Quantitative physical characterization of lipid–polycation–DNA lipopolyplexes

Abstract

Quantitative assays for the characterization of multi-component lipopolyplexes and their individual constituents are crucial for determining the consistency of formulation protocols which are ultimately reflected in biological activity. Lipid-polycation-DNA formulations consisting of lipids, polycations and DNA are of interest because they have been demonstrated to be efficient gene-delivery vehicles when administered systemically. We have developed a panel of analytical techniques to characterize these lipopolyplexes. Complexes were measured for size by dynamic light scattering and surface-charge characteristics by zeta potential. Interaction between DNA and the polycation, protamine sulphate, was determined using a PicoGreen dye-exclusion technique. Total DNA in the lipopolyplex was assayed through decomplexation of the formulation by addition of heparin sulphate and subsequent DNA quantification by PicoGreen reagent. Protamine sulphate in the lipopolyplex was determined using a novel Amido Black-staining protocol which is linearly sensitive in a range of 0.25-3 microg of protein. Lipids were quantified by HPLC after extraction in chloroform/methanol (2:1). In this method elution is conducted over 40 min, with 1,2-dioleoyl-3-trimethylammonium-propane and cholesterol being resolved by greater than 10 min. Such assays are essential for product characterization and release tests, as well as development of a better understanding of the correlation between physical structure and biological function.

The synergy between structural stability and DNA-binding controls the antibody production in EPC/DOTAP/DOPE liposomes and DOTAP/DOPE lipoplexes

“…The effectiveness of the Pico Green fluorescent probe was also explored by Tsai et al [53], who identified the quenching of the fluorescence signal at increasing complexing protamine/DNA ratios. The fluorescence signal is proportional to the accessibility, that means the availability of DNA to complexation to the probe.…”

Investigation on characterization and transfection of a novel multi‐polyplex gene delivery system

“…8 In 1997, Huang and coworkers 9 introduced cationic polypeptide protamine into liposome to form lipid-polycation-DNA lipopolyplexes (LPD), which have appeared promising as efficient gene-delivery vehicles for systemic administration. 10 Therefore, according to its constructional mechanism, the combined use of polycationic peptide and biodegradable macromolecular polymer to be a novel multi-polyplex gene delivery system is presented. PLGA microspheres containing PLL-pDNA were formulated by Capan et al 11 and PLGA-grafted PLL (PLL-g-PLGA) micelles were produced by Jeong.…”

Intracellular trafficking of nonviral vectors

“…[3][4][5] Combinations of cationic liposomes and polymers with DNA have been called lipopolyplexes. 6,7 Molecular conjugates, in this review, will refer to combinations of peptides or proteins with DNA. These vectors may also include combinations of peptides or proteins with either of vectors 1-3 in Table 1.…”