Cationic lipids preparation

In contrast, solid lipid microparticles consisting of a tripalmitin matrix and cationic lipids prepared using the micromixer-based solvent extraction process as described by Emi et al. [50] were of monomodal size, showing a narrow size distribution in the submicrometer range (Table 8.1). 

Most cationic lipids described in the literature are synthesised by solution synthesis [36-39]. Depending upon the complexity of the structure, the synthetic routes vary from just one or two chemical steps, as in the case of DOTMA (1) and DC-Choi (3), to longer convergent synthesis, as for DOGS (5) (these three compounds being the earliest examples of cationic lipids prepared for transfection purposes [41, 68, 69]) (see Fig. 2). 

The use of liposomes for transfection purposes was first described in 1987. Cationic lipids prepared for this purpose are commercially available i.e. Cytofectintrade or Lipofectintrade). However, failure of transfection in the presence of serum is possible, but much more important are side-effects such as lung inflammatory reactions.The main drawback of the use of conventional degradable polymers as delivery agents is their thermodynamic instability, which results in a short in vivo lifetime of the active species, besides their polydispersity. ... 

However, Emi et al. [50] have prepared LS with a method established in their laboratory using a solvent extraction. In particular, the method is based on the dissolution of the triglyceride (i.e., tripalmitin) and the cationic lipid in the organic solvent (i.e., dichloromethane), and on the addition of an aqueous polyvinyl alcohol (PVA)... 

FIGURE 8.1 Percentages of DFT association to CLS containing as cationic lipid. (A) DDAB18 prepared in the presence of PVA (open diamonds) or gelatin (closed diamonds) (B) DDAB12 (open circles) or CTAB (open squares). 

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). 

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).

Cationic lipids cannot be dissolved in water and form aggregates in aqueous solution, such as bilayers. To prepare a homogeneous reagent, in most cases liposomes were made from cationic lipids in a first step. When it is not possible to form stable lipid bilayers (i.e., liposomes) using a single lipid, then it may be necessary to combine the cationic lipid with one or more so-called helper lipids like cholesterol (Choi) (41) or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) (42). 

A DNA-lipid complex was prepared by simply mixing two aqueous solutions of DNA and cationic lipids as follows. An aqueous solution of DNA Na" from salmon testes (ca. 30 000 bps or 500 bps) and an aqueous solution of 1.1 eq. mol of cationic amphiphiles were mixed at room temperature and... 

Compared to viral vectors, the potential advantages of synthetic carriers (also called non-viral vectors) are apparent. Being synthetic, they could be made safe, non-immunogenic, easy to prepare and cost-effective. DNA delivered by these carriers may not be able to replicate or recombine into infectious forms. Among many reported non-viral carriers, including cationic polymers (Behr et al., 1989 Kukowska-Latallo et al., 1996 Wu and Wu, 1988) and cationic lipids (Feigner, 1990 Lee and Huang, 1997), the most frequently used form is cationic liposomes. 

The structure of cationic lipids and polymers is readily amenable to chemical modification [35, 36] allowing the exploration of a virtually unlimited number of combinations and strategies at the mercy of chemists creative abilities. Various reviews have been focused on cationic lipids, dendrimers and polymers in terms of their chemical structures and their transfection properties [36—41], in an attempt to shed some light on the chemical requirements necessary to mediate gene delivery. The focus of this chapter will be to explore these carriers from a synthetic perspective, with a description of the chemical strategies used for the preparation via synthetic organic chemistry (excluding polymer synthesis) of cationic lipids and dendrimers. 

A number of cationic lipids have been prepared using solid-phase methods [147— 159]. Along with the well-known advantages that solid-phase chemistry provide (e.g. mass action, simple purification, compatibility with microwave synthesis [ 160, 161]), the main reason to use this approach is that it facilitates parallel synthesis of libraries of compounds, allowing potential structure activity relationships to be rapidly determined by the systematic modification of the cationic lipid structure per domain. 

The helper effects of DOPE and cholesterol appear to be hydrocarbon chain-specific. This is demonstrated in studies of their mixtures with a series of alkyl acyl carnitine esters (alkyl 3-acyloxy-4-trimethylammonium butyrate chloride) tested with CV-1 cell culture (monkey fibroblast) [127]. The influence of the aliphatic chain length (n - 12-18) on transfection in vitro was determined using cationic liposomes prepared from these lipids and their mixtures with the helper lipids DOPE and cholesterol (Fig. 30). Both helper lipids provided for significant transfection enhancements in an apparently chain-specific manner, with the highest effects found for short-chain lipids with diC12 0 and diC14 0 chains in 1 1 mixtures with the respective helper lipid. 

Although the squalene emulsion was demonstrated to be the most potent carrier for gene delivery among the cationic lipid formulations, it is not yet as efficient as the use of a viral vector, thus requiring additional enhancement of its transfection activity. Since the cationic lipid formulations can be designed by various lipids to improve their in vitro and in vivo transfection activity, Kim et al.152 prepared various... 

In conclusion, we have designed a synthetic vesicular DNA carrier that physically and functionally mimics an enveloped virus particle. To achieve an acceptable degree of encapsulation within the vesicle, we use a process that is essentially inverse to the preparation of cationic lipid-DNA complexes. A suitable DNA condensing agent is introduced that, at a certain critical concentration, conveys a weak net cationic charge to the condensed DNA that then interacts spontaneously with a liposome containing one or more anionic components. These DNA formulations behave distinctly different from classic cationic liposome DNA complexes in vitro in as much as they have been shown to be nontoxic, to display a traditional linear dose response, and to be serum-insensitive. 

Cationic liposomes are a relatively new development in liposomal therapeutics, which demonstrate considerable potential for improving the delivery of genetic material. The cationic lipid components of the liposomes interact with, and neutralize, negatively charged DNA, thereby condensing the DNA into a more compact structure. Depending on the preparation method used, the complex may not be a simple aggregate, but an intricate structure in which the condensed DNA is surrounded by a lipid bilayer. These systems are discussed further in Chapter 14. 

The improved DNA binding and condensation provided by amino acids such as tryptophan suggests that the inclusion of hydrophobic interactions within DNA complexes may be beneficial. Peptides with moities that provide cooperative hydrophobic behavior of alkyl chains of cationic lipids would improve the stability of the peptide-based DNA delivery systems. Two general classes of lipopeptide analogs of Tyr-Lys-Ala-Lysn-Trp-Lys peptides have been prepared by including a hydrophobic anchor. The general structures are N, N-dialkyl-Gly-Tyr-Lys-Ala-Lysn-Trp-Lys and Na,Ne-diacyl-Lys-Lysn-Trp-Lys. These peptides differ from the parent structures in that they self-associate to form micelles in aqueous solutions. The inclusion of dialkyl or diacyl chains in the cationic peptides improves the peptide ability to bind DNA and reduces aggregation of the complexes in ionic media. 

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). 

Lipoplexes at charge ratios (+/-) of 1-11 of cationic lipid to DNA were formed by addition of 3.16-11.1 xL of liposome preparation (1-3.75 mg total lipid/ml water) to 1 tg of DNA in 5 p.L of water with 10 rounds of pipetting with gentle shaking and leaving at room temperature for 10-15 min. 

Lipoplexes were prepared in a Hepes/mes/pipes buffer at pH = 8.0, at different charge ratios from liposomes obtained with compounds 2 and 4 described above, in association with the co-lipid DOPE (cationic lipid/DOPE 1 1), and were compared to those obtained from stable cationic lipids 1 and 3. 

Fluorescence level was low and stable over time when considering lipoplexes prepared with stable cationic lipid 3 (or 1) at charge ratios 1.7 and 6. 

Cationic lipid pDNA complexes (lipoplexes) generally are prepared by the simple mixing together of the two components however, it is also important to consider that the applied protocols for complex formation and subsequent modifications strongly influence the properties of the transfection particle. Also, the order of addition of components to form the lipoplex affects considerably lipofection activity. 

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