Transfection Efficiency of Cationic Lipids with Different Hydrophobic

Bioconjugate Chem. 2010, 21, 563–577

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Transfection Efficiency of Cationic Lipids with Different Hydrophobic Domains in Gene Delivery DeFu Zhi,†,‡ ShuBiao Zhang,*,† Bing Wang,† YiNan Zhao,† BaoLing Yang,† and ShiJun Yu‡ Key Laboratory of Biotechnology and Bioresources Utilization, The State Ethnic Affairs Commission-Ministry of Education, College of Life Science, Dalian Nationalities University, Dalian, Liaoning, China 116600, and School of Chemistry and Chemical Engineering, Liaoning Normal University, Dalian, Liaoning, China 116029. Received September 7, 2009; Revised Manuscript Received January 4, 2010

The structure of cationic lipids is a major factor for their transfection activity. A cationic lipid generally contains four functional domains: a hydrophilic headgroup, a linker, a backbone domain, and a hydrophobic domain. The structure of the hydrophobic domain determines the phase transition temperature and the fluidity of the bilayer and influences the stability of liposomes, the DNA protection from nucleases, the endosomal escape, the DNA release from complex, and the nuclear penetration. Also, toxicity of the lipids is influenced by the hydrophobic domain. The compounds used for gene delivery are classified according to the structure of the hydrophobic domain as follows: aliphatic chains, steroid domain, and fluorinated domain. In this review, we summarized recent research results concerning the structures of the hydrophobic domain, in order to find the effect of the hydrophobic domain on transfection efficiency. Understanding these would be very important for scientists to prepare novel cationic lipids and design novel formulations with high transfection efficiency.

INTRODUCTION Over the past decades, gene therapy was expected to lead to new, powerful approaches for curing many diseases, which were currently being explored in worldwide clinical trials. Gene therapy offers extraordinary long-term potential for the treatment of human diseases, including inherited and acquired disorders, such as cancer, cystic fibrosis, and AIDS (1-3). This approach of traditional gene therapy is based on the delivery of genes by transfection vectors and subsequent expression of exogenous DNA into the host genome for long-term expression (4-7). Broadly speaking, transfection vectors commonly used in gene therapy are mainly classified into two categories: viral and nonviral (6, 8). In early clinical trials, although viral vectors such as adenovirus and retrovirus have achieved some success particularly in cancer gene therapy (9), they bring about a number of disadvantages, inducing adverse immunogenic responses, high cost, and so forth. Because of these reasons, people try to find a more efficient and flexible security system in the category of nonviral vectors (6). Nonviral vectors can be grouped into three main categoriesscationic lipids, cationic polymers, and peptides. Compared to their viral counterparts, these vectors are currently considerably less efficient, but their well-defined physical and chemical composition as well as their reduced immunogenicity and toxicity make them promising candidates for gene delivery (1, 10-14). Cationic lipids have been favored for many potential advantages compared with other nonviral vectors, including significant simplicity and ease of * Correspondence should be sent to ShuBiao Zhang, Key Laboratory of Biotechnology and Bioresources Utilization, The State Ethnic Affairs Commission-Ministry of Education, College of Life Science, Dalian Nationalities University, No. 18, Liaohe West Road, Economic & Technical Development Zone, Dalian, Liaoning, China 116600. +86411-87656141 (ph.), +86-411-87656141 (fax), [email protected] (email). † Dalian Nationalities University. ‡ Liaoning Normal University.

production, good repeatability and biodegradability, potential commercial value, and wide range of clinical application and safety (15). Felgner et al. (16) synthesized a glycerol backbone-based cationic transfection lipid-DOTMA1 (see Figure 1), which was used for introduction of plasmids into cells in 1987. The first in vivo experiments using cationic lipids were conducted in mice 1 Abbreviations: DOTMA, N-[1-(2,3-dioleyloxy)-propyl])-N,N,N-trimethylammonium chloride; GL-67, N4-spermine cholesterylcarbamate; DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate; DMRIE, N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)1-propanaminium bromide; EDMPC, dimyristoylglyceroethylphosphocholine; DOTIM, octadecenoylloxy (ethyl-2-heptadecenyl-3-hydroxyethyl) imidazolinium chloride; DOSPER, 3-dioleoyloxy-2-(6-carboxyspermyl)-propylamid; CFTR, cystic fibrosis transmembrane conductance regulator; IL, interleukin; HLA, Helen langridge associates; DOSPA, 2,3-dioleyloxyN-[2-(sperminecarboxyamido)ethyl]-N,N-dimethyl-1-propaniminium bromide; CTAB, cetyl trimethylammonium bromide; LHON, 6-lauroxyhexyl ornithinate; OLON, oleoyl ornithinate; BGDA, pentacosa-10,12-diynoicacid-{2[bis-(2-guanidinoethyl)-amino]-ethyl}-amide; BGTC, bis-guanidiniumtren-cholesterol; DOPE, dioleoyl phosphatidylethanolamine; DODAC, N,N-dioleyl-N,N-dimethylammonium chloride; TODMAC3, N,N,N′,N′tetraoelyl-N,N′-dimethyl-1,3-propanediammonium chloride; TODMAC6, N,N,N′,N′-tetraoelyl-N,N′-dimethyl-1,6-hexanediammonium chloride; SAINT2, N-methyl-4-(dioleyl)methylpyridinium-chloride; SAINT-5, N-methyl4-(distearyl)methylpyridiniumchloride; DLPE, dilauroylphosphatidylethanolamine; DMPE, dimiristoylphosphatidylethanolamine; DPPE, dipalmitoyl phosphatidylethanoiamine; DPyPE, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine; DSPE, distearoylphosphatidylethanolamine; GAP-DLRIE, N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-dodecyloxy)-1-propanaminium bromide; GAP-DMORIE, N-(3-aminopropyl)-N,N-dimethyl-2,3bis(cis-9-tetradecenyloxy)-1-propanaminium bromide; NADPH, nicotinamide adenine dinucleotide phosphate; GSH, glutathione; DC-Chol, 3β-[N-(N′,N′dimethylaminoethane)-carbamoyl]cholesterol hydrochloride; GL-67, N5cholesteryloxycarbonyl-5,10-diazatetradecane-1,14-diamine; F-DMAEA, N-[1-(2,3-di-11-(F-butyl)undecyloxy)-propyl]-N,N-dimethyl-N-(2-aminoethyl) ammonium hydroxide; F-DOSPA, N-[1-(2,3-di-11-(F-butyl)undecyloxy)-propyl]-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethylammonium; DOGS, N,N-dioctadecylamidoglycylspermine; GalSper, R-galactoω-spermine bolaamphiphile; PEI, polyethyleneimine; PE, polyethylene.

10.1021/bc900393r  2010 American Chemical Society Published on Web 02/01/2010

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Figure 1. Chemical structure of DOTMA.

in 1989 (17, 18). Since then, a series of efficient cationic transfection lipids have been synthesized (19-23) and used for delivery of nucleic acids into cells in culture and reported high transgene expression properties (both in vitro and in vivo). Many of these efficient cationic transfection lipids became commercially available as transfection reagents in the late 20th century, and a few liposome formulations have been used in gene therapy clinical trials for treatment of cancer and other genetic disorders (see Table 1). The basic structure of most of the cationic lipids employed for gene delivery is made up of four chemical functional domains: (i) a hydrophilic headgroup, which is positively charged and allow interactions with negatively charged DNA, usually via the protonation of one (monovalent lipid) or several (multivalent lipid) amino groups; (ii) a hydrophobic domain composed of a steroid or of alkyl chains (saturated or unsaturated); (iii) a linker bond which is between the hydrophobic domain and the backbone, between the cationic headgroup and the backbone or directly and between the hydrophobic domain and the cationic headgroup (often one or more amino acid, or ether, or ester bond); and (iv) a backbone domain which separates the headgroup from the hydrophobic domain, and acts as a scaffold on which the cationic lipid is built (the most common backbone is glycerol-based) (see Figure 2) (24). Cationic lipids have become a major research tool for transferring DNA into cells in vitro and are being investigated in a variety of in vivo gene therapy trials on human. As a vector, the geometry of the cationic lipid is an important property regarding its application. The hydrophobic domains represent the nonpolar hydrocarbon moieties of cationic lipids and can be grouped into three categories: aliphatic chains, steroid domain, and fluorinated domain. Before any discussion of cationic lipids, it should be noted that the separate analysis of each domain is somewhat artificial (24-28). Transfection efficiency can be affected by structural variations in the hydrophobic domain such as length, the specific type of chemical bonds, and the relative position of the hydrocarbon chains, which can also affect toxicity of cationic lipids. We shall not comment on the relationship of toxicity and structure of cationic lipids in gene delivery here, because we have already published a review about this (3). This review is chiefly concerned with the effect of the different hydrophobic domains of cationic lipids on transfection ef-

Figure 2. Schematic representation of DOTAP, a commonly used cationic lipid for gene delivery.

ficiency. The majority of the work studying cationic lipids structure/activity relationship has involved the in vitro delivery of genes.

ALIPHATIC CHAINS Cationic lipids with aliphatic chains have been thoroughly researched. The chains are either linear and saturated or linear and monounsaturated and used in liposomal vectors ranging from C5 to C25, but oleyl, lauryl, myristyl, palmityl, and stearyl have been the most researched ones (24). A common variation is the use of branched (29), acetylenic (30, 31) chains, and cismonounsaturated alkyl chains (32) have also been reported. Number of Aliphatic Chains. It is commonly believed that cationic lipids have one to four hydrocarbon chains. Several studies have also shown that incorporating aliphatic chains with different numbers can improve transfection efficiency potentially by promoting endosomal escape (16-21, 33-46). Since the pioneering work of Felgner et al. (16-18) who used the cationic lipid DOTMA with two aliphatic chains, several other related compounds (commercially available transfection reagents with two aliphatic chains, e.g., DOSPA, DOTAP, and DOSPER) have proved moderately successful for gene therapy of pulmonary diseases in animal models (28). DOTAP and DOSPER differ from the ether bond-containing reagents DOTMA and DOSPA because DOTAP and DOSPER contain ester bonds, which are endogenous to natural lipids. DOTAP and DOSPER offer higher transfection efficiency and lower cytotoxicity as compared with DOTMA, which may result from the fact that cellular lipases or esterases easily degrade the artificial lipid (19, 28). Most cationic lipids with linear hydrocarbons have two chains in the hydrophobic domain, but cationic lipids with one, three, or four chains are sometimes researched. For aliphatic chains, single-tailed cationic lipids were originally reported to have low transfection efficiency and high toxicity in plasmid delivery (33, 34). However, some examples showed that single-tailed cationic lipids may also function in plasmid transfection. Cameron et al. (34) described a new class of cationic lipids which offered various numbers of palmitoyl groups in the

Table 1. Clinical Trials Using Cationic Lipid cationic lipid

commercial name

DC-Chol GL-67/DOPE GL-67 DMRIE/Cholesterol 1:1 (M/M) DMRIE/DOPE EDMPC DOTIM-cholesterol DOTAP DOTAP/Cholesterol DOTAP/DOPE 1:1 (w/w) DOTMA/Cholesterol DOTMA/DOTAP DOSPER DOSPA/DOPE 3:1 (w/w)

DMRIE-C Reagent

DOTAP Escort LIPOFECTIN DOSPER LIPOFECTAMINE

gene (s)

supplier

phase

Ad5 E1A/HLA-B7/CFTR/ HLA-A2/HLA-B13/H-2K CFTR CFTR CFTR/HLA-B7 IL-2/HLA-B7 CFTR EV-CLDC CFTR Fus 1 CFTR IL-2 R1-Antitrypsin p53 IFN-γ

Xing et al., 1998; Nabel et al., 1994; Gill et al., 1997; Hui et al., 1997 Zabner et al., 1997 Ruiz et al., 2001 Invitrogen (Life Technologies Gibco BRL) Hersh et al., 1995 Noone et al., 2000 McHutchinson et al., 2008 Roche Molecular Charles et al., 2002 Sigma-Aldrich Zarrabi et al., 1999 Invitrogen(Life Technologies Gibco BRL) Roche Molecular Invitrogen (Life Technologies Gibco BRL)

I/II I I I/II I I/II I II I I/II II II I I

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Figure 3. Cationic lipids with one, two, or three palmitoyl chains. Figure 6. Chemical structures of BGDA.

Figure 4. Chemical structure of CTAB.

hydrophobic domain to prove that the transfection efficiency of the lipid with one chain was inferior to the di- or trisubstituted counterparts (see Figure 3). In a few examples, single-tailed cationic lipids were successful as transfection reagents. Huang et al. (33) compared the transfection properties of several quaternary ammonium detergents on fibroblasts and found that CTAB was most efficient (see Figure 4), but somewhat less than its double-tailed counterparts, such as DOTMA. Tang et al. (35) studied the transfection of liposomes produced from double-tailed 1′,2′-dioleyl-sn-glycero-3′-succinyl-1,6-hexanediol ornithine conjugate (DOGSHDO) with an ornithine headgroup, single-tailed OLON with an ornithine headgroup, single-tailed LHON with an ornithine headgroup, double-tailed DOTAP with a quaternary amine group, and single-tailed CTAB with a quaternary amine group. They found that OLON had more transfection activity than its double-tailed analogue, DOGSHDO, whereas double-tailed DOTAP had higher transfection activity than its single-tailed analogue, CTAB. This result shows that neither the headgroup nor the tail group by its own can be the determinant of transfection activity of cationic lipids (36, 37). The toxicity of cationic lipid was decreased by introducing a biodegradable ester linkage (LHON) (see Figure 5) and taking the place of the double bond in the middle of the chain without sacrificing the transfection activity. Then, Patel et al. (30) synthesized a cationic lipid, BGDA (see Figure 6), characterized by a bisguanidinium headgroup (actually identical to that in BGTC) and a chain with a diacetylene unit and 25 carbon atoms. It was efficient for in vitro transfection when used in formulations with a similar neutral diacetylene colipid or with DOPE. In the report that inclusion of DOPE may increase the stability of the lipoplexes, BGDA/DOPE liposomes containing high proportions of DOPE were able to completely condense the DNA at lower charge ratios than liposomes with low proportions of DOPE. Within the formulations, cationic lipids can adopt various structural phases, including the micellar, lamellar, cubic, and inverted

Figure 5. Chemical structures of LHON, OLON, and DOGSHDO.

Figure 7. Chemical structures of RPR 228091.

Figure 8. Chemical structures of O-alkyl dioleoylphosphatidylcholinium cationic analogues.

hexagonal phase, and arrange back-to-back in bilayers. High transfection activity will be acquired for inverted hexagonal structure when membrane fusion is dominant, but when serum is present, the lamellar structure lipoplexes show great superiority for their inhibition dissociation by serum during lipoplexes transporting. The mechanism by which DOPE exerts its catalytic activity is still poorly defined, but prevailing evidence suggests that its hexagonal-phase-forming propensity likely plays a major role. There are also reports that (38, 39) the inclusion of DOPE in the cationic lipid formulation increases transfection activity by enhancing mixing and fusion of liposomes and cell membranes. DOPE forms inverted hexagonal phase structures at ambient temperature (40); inclusion of DOPE is presumed to enhance endosomal escape of the lipoplexes into the cytoplasm, as DOPE is thought to have fusogenic properties important for endosomal membrane disruption. Another example of single-tailed cationic lipid is RPR 228091 (see Figure 7) that was composed of a spermine derived amino acid, coupled to a single C18 alkyl chain, and it was suitable for transfection (41). Cationic lipids with three aliphatic chains usually show lower transfection efficiency than lipids with two chains (34). However, the transfection can be greatly improved when the third chain is short or could be removed by intracellular reduction of a disulfide bond (42-44). Rosenzweig et al. (43) synthesized a series of O-alkyl dioleoylphosphatidylcholinium cationic analogues (see Figure 8) ranging from methyl to octadecyl, and tested them for transfection activity. These compounds could show phase behavior changing with the length of the third chain,


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Figure 9. Chemical structures of RPR 132776, PRP 132688, and PRP 120535.

Figure 11. Chemical structures of symmetrical cationic triglyceride. Figure 10. Chemical structures of TODMAC.

which was an alkyl group linked as phosphate triester. The shortchain (ethyl and propyl) derivatives, which formed a lamellar phase after hydration, were most effective in DNA transfection. The intermediate chain (hexyl) compound, which also formed a lamellar phase in aqueous solution, had approximately the same transfection efficiency as ethyl and propyl. However, hexyl compound assumed a cubic phase in high salt concentrations and became capable of mediating transfection only when vigorously dispersed by sonication. The octyl and octadecyl derivatives formed an inverted hexagonal phase in excess water and exhibited good transfection activity after sonication (43). Byk et al. (42, 44) prepared a series of lipopolyamines. They found that the lipid with a dithiol connecting the additional side chain to the vector body showed better transfection activity than the equivalent lipid missing the additional third side chain. Moreover, shorter chains (C5) showed better transfection activity than longer chain (C12) trilipids (see Figure 9). A few researchers were interested in novel tetraalkyl cationic lipids (45-47). Gaucheron et al. (46) synthesized a new class of divalent tetraalkyl cationic lipids. These cationic lipids are dimers of DODAC (see Figure 10) joined by a hydrocarbon of three or six carbons in length (TODMAC3 and TODMAC6, respectively). It was concluded that TODMAC6 exhibited potential as a transfection agent as compared to DODAC for in vitro and in vivo use and that the design of cationic lipids according to their ability to induce nonbilayer structure provided a useful guide for synthesis of new cationic lipids. However, TODMAC3 exhibited lower transfection efficiency than that achieved with DODAC. Generally speaking, for aliphatic chains, single-tailed and three-tailed cationic lipids are better known as surfactants because of their ability to form micelles in solution, but they are more toxic and less efficient than their double-tailed counterparts. Therefore, most of the aliphatic chains in the cationic lipids are double-tailed. Unsaturation and Branching of Aliphatic Chains. To rationally design and to optimize cationic lipid formulations for efficient gene delivery in therapeutics, many investigations aim at defining a structure-function relationship for cationic lipids. The solid geometry of the unsaturated aliphatic chains is an important variation. A few investigators found that the trans geometry was more favorable to transfection (48). However,

Figure 12. Chemical structures of SAINT-2 and SAINT-5 amphiphiles.

Obika et al. (49) obtained an opposite result by synthesizing and evaluating a series of triglycerides 1Aa-1Cb (see Figure 11). They found the cis oleoyl chain (1Ab) to be more active in transfection than the trans elaidoyl one (1Bb) in quaternary ammonium lipids. At the same time, they found that the transfection efficiency depends upon both the structure of the acyl chain at the 1- and 3-position of glycerol backbone and the length of the linker connecting the hydrophilic headgroup with the glycerol backbone. The transfection activity was increased when the unsaturated structure was introduced in the middle of a long acyl chain (1Aa > 1Bb > 1Cb). The overwhelming majority of the results showed that the unsaturated C18:1 oleyl was the optimal aliphatic chain, which was frequently the best choice for good transfection (31, 48, 50, 51). Zuhorn et al. (52) synthesized two pyridinium-based lipid analogues with identical headgroups but differing in alkyl chain unsaturation, i.e., SAINT-5 (C18:0) and SAINT-2 (C18:1) (see Figure 12). Their data indicated that at a charge ratio of 2.5/1 the enhancing effect of DOPE on transfection efficiency was highest and the unsaturated C18:1 oleyl SAINT-2/DOPE was more active in transfection than saturated C18:0 SAINT-5/ DOPE, which might rely on the relative efficiencies of DNA release from the lipoplexes upon encounter with (intra) cellular membranes. Koynova et al. (53) synthesized two cationic phospholipid derivatives with asymmetric hydrocarbon chains: ethyl esters of oleoyldecanoylethylphosphatidylcholine (C18:1/C10-EPC) and stearoyldecanoylethylphosphatidylcholine (C18:0/C10EPC). The former was more effective as a DNA transfection agent (human umbilical artery endothelial cells) than the latter, despite their similar chemical structure and virtually identical lipoplex organization. However, an opposite result was obtained by Roosgen and co-workers (54), showing that the saturated


Figure 15. Chemical structures of asymmetrical triglyceride.

Figure 13. Chemical structures of cationic lipids analogous to DOTMA and DMRIE.

C18:0 performed better than the equivalent C18:1 counterpart; it probably involved other effects like the easier hydrolysis of the headgroup-hydrophobic chain ester linker for saturated than for unsaturated chains. There are some examples that monoacetylenic or diacetylenic cationic lipids were useful as transfection agents (30, 31). Fletcher et al. (31) reported the modification of the hydrophobic region of the monovalent cationic lipid DOTAP, by substituting the cis double bonds of the oleate tails with C-C triple bonds in varying positions, in an effort to alter the membrane properties of the lipoplexes with minimal change to the liposome/ micelle-DNA interaction. Replacement of the cis double bonds of DOTAP with triple bonds forms more stable liposomes and lipoplex systems near physiological temperatures in comparison to DOTAP by X-ray diffraction studies, perhaps thereby providing enhanced protection to pDNA from extracellular DNA endonucleases. At the same time, the lipid transition is on the cusp of physiologically relevant temperatures, hence preventing overstability and excess rigidity that could otherwise hinder desired cell membrane-lipoplex interactions. It is anticipated that fatty acid tails with triple bonds rather than cis double bonds would exhibit a substantially reduced tangle and consequently afford more rigid liposomal bilayers. Membrane fluidity may be important for proper assembly of the lipoplexes and is thought to affect endosomal membrane fusion, an essential step required for the DNA to escape from the endosomes; thus, it also has influence on the transfection

Figure 14. Lipids with glycerol-type backbone.

efficiency (55-61). It is known that the membrane fluidity increased when an unsaturated structure was introduced into the hydrophobic chain of the lipid, by disrupting membrane packaging and facilitating DNA escape inside the cells. The best chains in terms of benefit to transfection are frequently the unsaturated ones: one reason lies in their effect on the membrane fluidity. A few typical cases have been enumerated in which unsaturated and branched chains help to fluidize the bilayer (29, 62). Arpicco et al. (62) synthesized a C15 dihydro farnesyl derivative (see Figure 13) with an unusual branched structure, which was less active than the C12 saturated lipids but, interestingly, more active than commercial lipids, such as DOTMA in almost all cell lines tested. Similarly, a formulation containing a short unsaturated C14:1 lipid and a branched C16 colipid, with very high fluidity, was found to be the best transfectant in vitro from a series of combinations of lipid and co-lipid chains by Ferrari et al. (29). These systems should be more fluid than their corresponding fully saturated analogues. A disadvantage of the unsaturation is the reduced stability in storage, because it is susceptible to oxidation (63). Asymmetry of the Hydrophobic Domain. One of the important factors which can affect the efficacy of cationic lipids is the asymmetry of the hydrophobic domain. Some researchers synthesized a series of cationic lipids that were based on glycerol backbone, which was constructed not only in symmetrical structure (see Figure 14) but asymmetrical structure as well (see Figure 14) (49). To compare the transfection ability of a symmetrical cationic triglyceride (1Ab) (see Figure 11) with that of an asymmetric triglyceride counterpart (see Figure 15), they were formulated into cationic liposomes with DOPE. It was found that symmetrical cationic triglycerides worked as well as the asymmetrical triglyceride counterpart for transfection efficiency, but showed an obvious advantage over their asymmetric counterparts in the synthesis (49). Several researchers found that the identical alkyl chains are liable to transfect in the lipid structure (48, 67, 68). However, most researchers have found that transfection efficiency can be increased either by using the different alkyl chains in the same lipid or by using cationic lipids of asymmetrical backbone (69). Cationic lipids which contain different alkyl chains were difficult to synthesize; therefore, only a few examples of cationic lipids with different alkyl chains can be seen in the literature. Heyes


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Scheme 1. Synthesis of Diether Lipids with Symmetric and Asymmetric Hydrophobic Domain

et al. (69) synthesized a series of diether lipids of identical or different alkyl chains with primary amino headgroups and showed a convenient route for the synthesis of asymmetric lipids (see Scheme 1). Two of the most effective diether lipids, hydrophobic domain with the shortest identical chains (RdR′dC12) and with particularly different chains (RdC12, R′dC18), were chosen. Ferrari et al. (29) found that the hydrocarbon chains of lipid-co-lipid pairs with a high level of asymmetry (C18:1 in lipid-C12:0 in co-lipid) and high symmetry (C16:0 in lipid-C16:0 in co-lipid) had the best transfection efficiency (see Figure 16). Regarding the above explanation, the asymmetry of the hydrophobic domains may afford better intermembrane mixing and the pDNA may be more weakly associated with high symmetry, thus leading to higher transfection levels. Numerous investigations have also succeeded in developing a highly efficient series of non-glycerol-based novel symmetrical transfection lipids (64-66). Recently, it was reported that the symmetrical disulfide lipid RPR 128522 (see Figure 17) without glycerol backbone formulated into cationic liposomes with DOPE had obtained significant transfection activity (62). Hyvonen et al. (64) synthesized a series of novel symmetrical cationic amphiphilic double-charged 1,4-dihydropyridine derivatives (1,4-DHP) (see Figure 18), which have been shown to condense DNA and efficiently transfect it into cells in vitro, and they were based on dihydropyridine ring backbone. Although there are many cationic lipids of the symmetrical structure for gene transfection, the in vitro activities of asymmetric lipids are usually superior to the symmetric analogues. Length of Aliphatic Chains. Much work has been carried out on modifying cationic lipids for use in gene transfection in order to determine if there is a “best” length of chain (29, 42, 45, 48, 51, 55, 60, 61, 64, 65, 70-77). However, solid conclusions are rarely obtained, for the conclusions of these studies are frequently contradictory. Most of the studies have indicated that cationic lipids with shorter chain length (for saturated chains) were generally important for acquiring high transfection efficiency, since they are responsible for membrane fluidity and good lipid mixing within the bilayer. For a series of 1,4-dihydropyridine amphiphiles with alkyl chains from C10 to C16 mentioned above, the C10 compound had the highest membrane destabilization activity and achieved the best delivery of DNA in the cytosol, but it did not transfect actively. The equivalent C12 compound, although a weaker endosomal disruptor, showed the highest transfection (64). In analyzing a series of cationic lipids that differ only by chain length (for saturated chains), most of the studies consider the suitable length to be C12 and C14.

Gilot et al. (77) synthesized a set of polyvalent cationic lipids (GB compounds) (see Figure 19) derived from natural glycine betaine compounds covalently linked to acyl chains by enzymatically hydrolyzable peptide and ester bonds. Their data indicated that GB compounds allowed high gene transfer efficiency in cultured hepatocytes without cytotoxicity, and

Figure 16. (A) Structures of the cytofectins used in this study (from top to bottom): GAP-DLRIE (C12:0); GAP-DLORIE (C12:1); GAPDMRIE (C14:0); GAP-DMORIE (C14:1); GAP-DPRIE (C16:0); GAPDPyRIE (16:0); GAP-DSRIE (C18:0); GAP-DORIE (C18:1). (B) Structures of co-lipids used in this study (from top to bottom): DLPE (C12:0); DMPE (C14:0); DPPE (C16:0); DPyPE (C16:0); DSPE (C18: 0); DOPE (C18:1).

Figure 17. Chemical structures of RPR 128522.


Figure 18. Chemical structures of 1,4-dihydropyridine derivatives. Figure 21. Chemical structures of the glutamide lipids.

Figure 19. Chemical structures of GB compounds.

Figure 20. Chemical structures of KD compounds.

unexpectedly, GB12/GB22 characterized by short acyl chains (C12) was most efficient. In the absence of serum, all cationic lipids tested with hydrocarbon chains in vicinal configuration esterified to a glycerol backbone (the respective N-(1,2-diacyldihydroxypropyl)-N,N,N-trimethylammonium chlorides or N-(1,2-diacyldihydroxypropyl)-N-(hydroxyethyl)-N,N-dimethylammonium chlorides, as well as N-(1,2-diacyldihydroxypropyl)-N-(1,2-dihydroxypropyl)-N,N-dimethylammonium chlorides with l-auroyl, myristoyl, palmitoyl, stearoyl, and erucoyl chains) were able to transfect cells when combined with DOPE. It was found that the cationic lipid with dimyristoyl chains demonstrated the highest transfection efficiency of plasmid DNA (78). A series of cationic lipids based on lysylaspartate (KD) (see Figure 20), diacyl derivatives with hydrocarbon chains of differing lengths (C12, C14, C16, and C18) exhibited different DNA-binding affinities which were correlated with their in vitro gene transferring capabilities. The KD derivatives with shorter acyl chains (C12 and C14) exhibited stronger DNA binding

affinity and more efficient transfection than those with longer (C16 and C18) acyl chains (79, 80). At the same time, the efficiency of cationic lipids with alkyls in the range 12-16 carbons in length was well-documented by Gopal et al. (81), and 14-carbon-length lipids were most efficient among several cationic lipids with different chemical structures (see Figure 21). Felgner et al. (59) reported the synthesis and assay results for several series of homologous lipids. They found that, as the length of the saturated aliphatic chain increased from 14 to 18 carbon atoms, the transfection activity of the resulting cationic lipids progressively declined. In order to understand the relationship between the length of aliphatic chains and transfection activity better, Floch et al. (82) synthesized a homologous series of cationic phosphonolipids with different alkyl chain substitutions. The order of transfection efficiency was disteryl (di-C18:0) < dipalmityl (di-C16:0) < dimyristyl (di-C14:0). The transfection efficiency and membrane fluidity of the analogues increased as the length of the aliphatic chains decreased. However, some degree of rigidity for the membrane is also required, in order to stabilize the liposome (74, 80). Long-chain lipids would probably strengthen the stability (42, 56, 74). From the interplay of these opposite trends, the optimal chain length depends on the specifics of a given overall cationic lipid structure to achieve the best balance. It should be mentioned that the shorter aliphatic chains allow for better transfection in vitro, whereas the longer aliphatic chains allow for better transfection in vivo (60, 82). Whatever the quaternary trimethylonium used for cationic phosphonolipids by using different neutral lipids in the transfection process, in vivo transfection efficiency of compounds involving a C18:1 chain was higher than with the C14:0 analogues and with the reverse in vitro (60, 82). It may be that the function of the neutral lipid is different in vitro from that in vivo in the formation of lipoplexes and depends on the nature of the quaternary trimethylonium used. In general, cationic lipids with the shorter aliphatic chains (C12 and C14) were more efficient than the longer (C16 and C18) counterparts in transfection efficiency. It is possible that shorter hydrocarbon chains increase the fluidity of the bilayer and favor a higher rate of intermembrane transfer of lipid monomers and lipid membrane mixing, resulting in potential disruption of the endosome and consequent DNA escape from endosomal degradation. Special Moieties at the Hydrophobic Chains. Some new vectors were designed to covalently connect some special moieties at the end of the hydrophobic chains, in order to get the relationship between hydrophobic chains and transfection efficiency. Jacopin et al. (83) synthesized a glycosylated analogue (see Figure 22) of the dialkylamidoglycylcarboxyspermines, which formed stable particles at low charge ratio and was efficient for gene delivery. Many groups also reported a few other glycosylated cationic bolaamphiphiles similar to the compound. Brunelle et al. (84)


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Figure 22. Glycosylated analogue of lipopolyamines.

Figure 23. Set of dissymmetric hemifluorocarbon bolaamphiphiles.

synthesized a new series of dissymmetric hemifluorocarbon bolaamphiphiles (see Figure 23), and the dissymmetric functionalization of diiodoperfluorooctane led to bolaamphiphile molecules composed of a partially fluorocarbon core end-capped with a glycoside and an ammonium salt. They found that the incorporation of two fluorinated segments in the molecular structure of the bolaamphiphiles is detrimental for an efficient DNA condensation. In contrast, a partial fluorination of the hydrophobic core of bolas increased not only the DNA complexation abilities, but also the DNA transfection efficiency. Relatively high transfection activity was obtained for the bolaamphiphile possessing a lysine headgroup and one fluorinated segment close to the carbohydrate moiety. This result could be explained by the small size and the positive surface charge of the bolaplex. Vierling’s group (85) described the synthesis of three new R-galacto-ω-polyamine double-chain bolaamphiphiles (Gal-CL) (see Figure 24), and their design combines a hydrophilic galactosyl residue (which serves as a molecular signal for cell targeting) linked through a long spacer

to various hydrophilic linear or branched polyamine polar heads (for the interactions with DNA and its compaction) and to a hydrophobic tail constituted by two long alkyl chains. The three new double-chain bolaamphiphiles, whether used with DOPE or with pcTG90/DOPE, were found to be more efficient nonspecific gene transfer agents than the single-chain head GalSper compound. A particular example has been found by Abe and co-workers, who reported two hydrophobic chains of a cationic lipid connected two ferrocenes, respectively (86, 87). Abbott et al. (88) demonstrated that a ferrocene-containing, redox-active cationic lipid (see Figure 25) can be used to offer either high (on) or very low (off) levels of cell transfection depending on a reduced or oxidized state. If successfully developed through future studies, the ability to activate lipoplexes toward transfection and “on demand” could create new opportunities to deliver DNA in vitro and in vivo. Balakirev et al. (89) synthesized a new cationic amphiphilic vector for gene delivery made from the natural provitamin, lipoic


Figure 24. Chemical structures of Gal-CL.

Figure 25. Chemical structures of ferrocene-containing dimethyldioctadecylammonium bromide.

acid. In addition, they demonstrated the principle of a redoxcontrolled gene delivery system that uses the reversibility of the thiol-disulfide exchange reaction. In the oxidized state, these amphiphiles condensed DNA into stable virus-sized complexes, which efficiently enter cells and released DNA in a reducing intracellular environment by various thiols as well as enzymatically, by thioredoxin reductase. Their data suggested that amphiphile-DNA complexes for gene transfection in vitro showed a several-fold increase of transgene expression compared with DOTAP. Their transfection efficiency can be further augmented by attachment of the nucleus-targeting peptide to

the amphiphile. Having been formed from the natural compound lipoic acid, these cationic amphiphiles might be biodegradable and nontoxic. Grinberg et al. (90) introduced the synthesis of a series of amphiphilic derivatives based on vernonia oil (see Figure 26). These compounds with three vernolic acid moieties bound together through a glycerol backbone proved to have good gene transfection properties, their structure is symmetrical, and their chains with some special moieties are branched and unsaturated. It is all these structure in cationic lipid that makes a combination


Figure 26. Chemical structures of amphiphilic derivatives based on vernonia oil.

Zhi et al.

Figure 28. Vitamin D-based cationic lipids.

Figure 27. Chemical structure of GL-67.

that facilitated the complexation of DNA and its transport across biological barriers.

STEROID HYDROPHOBIC DOMAIN In 1991, Gao et al. (58) reported the synthesis and application of the cholesterol-based cationic lipid DC-Chol, which was combined with DOPE to tranfect mammalian cells. Since then, considerable effort has been made in the synthesis of steroidal cationic lipids due to their potential applications in gene therapy (55, 57, 58, 91-100). Among the steroid groups, cholesteryl is by far the most frequently encountered. For cholesterol-containing cationic lipids, it has been shown that those with the cholesterol hydrophobic domain linked to a multivalent T-shaped headgroup were most efficient when instilled into murine lungs (91). An example is cationic lipid GL-67 (see Figure 27), one of the multivalent T-shaped headgroups, which has been found to be particularly efficient for gene transfer to cultured cells and in murine lungs (91). Then, Wu et al. (101) reported three cationic cholesterol derivatives differing in their types of amine and bearing a hydroxyethyl group at the amine group, synthesized and formulated into liposomes and nanoparticles as gene delivery vectors, and their formulations were optimized for gene transfection of the human lung adenocarcinoma A549 cell line. They found that the use of cationic lipids with a hydroxyethyl group at the tertiary amine headgroup showed the highest gene expression among cationic cholesterol derivatives lipids. Otherwise, Philippe Belmont and co-workers (102) demonstrated that they synthesized a cationic cholesterol derivative characterized by an aminoglycoside headgroup. The aminosugar-based cationic lipid was highly efficient for gene transfection into a variety of mammalian cell lines when used either alone or as a liposomal formulation with the neutral phospholipid DOPE. Their results showed the usefulness of cationic lipids composed of an aminoglycoside headgroup and cholesterol tail for gene transfection in vitro and in vivo. Other steroid compounds used as hydrophobic moieties for cationic lipids include vitamin D (103), bile acids (104), antibiotic (105), cholestane, and litocholic acid (63). Ren et al. (103) claimed that a new panel of steroidal cationic lipids (see Figure 26) using commercially available vitamin D2

Figure 29. Chlolestane and lithocholic acid-based cationic lipids.

(calciferol) or vitamin D3 (chole-calciferol) as hydrophobic motifs was synthesized for gene delivery. They found that the synthetic vitamin D-based cationic lipids 1d and 2d (see Figure 28) formulated with DOPE as a colipid exhibited superior transfection activity. Transfection results obtained from the experimentation suggested that these new vitamin D-based cationic lipids were useful for transfection reagents in vitro gene transfer studies. Kichler et al. (105) showed that a class of facial amphiphiles, which were made by conjugating different amines to a steroid nucleus, were able to efficiently mediate gene transfer when associated with DOPE and shown to possess significant antibacterial activity even on bacteria strains such as S. aureus while being poorly hemolytic. Therefore, they thought that this study could serve as a starting point for the development of new compounds which are able at the same time to deliver a gene medicine and kill bacteria. Fujiwara et al. (63) designed a novel potent class of DNA delivery agents based on cholestane and lithocolic acid, of which the former had a trans-decaline structure and the latter, a cis-decaline structure, different spacefilling character. The structure of the former was stretched, and the latter was angled-shaped. The former performed better in transfection efficiency than the latter (see Figure 29). A few examples showing cationic lipids using a bile acid scaffold had a similar shape to cholesterol but were more hydrophilic due to the presence of polyhydroxyl groups (104). A particular example has been found by Randazzo and coworkers (106), who reported that incorporation of a double bond in the bile acid moiety enhanced transfection efficiency significantly and produced two compounds with little cytotoxicity and transfection potency comparable to Lipofectamine 2000.


Figure 30. Chemical structure of the fluorinated and conventional cationic and helper lipids used and/or mentioned in this study. F-DOSPA, F-DMAEA, and F-DOGS F-PE are fluorinated lipids.

Lipids with steroid hydrophobic domain tend to rigidify the bilayers and are more stable in the bilayer structure.

FLUORINATED HYDROPHOBIC DOMAIN The fluorinated part of the hydrophobic chain can also influence the transfection efficiency of cationic lipids in vivo and in vitro. Many varieties of fluorinated cationic lipids have been developed as transfecting agents, which are very efficient in compacting DNA and delivering it to cells in vitro and in vivo cationic lipid-mediated gene delivery (107-111). Gaucheron et al. (111) described the synthesis of fluorinated glycerophosphoethanolamines (F-PEs) helper lipids, which showed a significantly higher transfection helper potential as compared with that of DOPE. Two fluorinated cationic lipids F-DMAEA and F-DOSPA (see Figure 28) were designed by Boulanger et al. (107). Their hydrophobic part consisted of two F(CF2)4(CH2)11 chains, which led to one of the most efficient gene transfer agents in the fluorinated DOGS series (with either both chains fluorinated to varying degrees or just a single chain fluorinated) (see Figure 30) (109, 110). Lipoplexes from the above compounds were very stable to storage and more resistant to detergent in vivo. Their data demonstrated that fluorinated lipids had higher transfection efficiency in vivo and in vitro. It also suggested that their enhanced hydrophobic and lipophobic character prevented them from disintegration and, consequently, from DNA degradation and from interactions with lipophilic and hydrophilic biocompounds. Vierling’s group (112) also described the synthesis of new perfluorinated dimerizable detergents with a tricationic or tetracationic (linear or branched spermine, respectively) polar head, which exhibited a higher in vitro and in vivo transfection potential than conventional lipoplexes or even than PEI polyplexes. Their production resulted in the development of tiny, monomolecular DNA nanoparticles that were obtained using new perfluorinated dimerizable detergents.

CONCLUSIONS In recent years, considerable progress has been made in defining conditions that favor or promote lipoplex-mediated transfection, particularly with regard to the structural features of cationic lipids. The extent of gene therapy using synthetic vectors depends on the development of new approaches to improve the features of cationic lipids for the purpose of facilitating their use in vivo. From the chemistry point of view,

the structure of cationic lipids is an important factor for their transfection activity and toxicity. Some common conclusions can be achieved by comparing the different structures and their transfection activity in the same family or different families of lipids. The transfection efficiency is not determined solely by one domain of cationic lipids but by the combination of them (35). The hydrophobic domain of cationic lipids mainly includes aliphatic chains, steroid domain, and fluorinated domain, which determines the phase transition temperature and the fluidity of the bilayer, and influences the stability of liposomes, the DNA protection from nucleases, amd the DNA release from complex. In the process of DNA transfer, cationic lipids protect DNA against degradation by nucleases and serum components, create a less negative surface charge, can be designed to target delivery to specific cell types through receptor-ligand interactions, and can also facilitate intracellular trafficking, which includes endosomal escape, cytoplasmic transport, and nuclear entry, while also having an important influence on the release of DNA to allow expression (113, 114). Some traditional cationic lipids have been used in gene therapy, many of which have two hydrophobic tails. The chain length, asymmetry, branching, and unsaturation affect transfection activity in vivo and in vitro. Most studies suggest that transfection increases with decrease of chain length or increase of chain asymmetry, branching, or unsaturation. It is possible that shorter, asymmetrical, branched, and unsaturated hydrocarbon chains decrease the phase transition temperature, increase the fluidity of the bilayer, and favor a higher intermembrane transfer rate and lipid mixing, resulting in potential disruption of the endosome and consequent DNA releasing from endosomal degradation (115). The introduction of some special groups (such as ferrocene, lipoic acid) in the tail of the lipid can improve the efficiency of transfection and reduce toxicity, and would be worth exploring further as an in vivo delivery system for gene therapy. There is a new strategy which is introducing some special chemical bounds (such as ester linkage) upon traditional hydrophobic tails of cationic lipids that show low toxicity level (3) and high transfection efficiency and may provide an optimistic outlook for the development of lipid-based systems that are more applicable for human gene therapy. It might be development trends for cationic lipids that hydrophobic chains with some special moieties are branched and unsaturated. Cationic lipids using cholesterol as a hydrophobic tail can also improve the efficiency of transfection. This is because the


formation of cholesterol offers rigidity and very stable bilayer structure, as well as its endogenous biodegradability and fusion activity. The hydrophobic domain of cationic lipids can not only directly affect the efficiency of transfection, but also indirectly influence transfection efficiency by the properties of the membrane (membrane fluidity and rigidity) that was formed from them. Further development in structure and properties of membrane will certainly rely on better understanding of vector interaction with plasmid and cellular components. Another trend of cationic lipid development is the substitution of some fluorinated groups for the hydrogens of two hydrophobic tails, which can improve transfection efficiency in vitro and in vivo. Fluorinated liposomes made from highly fluorinated doublechain phospho- or glycolipids, as well as fluorinated lipoplexes made from highly fluorinated polycationic lipospermines and reporter genes, have displayed a number of interesting physicochemical and biological properties (110). In general, it seems that, when researchers design cationic lipids for gene delivery, the balances between the opposing factors including fluidity and rigidity, symmetry and asymmetry, saturation and unsaturation, linearity and branching, short chain and long chain, and hydrophilicity and lipophilicity of lipids should be taken into great consideration. It would be very important for scientists to have a good understanding about the effect of the different hydrophobic domains of cationic lipids on transfection efficiency, which would be helpful to prepare novel cationic lipids and design novel formulations with high transfection efficiency.

ACKNOWLEDGMENT The study was supported by Program for New Century Excellent Talents in University (NCET-08-0654) and the National Natural Science Foundation of China (20876027).

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