DNA Delivery Systems Based on Peptide-Mimicking Cationic Lipids—The Effect of the Co-Lipid on the Structure and DNA Binding Capacity

In continuation of previous work, we present a new promising DNA carrier, OO4, a highly effective peptide-mimicking lysine-based cationic lipid. The structural characteristics of the polynucleotide carrier system OO4 mixed with the commonly used co-lipid DOPE and the saturated phospholipid DPPE have been studied in two-dimensional and three-dimensional model systems to understand their influence on the physical–chemical properties. The phase behavior of pure OO4 and its mixtures with DOPE and DPPE was studied at the air–water interface using a Langmuir film balance combined with infrared reflection-absorption spectroscopy. In bulk, the self-assembling structures in the presence and absence of DNA were determined by small-angle and wide-angle X-ray scattering. The amount of adsorbed DNA to cationic lipid bilayers was measured using a quartz crystal microbalance. The choice of the co-lipid has an enormous influence on the structure and capability of binding DNA. DOPE promotes the formation of nonlamellar lipoplexes (cubic and hexagonal structures), whereas DPPE promotes the formation of lamellar lipoplexes. The correlation of the observed structures with the transfection efficiency and serum stability indicates that OO4/DOPE 1:3 lipoplexes with a DNA-containing cubic phase encapsulated in multilamellar structures seem to be most promising.


Calculation of the theoretical mass for QCM-D
The measured data were compared with theoretically calculated values obtained by the assumption that a lipid bilayer consist of the monolayers, which means that two molecules in a bilayer (one above the other) require the same area such as one molecule in a monolayer. In this case, the molecular mass M is twice the value for a monolayer. According to the lateral pressure obtained in biological membranes, 1 the molecular area A M was taken from the π-A-isotherms at a surface pressure of 30 mN/m in order to estimate a theoretical mass deposition m theoretical . Since the number of molecules at one cm² can be calculated from its molecular mass and the molecular area via the basic relations below: Equation S1 Equation S1 represents in principle the lipid solution spread on the Langmuir trough with c being the concentration of the lipid solution [

Equation S2
The mass of a single molecule is defined by its molecular mass M [g/mol = 10 9 ng/mol] and the Avogadro constant (N A = 6.0221412927•10 23 mol -1 ) as shown in equation S3.

Equation S3
Finally, the theoretical mass deposition m theoretical can be used to interpret the results gained by QCM. In literature, a frequency shift of 13 Hz indicates a lipid monolayer, consequently, but also proofed, a lipid bilayer causes a frequency shift of 26 Hz, and for vesicle adsorption a frequency shift of 90 Hz was reported. 2 In this study the well-investigated phospholipid 1,2-dimyristoyl-sn-glycero-3phosphocholine (DMPC) was used as a reference for evaluation of obtained results.
The theoretical mass of calf thymus DNA was estimated assuming the DNA double-helix as a cylinder with the curved surface area A cs by using equation S4.

Equation S4
Here r is the radius of the cylinder and h is the height. Calf thymus DNA is B-Form DNA with a diameter d DNA = 20 Å and a distance of 3.4 Å between base pairs. 3,4 Referring to the provider (Sigma-Aldrich, St. Louis (MO), United States of America) the used calf thymus DNA (Typ 1) consists of > 13 kb (thirteen thousand base pairs). Hence this approach was used for the height h. Since the cylindrical DNA only attaches the lipid bilayer with on side, while the other side faces the aqueous solution, just half of the value for A cs was taken into account for the calculation of the theoretical mass deposition of DNA (25.7 ng/cm²).
According to Günter Sauerbrey, the weighting sensitivity of a quartz crystal with an electrode diameter of 4 mm in the center of the vibration region is about 10 10 Hz/g. Taking an error bare for S4 the frequency measurement ( ) into account the weighting accuracy can be determined to 0.1 ng.