**2.4 Cholesterol gemini-surfactants**

Recently, a new class of surfactants was discovered. These so-called 'dimeric' or 'geminisurfactants' attract a considerable amount of scientific attention as being of particular importance in biology. For instance, the ability to form "bilayer bridges"was demonstrated for some gemini-surfactants containing long hydrophobic spacers (Moss & Li, 1992). The structure of these gemini-surfactants resembles the structure of lipids found in the membrane of the thermophiles archaebacteria. Other gemini-surfactants were specially synthesized for the usage as nucleic acids carriers into the cells (Kirby et al., 2003;) and the effects of the nature of the spacer, hydrocarbon chains, and headgroups on the transfection activity of these compounds were studied in detail.

Dimeric lipids **14a-k**, differing in the length of the spacer and the type of the cationic headgroup, were synthesized, and the transfection activity of these compounds was compared with this parameter for the monomeric lipids **3d** and **3e** (Bajaj et al., 2007a; Biswas et al., 2011). Lipids formed different aggregates, depending on the structure of the cationic head. For instance, individual vesicles with sizes from 20 to 160 nm were formed by the compounds **14a-e**. In turn, for lipids **14f-k** formation of vesicular conglomerates of various lengths composed of the individual vesicles was observed. These conglomerates were formed due to the hydrogen bonding interactions imposed by the 2-hydroxyethylated headgroups of the lipids of each vesicle of geminies **14f-k** (Biswas et al., 2011). The maximal TE was observed for the lipid **14с**, containing pentamethylene spacer, and, on the contrary, lipid **14e** was not active in the absence of serum in the culture media. A different morphology of the lipoplexes formed by these lipids and DNA was subjected to further study. Transmission electron microscopy showed the presence of the aggregates with the size range from 100 to 200 nm for the lipoplexes formed by **14c**, while lipid **14e** was found to form lipoplexes of irregular shapes and sizes.

The replacement of the hydrophobic spacer by hydrophilic one led to the new set of dimeric amphiphils **15a-d** possessing low toxicity. The amphiphiles **15a,b** were able to transfect the cells at the low N/P ratio, as well as in the presence of 30 and 50% serum (Bajaj et al., 2007b).

Due to the fact that lipids with ether bond are poorly degraded in the organism, new disulfide bond containing dimeric lipids **16a-c** were synthesized, able to degrade under the influence of intracellular reducing agents (Bajaj et al., 2008a). These lipids contain flexible hydrophobic and hydrophilic spacers, in addition to rigid hydrophobic spacers. The comparative analysis indicated that the TE of the lipids is decreases in the range **16с** > **16a** > **16b**. Thus, the hydrophilic flexible spacer in the lipid structure is essential for an effective transfection. The serum inhibited the activity of the lipids **16a-c**, as its negatively charged components compete with DNA for the binding to the cationic lipids, which results in the dissociation of the complexes and a decrease of the TE. In recent times, a new method for the synthesis of the dimeric lipids **17а-с** that contain cholesterol and spermine moieties and potentially posses the high TE was described (Petukhov et al., 2010).

**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

Recently, a new class of surfactants was discovered. These so-called 'dimeric' or 'geminisurfactants' attract a considerable amount of scientific attention as being of particular importance in biology. For instance, the ability to form "bilayer bridges"was demonstrated for some gemini-surfactants containing long hydrophobic spacers (Moss & Li, 1992). The structure of these gemini-surfactants resembles the structure of lipids found in the membrane of the thermophiles archaebacteria. Other gemini-surfactants were specially synthesized for the usage as nucleic acids carriers into the cells (Kirby et al., 2003;) and the effects of the nature of the spacer, hydrocarbon chains, and headgroups on the transfection

Dimeric lipids **14a-k**, differing in the length of the spacer and the type of the cationic headgroup, were synthesized, and the transfection activity of these compounds was compared with this parameter for the monomeric lipids **3d** and **3e** (Bajaj et al., 2007a; Biswas et al., 2011). Lipids formed different aggregates, depending on the structure of the cationic head. For instance, individual vesicles with sizes from 20 to 160 nm were formed by the compounds **14a-e**. In turn, for lipids **14f-k** formation of vesicular conglomerates of various lengths composed of the individual vesicles was observed. These conglomerates were formed due to the hydrogen bonding interactions imposed by the 2-hydroxyethylated headgroups of the lipids of each vesicle of geminies **14f-k** (Biswas et al., 2011). The maximal TE was observed for the lipid **14с**, containing pentamethylene spacer, and, on the contrary, lipid **14e** was not active in the absence of serum in the culture media. A different morphology of the lipoplexes formed by these lipids and DNA was subjected to further study. Transmission electron microscopy showed the presence of the aggregates with the size range from 100 to 200 nm for the lipoplexes formed by **14c**, while lipid **14e** was found to

The replacement of the hydrophobic spacer by hydrophilic one led to the new set of dimeric amphiphils **15a-d** possessing low toxicity. The amphiphiles **15a,b** were able to transfect the cells at the low N/P ratio, as well as in the presence of 30 and 50% serum (Bajaj et

Due to the fact that lipids with ether bond are poorly degraded in the organism, new disulfide bond containing dimeric lipids **16a-c** were synthesized, able to degrade under the influence of intracellular reducing agents (Bajaj et al., 2008a). These lipids contain flexible hydrophobic and hydrophilic spacers, in addition to rigid hydrophobic spacers. The comparative analysis indicated that the TE of the lipids is decreases in the range **16с** > **16a** > **16b**. Thus, the hydrophilic flexible spacer in the lipid structure is essential for an effective transfection. The serum inhibited the activity of the lipids **16a-c**, as its negatively charged components compete with DNA for the binding to the cationic lipids, which results in the dissociation of the complexes and a decrease of the TE. In recent times, a new method for the synthesis of the dimeric lipids **17а-с** that contain cholesterol and spermine moieties and

potentially posses the high TE was described (Petukhov et al., 2010).

"naked" DNA.

**2.4 Cholesterol gemini-surfactants** 

activity of these compounds were studied in detail.

form lipoplexes of irregular shapes and sizes.

al., 2007b).

#### **2.5 Polycationic lipids**

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

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

Non-Viral Gene Delivery Systems Based on

al., 2004).

(40:50:10 molar ratio).

Cholesterol Cationic Lipids: Structure-Activity Relationships 363

confer low toxicity to mammalian cells. CDAN/DOPE-siRNA complexes exhibited a slower cellular uptake than Lipofectamine2000 based formulations. Intracellularly CDAN/DOPEsiRNA complexes appear to behave in a different fashion, accumulating in distinct but diffuse small non-lysosomal compartments for at least 5 h after transfection (Spagnou et

Recently, a new hepatotropic nontoxic lipid-based vector system for delivering chemically unmodified siRNA to the liver to inhibit Hepatitis B virus (HBV) propagation was described (Carmona et al., 2008). These anti-HBV formulations were created based on synthetic, selfassembly ABCD nanoparticle paradigm (Kostarelos & Miller, 2005). ABCD nanoparticles comprising nucleic acids, such as plasmid DNA (pDNA) or siRNA (A component) which are condensed with cationic liposomes (B component) to form AB core nanoparticles. An important feature of the assembly is the incorporation of an aminoxy cholesteryl lipid into these AB core nanoparticles in order to enable the quantitative chemoselective postcoupling of biocompatibility polymers (C component) and optional tissue-targeting ligands (D component) to the core nanoparticles. (Carmona et al., 2008). AB nanoparticles (70-80 nm in diameter) were initially formulated in an aqueous solution by mixing of siRNAs with cationic liposomes CDAN (**18d**)/DOPE/**19a** (40:50:10 molar ratio) or CDAN/DOPE/**19b**

Polyethylene glycol2000-dialdehyde (C component), was coupled to AB particles under aqueous acidic conditions (pH 4). This surface post coupling was facilitated by a rapid, quantitative chemoselective aminoxy-aldehyde conjugation between the aminoxy functional group of aminoxy cholesteryl lipid and of the aldehyde functional groups of the Ccomponent. The resulting oxime linkages are robust at pH 7; however at a level of pH 5.5 and below, they are prone to decomposition. The developed vectors administered intravenously, efficiently deliver unmodified siRNAs to murine livers leading to the strong

The conjugates of guanidinium and cholesterol were synthesized with the yields of up to 61%, with the purpose of further improving the polar domain of cationic lipids (Vigneron et al., 1996). The guanidinium group can form with phosphate anions characteristic pairs stabilized by parallel zwitterionic hydrogen bonds. Moreover, the guanidinium group is also able to develop hydrogen bonding with nucleic bases, especially with guanine. The tertiary amine of compound **13b**, which is situated between two positive guanidinium groups and has probably a lower pKa, could also be able to buffer the acidic environment of late endosomes and of lysosomes, hence protecting the DNA against degradation. The lipid **13b** can be used *in vitro* without DOPE, and this permits the avoidance of the liposomes preparation step. Commonly

suppression of HBV replication (Carmona et al., 2008).

polyamines (determined by the p*K*a of each amino group), and the local salt concentration. A series of polyamine carbamates of cholesterol was prepared where both the charge and its regiochemical distribution have been varied along the polyamine moiety (**18a-f**) (Geall et al., 1999). Lipids **18a-f** were tested for transfection competence at three different N/P ratios (0.5:1, 1:1, 4:1), calculated taking into account the average charge per molecule at pH 7.4. It was found that the spermine based lipid **18a**, incorporating 3-4-3 methylene spacing along the polyamine moiety, has the highest TE, while lipid **18f** display the lowest TE. Geall and co-workers supposed that the four methylene spacing found in spermine, could have significant implications for DNA polyamine association and lipoplex formation.

Recently second generation cationic liposomal systems that were formulated from polyamine analogues of DC-Chol and DOPE have been described (CDAN (**18d**), CDAD (**18a**), CTAP (**18g**), CTAH (**18h**)) (Cooper et al., 1998). Among the liposomal formulation tested the formulation from the novel pentaamine amphiphile CTAP (**18g**) and DOPE were shown to be approximately 400 times more efficient at mediating gene delivery to a mouse lung *in vivo* than DC-Chol/DOPE liposomes. CDAN (**18d**) is another cholesterol-based polyamine lipid with an unnaturally occurring 2–3–2 methylene spacing, which, in combination with DOPE forms an exceptionally effective transfection agent. Biophysical analyses show that CTAP/DOPE liposomes are effective *in vivo* because these liposomes are able to efficiently neutralise, condense and encapsulate nucleic acids into lipoplex particles and the unprotonated amine groups (p*K***a** < 8) presented in the polyamine at neutral pH that could have the capacity for endosome buffering, thereby facilitating nucleic acid escape from endosomes into the cytosol, like polyethylenimine (Stewart et al., 2001).

Recently a new solid-phase strategy to synthesize a library of cholesterol-based polyamine lipids in excellent yields (>87%) and purity was set forth (Oliver et al., 2004). The strategy employs 2-chlorotrityl chloride resin as a solid support and protecting group for one primary amine on the starting material, utilizing the high selectivity of 2-acetyldimedone as a protecting group for the second primary amine (Oliver et al., 2004).

Cationic liposomes CDAN (**18d**)/DOPE were tested as delivery systems for siRNAs (Spagnou et al., 2004). The results show that CDAN and DOPE with and without siRNA

polyamines (determined by the p*K*a of each amino group), and the local salt concentration. A series of polyamine carbamates of cholesterol was prepared where both the charge and its regiochemical distribution have been varied along the polyamine moiety (**18a-f**) (Geall et al., 1999). Lipids **18a-f** were tested for transfection competence at three different N/P ratios (0.5:1, 1:1, 4:1), calculated taking into account the average charge per molecule at pH 7.4. It was found that the spermine based lipid **18a**, incorporating 3-4-3 methylene spacing along the polyamine moiety, has the highest TE, while lipid **18f** display the lowest TE. Geall and co-workers supposed that the four methylene spacing found in spermine, could have

Recently second generation cationic liposomal systems that were formulated from polyamine analogues of DC-Chol and DOPE have been described (CDAN (**18d**), CDAD (**18a**), CTAP (**18g**), CTAH (**18h**)) (Cooper et al., 1998). Among the liposomal formulation tested the formulation from the novel pentaamine amphiphile CTAP (**18g**) and DOPE were shown to be approximately 400 times more efficient at mediating gene delivery to a mouse lung *in vivo* than DC-Chol/DOPE liposomes. CDAN (**18d**) is another cholesterol-based polyamine lipid with an unnaturally occurring 2–3–2 methylene spacing, which, in combination with DOPE forms an exceptionally effective transfection agent. Biophysical analyses show that CTAP/DOPE liposomes are effective *in vivo* because these liposomes are able to efficiently neutralise, condense and encapsulate nucleic acids into lipoplex particles and the unprotonated amine groups (p*K***a** < 8) presented in the polyamine at neutral pH that could have the capacity for endosome buffering, thereby facilitating nucleic acid escape

Recently a new solid-phase strategy to synthesize a library of cholesterol-based polyamine lipids in excellent yields (>87%) and purity was set forth (Oliver et al., 2004). The strategy employs 2-chlorotrityl chloride resin as a solid support and protecting group for one primary amine on the starting material, utilizing the high selectivity of 2-acetyldimedone as

Cationic liposomes CDAN (**18d**)/DOPE were tested as delivery systems for siRNAs (Spagnou et al., 2004). The results show that CDAN and DOPE with and without siRNA

from endosomes into the cytosol, like polyethylenimine (Stewart et al., 2001).

a protecting group for the second primary amine (Oliver et al., 2004).

significant implications for DNA polyamine association and lipoplex formation.

confer low toxicity to mammalian cells. CDAN/DOPE-siRNA complexes exhibited a slower cellular uptake than Lipofectamine2000 based formulations. Intracellularly CDAN/DOPEsiRNA complexes appear to behave in a different fashion, accumulating in distinct but diffuse small non-lysosomal compartments for at least 5 h after transfection (Spagnou et al., 2004).

Recently, a new hepatotropic nontoxic lipid-based vector system for delivering chemically unmodified siRNA to the liver to inhibit Hepatitis B virus (HBV) propagation was described (Carmona et al., 2008). These anti-HBV formulations were created based on synthetic, selfassembly ABCD nanoparticle paradigm (Kostarelos & Miller, 2005). ABCD nanoparticles comprising nucleic acids, such as plasmid DNA (pDNA) or siRNA (A component) which are condensed with cationic liposomes (B component) to form AB core nanoparticles. An important feature of the assembly is the incorporation of an aminoxy cholesteryl lipid into these AB core nanoparticles in order to enable the quantitative chemoselective postcoupling of biocompatibility polymers (C component) and optional tissue-targeting ligands (D component) to the core nanoparticles. (Carmona et al., 2008). AB nanoparticles (70-80 nm in diameter) were initially formulated in an aqueous solution by mixing of siRNAs with cationic liposomes CDAN (**18d**)/DOPE/**19a** (40:50:10 molar ratio) or CDAN/DOPE/**19b** (40:50:10 molar ratio).

Polyethylene glycol2000-dialdehyde (C component), was coupled to AB particles under aqueous acidic conditions (pH 4). This surface post coupling was facilitated by a rapid, quantitative chemoselective aminoxy-aldehyde conjugation between the aminoxy functional group of aminoxy cholesteryl lipid and of the aldehyde functional groups of the Ccomponent. The resulting oxime linkages are robust at pH 7; however at a level of pH 5.5 and below, they are prone to decomposition. The developed vectors administered intravenously, efficiently deliver unmodified siRNAs to murine livers leading to the strong suppression of HBV replication (Carmona et al., 2008).

The conjugates of guanidinium and cholesterol were synthesized with the yields of up to 61%, with the purpose of further improving the polar domain of cationic lipids (Vigneron et al., 1996). The guanidinium group can form with phosphate anions characteristic pairs stabilized by parallel zwitterionic hydrogen bonds. Moreover, the guanidinium group is also able to develop hydrogen bonding with nucleic bases, especially with guanine. The tertiary amine of compound **13b**, which is situated between two positive guanidinium groups and has probably a lower pKa, could also be able to buffer the acidic environment of late endosomes and of lysosomes, hence protecting the DNA against degradation. The lipid **13b** can be used *in vitro* without DOPE, and this permits the avoidance of the liposomes preparation step. Commonly

Non-Viral Gene Delivery Systems Based on

in the early endosomes (Kichler, 2004).

*vivo* were obtained (Kim et al., 2007).

fold) of the TE (Wang et al., 2002).

demonstrated the efficient inhibition of the tumor growth.

Cholesterol Cationic Lipids: Structure-Activity Relationships 365

et al., 1995). PEI is well-known for its ability to compact DNA and to facilitate its early endosomal release, preventing the delivered DNA from degradation in the late endosomes (Kichler, 2004). The reason for the good transfection activity of PEI is the presence of the primary, secondary, and tertiary amino groups. These groups have different p*K*a values, which give the PEI a good buffer capacity, therefore not allowing a decrease in the pH value

In literature several kinds of the PEI-cholesterol conjugates have been described. Kim and co-workers (Han et al., 2001) synthesized water-soluble lipopolymer (WLSP (**21a**)) in the reaction of the branched PEI (mw 1.8 kDa) and cholesteryl chloroformate. The average molecular weight of WSLP (**21a**) was approximately 2 kDa and the extent of modification with cholesterol was ~ 0.5. The WSLP (**21a**)-pDNA complexes were characterized by low toxicity *in vitro* and they induced the aggregation of erythrocytes to a lesser extent, as compared to PEI 25 kDa. WSLP (**21a**)-pDNA complexes demonstrated higher TE in both CT-26 and 293 T cells compared to PEI 25 kDa- or PEI 1.8 kDa-based formulations. As an experimental model for the estimation *in vivo* of the biological activity the authors used the antitumor activity of IL-12 coded by the pDNA transfected into the tumor cells. As a result of the injections of the WSLP (**21a**)-pDNA complexes into mice tumors a significant decrease in the tumor growth rate (~20% higher, as compared to the administration of the *naked DNA*) and of the level of metastasis formation were observed; resulting in higher survival rates of the animals after treatment (Mahato et al., 2001; Janat-Amsbury et al., 2005*)*. Later data confirming the high efficacy of WSLP (**21a**) for the transfection of siRNA *in vitro* and *in* 

The same authors endeavored to design a more effective transfection reagent based on branched PEI (bPEI) using chemically protected primary amine and conjugation of cholesterol at the secondary amino groups of PEI. However, contrary to expectations, the presence of the non-modified primary amino groups only resulted in a slight increase (1.5-

Kim and co-workers obtained partially contradictory data (Kim et al 2001). In this work a simple one-step synthetic procedure was used to yield myristyl and cholesterol derivatives of PEI having molecular mass 2 kDa. According to the obtained data these modified PEI efficiently transfected cell *in vitro* and displayed lower toxicity when compared to bPEI 2 kDa; however the compounds were less tolerated by cells than the parent bPEI 2 kDa. Fewell and co-workers studied the properties of the PPC conjugates, containing the bPEI 1.8 kDa, cholesterol and PEG, where the PEGylation extent varied from 0.6 to 20 PEG molecules to one bPEI molecule (Fewell et al., 2005*)*. The highest transfection efficiency *in vivo* was observed for the PPC conjugate, where the molar ratio bPEI:cholesterol:PEG was 1:1:2. When using the conjugates containing a large quantity of PEG molecules, a decrease of the reporter gene expression was observed. The variation of the number of the cholesterol residues in the conjugates was not performed: all the tested conjugates contained the bPEI and cholesterol at the ratio of 1:1. In order to study the TE of these molecules *in vivo* the authors used a biological model similar to the one described in (Mahato et al., 2001) and also

Furgeson and co-workers also used linear PEI for the synthesis of new cholesterol-containing conjugates apart from bPEI. The polymer, obtained in the reaction of a low molecular weight lPEI (423 Da) with cholesterol chloroformate, was used for the preparation of the water insoluble liponanoparticles using DOPE as a lipid-helper (Furgeson et al., 2002). The *in vitro* TE of nanoparticle-pDNA complexes was ~4-fold higher, in comparison with bPEI 25 kDa.

used for the transfection **13b**-DNA complexes were found to form ordered aggregates characterized by a fingerprint-like structures, but not the concentric multilamellar vesicles demonstrated for the **13b**/DOPE–DNA complexes (Pitard et al., 1999).

The analysis of transfection of a wide range of cell lines with **13b**/DOPE-DNA complexes revealed the high efficiency of this process, which is comparable with the commercially available transfectants and 10–20-fold exceeds the calcium phosphate precipitation protocol (Vigneron et al., 1996; Ouderhiri et al., 1997). Mediated by guanidinium-cholesterol lipids gene transfection is appropriate for the mammalian airway epithelium (Ouderhiri et al., 1997). The positive results of the transfection into primary human cells *in vitro* and into the mouse airways cells *in vivo* confirm the potential of the cationic lipids in relation to lungdirected gene therapy.

Aminoglycosides, natural polyamines that are known to bind to nucleic acids, represent a favorable scaffold for the synthesis of a variety of cationic lipids. The synthesis of a cationic cholesterol derivative of kanamycin A and its polyguanidinylated derivative was recently described (Belmont et al., 2002). The amino-sugar-based cationic lipid **20a** demonstrated a high level of TE in terms of gene transfection of a variety of mammalian cell lines when used either alone or as a part of a liposomal formulation with helper-lipid. In addition, colloidally stable kanamycin-cholesterol/DOPE lipoplexes were found to be efficient for gene transfection into the mouse airways *in vivo*.

Designed cholesterol-based kanamycin A analogues (**20b,c**) bearing various linkers between the aminoglycoside headgroup and the cholesterol moiety were prepared (Sainlos et al., 2005). It was successfully shown, that the length and nature of the spacer can affect the physicochemical and biological properties of the lipoplexes. The incorporation of a longer spacer into the structures of cholesterol derivatives of kanamycin A can yield the lipoplexes with improved in terms of TE physicochemical properties. The cholesterol derivatives **20b,c** were successfully used for the transfection of mouse airway epithelium *in vivo*. But the beneficial effects of the longer spacers observed *in vitro* with **20b,c** were not found *in vivo*, as only **20c** yielded levels of transgene expression higher than those obtained with **20a**.

#### **2.6 Colesterol-PEI conjugates**

Polyethyleneimine (PEI) is polycation widely used for DNA compaction and delivery. The high transfection efficiency of PEI was firstly demonstrated by Behr and co-workers (Boussif

used for the transfection **13b**-DNA complexes were found to form ordered aggregates characterized by a fingerprint-like structures, but not the concentric multilamellar vesicles

demonstrated for the **13b**/DOPE–DNA complexes (Pitard et al., 1999).

H2N NH2

**20a**,X=

**20b**,X=

HO

O

O

**20c**,X= N

NH2

OH

OH HN **X** O

> N H

> > H

O O

O O

The analysis of transfection of a wide range of cell lines with **13b**/DOPE-DNA complexes revealed the high efficiency of this process, which is comparable with the commercially available transfectants and 10–20-fold exceeds the calcium phosphate precipitation protocol (Vigneron et al., 1996; Ouderhiri et al., 1997). Mediated by guanidinium-cholesterol lipids gene transfection is appropriate for the mammalian airway epithelium (Ouderhiri et al., 1997). The positive results of the transfection into primary human cells *in vitro* and into the mouse airways cells *in vivo* confirm the potential of the cationic lipids in relation to lung-

Aminoglycosides, natural polyamines that are known to bind to nucleic acids, represent a favorable scaffold for the synthesis of a variety of cationic lipids. The synthesis of a cationic cholesterol derivative of kanamycin A and its polyguanidinylated derivative was recently described (Belmont et al., 2002). The amino-sugar-based cationic lipid **20a** demonstrated a high level of TE in terms of gene transfection of a variety of mammalian cell lines when used either alone or as a part of a liposomal formulation with helper-lipid. In addition, colloidally stable kanamycin-cholesterol/DOPE lipoplexes were found to be efficient for gene

Designed cholesterol-based kanamycin A analogues (**20b,c**) bearing various linkers between the aminoglycoside headgroup and the cholesterol moiety were prepared (Sainlos et al., 2005). It was successfully shown, that the length and nature of the spacer can affect the physicochemical and biological properties of the lipoplexes. The incorporation of a longer spacer into the structures of cholesterol derivatives of kanamycin A can yield the lipoplexes with improved in terms of TE physicochemical properties. The cholesterol derivatives **20b,c** were successfully used for the transfection of mouse airway epithelium *in vivo*. But the beneficial effects of the longer spacers observed *in vitro* with **20b,c** were not found *in vivo*, as

only **20c** yielded levels of transgene expression higher than those obtained with **20a**.

Polyethyleneimine (PEI) is polycation widely used for DNA compaction and delivery. The high transfection efficiency of PEI was firstly demonstrated by Behr and co-workers (Boussif

HO O O

O

HO

OH

H2N

HO

directed gene therapy.

transfection into the mouse airways *in vivo*.

**2.6 Colesterol-PEI conjugates** 

et al., 1995). PEI is well-known for its ability to compact DNA and to facilitate its early endosomal release, preventing the delivered DNA from degradation in the late endosomes (Kichler, 2004). The reason for the good transfection activity of PEI is the presence of the primary, secondary, and tertiary amino groups. These groups have different p*K*a values, which give the PEI a good buffer capacity, therefore not allowing a decrease in the pH value in the early endosomes (Kichler, 2004).

In literature several kinds of the PEI-cholesterol conjugates have been described. Kim and co-workers (Han et al., 2001) synthesized water-soluble lipopolymer (WLSP (**21a**)) in the reaction of the branched PEI (mw 1.8 kDa) and cholesteryl chloroformate. The average molecular weight of WSLP (**21a**) was approximately 2 kDa and the extent of modification with cholesterol was ~ 0.5. The WSLP (**21a**)-pDNA complexes were characterized by low toxicity *in vitro* and they induced the aggregation of erythrocytes to a lesser extent, as compared to PEI 25 kDa. WSLP (**21a**)-pDNA complexes demonstrated higher TE in both CT-26 and 293 T cells compared to PEI 25 kDa- or PEI 1.8 kDa-based formulations. As an experimental model for the estimation *in vivo* of the biological activity the authors used the antitumor activity of IL-12 coded by the pDNA transfected into the tumor cells. As a result of the injections of the WSLP (**21a**)-pDNA complexes into mice tumors a significant decrease in the tumor growth rate (~20% higher, as compared to the administration of the *naked DNA*) and of the level of metastasis formation were observed; resulting in higher survival rates of the animals after treatment (Mahato et al., 2001; Janat-Amsbury et al., 2005*)*. Later data confirming the high efficacy of WSLP (**21a**) for the transfection of siRNA *in vitro* and *in vivo* were obtained (Kim et al., 2007).

The same authors endeavored to design a more effective transfection reagent based on branched PEI (bPEI) using chemically protected primary amine and conjugation of cholesterol at the secondary amino groups of PEI. However, contrary to expectations, the presence of the non-modified primary amino groups only resulted in a slight increase (1.5 fold) of the TE (Wang et al., 2002).

Kim and co-workers obtained partially contradictory data (Kim et al 2001). In this work a simple one-step synthetic procedure was used to yield myristyl and cholesterol derivatives of PEI having molecular mass 2 kDa. According to the obtained data these modified PEI efficiently transfected cell *in vitro* and displayed lower toxicity when compared to bPEI 2 kDa; however the compounds were less tolerated by cells than the parent bPEI 2 kDa.

Fewell and co-workers studied the properties of the PPC conjugates, containing the bPEI 1.8 kDa, cholesterol and PEG, where the PEGylation extent varied from 0.6 to 20 PEG molecules to one bPEI molecule (Fewell et al., 2005*)*. The highest transfection efficiency *in vivo* was observed for the PPC conjugate, where the molar ratio bPEI:cholesterol:PEG was 1:1:2. When using the conjugates containing a large quantity of PEG molecules, a decrease of the reporter gene expression was observed. The variation of the number of the cholesterol residues in the conjugates was not performed: all the tested conjugates contained the bPEI and cholesterol at the ratio of 1:1. In order to study the TE of these molecules *in vivo* the authors used a biological model similar to the one described in (Mahato et al., 2001) and also demonstrated the efficient inhibition of the tumor growth.

Furgeson and co-workers also used linear PEI for the synthesis of new cholesterol-containing conjugates apart from bPEI. The polymer, obtained in the reaction of a low molecular weight lPEI (423 Da) with cholesterol chloroformate, was used for the preparation of the water insoluble liponanoparticles using DOPE as a lipid-helper (Furgeson et al., 2002). The *in vitro* TE of nanoparticle-pDNA complexes was ~4-fold higher, in comparison with bPEI 25 kDa.

Non-Viral Gene Delivery Systems Based on

systems.

**transfection efficiency** 

(Keller et al., 2003).

of physico-chemical parameters of the lipoplexes.

Cholesterol Cationic Lipids: Structure-Activity Relationships 367

effective, even in the presence of 50% of serum. The TE and cytotoxicity of the lipopolymers were found to depend on the percentage of cholesterol moieties and the molecular weight of PEI used for the synthesis of lipopolymers. Optimized lipopolymer-DOPE formulations exhobited a higher cell viability and high TE, which was unaffected by serum: the TE of the lipopolymers was obviously one of the highest among known non-viral delivery

**3. Physico-chemical properties of lipoplexes and their influence on the** 

It is commonly known, that the efficiency of liposome-mediated gene delivery is determined not only by the structure of cationic and helper lipids or properties of the transfected plasmids, but also by the size of the lipoplex and its ζ-potential. The structure of the supramolecular DNA–lipid complexes is dependent upon both the external (pH, degree of hydration, temperature, and the presence of doubly charged cations, i.e., Ca2+, Mg2+) and internal factors. The physico-chemical properties can alter the lifetime, distribution, and the TE efficacy of lipoplexes. Thus, in order to shed more light on the mechanism of transfection and to elucidate the structure-activity relationships, it is necessary to investigate a number

Using a set of physico-chemical methods, it was demonstrated that condensation and compactization of DNA by DC-Chol (**1a**)/DOPE cationic liposomes is a result of a strong entropically-driven surface electrostatic interactions. Fluorescence anisotropy results have revealed that low cationic lipid contents in the liposomes tend to favor more fluid bilayers; which in turn are potential advantages for transfecting cells. DC-Chol/DOPE-DNA lipoplexes are represented by supramolecular complexes with a different morphology: DNA-coated unilamellar lipoplexes, lipoplex nanostructures with thickened, flattened, and deformed walls, and also multilamellar lipoplexes with or without open bilayers (Rodriguez-Pulido et al., 2008). The effects of hydration and temperature on the structure of DC-Chol/DOPE–DNA lamellar lipoplexes were also investigated (Pozzi et al., 2006). The DNA complexation and condensation properties of two established cationic liposome formulations, CDAN (**18d**)/DOPE (50:50, *m*/*m*; TrojeneTM) and DC-Chol (**1a**)/DOPE (60:40, *m*/*m*), were studied by means of biophysical methods (Keller et al., 2003). The results provide a suitable framework for the understanding of why CDAN/DOPE cationic liposomes are exceptionally efficient, in comparison with other cationic lipid-based systems, at mediating cell transfection. The liposomes CDAN (**18d**)/DOPE formed the metastable lipoplexes, exhibiting greater transfection efficiency *in vitro* in the presence of 10% serum, in comparison to DC-Chol/DOPE liposomes. This metastability may be related to the unusually low p*K*a value of 5.7 of amino groups. In addition, it was supposed that CDAN (**18d**)/DOPE-pDNA particles may have a greater tendency to interact with negatively charged serum components and facilitate the DNA release from endosomes

A critical factor in the lipid-mediated gene delivery is the structural and phase evolution of lipoplexes upon interaction and mixing with anionic cellular lipids (Tarahovsky et al., 2004; Koynova et al., 2005, 2006; Koynova & MacDonald, 2007). Such a structural rearrangement is supposed to play a central role in the DNA escape process; i.e. how DNA dissociates from lipoplexes and is released into the cytoplasm. The structural and phase evolution of lipoplexes upon interaction with lipid mixtures similar to real membranes and DNA release

T-shaped and L- (linear) shaped PEI 25 kDa conjugates with cholesterol (PEIC) were synthesized and thoroughly studied, revealing the improved transfection and reduced cytotoxic effect, partially a result of the sequestering of charged secondary amines of PEI, in the presence of cholesterol moiety (Furgeson et al., 2003*)*. The modification extent of the PEI carrier in this study ranged from 1 to 2 cholesterol molecules per PEI. Polyplexes, formed by L- and T shapes PEICs with DNA possessed a higher TE *in vitro*, in comparison with the initial linear polymer; as well as the bPEI with the same molecular weight. The highest TE among the compounds tested was observed for L-PEIC: the expression of the reporter gene, delivered into the Renca cells using this conjugate was 32-fold higher in comparison with the expression observed in the presence of bPEI. The authors proposed that the differences in the TE depended on the conformational changes of the PEI molecule in the presence of the hydrophobic substitues. It was shown that the PEIC conjugates penetrated into the cells *via* the interaction with LDL receptors. The high TE of the LPC was confirmed *in vivo* in experiments with systemic and local administration of the polyplexes (Furgeson et al., 2004*)*  Multiple modifications of PEI's amines were commonly avoided, due to the importance of the cationic charge for DNA condensation and buffering capacity of the polymer. Therefore quantitative data characterizing the impact of cholesterol conjugation was not yet available. In connection with this, we attempted to find the optimal extent of modification of the 25 kDa PEI with cholesterol. Conjugates of PEI bearing a different number of cholesterol residues with 0.5 to 20% of amines modified were synthesized. We found that a small number of cholesterols attached to PEI (extent of modification was 0.5 or 1%) significantly increased the TE of the polymer, while extensively modified PEI-cholesterol conjugates demonstrated reduced TE, although possessing lower cytotoxicity (Gusachenko (Simonova) et al, 2009). TE studies were performed using different types of biologically active nucleic acids: single stranded oligonucleotide, plasmid DNA and siRNA duplex. The most promising conjugates in the series were found to be PEIC 0.5 and PEIC 1 demonstrating the best combination of TE with lower cytotoxicity.

The aforementioned PEI-cholesterol conjugates were prepared in the reaction of PEI, having various molecular weights and cholesteryl chloroformate. The PEI-lipid conjugates (**21b**) based on ether-linked cholesterol units were first described by Bhattacharya and co-workers (Bajaj et al., 2008b). Nine PEI-cholesterol-based conjugates having polymer amine backbone linked to the cholesterol unit via the ether link were synthesized. Three low molecular weight PEIs were used for the synthesis of these lipopolymers. The TE studies in HeLa cells showed a high potency and low cytotoxicity of these lipopolymers in comparison with the commercially available PEI. The TE of PEI 25 kDA decreased in the presence of a high percentage of serum, whereas PEI-cholesterol-based lipopolymers were discovered to be

T-shaped and L- (linear) shaped PEI 25 kDa conjugates with cholesterol (PEIC) were synthesized and thoroughly studied, revealing the improved transfection and reduced cytotoxic effect, partially a result of the sequestering of charged secondary amines of PEI, in the presence of cholesterol moiety (Furgeson et al., 2003*)*. The modification extent of the PEI carrier in this study ranged from 1 to 2 cholesterol molecules per PEI. Polyplexes, formed by L- and T shapes PEICs with DNA possessed a higher TE *in vitro*, in comparison with the initial linear polymer; as well as the bPEI with the same molecular weight. The highest TE among the compounds tested was observed for L-PEIC: the expression of the reporter gene, delivered into the Renca cells using this conjugate was 32-fold higher in comparison with the expression observed in the presence of bPEI. The authors proposed that the differences in the TE depended on the conformational changes of the PEI molecule in the presence of the hydrophobic substitues. It was shown that the PEIC conjugates penetrated into the cells *via* the interaction with LDL receptors. The high TE of the LPC was confirmed *in vivo* in experiments with systemic and local administration of the polyplexes (Furgeson et al., 2004*)*  Multiple modifications of PEI's amines were commonly avoided, due to the importance of the cationic charge for DNA condensation and buffering capacity of the polymer. Therefore quantitative data characterizing the impact of cholesterol conjugation was not yet available. In connection with this, we attempted to find the optimal extent of modification of the 25 kDa PEI with cholesterol. Conjugates of PEI bearing a different number of cholesterol residues with 0.5 to 20% of amines modified were synthesized. We found that a small number of cholesterols attached to PEI (extent of modification was 0.5 or 1%) significantly increased the TE of the polymer, while extensively modified PEI-cholesterol conjugates demonstrated reduced TE, although possessing lower cytotoxicity (Gusachenko (Simonova) et al, 2009). TE studies were performed using different types of biologically active nucleic acids: single stranded oligonucleotide, plasmid DNA and siRNA duplex. The most promising conjugates in the series were found to be PEIC 0.5 and PEIC 1 demonstrating the

The aforementioned PEI-cholesterol conjugates were prepared in the reaction of PEI, having various molecular weights and cholesteryl chloroformate. The PEI-lipid conjugates (**21b**) based on ether-linked cholesterol units were first described by Bhattacharya and co-workers (Bajaj et al., 2008b). Nine PEI-cholesterol-based conjugates having polymer amine backbone linked to the cholesterol unit via the ether link were synthesized. Three low molecular weight PEIs were used for the synthesis of these lipopolymers. The TE studies in HeLa cells showed a high potency and low cytotoxicity of these lipopolymers in comparison with the commercially available PEI. The TE of PEI 25 kDA decreased in the presence of a high percentage of serum, whereas PEI-cholesterol-based lipopolymers were discovered to be

best combination of TE with lower cytotoxicity.

effective, even in the presence of 50% of serum. The TE and cytotoxicity of the lipopolymers were found to depend on the percentage of cholesterol moieties and the molecular weight of PEI used for the synthesis of lipopolymers. Optimized lipopolymer-DOPE formulations exhobited a higher cell viability and high TE, which was unaffected by serum: the TE of the lipopolymers was obviously one of the highest among known non-viral delivery systems.
