Liposomes and Lipoplexes

Liposomes and lipoplexes are usually self-assembling, easy to prepare and biodegradable. They allow increased uptake of naked DNA and DNA NPs. They can also be combined with polycations to form lipid-DNA NPs. Caracciola et al. [8] observed that lipid-protamine-DNA (LPD) NPs were more efficient than lipoplexes for gene delivery in CHO (Chinese hamster ovary cells), HEK293 (human embryonic kidney cells), NIH 3T3 (mouse embryraial cells) and A17 (murine cancer cells) cells. Unfortunately, cationic liposomes exhibit significant variability in gene delivery efficiency and are often toxic to cells. 

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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. 

We reported that greater transfection efficiency in medium with serum was obtained in human cervical carcinoma HeLa cells, using (I) DC-Chol/DOPE liposomes (molar ratio, 1 2) than liposomes (1 1 or 3 2), (2) a modified ethanol injection (MEI) method to prepare liposomes than the dry-film method (13, 14), and (3) a dilution method to form lipoplex than direct mixing. The physicochemical properties of liposomes and lipoplexes can be examined by measuring particle size. Transfection efficiency was evaluated by using plasmid DNA encoding luciferase gene and the cells. 

In the process of formation of lipoplex, the size of liposomes and lipoplexes was determined to be diluted in water within 5 min and 20 min after incubating liposomes and lipoplexes in optiMEM, respectively as shown in Fig. 5a, b (see Note 9). 

The zeta potential of AF-liposomes and lipoplexes showed clearly positive values by using amounts of AF lower than I pg. AF-complexes aggregated at 4.5 and 9 pg AF/pg DNA, which corresponds to a value of the zeta potential close to the electroneutrality (Fig. I). 

LPDI nanoparticles are homogenous, self-forming spheres between 100 and 200 nm in diameter that are formed from the spontaneous rearrangement of a lipid bilayer around a polycation condensed DNA core. The LPDI particles (lipopolyplexes) have benefits over lipoplexes, which are composed of liposomes and DNA. Homogenous particles are formed during preparation and thus allow a more consistent production of particles, as required by the FDA for clinical use. The LPDI particles also have a lower toxicity associated with them as opposed to lipoplexes, which can generate severe systemic inflammatory responses, most likely to the increased DNA content on the surface of the particles. The internalization of DNA inside the LPDI also has a benefit of DNA protection. The DNA is not nearly as accessible to nuclease attack and mechanical stress. Therefore, a lower quantity of DNA is used because it is protected inside of the LPDI for delivery. 

Liposome-mediated gene delivery is dependent on numerous factors, such as, the formulation of the liposomes including the cationic lipid/neutral lipid ratio, how the liposomes are prepared, the cationic liposome/DNA charge ratio of the complex of cationic liposome and DNA (lipoplex), and the method used to produce the lipoplex. Recently, it was reported that the way in which a liposome was prepared affected transfection efficiency (1), and formation method of lipoplex affected size of lipoplex in which large ones increased the efficiency of transfection (2-7). 

In regards of formation of lipoplexes, we used direct mixing of liposomes and DNA in water (non-dilution method) or dilution of liposomes and DNA separately in optiMEM (dilution method) for the formation of lipoplexes. 

Volume of concentrated stocks (cationic liposome and siRNA) calculated according to desired N/P ratio for preparing their dilutions and the siRNA-lipoplex at a desired N/P ratio... 

This is a general procedure for lipoplex formation based on the electrostatic interaction between the positively charged lipid in the liposome and the negatively charged phosphate backbone of the nucleic acid (pDNA or siRNA). 

DOPC). Neutral lipids are used to facilitate the formation of liposomes and to ease disassembly of the lipoplexes after internalization [62]. The choice of neutral lipid is also important for example, cationic liposomes made with DOTAP DOPE have shown higher transfection efficiency in mammalian cell culture than those made with DOTAP DOPC [63]. Although in vivo work has shown lower transfection efficiency of DOTAP DOPE than DOTAP alone following i.v. administration in mice [115]. 

Delivery of recombinant DNA to eukaryotic cells is not a trivial procedure. A method that has been used to help in the transformation includes presenting DNA in the form of complexes (lipoplexes) with cationic lipids, e.g. DOTAP A-([ 1 -(2,3-dioleoyloxy)propyl])-A,A,A-trimethylammonium chloride. Lipoplexes form spontaneously when cationic liposomes and DNA come into contact. One important parameter of lipoplexes, namely hydration, has been measured by D. Hirsch-Lemer and Y. Barenholz using DSC. In this case free water content is computed from the large ice-water transition endotherm at ca. 0 °C. The endotherm for the lipoplex is smaller (as low as 50 per cent in some cases) than the sum of the endotherms for each of the components, showing that dehydration occurs during lipoplex formation. Dehydration happens to be a prerequisite for the intimate contact between cationic lipids and DNA, and is probably instrumental in facilitating DNA transport across the cell membrane. 

Box 2 Thermograms of cationic liposomes, DNA, and lipoplexes. (a) DOTAP-DOPE (mole ratio 1 1) in H2O. Peak 1 gel-fluid transition of the lipid. Peak 2 melting of free water, (b) Plasmid DNA (0.95 mg) in excess water (1.31 mg), (c) Lipoplex with a DNA4ipid charge ratio of 1.5. The above thermograms indicate an amount of bound water of (a) 14 H2O molecules per lipid molecule, (b) 12 H2O molecules per DNA phosphate group, (c) 12.5 H2O molecules per (DNA phosphate group + lipid molecule). [Adapted from D. Hirsch-Lerner and Y. Barenholz (1999) Biochim. Biophys. Acta 1461 47-57, with permission.]... 

Sakaguchi N, Kojima C, Harada A, Koiwai K, Kono K (2008) The correlation between fusion capability and transfection activity in hybrid complexes of lipoplexes and pH-sensitive liposomes. Biomaterials 29 4029 1036... 

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 ( ).

Li S, Huang L. Functional polymorphism of liposomal gene delivery vectors lipoplex and lipopolyplex. In Janoff AS, ed. Liposomes Rational Design. New York Marcel Dekker, Inc., 1999 89. 

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).

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