Using liposomes as carriers for polyphenolic compounds: the case of trans-resveratrol

Abstract

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a polyphenol found in various plants, especially in the skin of red grapes. The effect of resveratrol on human health is the topic of numerous studies. In fact this molecule has shown anti-cancer, anti-inflammatory, blood-sugar-lowering ability and beneficial cardiovascular effects. However, for many polyphenol compounds of natural origin bioavailability is limited by low solubility in biological fluids, as well as by rapid metabolization in vivo. Therefore, appropriate carriers are required to obtain efficient therapeutics along with low administration doses.Liposomes are excellent candidates for drug delivery purposes, due to their biocompatibility, wide choice of physico-chemical properties and easy preparation.In this paper liposome formulations made by a saturated phosphatidyl-choline (DPPC) and cholesterol (or its positively charged derivative DC-CHOL) were chosen to optimize the loading of a rigid hydrophobic molecule such as resveratrol.Plain and resveratrol loaded liposomes were characterized for size, surface charge and structural details by complementary techniques, i.e. Dynamic Light Scattering (DLS), Zeta potential and Small Angle X-ray Scattering (SAXS). Nuclear and Electron Spin magnetic resonances (NMR and ESR, respectively) were also used to gain information at the molecular scale.The obtained results allowed to give an account of loaded liposomes in which resveratrol interacted with the bilayer, being more deeply inserted in cationic liposomes than in zwitterionic liposomes. Relevant properties such as the mean size and the presence of oligolamellar structures were influenced by the loading of RESV guest molecules.The toxicity of all these systems was tested on stabilized cell lines (mouse fibroblast NIH-3T3 and human astrocytes U373-MG), showing that cell viability was not affected by the administration of liposomial resveratrol.

Figure 1. SAXS diagrams of plain and resveratrol loaded zwitterionic liposome.

Figure 2. Experimental SAXS diagrams (symbols) and fitting (continuous lines) of DPPC/CHOL 75/25 plain and RESV loaded liposomes (a and b, respectively).

The best fit parameters used are listed in table 3. The curves of RESV loaded liposomes have been vertically shifted for the sake of clarity.

Figure 3. SAXS diagrams of plain and resveratrol loaded cationic liposomes.

Figure 4. ¹H spectrum of trans-Resveratrol in D₂O/DMSO (2/3) recorded at 600 MHz and 298 K.

Figure 5. NMR proton spectrum in the range 0–4.5 ppm for: a) DPPC/Chol liposome in D₂O and b) DPPC/DC-CHOL liposome in D₂O.

Figure 6. ¹H NMR spectrum of zwitterionic liposomes and corresponding enlargement of the aromatic region.

Figure 7. ¹H NMR spectrum of cationic liposomes and corresponding enlargement in the aromatic region.

Figure 8. NOESY spectrum of DPPC/DC-CHOL RESV loaded liposomes in D₂O recorded at 600 MHz and 298 K.

Figure 9. Sketch of the interbilayer location of the 5-DSA (left) and 16-DSA (right) spin probes.

Figure 10. ESR spectra of 5-DSA, 12-DSA and 16-DSA (a,b and c, respectively) inserted in the bilayer of plain and RESV loaded cationic liposomes.

The three probes have their paragnetic unit located progressively deeper in the hydrocarbon region.

Figure 11. Percentage of viable NIH3T3 (a) and U373-MG (b) after 24 h of contact with plain and RESV loaded liposomes, as determined by the Neutral Red Uptake.

Data are the mean ± SD of three experiments run in six replicates. No value was statistically different versus control (complete medium).

Figure 12. Sketch of RESV insertion in the bilayer of DPPC/CHOL (a) and DPPC/DC-CHOL(b) liposomes.