Lipoplexes cationic

Amplicon-based Aptamers Ribozyme Naked DNA Lipoplexes Cationic lipids Molecular conjugates... 

Cationic liposomes composed of 3(3- [ N- (N N-dimethylaminoethane (carbamoyl] cholesterol (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) (DC-Chol/DOPE liposome, molar ratio, 1 1 or 3 2) prepared by the dry-film method have been often used as non-vtral gene delivery vectors. We have shown that a more efficient transfection in medium with serum was achieved using DC-Chol/DOPE liposomes (molar ratio, 1 2) than those (3 2), and preparation method by a modified ethanol injection than the dry-film. The most efficient DC-Chol/DOPE liposome for gene transfer was molar ratio (1 2) and prepared by a modified ethanol injection method. The enhanced transfection is related to an increase in the release of DNA in the cytoplasm by the large lipoplex during incubation in opti-MEM 1 reduced-serum medium (optiMEM), not to an increased cellular association with the lipoplex. Cationic liposomes rich in DOPE prepared by a modified ethanol injection method will help to improve the efficacy of liposome vector systems for gene delivery. 

Figure 7 Pharmacokinetic properties and in vivo gene expression of stabilized plasmid-lipid particles (SPLP). (A) The levels of intact plasmid DNA (pDNA) in the circulation resulting from IV injection of naked plamid pDNA ( ), lipoplexes (O), and SPLP ( ) were determined by Southern blot analysis of plasma samples (100 pg pDNA/mouse). (B) Transgene expression at a distal tumor site resulting from rv injection of naked plamid pDNA ( ), plamid pDNA-cationic liposome complexes (O), and SPLP ( ).

When discussing the ability of nucleic acid to enter the nucleus, size is the most important consideration (4). Oligonucleotides less than 100 base pairs in length can freely diffuse into the nucleus and oligos of 20 to 30 base pairs in length will accumulate in the nucleus when administered by either cationic lipoplex or cytoplasmic injection (5). 

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. 

Figure 1 The principles and variant parameters of lipofection. (i) Preparation of a lipofection reagent cationic liposomes were prepared from cationic lipids and helper (if required), (ii) Formation of positively charged lipoplexes by addition of DNA (e.g., reporter plasmid carrying the firefly luciferase gene) to the cationic liposomes, (iii) Transfection (lipofection) by incubation cells with the preformed lipoplexes. The efficiency of gene transfer (lipofection efficiency) can be determined from reporter gene amount or activity (e.g., luciferase activity). Most of the steps of a lipofection experiment can be varied and optimized (grey spots).

Figure 6 Lipofection results (lipofection profiles) of lipoplexes from the R-configu-rated cationic lipids KL-1-1 to KL-1-17 (Table 1) in a mixture with equimolar amounts of l,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) (counterion chloride) and the pCMVluc-plasmid. Each bar represents the mean ( S.D.) of three wells of a 96-well microtiter plate. T-axis (left) represents the transfection efficiencies expressed in relative light units (RLU) (lu/pg protein). X-axis (right) represents the viability of the cells compared to nontreated control cells. F-axis represents the different cationic lipid/plasmid DNA-charge ratios from 1 to 15.

Opsonization by complement components also represents a potential barrier for intravenous gene delivery. Cationic charges of the particles activate the complement, which then takes part in particle elimination. This hurdle is possibly limited by using short hydrophobic chains, reducing the particle size, and eventually PEG insertion into lipoplexes (18). The interaction effect between the lipoplex and the complement might not be such a limitation. Indeed, it was reported that depletion of complement by injection of cobra venom factor and anti-C3 antibodies in mice indicated no differences upon intravenous injection of lipoplexes, neither in terms of tissue distribution nor in lipofection efficiency (19). 

In the case of local administration, lipoplexes are generally retained at the site of injection, with poor dispersion (22). In contrast to small emulsions or neutral liposomes, which immediately appear in the venous outflow perfusate following intratumoral injection, the appearance of cationic liposomes is highly restricted to the injection zone (22). The authors deduced that the determining factor altering the pharmacokinetic properties is not the rate of transfer from the interstitial space to the vascular site but rather the rate of transfer from the injection site to the well-vascularized region (23). 

An alternative to the conventional PEG-PE was to incorporate between the PEG and the lipidic chain, a linker bearing anionic charges conveniently positioned in order to interact with the free amines of the cationic lipid within the lipoplexes (8). 

Figure 4 Effect on the serum stability of the incorporation of 5% cholesterol poly(ethy-lene glycol) (PEG) into cationic lipoplexes [hpopolyamine RPR209120/DOPE1/1, ratio (mol) lipid/DNA = 10 in 150 mM NaCl]. Lipoplexes were incubated in DMEM + 10% SVF, at 37°C, aliquots were regularly sampled and monitored by dynamic diffusion. Results represent a mean between three measurements. Error bars are not presented to simplify the graph, but differences among PEG, PEG-1, and PEG-2 are significant. Abbreviations PEG, poly(ethylene glycol) DOPE, dioleylphosphatidylethanolamine DMEM, Dulbecco s Modified Eagle Medium.

In addition to extracellular degradation in tissues, endosomal acidification might also trigger PEG-lipid cleavage. We showed that despite the presence of the PEG, which slightly reduces lipoplex internalization into the cells, DNA transfection level almost reaches the level of the cationic lipo-plex (31). Cholesterol PEG incorporation into lipoplexes not only reduces lipoplex internalization, but also inhibits the transfection efficiency. 

This strategy applied to lipoplexes did not reach the expectations. Complete masking of the cationic charges was not achieved by PEG shielding on the surface of the cationic particles. Hence, the distribution profile that can be found in the literature do not impress much as compared to... 

Nicolazzi C, et al. Anionic polyethyleneglycol lipids added to cationic lipoplexes increase their plasmatic circulation time. J Control Rel 2003 429. 

Tandia B, et al. Lipid mixing between lipoplexes and plasma lipoproteins is a major barrier for intravenous transfection mediated by cationic lipids. J Biol Chem 2005 280 12255. 

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. 

Zhdanov, R.I., Podobed, O.V., Vlassov, V.V. (2002). Cationic lipid-DNA complexes-lipoplexes-for gene transfer and therapy. Bioelectrochemistry, 58(1), 53-64. 

Figure 11.1 The intracellular trafficking pathway of plasmid DNA complexed by poly cationic lipid (lipoplex). Critical steps are indicated by numbers (1) endocytosis, sorting and recycling via vesicular compartments comprising the early (EE) and sorting endosomes (2) entrapment and degradation in the late-endosomes (LE) and lysosomes (3) destabilization of the endo-lysosomal membrane and release into the cytosol, (the precise location of this step is not known) (4) diffusion toward the nuclear pore complex (NPC) and degradation in the cytoplasm, and (5) nuclear translocation across the NPC.

Cationic lipids and polymers (including dendrimers) deliver DNA and siRNA into cells via cargo compaction to give lipoplexes or polyplexes (or dendriplexes), respectively, of various sizes which are then taken up via non-specific endocytosis (see Fig. 1). Once these complexes have been endocytosed the cargo has to be released into the cytoplasm to avoid degradation by the lysosomes. This escape is based upon the ability of the carrier to dismpt endosomal integrity [29-33] and is a critical step in the process. Once in the cytoplasm, the genetic material needs to be... 

Keywords Cationic lipid, Lipoplex, Nucleic acid, Phase transition, Transfection... 

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