Lipids Mediated Gene Transfection

Introduction

The effort to bring membrane-impermeable DNA into cells, commonly called transfection. Cationic lipids have attracted much attention as transfection reagents and carriers for gene therapy^[1]. The cationic lipids as transfection reagents own many advantages, including their: (a) robust manufacture; (b) ease in handling & preparation techniques; (c) ability to inject large lipid-DNA complexes and (d) low immunogenic response^[2]. Alfa Chemistry briefly introduced and summarized the related knowledge of cationic lipid-mediated gene transfection, in order to provide some theoretical basis and help for the researchers who study the system.

Why Cationic Lipids?

Cationic lipids are amphiphiles, which can spontaneously form the large spherical structures known as liposomes when exposed to an aqueous environment. Because of their opposite surface charge, cationic liposomes can form an overall positively charged complex with negatively charged DNA molecules. The complex is lipid-DNA complexes, also known as lipoplex. Lipoplex do not experience the electrostatic barrier faced by the naked DNA in entering biological cells and gets endocytosed by the cell plasma membrane. In addition, it protects DNA from attack by the enroute DNases and may effectively deliver DNA to the target cells^[2].

Fig. 1. Structures of some efficient cationic transfection lipids.

Lipoplex Formation

The step of lipoplex formation is usually rather simple, since only one needs to mix the cationic lipid-containing formulation with a given concentration of the polynucleic acid. Because DNA is a polyanionic entity that can occur a self association with the positively charged cationic lipid formulation and then the supramolecular assemblies are formed. However, preparation of lipoplex of homogenous particle size is an art. The concentrations of lipids and DNA, the ionic strength, the temperature of solution, the order of addition, and the rate of mixing all affect the size and homogeneity of the lipoplex. Empirically, when the limited component is added to the one in excess, the most uniform lipoplex can be obtained. In addition, with a carefully controlled mixing procedure in the presence of detergents and using the dialysis method, uniform lipoplex structure can be obtained^[3].

Lipoplex Characterization

• Basic characterization: lipoplex has been generally characterized by three major parameters: the size of the particle formed, the percentage of DNA compaction and the particle zeta potential. The diameter of the lipoplex is measured by dynamic light scattering. The percentage of DNA compaction is performed via a fluorescence test consisting by measuring the reduction in the fluorescence intensity of the DNA-bound ethidium. And evaluation of the charge on the surface of the lipoplex may be realized by a direct measurement of their electrophoretic mobility, which is directly correlated to their zeta potential value^[3]. By the way, the diameter and zeta potential of lipoplex are related to lipid/DNA charge ratio, as shown in Fig. 2.

Fig. 2. Diameter (nm) (square) and zeta potential (mV) (triangles) of lipid/DNA assemblies as a function of the charge ratio lipid/DNA.

• Structural characterization: Transmission electron microscopy (TEM) and its derivative techniques have been used to characterize the structural features of the lipoplex. A variety of polymorphic and metastable structures, oligolamellar and tubular structures have been described by these technologies. In addition, high resolution synchrotron small-angle X-ray scattering (SAXS) and ³¹P NMR are often used for structural characterization of lipoplex.

Improvement of Transfection Efficiency of Lipoplex

The chemical structure of the cationic lipids has a huge impact on the transfection efficiency of lipoplex. Even small structural changes can lead to vastly different transfection efficiency. In order to improve the transfection efficiency, two approaches to optimize the chemical structure of cationic lipids have been pursued. One approach is to carry out systematic modification of the cationic lipid structure in order to discover a structure activity relationship. This semi-empirical feedback method can be used to screen the cationic lipids with good transfection efficiency. Another approach is based on the rational design of the structure, in order to address one or several biological or practical barriers affecting the transfection. For examples, an ester linker, or a substrate for endogenous esterases are used to modify the lipids to decrease the cytotoxicity of the vector. And lipids coupled to poly(ethylene glycol) (PEG) can evade the immune system and also to prevent aggregation of lipoplex^[4]. Both of two examples can improve the transfection efficiency of lipoplex.

Lipoplex Transfection Barriers

The barriers to successful gene delivery of lipoplex can be broadly divided into two classes: cellular barriers and systemic barriers.

• Cellular barriers: As shown in Fig. 3, the steps involved in cationic lipid-mediated gene delivery are A, adhesion of the lipoplex on cell membrane; B, cell uptake; C, liberation of the particles in the cytosol and intracellular traffic; D, nuclear localization or uptake; E, transcription and F, translation^[3]. In order to achieve the purpose of gene delivery, each of steps is an obstacle that needs to be successfully overcome by lipoplex.

Fig. 3. Generally recognized process of cellular transfection by lipoplex.

• Systemic barriers: Systemic barriers, also known as biological barriers, are the factors that hamper the specific transport of the transfection vectors to the affected organ sites. The biological barriers include blood compartment, serum proteins, cellular elements like erythrocytes, platelets, etc.

A Lipoplex can effectively overcome the above cell barriers and system barriers to successfully complete the gene delivery, indicating that lipoplex has excellent transfection efficiency.

As a leading global supplier of lipids, Alfa Chemistry has accumulated extensive knowledge and experience in lipids mediated gene transfection. If you need any help, you can contact us and we will provide you with professional advice and solution. In addition, we deliver a wide range of high-quality lipids and offer superior lipid-related services. Please feel free to contact us if necessary.

References

1. Liu D., et al. Cationic transfection lipids[J]. Current medicinal chemistry, 2003, 10(14): 1307-1315.
2. Kumar V., et al. Cationic transfection lipids in gene therapy: successes, set-backs, challenges and promises[J]. Current medicinal chemistry, 2003, 10(14): 1297-1306.
3. Nicolazzi C., et al. Cationic lipids for transfection[J]. Current medicinal chemistry, 2003, 10(14): 1263-1277.
4. Niculescu-Duvaz D., et al. Structure-activity relationship in cationic lipid mediated gene transfection[J]. Current medicinal chemistry, 2003, 10(14): 1233-1261.