Effect of DMSO on Structural Properties of DMPC and DPPC Liposome Suspensions

Abstract

The study and characterization of the biophysical properties of membranes and drug-membrane interactions represent a critical step in drug development, as biological membranes act as a barrier that the drug must overcome to reach its active site. Liposomes are widely used in drug delivery to circumvent the poor aqueous solubility of most drugs, improving systemic bioavailability and pharmacokinetics. Further, they can be targeted to deliver to specific disease sites, thus decreasing drug load, and reducing side effects and poor adherence to treatment. To improve drug solubility during liposome preparation, DMSO is the most widely used solvent. This raises concern about the potential effect of DMSO on membranes and leads us to investigate, using DSC and EPR, the influence of DMSO on the behavior of lipid model membranes of DMPC and DPPC. In addition, we tested the influence of DMSO on drug-membrane interaction, using compounds with different hydrophobicity and varying DMSO content, using the same experimental techniques. Overall, it was found that with up to 10% DMSO, changes in the bilayer fluidity or the thermotropic properties of the studied liposomes were not significant, within the experimental uncertainty. For higher concentrations of DMSO, there is a stabilization of both the gel and the rippled gel phases, and increased bilayer fluidity of DMPC and DPPC liposomes leading to an increase in membrane permeability.

Keywords: differential scanning calorimetry (DSC); dimethyl sulfoxide (DMSO); dimyristoylphosphatidylcholine (DMPC); dipalmitoylphosphatidylcholine (DPPC); electron paramagnetic resonance (EPR); liposomes.

Scheme 1

Methodology adopted for liposome samples preparation. (a) LUVs with “DMSO added after preparation”, (b) MLVS with DMSO added to already formed liposomes, and (c) MLVs with “DMSO added on liposome preparation, incorporation method”. In sample (a,b) DMSO is added to the liposome suspensions just prior to the DSC experiment.

Figure 1

Typical EPR spectra of 5-DSA in: (A) in solution, (B) DMPC liposome at T = 310 K, where 2A_max is the maximum hyperfine splitting. The structure of 5-DSA is shown below.

Figure 2

The DSC heating thermograms for DMPC (A,B) and DPPC (C,D) unilamellar vesicles (LUVs), with increasing amounts of DMSO, added at room temperature (~22 °C) after liposome preparation, immediately prior to the DSC run (“DMSO added after preparation”). The DMSO content is expressed in the legends, in (v/v) %. The curves are already corrected for blank (buffer/buffer run), and baseline subtracted.

Figure 3

DSC heating thermograms of DMPC multilamellar vesicles, MLVs, “with DMSO added to already formed liposomes” plotted as a function of DMSO content. The DMSO content is expressed in the legends, in (v/v)%. The curves are already corrected for blank (buffer/buffer run) and baseline subtracted. Insert: enlargement of the pre-transition for the different DMSO contents.

Figure 4

DSC heating thermograms of the DMPC multilamellar vesicles, MLVs, with increasing amounts of DMSO, using “DMSO added on liposome preparation, incorporation method”. Insert: enlargement of the pre-transition for the various DMSO contents. The DMSO content is expressed in the legends, in (v/v)%. The curves are already corrected for blank (buffer/buffer run) and baseline subtracted.

Figure 5

Effect of increasing amounts of DMSO on 5-DSA degree of anisotropy (2A_max) in DMPC liposomes at T = 310 K (fluid phase, Lα). ** p ≤ 0.01; **** p ≤ 0.0001.

Figure 6

EPR spectrum of spin labelled DPPC liposomes at T = 323 K, with DMSO contents: (A)—0%; (B)—40%; (C)—5-DSA in HEPES/DMSO solution.