Avocado-derived polyols for use as novel co-surfactants in low energy self-emulsifying microemulsions

Abstract

Avocado (Persea americana Mill.; Lauraceae) seed-derived polyhydroxylated fatty alcohols (PFAs) or polyols (i.e., avocadene and avocadyne) are metabolic modulators that selectively induce apoptosis of leukemia stem cells and reverse pathologies associated with diet-induced obesity. Delivery systems containing avocado polyols have not been described. Herein, natural surface active properties of these polyols are characterized and incorporated into self-emulsifying drug delivery systems (SEDDS) that rely on molecular self-assembly to form fine, transparent, oil-in-water (O/W) microemulsions as small as 20 nanometers in diameter. Mechanistically, a 1:1 molar ratio of avocadene and avocadyne (i.e., avocatin B or AVO was shown to be a eutectic mixture which can be employed as a novel, bioactive, co-surfactant that significantly reduces droplet size of medium-chain triglyceride O/W emulsions stabilized with polysorbate 80. In vitro cytotoxicity of avocado polyol-SEDDS in acute myeloid leukemia cell lines indicated significant increases in potency and bioactivity compared to conventional cell culture delivery systems. A pilot pharmacokinetic evaluation of AVO SEDDS in C57BL/6J mice revealed appreciable accumulation in whole blood and biodistribution in key target tissues. Lastly, incorporation of AVO in SEDDS significantly improved encapsulation of the poorly water-soluble drugs naproxen and curcumin.

Figure 1

Avocatin B shows eutectic phase behaviour. (A) Chemical structures of avocadene and avocadyne. (B) Representative DSC melting thermograms for avocadene, avocadyne and their mixtures. Plots have been nudged for illustration. (C) Melting temperatures (left Y-axis) and melting enthalpies (right Y-axis) as a function of avocadene and avocadyne composition. (D) Domain size (left Y-axis) and melting entropy (right Y-axis), ΔS_m as a function of avocadene and avocadyne composition. For (C,D) values are means ± SD from three independent experiments.

Figure 2

Avocatin B reduces droplet size of a MCT oil— polysorbate 80 based SEDDS. (A) SEDDs method of preparation. (B) Effect of avocatin B concentration on average hydrodynamic diameter (Z-average) of NeoBee—Tween 80 SEDDs over time. Inset: visual appearance of control (blank SEDDS) and avocatin B containing SEDDS on day 0. (C) Polydispersity index of SEDDS described in (B). Values are means ± SEM of three independent experiments. (D) Cryo-TEM images of control (blank SEDDS) and 20 mg/mL avocatin B containing SEDDs. Scale bar represents 100 nm.

Figure 3

The ratio of avocadene and avocadyne dictates SEDDS droplet size and stability. (A) Effect of 3:1 avocadene-avocadyne concentration on average hydrodynamic diameter (Z-average) of NeoBee—Tween 80 SEDDS over time. Inset: visual appearance of control (blank SEDDs) and 3:1 avocadene-avocadyne containing SEDDS on day 0. (B) Polydispersity index of SEDDS described in (A). (C) Effect of 3:2 avocadene-avocadyne concentration on Z-average of NeoBee—Tween 80 SEDDS over time. Inset: visual appearance of control (blank SEDDs) and 3:2 avocadene-avocadyne containing SEDDs on day 0. (D) Polydispersity index of SEDDs described in (C). For A–D, values are means ± SEM of three independent experiments. Note: size and polydispersity data for both 3:1 and 3:2 avocadene-avocadyne at a concentration of 20 mg/mL are missing on day 3 and onwards due to emulsion destabilization.

Figure 4

Avocadene and avocadyne behave differently in SEDDS. (A) Effect of avocadene concentration on average hydrodynamic diameter (Z-average) of NeoBee—Tween 80 SEDDs over time. Inset: visual appearance of control (blank SEDDS) and avocadene containing SEDDS on day 0. (B) Polydispersity index of SEDDS described in (A). (C) Effect of avocadyne concentration on Z-average of NeoBee—Tween 80 SEDDS when final emulsions are heated for 3–5 min and droplet size is measured at 37 °C. Inset: visual appearance of control (blank SEDDs) and avocadyne containing SEDDS before and after application of heat (emulsion formation). (D) Polydispersity index of SEDDs described in (C). For A–D, values are means ± SEM of three independent experiments. Note: size and polydispersity data for avocadene at a concentration of 20 mg/mL is missing on day 7 and onwards due to emulsion destabilization.

Figure 5

Avocado polyol SEDDS selectively reduce viability of AML cells with enhanced potency compared to DMSO delivery. In vitro activity of avocado polyol SEDDS was evaluated in AML cell lines OCI-AML-2 and TEX. Cells were incubated with varying concentrations of avocado polyols dissolved in DMSO or as SEDDS. After 72 hours, cell viability was measured by the MTS assay. For avocadyne (A), avocadene (B), avocatin B (C), and 3:1 avocadene-avocadyne (D), 20 mg/mL (2% w/w) SEDDS were freshly prepared and used for serial dilution. For 3:2 avocadene-avocadyne (E), 15 mg/mL (1.5% w/w) SEDDS were freshly prepared and used. Data represent logarithmic transformation of avocado polyol concentrations (µg/mL) and cell viability data that was fit to a nonlinear four-parameter logistic curve to determine inhibitory concentration 50 (IC50). All data represents mean ± SEM from three independent experiments performed in triplicate.

Figure 6

Avocado polyols exert less toxicity in non-AML cell lines when delivered in either DMSO or SEDDS. In vitro cytotoxicity of avocado polyols was evaluated in non-AML cell lines (Caco-2, INS-1 (832/13), C2C12 myotubes and HepG2). Cell lines were incubated with varying concentrations of avocado polyols dissolved in DMSO or as SEDDS. After 24 hours, cell viability was measured by the MTS assay. For avocadyne (A), avocadene (B), avocatin B (C) and 3:1 avocadene-avocadyne (D), 20 mg/mL (2% w/w) SEDDS were freshly prepared and used. For 3:2 avocadene-avocadyne (E), 15 mg/mL (1.5% w/w) SEDDS were freshly prepared and used. Data fit to nonlinear regression curve as described for Fig. 5. All data represents mean ± SEM from three independent experiments performed in triplicate.

Figure 7

Avocatin B SEDDS show bioavailability and biodistribution in a pilot in vivo pharmacokinetic study. Avocatin B SEDDS (2% w/w) was delivered via gavage (100 mg/kg body weight (b.w.)) to 6–8 week old female C57BL/6J mice. (A) 50–100 µL blood was collected via tail-snips at 2 hr, 6 hr and endpoint (24 hr). Avocadene and avocadyne were quantified in blood using a validated LC-MS bio-analytical method. (B) Tissues (bone marrow, heart, pancreas, liver, gonadal fat pad, inguinal fat pad, and brain) were collected at endpoint for avocadene and avocadyne quantification. Data are shown as mean ± S.D., N = 3 in each group.

Figure 8

AVO improves encapsulation of poorly water soluble drugs. (A) Histograms represent Z-average (left Y-axis) of Naproxen (0.5% (w/w)) encapsulated in NeoBee—Tween 80 SEDDS (control SEDDS) and AVO (1% (w/w)) SEDDS. Symbol represents PDI (right Y-axis) (B) Visual appearance of SEDDS described in (A). (C) Z-average and PDI of curcumin (0.5% (w/w)) in control or AVO (1% (w/w)) SEDDS. (D) Visual appearance of SEDDS described in (C). (E) In vitro cytotoxicity of curcumin formulated in DMSO or SEDDS with or without AVO was evaluated in OCI-AML-2 cells. Data fit to nonlinear regression curve described for Fig. 5. For (A,C) data represents mean ± SEM of two independent experiments. For (E), data represents mean ± SEM from three independent experiments performed in triplicate.