**) linkage. (Han et al., 2008). The cellular accumulation of the fluorescein-labeled siRNA mediated by liposome **1с**/DOPE was inhibited by serum, while liposomes **3g/**DOPE had the ability to efficiently deliver siRNA into the cell in the presence of serum. Liposomes **3g/**DOPE were successfully applied

A comparative study of the TE of DC-Chol (**1a**), and lipids **1c-e** was performed in order to estimate the effect of the cationic headgroup in the cholesterol lipids containing the urethane linker group (Kearns et al., 2008). The lipids **1с** and **1d,** containing both primary and secondary amino groups, were able to transport DNA into the melanoma B16-F10 cells. These data differ from the results received for the 3-deoxycholesterol derivatives **2af**, where the lipid **2f** with primary amino group display extremely low TE in comparison of the analogue **2a** with the tertiary amino group. It was observed that lipids **1с** and **1d** containing primary and secondary amino groups are less toxic, in comparison to the lipids with tertiary (**1a**) and quaternary (**1e**) amino groups. The highest TE achieved for liposomes with lipids **1c** and **1d** could be a result of the ability of these liposomes to penetrate into the cells, to interact with the endosomal membrane and to release the

Cationic liposomes formed by lipid **1f** in combination with DOPE were able to deliver DNA into the cells (Reynier et al., 2002**;** Lesage, 2002**)**. The TE of these liposomes was 2-fold higher in comparison with DC-Chol (**1a)**/DOPE liposomes. What is more, the TE increased in the presence of 4% PEG 8000. Using the liposomes **1e**/DOPE and **1f**/DOPE (1:1) the relocation of DNA within the lipoplexes upon the transfection was monitored in addition to the localization of the plasmid DNA inside the cell nucleus was visualized using immunogold

As mentioned above, the introduction of the 2-hydroxyethyl substituent into the cationic headgroup can increase the TE (Okayama et al., 1997; Hasegawa et al, 2002). The cationic lipid **1h** was synthesized and formed stable liposomes with DOPE with the monomodal size distribution (Percot et al, 2004). *In vivo* direct injection into the tumor of the liposomes **1h/**DOPE and **1g/**DOPE demonstrated that the TE of the 2-hydroxyethyl-containing lipid **1h** was slightly lower than the TE of the lipid **1g** containing trimethylammonium headgroup. In order to study the effect of 2-hydroxyethyl group on the TE more thoroughly, the activity the lipids **1b** and **1h-k** was tested and compared with the activity of DC-Chol (**1a**) and 3 deoxycholesterol derivative **2d** (Ding, et al., 2008). It was discovered that lipids without ОНgroups (DC-Chol and **1b**) had the higher TE in comparison with the 2-hydroxyethylcontaining lipids **1h-k**, excluding lipid **2d**. Liposomes **2d**/DOPE possessed the highest TE *in vitro* (comparable with Lipofectamine2000) when N/P ratio was equal to 3, and were characterized by the big sizes of both liposomes itself (400 nm) and lipoplexes. The maximal level of the luciferase gene expression *in vivo* after intratracheal administration of the lipoplexes **1k/**DOPE-pDNA was 2-fold higher than the levels for the **1h/**DOPE, **1j/**DOPE and 4-fold higher than for DC-Chol (**1a**)/DOPE. The liposomes containing lipid **2d** were the

for silencing survivin and green fluorescent protein (GFP).

nucleic acid into the cytoplasm.

labeling (Briane, et al., 2002).

least active.

It was found that heterocyclic cationic lipids containing imidazolinium (Solodin et al., 1995) or pyridinium polar heads (Ilies et al., 2006) reveal a higher TE and a reduced level of cytotoxicity in comparison with the classical transfectants. To study the influence of this type of heterocyclic polar head on the TE, the cholesterol-based lipids containing heterocyclic amine connected to the cholesterol residue *via* urethane (**4a-f**, **6a**), ether (**5a-5h**) and ester linkers (**6b-f**) were synthesized (Gao & Hui, 2001; Bajaj et al., 2008c; Medvedeva et al., 2009).

The study of the transfection activity for the lipids **4a-f** revealed that liposomes **4c**/DOPE and **4f**/DOPE displayed the highest TE, which from 3 to 6-fold exceeded the TE of DC-Chol (**1а**)/DOPE and Lipofectamine 2000 (Gao & Hui, 2001). The serum (from 1 to 10%) have no effect on cells transfection mediated by these cationic liposomes at different N/P ratios. It was observed that these liposomes gained a negative charge in the presence of serum. *In* 

Non-Viral Gene Delivery Systems Based on

O HO O

R

OH

**2.3 Stimuli-responsive cationic lipids** 

presence of the intracellular reducing agents.

**2.3.1 Redox-responsive disulfide cationic lipids** 

**7a-d**

media.

HO

Cholesterol Cationic Lipids: Structure-Activity Relationships 357

not possible using individual lipids. The delivery of fluorescein-labeled oligonucleotide was comparable for the liposomes **7d/**DOPE and Lipofectamine 2000. In the case of the siRNA delivery, the highest TE was observed for the liposomes **7c/**DOPE and the GFP genesilencing was observed both in the absence and in the presence of serum in the culture

It was revealed that lipoplexes enter the cells *via* nonspecific endocytosis, which occurs after the electrostatic binding of the positively charged lipoplexes to the negatively charged components of the cell membrane (Rejman, et al., 2006; Belting et al., 2005). There are a number of obstacles, hindering the efficient cationic liposome-mediated gene transfection: DNA release from the endosomes, DNA dissociation from the lipoplexes (Escriou et al., 1998; Rolland, 1998; Zabner et al., 1995) as well as insufficient release of DNA from endosomes. The use of stimuli-responsive delivery systems offers a new opportunity for the improvement of the delivery of nucleic acid (Ganta et al., 2008). Therefore, pH and redox microenvironment can be used as biological stimuli to improve the TE of lipoplexes. To achieve a stimuli-responsive release of DNA it is necessary to design cationic lipids containing trigger-groups, which specifically react to the alteration of the pH value, or to the

Thiol-disulfide exchange reactions play an important role in the biological functions of living cells; notably in the stabilization of the protein structure and redox cycles. The strong intracellular reductive micro-environment can stimulate the disintegration of the lipoplexes if these compounds contain the disulfide cationic lipids that are stable outside the cells, but could be reduced in the cells by intracellular reductive agents, *e.g.* glutathione (Tang & Hughes, 1998). The reduction of the disulfide bond will enhance the release of DNA from the DNA-liposomes complexes. Previously, it was demonstrated that the transfection of the plasmid DNA by the glycerolipids containing disulfide bonds was higher compared to the

Lipid **8a** containing cholesterol, was synthesized and its activity to mediate DNA transfer was compared with the activity of both non-disulfide analogue **8b** and DC-Chol (**1a**) (Tang & Hughes, 1999). In the presence of glutathione (10 mM) a 50% DNA release from the complex with liposomes **8а/**DOPE was observed, while the lipoplexes formed by lipid **8b** did not release DNA. The TE for the disulfide lipid **8а** was 100-fold higher, in comparison with the DC-Chol (**1a**) and 7-fold higher when compared to the lipid **8b,** in spite of the fact,

Lipids **9a-c** based on thiocholesterol (in the mixture with DOPE) were more active in comparison to PEI and DOTAP/DOPE when transfecting CV-1 cells (Huang et al., 2005).

transfection activity of its non-disulfide analogue (Tang & Hughes, 1998).

that the amount of DNA internalized by cells was lower in the case of **8а**.

N

MsO-

MsO-

**7a**,R=

**7b**,R=

<sup>N</sup> <sup>N</sup> Me

I -

Me

**7c**,R=

**7d**,R=

N Me

I -

N O

*vivo* DNA delivery (direct administration into the spleen) was efficient at low N/P ratios, but as yet, no reasonable explanation for this occurrence has been found.

Liposomes formed from lipids **5a-g** and DOPE were able to transfect 50-80% of cells, and the TE increased when the N/P ratio increases (Bajaj et al., 2008c). The highest TE was observed for lipid **5f** containing *N,N*-dimethylaminopyridinium headgroup. This lipid transfected cells in the presence of serum without any loss of activity. The experiments with sodium dodecylsulphate (SDS)-induced DNA release from the lipoplexes demonstrated that DNA is released from the complex with liposomes 5f/DOPE in an unhurried manner. This could possibly be a result of a more effective lipid shielding of DNA. This is probably a reason for the high TE displayed by liposomes **5f**/DOPE, even in the presence of the serum.

The structure-activity relationships study for the series of lipids **6a-f**, containing different heterocyclic cationic groups and linkers, permitted us to determine that lipids containing pyridinium (**6a,b**) or *N*-methylimidazium (**6d**) heads and ester or urethane linkers are the most promising in terms of transfection (Medvedeva et al., 2009). It was also revealed, that the ability of these lipids to deliver the oligodeoxyribonucleotides and pDNA into cells, correlates positively with their ability to form lipoplexes with the size not exceeding 100 nm. Cholesterol-based lipids containing heterocyclic polar heads linked *via* biodegradable β*-*glucosyl spacer were prepared (Maslov et al., 2010).

The study of the biological activity of the cationic glycolipids **7a-d** demonstrated that nucleic acids could be efficiently delivered only by means of cationic liposomes; however this was

*vivo* DNA delivery (direct administration into the spleen) was efficient at low N/P ratios,

Liposomes formed from lipids **5a-g** and DOPE were able to transfect 50-80% of cells, and the TE increased when the N/P ratio increases (Bajaj et al., 2008c). The highest TE was observed for lipid **5f** containing *N,N*-dimethylaminopyridinium headgroup. This lipid transfected cells in the presence of serum without any loss of activity. The experiments with sodium dodecylsulphate (SDS)-induced DNA release from the lipoplexes demonstrated that DNA is released from the complex with liposomes 5f/DOPE in an unhurried manner. This could possibly be a result of a more effective lipid shielding of DNA. This is probably a reason for

but as yet, no reasonable explanation for this occurrence has been found.

the high TE displayed by liposomes **5f**/DOPE, even in the presence of the serum.

 **R R R** 

**4a 5a 6a** 

**4b 5b 6b**

**4c 5c 6c** 

**4d 5d 6d**

**4e 5e 6e** 

**4f 5f 6f** 

β*-*glucosyl spacer were prepared (Maslov et al., 2010).

**5g** 

The structure-activity relationships study for the series of lipids **6a-f**, containing different heterocyclic cationic groups and linkers, permitted us to determine that lipids containing pyridinium (**6a,b**) or *N*-methylimidazium (**6d**) heads and ester or urethane linkers are the most promising in terms of transfection (Medvedeva et al., 2009). It was also revealed, that the ability of these lipids to deliver the oligodeoxyribonucleotides and pDNA into cells, correlates positively with their ability to form lipoplexes with the size not exceeding 100 nm. Cholesterol-based lipids containing heterocyclic polar heads linked *via* biodegradable

The study of the biological activity of the cationic glycolipids **7a-d** demonstrated that nucleic acids could be efficiently delivered only by means of cationic liposomes; however this was not possible using individual lipids. The delivery of fluorescein-labeled oligonucleotide was comparable for the liposomes **7d/**DOPE and Lipofectamine 2000. In the case of the siRNA delivery, the highest TE was observed for the liposomes **7c/**DOPE and the GFP genesilencing was observed both in the absence and in the presence of serum in the culture media.
