Transporting antitumor drug tamoxifen and its metabolites, 4-hydroxytamoxifen and endoxifen by chitosan nanoparticles.

Synthetic and natural polymers are often used as drug delivery systems in vitro and in vivo. Biodegradable chitosan of different sizes were used to encapsulate antitumor drug tamoxifen (Tam) and its metabolites 4-hydroxytamoxifen (4-Hydroxytam) and endoxifen (Endox). The interactions of tamoxifen and its metabolites with chitosan 15, 100 and 200 KD were investigated in aqueous solution, using FTIR, fluorescence spectroscopic methods and molecular modeling. The structural analysis showed that tamoxifen and its metabolites bind chitosan via both hydrophilic and hydrophobic contacts with overall binding constants of K(tam-ch-15) = 8.7 ( ± 0.5) × 10(3) M(-1), K(tam-ch-100) = 5.9 (± 0.4) × 10(5) M(-1), K(tam-ch-200) = 2.4 (± 0.4) × 10(5) M(-1) and K(hydroxytam-ch-15) = 2.6(± 0.3) × 10(4) M(-1), K(hydroxytam - ch-100) = 5.2 ( ± 0.7) × 10(6) M(-1) and K(hydroxytam-ch-200) = 5.1 (± 0.5) × 10(5) M(-1), K(endox-ch-15) = 4.1 (± 0.4) × 10(3) M(-1), K(endox-ch-100) = 1.2 (± 0.3) × 10(6) M(-1) and K(endox-ch-200) = 4.7 (± 0.5) × 10(5) M(-1) with the number of drug molecules bound per chitosan (n) 2.8 to 0.5. The order of binding is ch-100>200>15 KD with stronger complexes formed with 4-hydroxytamoxifen than tamoxifen and endoxifen. The molecular modeling showed the participation of polymer charged NH2 residues with drug OH and NH2 groups in the drug-polymer adducts. The free binding energies of -3.46 kcal/mol for tamoxifen, -3.54 kcal/mol for 4-hydroxytamoxifen and -3.47 kcal/mol for endoxifen were estimated for these drug-polymer complexes. The results show chitosan 100 KD is stronger carrier for drug delivery than chitosan-15 and chitosan-200 KD

Chemical structures of tamoxifen, 4-hydroxtamoxifen, endoxifen and chitosan.

<p>Chemical structures of tamoxifen, 4-hydroxtamoxifen, endoxifen and chitosan.</p

FTIR spectra in the region of 1800–600 cm⁻¹ of hydrated films (pH 6) for free chitosan (60 µM) and its 4-hydroxytamoxifen complexes for (A) chitosan-15 KD, (B) chitosan-100 KD and (C) chitosan-200 KD with difference spectra (diff.) (bottom two curves) obtained at different drug concentrations (indicated on the figure).

<p>FTIR spectra in the region of 1800–600 cm⁻¹ of hydrated films (pH 6) for free chitosan (60 µM) and its 4-hydroxytamoxifen complexes for (A) chitosan-15 KD, (B) chitosan-100 KD and (C) chitosan-200 KD with difference spectra (diff.) (bottom two curves) obtained at different drug concentrations (indicated on the figure).</p

Binding parameters for drug-chitosan complexes.

<p>Binding parameters for drug-chitosan complexes.</p

Fluorescence emission spectra of drug-chitosan systems in 10 mM acetate buffer pH 6 at 25°C presented for (A) endox-ch-15: (a) free endox (30 µM), (b-i) with chitosan at 30, 40, 50, 60, 80, 100, 120, 140 and 160 µM; (B) endox-chitosan-100: (a) free endox (30 µM), (b–h) chitosan at 1, 3, 5, 7, 10, 15, 20 and 30 µM; (C) endox-ch-200: (a) free endox (30 ) (b–g) with chitosan at 3, 5, 7, 10, 20 and 30 µM; Inset: <i>K</i> values calculated by <i>F</i>₀/(<i>F</i>₀ – <i>F</i>) vs 1/[chitosan] for A' (endox-chitosan-15), B' (endox- chitosan 100) and C' (endox-chitosan-200).

<p>Fluorescence emission spectra of drug-chitosan systems in 10 mM acetate buffer pH 6 at 25°C presented for (A) endox-ch-15: (a) free endox (30 µM), (b-i) with chitosan at 30, 40, 50, 60, 80, 100, 120, 140 and 160 µM; (B) endox-chitosan-100: (a) free endox (30 µM), (b–h) chitosan at 1, 3, 5, 7, 10, 15, 20 and 30 µM; (C) endox-ch-200: (a) free endox (30 ) (b–g) with chitosan at 3, 5, 7, 10, 20 and 30 µM; Inset: <i>K</i> values calculated by <i>F</i>₀/(<i>F</i>₀ – <i>F</i>) vs 1/[chitosan] for A' (endox-chitosan-15), B' (endox- chitosan 100) and C' (endox-chitosan-200).</p

Best docked conformations of drug–chitosan complexes.

<p>(<b>A</b>) for tamoxifen bound to chitosan and (<b>B</b>) for 4-hydroxytamoxifen bound to chitosan (<b>C</b>) for endoxifen bound to chitosan with free binding energy.</p

Fluorescence emission spectra of drug-chitosan systems in 10 mM acetate buffer pH 6 at 25°C presented for (A) 4-hydroxytam-ch-15: (a) free 4-hydroxytam (30 µM), (b–h) with chitosan at 30, 40, 50, 60, 80, 100, 120 and 140 µM; (B) 4-hydroxytamx-chitosan-100: (a) free 4.hydroxytam (30 µM), (b–h) chitosan at 1, 3, 5, 7, 10, 15, 20 and 30 µM; (C) 4-hydroxytam-ch-200: (a) free 4-hydroxytam (30 ) (b–f) with chitosan at 3, 5, 7, 20 and 30 µM; Inset: K values calculated by <i>F</i>₀/(<i>F</i>₀ –<i>F</i>) vs 1/[chitosan] for A' (4-hydroxytam -chitosan-15), B' (4 hydroxytam – chitosan 100) and C' (4-hydroxytam -chitosan-200).

<p>Fluorescence emission spectra of drug-chitosan systems in 10 mM acetate buffer pH 6 at 25°C presented for (A) 4-hydroxytam-ch-15: (a) free 4-hydroxytam (30 µM), (b–h) with chitosan at 30, 40, 50, 60, 80, 100, 120 and 140 µM; (B) 4-hydroxytamx-chitosan-100: (a) free 4.hydroxytam (30 µM), (b–h) chitosan at 1, 3, 5, 7, 10, 15, 20 and 30 µM; (C) 4-hydroxytam-ch-200: (a) free 4-hydroxytam (30 ) (b–f) with chitosan at 3, 5, 7, 20 and 30 µM; Inset: K values calculated by <i>F</i>₀/(<i>F</i>₀ –<i>F</i>) vs 1/[chitosan] for A' (4-hydroxytam -chitosan-15), B' (4 hydroxytam – chitosan 100) and C' (4-hydroxytam -chitosan-200).</p

FTIR spectra in the region of 1800–600 cm⁻¹ of hydrated films (pH 6) for free chitosan (60 µM) and its tamoxifen complexes for (A) chitosan-15 KD, (B) chitosan-100 KD and (C) chitosan-200 KD with difference spectra (diff.)

<p>(bottom two curves) obtained at different drug concentrations (indicated on the figure).</p

FTIR spectra in the region of 3500-2800 cm⁻¹ of hydrated films (pH 6.0) for free chitosan and their tamox, 4-hydroxytamox and endoxifen complexes obtained with 60 µM polymer and 60 <b>µ M drug concentrations.</b>

<p>FTIR spectra in the region of 3500-2800 cm⁻¹ of hydrated films (pH 6.0) for free chitosan and their tamox, 4-hydroxytamox and endoxifen complexes obtained with 60 µM polymer and 60 <b>µ M drug concentrations.</b></p

FTIR spectra in the region of 1800–600 cm⁻¹ of hydrated films (pH 6) for free chitosan (60 µM) and its endoxifen complexes for (A) chitosan-15 KD, (B) chitosan-100 KD and (C) chitosan-200 KD with difference spectra (diff.) (bottom two curves) obtained at different drug concentrations (indicated on the figure).

<p>FTIR spectra in the region of 1800–600 cm⁻¹ of hydrated films (pH 6) for free chitosan (60 µM) and its endoxifen complexes for (A) chitosan-15 KD, (B) chitosan-100 KD and (C) chitosan-200 KD with difference spectra (diff.) (bottom two curves) obtained at different drug concentrations (indicated on the figure).</p