Lipoplex

Zelphati, O., Nguyen, C., Ferrari, M., Feigner, J., Tsai, Y., and Feigner, P.L., Stable and monodisperse lipoplex formulations for gene delivery, Gene Therapy, 1998, 5, 1272-1282. 

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... 

Brunner S, Sauer T, Carotta S, Cotten M, Saltik M, Wagner E (2000) Cell cycle dependence of gene transfer by lipoplex, polyplex and recombinant adenovirus. Gene Ther 7 401 107... 

Whereas lipoplexes/polyplexes generally protect the plasmid from serum nucleases, the overall positive charge characteristic of these structures leads to their non-specific interactions with cells... 

Figure 14.10 Overview of cellular entry of (non-viral) gene delivery systems, with subsequent plasmid relocation to the nucleus. The delivery systems (e.g. lipoplexes and polyplexes) initially enter the cell via endocytosis (the invagination of a small section of plasma membrane to form small membrane-bound vesicles termed endosomes). Endosomes subsequently fuse with golgi-derived vesicles, forming lysosomes. Golgi-derived hydrolytic lysosomal enzymes then degrade the lysosomal contents. A proportion of the plasmid DNA must escape lysosomal destruction via entry into the cytoplasm. Some plasmids subsequently enter the nucleus. Refer to text for further details...

The administration of SPLP results in reporter gene expression at the tumor site (Fig. 7B). Injection of free plasmid or lipoplexes resulted in no detectable gene expression at the tumor site. However, transfection was observed in the limg, liver, and spleen. SPLP, on the other hand, did not show detectable levels of gene expression in these organs. 

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). 

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. 

The LPDI can cause release of Thl cytokines, most notably TNF-alpha, IFN-gamma, and IL-12 but to a much lower degree than lipoplexes. Also, LPDI nanoparticles can activate the professional APCs, macrophages, and dendritic cells. 

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. 

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. 

As indicated in Figure 1, the process of lipofection can be divided into independent steps (i) preparation of a lipofection reagent, (ii) formation of lipoplexes, and (iii) the transfection itself. 

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).

Once inside the cell, the lipoplexes are located in the endosomes, which apparently do not fuse with lysosomes. Rather, a considerable number of endocytotic vesicles accumulate in the vicinity of the nucleus after a few hours (52,53). Investigations carried out using fluorescence-labeled lipoplexes show that lipoplexes can be detected in the cytosol in almost every cell that has been treated. 

It is discussed that the addition of the helper lipid DOPE (see above) increases the release of DNA from the lipoplexes in the endosomes and... 

COS-7 or CHO cells (for initial transfection screening) or cells of therapeutic interest (e.g., dendritic cells and various cancer cells) at a confluence of 50%, grown in 96-well culture plates, were placed into the robot by the robotic conveyor. In a fully automated process, the robot removes the lid from the cell culture microtiter plate, dispenses lipoplexes into the wells (triplicates), replaces the lid and returns the plate to the incubator. After four hours, the cells are automatically retrieved, the cell monolayers are carefully washed using a special drop mode of the integrated plate washer, fresh medium is added, and the cells are incubated for further 42 hours before harvesting. 

The previous screening experiments were performed with lipoplexes containing equimolar amounts of the helper lipid DOPE. Here, the influence of different ratios of the helper lipids DOPE and Choi on TE of KL-1-14 were tested. The transfection behavior of KL-1-14 without any helper lipid was tested as well. 

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). 

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.

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. 

Dass CR, Burton MA. Lipoplexes and tumours. A review. J Pharm Pharmacol 1999 51(7) 755-770. 

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