2-Hydroxyoleic Acid as a Self-Assembly Inducer for Anti-Cancer Drug-Centered Nanoparticles

Abstract

A potent nontoxic antitumor drug, 2-hydroxyoleic acid (6, 2OHOA) used for membrane lipid therapy, was selected as a self-assembly inducer due to its ability to form nanoparticles (NPs) in water. For this purpose, it was conjugated with a series of anticancer drugs through a disulfide-containing linker to enhance cell penetration and to secure drug release inside the cell. The antiproliferative evaluation of the synthesized NP formulations against three human tumor cell lines (biphasic mesothelioma MSTO-211H, colorectal adenocarcinoma HT-29, and glioblastoma LN-229) showed that nanoassemblies 16-22a,bNPs exhibit antiproliferative activity at micromolar and submicromolar concentrations. Furthermore, the ability of the disulfide-containing linker to promote cellular effects was confirmed for most nanoformulations. Finally, 17bNP induced intracellular ROS increase in glioblastoma LN-229 cells similarly to free drug 8, and such elevated production was decreased by pretreatment with the antioxidant N-acetylcysteine. Also, nanoformulations 18bNP and 21bNP confirmed the mechanism of action of the free drugs.

Keywords: 2-hydroxyoleic acid (2OHOA); anticancer drugs; biphasic mesothelioma (MSTO-211H); colorectal adenocarcinoma (HT-29); conjugates; glioblastoma (LN-229); methyl 2-hydroxyoleate; nanoassemblies.

Figure 1

Chemical structures of self-assembling inducers 1–6.

Figure 2

Schematic presentation of internalization and drug release of disulfide-2OHOA-drugnanoparticles.

Figure 3

Chemical structures of anticancer drugs 7–13 used for conjugation with 2OHOA; the anchor point is shown in red.

Scheme 1

Synthesis of conjugates 16–21a,b: (i) (a) DMPU, LDA, THF, rt to 50-55 °C, 30 min (b) O₂, rt, 30 min (c) HCl, 45%; (ii) B(OH)₃, MeOH, overnight, rt, 80%; (iii) sebacic acid or 4,4′-dithiodibutyric acid, EDC·HCl, DMAP, Et₃N, DCM, 0 °C to rt, overnight, 64% for 15a and 68% for 15b; (iv) EDC·HCl, DMAP, DCM, rt, 1-12 h, 36–99%.

Figure 4

Chemical structures of drug-2OHOA conjugates 16–21a,b.

Scheme 2

Synthesis of N-desacetyl thiocolchicine conjugates 22a,b: (i) 2N HCl, MeOH, reflux, 48 h, 86%; (ii) 15a or 15b, EDC·HCl, DMAP, Et₃N, DCM, 0 °C to rt, 22–46 h, 72–86.

Figure 5

TEM micrographs of 17bNP and 18bNP, with a mean diameter of, respectively, 14 and 19 nm. The yellow arrows in the upper panel show 17bNPs, which are disappearing under the electron beam.

Figure 6

Cytotoxicity curves for 17aNP (blu line, panel A) 17bNP (green line, panel A), 18aNP (blue line, panel B) and 18bNP (green line, panel B) on MSTO 211-H, HT-29 and LN-229 human tumor cell lines. The insets show the cytotoxicity curves of the free drug 8 (red line, panel A) and 9 (red line, panel B).

Figure 7

Intracellular ROS production by free 8 and 17bNP in LN-229 cells determined by the fluorogenic probe 2′,7′-dichlorofluorescein diacetate. NAC = N-acetylcysteine.

Figure 8

Cell cycle analysis of LN229 cells incubated for 24 h with free 9 and 18bNP (panel A) and with free 12 and 21bNP(panel B). Data are expressed as the mean ± SD from four independent experiments.