All-trans-retinoic acid nanodisks

Abstract

Nanodisks are nanoscale, disk-shaped phospholipid bilayers whose edge is stabilized by association of apolipoprotein molecules. Self-assembled ND particles enriched with all-trans-retinoic acid (ATRA) (phospholipid:ATRA molar ratio = 5.5:1) were generated wherein all reaction components were solubilized. ATRA-ND migrated as a single band (Stokes' diameter approximately 20 nm) on native gradient polyacrylamide gel electrophoresis. ATRA, phospholipid and apolipoprotein co-eluted from a Sepharose 6B gel filtration column, consistent with stable integration of ATRA into the ND particle milieu. Spectroscopic analysis of ATRA-ND in buffer yielded an absorbance spectrum characteristic of ATRA. ATRA-ND mediated time-dependent inhibition of cultured HepG2 cell growth more effectively than free ATRA. The nanoscale size of the formulation particles and the stable integration of biologically active ATRA suggest ND represent a potentially useful vehicle for solubilization and in vivo delivery of ATRA.

Figure 1. ATRA-ND formulation scheme and structure model

Phospholipid vesicles, ATRA and apoE3-NT (as a lipid-free 4-helix bundle; Wilson et al., 1991) were combined to form ATRA-ND. Lipid interaction converts apo E3-NT from a soluble 4-helix bundle into an open, extended lipid bound conformation (Narayanaswami et al., 2004). ATRA ND are comprised of a disk-shaped phospholipid bilayer in which ATRA molecules (yellow dots) are integrated. The edge of the ND bilayer is stabilized by the apolipoprotein “scaffold”. Component images are not to scale.

Figure 2. UV/visible absorbance spectra of ATRA

Scans were recorded from 250 – 450 nm. Samples included free ATRA (5 μg/ml ) in a) ethanol c) PBS and b) ATRA-ND in PBS.

Figure 3. Native PAGE of ND

Lane 1) protein standards with known Stokes’ diameters [from top to bottom: thyroglobulin (17 nm), horse ferritin (12.2 nm), catalase (9.2 nm) and lactate dehydrogenase (8.2 nm)]; Lane 2) empty ND; Lane 3) ATRA-ND.

Figure 4. filtration chromatography of ATRA-ND

ATRA-ND were applied to a Sepharose 6B column equilibrated in PBS and eluted with collection of 2 ml fractions. The phospholipid (filled circles), protein (open circles) and ATRA (open squares) content in each fraction were determined as described in Material and Methods.

Figure 5. Effect of ATRA on viability of cultured hepatoma cells

HepG2 cells were treated with buffer (control), free ATRA (5 μg/ml from ethanol stock solution) or ATRA-ND in PBS (5 μg ATRA/ml). After 72 h (panel A) and 120 h (panel B), cell viability was measured using the MTT assay. Values reported are the mean ± S.D. of 3 determinations.