Docosahexaenoic acid liposomes for targeting chronic inflammatory diseases and cancer: an in vitro assessment

Abstract

Inflammation, oxidative stress, and uncontrolled cell proliferation are common key features of chronic inflammatory diseases, such as atherosclerosis and cancer. ω3 polyunsaturated fatty acids (PUFAs; also known as omega3 fatty acids or fish oil) have beneficial effects against inflammation upon dietary consumption. However, these effects cannot be fully exploited unless diets are enriched with high concentrations of fish oil supplements over long periods of time. Here, a nanomedicine-based approach is presented for delivering effective levels of PUFAs to inflammatory cells. Nanoparticles are internalized by immune cells, and hence can adequately deliver bioactive lipids into these target cells. The ω3 FA docosahexaenoic acid was formulated into liposomes (ω-liposomes), and evaluated for anti-inflammatory effects in different types of immune cells. ω-Liposomes strongly inhibited the release of reactive oxygen species and reactive nitrogen species from human neutrophils and murine macrophages, and also inhibited the production of the proinflammatory cytokines TNFα and MCP1. Moreover, ω-liposomes inhibited tumor-cell proliferation when evaluated in FaDu head and neck squamous carcinoma and 4T1 breast cancer cells in in vitro cultures. We propose that ω-liposomes are a promising nanonutraceutical formulation for intravenous delivery of fish oil FAs, which may be beneficial in the treatment of inflammatory disorders and cancer.

Keywords:

• nanomedicine
• PUFA
• inflammation
• cancer
• fish oil
• delivery

Supplementary materials

Figure S1 Physical appearance and calculation of D₉₀ of ω-liposomes and control liposomes (C-liposomes) before (A) and after (B) extrusion.

Abbreviations: C-LMV, control large, multilamellar vesicles; D₉₀, size distribution at 90%; PDI, polydispersity index; ω-liposomes, docosahexaenoic acid-loaded liposomes; ω-LMV, docosahexaenoic acid-loaded large, multilamellar vesicles.

Figure S2 Dynamic light scattering-generated histograms demonstrating the particle-size distribution of ω-liposomes (B) and control liposomes (C-liposomes) (A) after extrusion.

Abbreviation: ω-liposomes, docosahexaenoic acid-loaded liposomes.

Figure S3 Colloidal stability of ω-liposomes and control liposomes (C-liposomes) at room temperature (RT) and 37°C.

Note: Particle size (A and C) and polydispersity index (B and D) were monitored with dynamic light scattering over 72 hours.

Abbreviation: ω-liposomes, docosahexaenoic acid-loaded liposomes.

Figure S4 Thermotropic stability of liposomes.

Notes: The thermotropic stability of the liposomes was analyzed using discovery differential scanning calorimetry and HEPES buffered saline as reference. The thermal behavior of the liposomes was monitored between 4°C and 60°C, with increasing steps of 0.5°C per minute.

Abbreviations: C-liposomes, control liposomes; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; ω-liposomes, docosahexaenoic acid-loaded liposomes.

Figure S5 Effect of ω-liposomes and control liposomes (C-liposomes) on lipopolysaccharide (LPS)-induced nitric oxide production in prestimulated murine macrophages.

Notes: (A) RAW264.7 cells were stimulated with LPS for 2 or 4 hours (B, C). Afterward, LPS was washed (C) or not (A and B), and cells were treated with ω-liposomes or C-liposomes. NO production was measured in the supernatant with Griess reagent. Data presented as mean ± standard error of mean from a representative experiment (n=4).

Abbreviations: NT, nontreated; TL, total lipid; ω-liposomes, docosahexaenoic acid-loaded liposomes.

Figure S6 Effect of ω-liposomes and control liposomes (C-liposomes) on proliferation of human umbilical vein endothelial cells (HUVECs).

Notes: HUVECs were seeded at 4,000 cells/well in a 96-well plate. Cells were exposed for 24 hours to ω-liposomes and C-liposomes, after which the medium was replaced with medium containing bromodeoxyuridine (BrdU) and cells incubated for an additional 4–6 hours. Afterward, BrdU incorporation was determined by enzyme-linked immunosorbent assay. Data presented as mean ± standard error of mean from a representative experiment (n=6).

Abbreviations: NT, nontreated; TL, total lipid; ω-liposomes, docosahexaenoic acid-loaded liposomes.

Acknowledgments

This work was supported by the European Framework Program 7 grant FP7-Health 309820: Nano-Athero.

Disclosure

The authors report no conflicts of interest in this work.