DOTAP

Cationic liposomes form complexes with plasmid DNA. Also, in this case DOPE affects the architecture of the complexes. DOTAP liposomes without DOPE are able to condense DNA, while DOTAP/DOPE liposomes do not condense DNA, presumably due to Hn-phase formation [230,233]. Interestingly, multivalent cationic liposomes, such as DOGS, can condense DNA more efficiently than monovalent cationic liposomes [233]. If the complexation is carried out at high ionic strength the DNA condensation is less prominent than in plain water. 

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). 

Figure 2 Anti-HBsAg IgG titres ( SD) (Y-axis) in mice immunized with a single subcutaneous injection of small DRV liposomes composed of PC, DOPE, DOTAP, and DOGMann (molar ratios 4 2 1 1) containing pRc/CMV HBS DNA and the encoded antigen HBsAg ( ) or with HBsAg in the form of Engerix ( ). For other details, see the text. Abbreviations DRV, dehydration-rehydration vesicles PC, phosphatidylcholine DOPE, dioleoyl phosphatidylcholine DOTAP, 1,2-dioleyloxy-3-(trimethylamonium propane).

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. 

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.

Figure 4 Standard transfection profile of the cationic lipid 10-(cholesteryloxycarbo-nyl-methyl)-l,4,10-triazadecane acetate (CholAc43) (64) on COS-7-cells is shown. A standard transfection experiment comprised eight different lipid/DNA-charge ratios from 1 1 to 1 15 (x-axis). TE (luciferase activity) is expressed in relation to the TE of a standard lipid (DOTAP), determined in the same experiment (DOTAP = 100%, left bar). As a measure of toxicity, the protein content after the transfection experiment is shown in the same diagram (left y-axis). Abbreviation DOTAP, N-[l-2,3-dioleoyloxy)propyl-N,N,N-trimethyl-ammoniumchloride.

TE of KL-1-14 without helper lipids was very low and reached only about twice the TE, which was found for the standard lipid DOTAP. Independent of the amount of DOPE incorporated in the lipoplexes (ratio of DOPE/KL-1-14 0.3, 0.5, 0.6, 0.7, 0.8, 0.8, 1.0, and 1.2), transfection behaviors (maximum transfection efficiencies and transfection profiles) of all mixtures were similar and comparable to the profile of KL-1-14/DOPE (1 1) as shown in Figure 2 (individual data for all mixtures are not shown). 

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). 

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). 

Transfection efficiencies of the KL-1-14 lipoplexes were compared to the TE achieved with the standard transfection lipid DOTAP. Results were given in RLU (lu/pg protein) and, for easier comparison, standardized on the lipofection efficiency of DOTAP-lipoplexes, which was set to 100% Compared to the respective DOTAP-value. 

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. 

Porteous DJ, Dorin JR, McLachlan G, et al. Evidence for safety and efficacy of DOTAP cationic liposome mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis. Gene Ther 1997 4(3) 210-218. 

Simberg D, Weisman S, Talmon Y, Barenholz Y. DOTAP (and other cationic lipids) chemistry, biophysics, and transfection. Crit Rev Ther Drug Carrier Syst 2004 21(4) 257-317. 

Regelin AE, Fankhaenel S, Gurtesch L, Prinz C, von Kiedrowski G, Massing U. Biophysical and lipofection studies of DOTAP analogs. Biochim Biophys Acta 2000 1464(1) 151-164. 

Massing U, Kley JT, Gurtesch L, Fankhaenel S. A simple approach to DOTAP and its analogs bearing different fatty acids. Chem Phys Lipids 2000 105(2) 189-191. 

Crook K, Stevenson BJ, Dubouchet M, Porteous DJ. Inclusion of cholesterol in DOTAP transfection complexes increases the delivery of DNA to cells in vitro in the presence of serum. Gene Ther 1998 5(1) 137-143. 

The therapeutic efficacy of either systemic or local pulmonary delivery of the IFN-y gene was evaluated in a murine allergen-induced airway hyperresponsiveness (AHR) model (Dow et al. 1999) and it was found that a high efficiency of gene transfer could be achieved. Intratracheal administered cationic liposomes were prepared from a mixture of l,2-diacylglycero-3-ethylphosphocholine (EDMPC) and cholesterol. Intravenous injections were prepared from l,2-dioleyl-3-trimethylammo-ninm propane (DOTAP) and cholesterol and compared with pulmonary administered... 

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). 

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