Synthesis, characterization, and in vitro transfection activity of charge-reversal amphiphiles for DNA delivery

Abstract

A series of charge-reversal lipids were synthesized that possess varying chain lengths and end functionalities. These lipids were designed to bind and then release DNA based on a change in electrostatic interaction with DNA. Specifically, a cleavable ester linkage is located at the ends of the hydrocarbon chains. The DNA release from the amphiphile was tuned by altering the length and position of the ester linkage in the hydrophobic chains of the lipids through the preparation of five new amphiphiles. The amphiphiles and corresponding lipoplexes were characterized by DSC, TEM, and X-ray, as well as evaluated for DNA binding and DNA transfection. For one specific charge-reversal lipid, stable lipoplexes of approximately 550 nm were formed, and with this amphiphile, effective in vitro DNA transfection activities was observed.

Figure 1

Chemical structures of several common cationic lipids.

Figure 2

Charge-reversal or charge- switchable effect of the amphiphiles.

Figure 3

Structures of the charge-reversal amphiphiles under investigation for gene delivery.

Figure 4

(top) Fluorescence intensity as a function of amphiphile/DNA charge ratio. (bottom) The fluorescence intensity as a function of amphiphile/DNA charge ratio for amphiphiles 1, 6-10, and DOTAP. N=3 Avg ± SD

Figure 5

Fluorescence intensity as a function of amphiphile/DNA charge ratio in function of the pH. N=3 Avg ± SD

Figure 6

Ethidium bromide intercalation assay showing the fluorescence intensity as a function of time in the presence of a porcine liver esterase (1000 units/mL). N=3 Avg ± SD

Figure 7

Ethidium bromide intercalation assay showing the fluorescence intensity as a function of time in the presence of a porcine liver esterase. N = 3 Avg ± SD

Figure 8

Transfection efficiency of the different amphiphiles in CHO cells. N=3 Avg ± SD

Figure 9

Photograph of β-galactosidase transfected cells using amphiphile 1 at low and high magnification. Dark blue stain represents B-gal expression.

Figure 10

Cytotoxicity of the different lipids in CHO cells. N=3 Avg ± SD

Figure 11

Transfection efficiency of amphiphile 1 with HEK293 and K562 cell lines. N=3 Avg ± SD. First data set with 1 is at 15:1 and the second data set is at 45:1 charge ratio.

Figure 12

TEM micrographs of amphiphile 1 in aqueous solution, negative staining with ammonium molybdate 1% in water.

Figure 13

Flow cytometry histogram for CHO cells transfected with rhodamine-labeled DNA using either 1 (blue) or 6 (red). Negative control (black).

Scheme 1

Synthesis of the charge-reversal amphiphiles.