**2.3.1 Redox-responsive disulfide cationic lipids**

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

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

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).

Non-Viral Gene Delivery Systems Based on

promising transfection agents.

Cholesterol Cationic Lipids: Structure-Activity Relationships 359

such hydrolytic degradation and enhance the release of DNA from endosome pH-sensitive cationic lipids were synthesized (Budker et al., 1996). These lipids contain weakly basic lysosomotropic imidazole head group which acts as a proton sponge, preventing the acidification of endosomal environment and inhibiting the degradative hydrolysis. Another approach to enhance the DNA release is the incorporation of the chemical trigger-bond into lipid that could be hydrolyzed at a specific pH gradient. Therefore, incorporation of acidlabile bonds into the lipid structure favors the lipoplex destabilization and facilitates the DNA release from the endosomal compartment to the cytoplasm, thus improving the transfection efficiency (Boomer et al., 2002; Guo & Szoka, 2003). Furthermore, the use of biodegradable cationic lipids lowers the cytotoxicity of cationic liposomes, making them

Cholesterol-based, endosomal, pH-sensitive, histidylated, cationic lipid (**11a**), its less pH-sensitive analogue with the electron-deficient head group (**11b**) and cationic lipid, which does not contain histidine headgroup (**11c**) were synthesized (Singh, et al., 2004). Lipid **11b** exhibited lower TE than lipid **11a** in relation to 293T7 cells. The activities of both lipids were inhibited in the presence of Bafilomycin A1, demonstrating the involvement of imidazole ring protonation in the endosomal escape of DNA. However, the TE of histidinylated lipid **11a** did not exceed this value for lipid **11с**. The lipid **12** with an acid-sensitive ketal bond was hydrolyzed in acidic medium where an ether analogue remained undegraded (Zhu, et al., 2002). Lipid (**12**) achieved levels of gene delivery similar

In recent time a new series of cationic steroid derivatives, containing guanidinium headgroup connected with the hydrophobic cholesterol *via* the acid-sensitive acylhydrazone linker have been developed (Aissaoui et al., 2004). The lipid **13a** was found to possess a low cytotoxicity and was able to mediate the efficient gene transfection into various mammalian cell lines *in vitro*. The TE of the lipid **13а** was comparable with TE of its analogue **13b**, which did not contain the acid-labile group. Colloidally stable

to DC-Chol (**1a**), but the toxicity was correspondingly low.

Nanolipoparticles (NLP) were formed from lipids **9a-c** and PEG was added for the steric stabilization. The treatment of NLP with cysteine or glutathione changed the surface charge of the particles; the modification of the NLP surface with Tat-protein resulted in the increase of the TE of the neutral and negatively charged NLP.

To find new efficient transfectants, the water-soluble low-toxic cholesterol-based lipids **10a-d** were synthesized. The lipids contain the positively-charged headgroups connected to the cholesterol backbone *via* the disulfide and carbonate linkers (Sheng et al., 2011). The atomic force microscopy indicated that mixing the cationic lipids and DNA gave compact, condensed lipoplexes with a size 200-300 nm. The addition of dithiotreitol (10 mM) resulted in the disassembly of these complexes into tiny, irregular-shaped fragments and small sized pieces, confirming the cleavage of disulfide bonds and distortion of the stable lipoplexes. Lipid **10c**, which contained the natural aminoacid lysine, displayed the highest TE in respect to COS-7 cells, both in the presence and absence of serum. The least active was lipid **10b** with the quaternary amino group. It is worthy of note, that lipid **10a** was almost as active as **10c** at low N/P ratio (up to 5). This ratio is characterized by the formation of the small lipoplexes (approximately 250 nm) with the negative ξ-potential.

#### **2.3.2 pH-responsive cationic lipids**

It is known that endocytosis is accompanied by a noticeable increase in the environment acidity from the physiological value of pH 7.4 to 6.5–6.0 in endosomes and to 5.0 in primary or secondary lysosomes (Mukherjee et al., 1997). Endocytosed lipoplexes may be digested by acidic hydrolases, active at the acidic pH of the lysosome. In order to protect DNA from

Nanolipoparticles (NLP) were formed from lipids **9a-c** and PEG was added for the steric stabilization. The treatment of NLP with cysteine or glutathione changed the surface charge of the particles; the modification of the NLP surface with Tat-protein resulted in the increase

of the TE of the neutral and negatively charged NLP.

**8a 10a**

**8b 10b**

Me Me

**9a** <sup>S</sup> <sup>S</sup> <sup>N</sup>

**9b** 

**9c** 

ξ-potential.

**2.3.2 pH-responsive cationic lipids** 

 **R R** 

HCl **10c**

**10d**

To find new efficient transfectants, the water-soluble low-toxic cholesterol-based lipids **10a-d** were synthesized. The lipids contain the positively-charged headgroups connected to the cholesterol backbone *via* the disulfide and carbonate linkers (Sheng et al., 2011). The atomic force microscopy indicated that mixing the cationic lipids and DNA gave compact, condensed lipoplexes with a size 200-300 nm. The addition of dithiotreitol (10 mM) resulted in the disassembly of these complexes into tiny, irregular-shaped fragments and small sized pieces, confirming the cleavage of disulfide bonds and distortion of the stable lipoplexes. Lipid **10c**, which contained the natural aminoacid lysine, displayed the highest TE in respect to COS-7 cells, both in the presence and absence of serum. The least active was lipid **10b** with the quaternary amino group. It is worthy of note, that lipid **10a** was almost as active as **10c** at low N/P ratio (up to 5). This ratio is characterized by the formation of the small lipoplexes (approximately 250 nm) with the negative

It is known that endocytosis is accompanied by a noticeable increase in the environment acidity from the physiological value of pH 7.4 to 6.5–6.0 in endosomes and to 5.0 in primary or secondary lysosomes (Mukherjee et al., 1997). Endocytosed lipoplexes may be digested by acidic hydrolases, active at the acidic pH of the lysosome. In order to protect DNA from such hydrolytic degradation and enhance the release of DNA from endosome pH-sensitive cationic lipids were synthesized (Budker et al., 1996). These lipids contain weakly basic lysosomotropic imidazole head group which acts as a proton sponge, preventing the acidification of endosomal environment and inhibiting the degradative hydrolysis. Another approach to enhance the DNA release is the incorporation of the chemical trigger-bond into lipid that could be hydrolyzed at a specific pH gradient. Therefore, incorporation of acidlabile bonds into the lipid structure favors the lipoplex destabilization and facilitates the DNA release from the endosomal compartment to the cytoplasm, thus improving the transfection efficiency (Boomer et al., 2002; Guo & Szoka, 2003). Furthermore, the use of biodegradable cationic lipids lowers the cytotoxicity of cationic liposomes, making them promising transfection agents.

Cholesterol-based, endosomal, pH-sensitive, histidylated, cationic lipid (**11a**), its less pH-sensitive analogue with the electron-deficient head group (**11b**) and cationic lipid, which does not contain histidine headgroup (**11c**) were synthesized (Singh, et al., 2004). Lipid **11b** exhibited lower TE than lipid **11a** in relation to 293T7 cells. The activities of both lipids were inhibited in the presence of Bafilomycin A1, demonstrating the involvement of imidazole ring protonation in the endosomal escape of DNA. However, the TE of histidinylated lipid **11a** did not exceed this value for lipid **11с**. The lipid **12** with an acid-sensitive ketal bond was hydrolyzed in acidic medium where an ether analogue remained undegraded (Zhu, et al., 2002). Lipid (**12**) achieved levels of gene delivery similar to DC-Chol (**1a**), but the toxicity was correspondingly low.

In recent time a new series of cationic steroid derivatives, containing guanidinium headgroup connected with the hydrophobic cholesterol *via* the acid-sensitive acylhydrazone linker have been developed (Aissaoui et al., 2004). The lipid **13a** was found to possess a low cytotoxicity and was able to mediate the efficient gene transfection into various mammalian cell lines *in vitro*. The TE of the lipid **13а** was comparable with TE of its analogue **13b**, which did not contain the acid-labile group. Colloidally stable

Non-Viral Gene Delivery Systems Based on

**14e** 12

**14g** 4

**14i** 6

Cholesterol Cationic Lipids: Structure-Activity Relationships 361

**# n X # n X** 

4 HCl

HN N

( )n

X O

Polycationic lipids contain a polar head that bears either several or multiple positive charges increasing their affinity to nucleic acids. Polyamines were successfully used as a component of polycationic lipids (Geall et al., 2000; Blagbrough et al., 2003; 2004; Oliver et al., 2004). Polyamines are a class of naturally occurring compounds that display excellent nucleic acid binding and condensing properties. It is well known that the overall positive charge of the lipoplexes is important for initiating cell entry and release of the complexed nucleic acid into the cell cytoplasm. Although the exact mechanism by which polycationic lipids mediate transfection requires more detailed investigation evidence in literature points towards the notion that the success of these reagents arises from a couple of factors: the abnormally low pKa's (pKa <7) of the polyamines, a direct result of the number of amino groups present and the methylene spacings between them (Stewart et al., 2001; Keller et al.,

The effects of the regiochemical distribution of positive charges along the polyamine moiety in DNA condensing agents were studied (Geall et al., 2000). DNA condensation is dependent upon the number of positive charges, the regiochemical distribution of charges of

**17a**, X = C(O), n = 4 **17b**, X = C(O)NH, n = 4 **17c**, X = C(O)NH, n = 6

H

**14a** 3 **15a** 1 **14b** 4 **15b** 2 **14c** 5 **15c** 3 **14d** 6 **15d** 5

**16a 14f** <sup>3</sup>

**16b 14h** <sup>5</sup>

**16c 14k** <sup>12</sup>

( )n O X NH

> H N

**2.5 Polycationic lipids** 

2003; Geall et al., 1999; 2000).

**13a**/DOPE-DNA complexes were prepared and administered *via* nasal instillation into the mouse airways. A significant expression of the reporter protein in the lung homogenates was subsequently detected. It should be noted, the TE in the experimental groups was much higher compared to the control group of mice receiving the identical dose of "naked" DNA.
