Cationic lipid structure

Ferrari M, et al. Trends in lipoplex physical properties dependent on cationic lipid structure, vehicle and complexation procedure do not correlate with biological activity. Nucl Acids Res 2001 29 1539. 

Attempting to improve transfection efficiency, the inclusion of enzyme, pH or redox susceptible chemical groups in the cationic lipid structure has been investigated [126-129] with, for example the synthesis of lipid moieties connected through ester bonds sensitive to intracellular esterases [64, 70, 71, 126]. 

A number of cationic lipids have been prepared using solid-phase methods [147— 159]. Along with the well-known advantages that solid-phase chemistry provide (e.g. mass action, simple purification, compatibility with microwave synthesis [ 160, 161]), the main reason to use this approach is that it facilitates parallel synthesis of libraries of compounds, allowing potential structure activity relationships to be rapidly determined by the systematic modification of the cationic lipid structure per domain. 

Fig. 1 Examples of cationic lipid structural components polar headgroups, linkers, and hydro-phobic moieties...

Lipid. Lipids or surfactants had been used widely in the delivery of pharmacologically active materials in the form of liposomes, emulsions, or micelles. Since the first description of their potential for exogenous gene transfer, much progress has been made in the development of improved cationic lipid structures and formulations with enhanced gene transfection activity. 

Subramanian G, Hjehn RP, Deming TJ, Smith GS, Li Y, Safinya CR (2000) Structure of complexes of cationic lipids and poly(glutamic acid) polypeptides a pinched lamellar phase. J Am Chem Soc 122 26-34... 

Figure 14.8 Structure of some cationic lipids and polylysine...

Initial vaccination studies with LPDI nanoparticles were completed using liposomes prepared with both 1,2-dioleyltriammonium propane (DOTAP) and cholesterol. After it was determined that cholesterol played only a small structural role and was not necessary for activity, the liposomes were then prepared using only DOTAP to become an LPDI type of formulation. Regardless of the lipid used, the ratio of cationic lipid, polycation, and DNA must be maintained to have all properties associated with LPDI particles (2). 

Despite the fact that many different cationic lipids have been synthesized and tested for transfection (25 34), relatively few systematic structure activity TE-relationship studies have been performed (35 39). As a result, no general relationship between chemical structure and TE could be drawn from these studies. One reason for this is that the chemical structure of a cationic lipid is not directly responsible for TE. TE rather depends on the biophysical characteristics of the cationic lipid aggregate (e.g., liposomes and lipoplexes), which, for its part, is dependent on the chemical structure of the lipids. In a previous study with analogs of the transfection lipid A-[l-(2,3-dioleoyloxy) propyl]-A,A,A-trimethylammoniumchloride (DOTAP) (40) which differ in their nonpolar hydrocarbon chains, it could be shown that the TE strongly depended on the biophysical properties of the resulting liposomes and lipoplexes (35). Minimal alterations of biophysical properties by using lipids with different hydrocarbon chains or by mixing the lipid with different neutral helper lipids could completely allow or prevent transfection. 

The structure of a cationic lipid can be broken-down into three structural elements a lipophilic lipid anchor comprising one or—mostly—two long alkyl chains or Choi, a spacer, and a polar, positively charged head group consisting of one or more quaternised or protonatable amino groups. Figure 2 shows a few of the well-known, older cationic lipids, which can be classified as either monocationic or polycationic lipids. A series of recently synthesized cationic lipids will be discussed later. 

Figure 2 Examples of cationic lipids, differing in the head group structure (mono/ poly cationic) and the nonpolar lipid anchor (Chol/hydrocarbon chains). Abbreviations DOTAP, A-[l-(2,3-dioleoyloxy)propyl]-A,A,7V-trimethyl-ammoniumchloride DOTMA, A-[l -(2,3-dioleyloxy)propyl]-A A, A-trimethylammoniumchloride DC-Chol, 3 P"[A-(A, A -dimethylaminoethyl)carbamoyl]-cholesterol DOGS, A, A-dioctodecyl-amidoglycylspermin DORI, A-(l, 2-dioleoyloxypropyl)-A,A-dimethyl-A-hydroxyethyl-ammoniumbromide SpdC, spermidin-cholesterol.

In this section, an example will be given in which a (small) library of a new type of cationic lipids was synthesized and screened for TE (63). For synthesis, combinatorial solid phase chemistry was used. All cationic lipids of the example library are structurally based on 3-methylamino-1,2-dihydroxy-propane as the polar, cationic lipid part. As nonpolar lipid part, different hydrocarbon chains are boimd to the amino group of the scaffold and the amino group was further methylated to get constantly cationic-charged lipids. Lipids were synthesized in both configurations and as racemats, and the counterions were varied as well. Table 1 summarizes the structural features of these lipids. 

The solid-phase synthesis strategy was based on the utilization of 4-methoxy-trityl chloride resin. To gain access to a large number of compounds, only commercially available building blocks were used and protective groups were omitted if possible. The synthesis strategy resulted in a new class of cationic lipids as shown in Figure 5 (compound 6). The structure bases on... 

Methylation of KL-1-14 was an important prerequisite for its transfection properties. A KL-1-14-analog, which was not methylated, did not transfect at all. It could be assumed that the nonmethylated KL-1-14 was not sufficiently protonated at physiological pH, so that the formation of a bilayer structure from these lipids is not possible. As previously shown for DOTAP-analogs, formation of lipid bilayer is an important prerequisites for a cationic lipid to be a transfection lipid (35,47). 

Lee ER, Marshall J, Siegel CS, et al. Detailed analysis of structures and formulations of cationic lipids for efficient gene transfer to the lung. Hum Gene Ther 1996 7(14) 1701-1717. 

Byk G, Dubertret C, Escriou V, et al. Synthesis, activity, and structure-activity relationship studies of novel cationic lipids for DNA transfer. J Med Chem 1998 41(2) 229-235. 

It was believed that the main factors affecting transfection efficiency were the structure of the cationic lipid, the type of helper lipid used and their susceptibility to disruption by serum proteins. For gene transfer in vivo, apart from DOTMA-based liposomes, other complexes (in equimolar ratios) are also used—such as dioctade-cylamidoglicylspermidin (DLS)/DOPE (137), DOPE/DOTMA (1 1), DOPE/DOTAP (1 1) (138, 139), dimethyloctadecylammonium bromide (DDAB), and DOTAP with cholesterol (1 1) (mol/mol) (139). 

Structure and structure-activity correlations of cationic lipid/DNA complexes supramolecular assembly and gene... 

DOTAP ratios. SAXS data of complexes with 4>DOPE=0.26 and 0.70 clearly show the presence of two different structures. At DOpE=0.26 SAXS of the lamellar complex shows sharp peaks at f/um =0.099 A 1 and oo2=0198 A-1 resulting from the lamellar periodic structure (d =2%lqom=63A1 A), with DNA intercalated between cationic lipid analogous to the structure in DOPC/DOTAP-DNA complexes (Figure 10.1, left). 

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