DOTAP:Chol

Asialofetuin is an asialoglycoprotein containing terminal galactosyl residues that have been used to target liposomes to the liver. There was seven-fold enhancement in CAT expression in the liver when succinylatcd asialofetuin was added to preformed plasmid/ DOTAP Chol complexes to provide a ligand for hepatic asialoglycoprotein receptor. 

DOTAP/Chol (1 0.9 molar ratio) liposomes were prepared by drying a chloroform solution of the lipids by rotary evaporation under reduced pressure. 

DOTAP Chol Cancer DNA Mda-7/IL-24 Mice/intravenous, local [89]... 

Cholesterol-fusl Liposome Complex (DOTAP Chol-fusl) fusl plasmid DNA Non-small cell lung cancer I NCT00059605... 

PS (anionic) or 4 nmol of SA, BisHOP, DOTMA, DOTAP, DODAB, or DC-CHOL (cationic) are also added (molar ratio 4 2 1). For cationic man-nosylated liposomes, 4 pmol of DOGP-4 a Man was included at the molar ratio of 4 2 1 1. Greater amounts of charged lipids can be added depending on the amount of vesicle surface charge required. 

S-labeled plasmid DNA (10-500 pg) was incorporated into( or mixed ( ) with neutral (PC, DOPE), anionic (PC, DOPE, PS, or PG), or cationic (PC, DOPE, SA, BisHOP, DOTMA, DC-CHOL, DOTAP, or DODAP) DRV. Incorporation values of the different amounts of DNA used for each of the liposomal formulations did not differ significantly and were therefore pooled (values shown are means of values obtained from three to five experiments). PC (16pmol) was used in molar ratios of 1 0.5 (neutral) and 1 0.5 0.25 (anionic and cationic liposomes). 

Abbreviations. HA, hemagglutinin DRV, dehydration-rehydration vesicles PC, phosphatidylcholine DOPE, dioleoyl phosphatidylcholine DOTAP, l,2-dioleyloxy-3-(trimethylamonium propane) BisHOP,, 2-bis (hexadecylcycloxy)-3-trimethylamino propane DC-CHOL, 3p(V,V,-dimethylami-noethane)-carbamyl cholesterol (DC-CHOL) DOTMA, V-[l-(2,3-dioleyloxy) propyl]-iV,V,V-triethylammonium PG, phosphatidyl glycerol PS, phosphatidylserine SA, stearylamine DODAP, l,2-dioleyloxy-3-(dimethylamonium propane). 

Cholesterol as well as unsaturated or saturated hydrocarbon chains are used as lipophilic lipid anchors. Although Cl 8-hydrocarbon chains (oleoyl or oleyl unit) are only used in unsaturated compounds, structural variations with C14-, C16-, or even Cl 8-hydrocarbon chains in saturated compounds are known (27). The lipophilic units are linked with a parent structure (usually glycerol) via ether (e.g., DOTMA) or ester bridges (e.g., DOTAP). Ester bridges are often used to create the linkage to avoid cytotoxicity, because ether bonds are more difficult to break down biologically than ester bonds (58). Substances that are easy to decompose and are therefore often used as a spacer are carbamate units (29) [e.g., 3p-[A-(A, A -dimethylaminoethyl)carbamoyl]-cholesterol (DC-Chol)], amide units, or phosphate esters. However, a direct correlation between toxicity and the... 

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.

For testing the transfection properties of KL-1-14 toward the mamma carcinoma cell lines MDA-MB-468 and MCF-7, the polarized cell line MDCK-C7, and the primary dendritic cells KL-1-14 was used in its racemic form with chloride as counterion and as a mixture with 60 mol% Choi. In comparison to the transfection efficiencies found for COS-7-cells (see above), the transfection efficiencies were generally lower (Table 2). For MDA-MB-468 and MCF-7, transfection efficiencies were reduced by a factor of about 10, the MDCK-C7-cells by a factor of about 80, and the dendritic cells by a factor of about 500. Nevertheless, the transfection efficiencies found for KL-l-14/Chol (1 0.6) were generally higher than for DOTAP, respectively. For the mamma carcinoma cell lines, transfection efficiencies with KL-1-14/ Choi (1 0.6) were four times higher than for DOTAP. For MDCK-C7 and dendritic cells, the increase was from 1.9- to 2.5-fold. We also tested KL-1-14 as equimolar mixture with DOPE for its transfection efficiencies toward the mamma carcinoma cell lines and the dendritic cells. Again, transfection efficiencies were greatly reduced even for the KL-1-14/DOPE mixture and were similar to the values found for KL-l-14/Chol (1 0.6). 

Abbreviations DOPE, l,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine DOTAP, iV-[l-(2, 3-dioleoyloxy)propyl]-A,iV,JV-trrmethylanimoniumchloride TE, transfection efficiency DC, dendritic cells RLU, relative light units chol, cholesterol. 

Ciani, L., Ristori, S., Salvati, A., Calamai, L., Martini, G. (2004) DOTAP/DOPE and DC-Chol/DOPE lipoplexes for gene delivery zeta potential measurements and electron spin resonance spectra. Biochim. Biophys. Acta., 1664(1), 70-79. 

Lipid moieties are usually integrated into the framework by alkylation or acylation reactions of amino and hydroxyl groups from the linking moiety. In solution approaches, this incorporation takes place at an early stage of the synthetic route (e.g. DOTMA (1) [42], DOTAP [70], DORI and DORIE [71], DC-Chol (3) [68], DOGS (5) [69]) (see Fig. 2). 

Headgroups with multiple cationic charge, e.g., DOSPA, DOGS [31], are claimed to be more efficient than single-charged lipids such as DOTMA, DOTAP, DC-Chol, DMRIE [32]. This may be related to the greater ability of... 

Motivated by its important role in gene delivery, we have studied the effect of cholesterol (chol) and several analogs on the transfection efficiency of lamellar CL-DNA complexes in vitro [27]. As evident from the results on DOPC/DOTAP and DOPE/DOTAP vectors, the nature of the neutral lipid component is an important parameter that is worth further exploration. Conveniently, a number of neutral lipids are commercially available. In addition, modifying the neutral lipid component has the potential to improve TE in a regime (at low aM) where DNA dissociation from the complex in the cytosol is not yet a barrier to transfection. 

As expected, due to the small headgroup area of cholesterol (Achol = 40 A2, while ADOpc = 72 A2) [40, 41], the membrane charge density of DOTAP/DOPC/ Chol-DNA complexes increases with cholesterol content. Exchanging DOPC for cholesterol reduces the total membrane area while the membrane charge, given by dotap = 0-3, remains constant thus cM increases. A particularly strong increase in cM occurs for Choi > 0.4, where part of the cholesterol is not incorporated in the complex. This results in an increased <2>DOTap and thus aM. 

DOTAP/DOPC MVL2/DOPC MVL DOPC MVL5/DOPC TMVL5/DOPC5 Chol/DOTAP/DOPC Universal Curve fit)... 

Fig. 5 (a) TE of DNA complexes of binary DOTAP/DOPC lipid mixtures (black circles). Their TE increases over several orders of magnitude with increasing molar fraction of monovalent DOTAP (lipid mixtures with constant <1>dotap = 0.3. Different symbol shapes correspond to different choi (cf. legend), (b) The TE of the DNA complexes of ternary DOTAP/DOPC/Chol lipid mixtures (empty circles) plotted against aM significantly deviates from the universal bell shaped curve observed for binary systems [21]. Reprinted with permission from [27]. Copyright 2009 American Chemical Society... 

Using the experimentally obtained values of aM calculated using (1), Fig. 5b plots TE of DOTAP/DOPC/Chol-DNA complexes (empty circles) as a function of membrane charge density, together with the universal curve and the TE data used for its derivation [21],... 

TE of the DOTAP/DOPC/Chol-DNA complexes strongly deviates from the universal bell-shaped curve observed for binary systems. The TE of cholesterol-containing complexes increases more rapidly with increasing cholesterol content than the increase in membrane charge density predicts for 0 < 4>chol < 0.4. No further TE increase is seen for 4>chol > 0.4 (where the membrane is saturated with cholesterol [Pg.201]

DOTAP A-(l-(2,3-dioleoyloxy)propyl)-jV,jV,ALtrimethylammonium chloride DC-Chol 3 3[jV-(AUV -di methylaminoethane)carbamoyl]-cholesterol CCS ceramide carbomoyl spermine. 

Positively charged liposomes containing 3.2-8 mmol of SA, BisHOP, DOTMA, DOTAP, or DC-CHOL. 

DOTAP l,2-diacyl-3-trimethylammonium propane [29a] DC-Chol 3[N-(N, N -dimethylaminoethane)-carbamoyl] cholesterol [28]... 

Fig. 6.34. Chemical structures of common cationic lipids 1,2-dioleyl-oxyproply-3-trimethylammonium bromide (DOTMA), 1,2,-dimyristyl-oxypropyl-3,3-dimethyl-3(2-hydroxyethyl) ammonium bromide (DMRIE), N,N-dimethyl-N,N-dioctadecylammonium bromide (DDAB), 1,2-dioleoyloxy-proply-3-trimethylammonium bromide (DOTAP), 3p-[N-(N,N-dimethylaminoethyl)carbamoyl] cholesterol hydrobromide (DC-Chol), and 1,2-dioleoylpropyloxyphosphatidylethanolamine (DOPE).

Fig. 4 Cationic lipids DOSPA 2,3-Dioleoyloxy-/ /-[2-(spermincarboxyamido)ethyl]-/V./V-dimethyl-l-propanamminiumchloride DOTAP 1,2-Dioleoyloxypropyl-3-N,N,N-trimethylamnnoniumchloride DC-chol 3/S-[N-(N, N -dimethyl-aminoethane)carbamoyl]-cholesterol DMRIE l,2-Dimyristyloxypropyl-3-/ /, N-dimethylhydroxyammoniumbromide [42, 43].

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