Synthesis of Dextran-Phenoxodiol and Evaluation of Its Physical Stability and Biological Activity

Abstract

Phenoxodiol, an isoflavene anti-tumor agent, was conjugated on the polysaccharide dextran using immobilized laccase as biocatalyst. The success of the enzymatic conjugation was determined by UV-vis spectrophotometry and its functionalization degree was assessed by ¹H NMR and was found to be 3.25 mg phenoxodiol/g of conjugate. An accelerated stability test showed that the resultant conjugate was nine times more stable than the free phenoxodiol when tested for its residual anti-oxidant activity with the Folin-Ciocalteu assay. The in vitro anti-proliferative activity of the conjugate was evaluated against neuroblastoma SKN-BE(2)C, triple-negative breast cancer MDA-MB-231, and glioblastoma U87 cancer cells. The conjugate was shown to be generally more potent than phenoxodiol against all three cell types tested. Additionally, the cytotoxicity and anti-angiogenic activity of the conjugate were also evaluated against non-malignant human lung fibroblast MRC-5 and human microvascular endothelial cells HMEC-1, respectively. The conjugate was found to be 1.5 times less toxic than phenoxodiol while mostly retaining 62% of its anti-angiogenic activity in the conjugate form. This study provides further evidence that the conjugation of natural product-derived drugs onto polysaccharide molecules such as dextran can lead to better stability and enhanced biological activity of the conjugate compared to the free drug alone.

Keywords: anti-angiogenic; anti-tumor; conjugate; dextran; phenoxodiol.

Scheme 1

Conjugation of phenoxodiol 1 with dextran.

Figure 1

UV-vis spectrum of phenoxodiol 1 and dextran–phenoxodiol 2 in 50% aqueous MeOH.

Figure 2

¹H NMR spectrum of phenoxodiol 1 (A) and 0.25 mg/ml dextran–phenoxodiol 2 (B) in DMSO-d₆.

Figure 3

Residual activity of phenoxodiol 1 and dextran–phenoxodiol 2 after undergoing accelerated degradation conditions. n = 3; bars, SEM.

Figure 4

In vitro anti-proliferative and cytotoxicity activity of phenoxodiol 1 and dextran–phenoxodiol 2 against cancer, endothelial and non-malignant cells. Cell viability is measured on SKN-BE(2)C, MDA-MB-231, U87, HMEC-1, and MRC-5 cells using the Alamar Blue assay after 72-h incubation with a range of drug concentrations. Points as a % of cell proliferation as compared to untreated cells, n = 3; bars, SEM (**p < 0.05, ***p < 0.005).

Figure 5

Effect of 10 μM phenoxodiol 1 and dextran–phenoxodiol 2 treatment on HMEC-1 angiogenic activity assessed with the Matrigel™ assays. (A) Representative photographs of HMEC-1 cells following drug treatment. Scale bar, 200 μm. (B) Total surface area of vascular structure following drug treatment. n = 3; bars, SEM (****p < 0.0001).