Anticancer efficacy of a supramolecular complex of a 2-diethylaminoethyl-dextran-MMA graft copolymer and paclitaxel used as an artificial enzyme

Abstract

The anticancer efficacy of a supramolecular complex that was used as an artificial enzyme against multi-drug-resistant cancer cells was confirmed. A complex of diethylaminoethyl-dextran-methacrylic acid methylester copolymer (DDMC)/paclitaxel (PTX), obtained with PTX as the guest and DDMC as the host, formed a nanoparticle 50-300 nm in size. This complex is considered to be useful as a drug delivery system (DDS) for anticancer compounds since it formed a stable polymeric micelle in water. The resistance of B16F10 melanoma cells to PTX was shown clearly through a maximum survival curve. Conversely, the DDMC/PTX complex showed a superior anticancer efficacy and cell killing rate, as determined through a Michaelis-Menten-type equation, which may promote an allosteric supramolecular reaction to tubulin, in the same manner as an enzymatic reaction. The DDMC/PTX complex showed significantly higher anticancer activity compared to PTX alone in mouse skin in vivo. The median survival times of the saline, PTX, DDMC/PTX4 (particle size 50 nm), and DDMC/PTX5 (particle size 290 nm) groups were 120 h (treatment (T)/control (C), 1.0), 176 h (T/C, 1.46), 328 h (T/C, 2.73), and 280 h (T/C, 2.33), respectively. The supramolecular DDMC/PTX complex showed twice the effectiveness of PTX alone (p < 0.036). Above all, the DDMC/PTX complex is not degraded in cells and acts as an intact supramolecular assembly, which adds a new species to the range of DDS.

Keywords: artificial enzyme; diethylaminoethyl–dextran–MMA; graft copolymer; multi-drug resistance of cancer cells; paclitaxel; supramolecular complex.

Figure 1

Non-viral vectoring of DEAE–dextran and DEAE–dextran–MMA graft copolymer (DDMC). (a) Transfection of DDMC into HEK293 cell lines. The grafting rate is 130% for sample 2 at 10 mg/mL, sample 3 at 20 mg/mL, and sample 4 at 28.6 mg/mL. Expression of the LacZ gene is shown at 48 h after transfection. (b) DNase degradation (1: DEAE–dextran, 2 DDMC): To the samples was added 4 mL of distilled water, then 10 u of DNase (RQ1 RNase-free DNase, Promega) and 0.1 mL of 10× reaction buffer (400 mM Tris-HCl, 100 mM MgSO₄, 10 mM CaCl₂, pH 8) and the samples were incubated at 37 °C. The wavelength used for this experiment was 633 nm for toluidine blue isolated from DNA. (b): Reprinted from [18].

Figure 2

Differential scanning calorimetry (DSC) with DDMC–paclitaxel complex and paclitaxel: (1) DDMC, (2) DDMC/PTX complex (DDMC 9.6 mg/PTX 0.385 mg), (3) DDMC/PTX complex (DDMC 9.6 mg/PTX 0.709 mg), and (4) PTX. Reprinted from [62].

Figure 3

IR absorption spectra of the DDMC–paclitaxel complex and paclitaxel. (a) Mid-infrared region (4.000–400 cm⁻¹), (b) X–H stretching region: (1) DDMC, (2) DDMC/PTX complex (DDMC 9.6 mg/PTX 0.385 mg), (3) DDMC/PTX complex (DDMC 9.6 mg/PTX 0.709 mg), and (4) PTX. (b): Reprinted from [62].

Figure 4

Characteristics of the DDMC–paclitaxel complex. (a) Particle size distribution and ζ-potential of the DDMC–paclitaxel complex determined by dynamic light scattering and (b) particle electrophoretic mobility measurements. (c) A Scanning electron microscopic image (HITACHI S-4800) of the freeze-dried DDMC–paclitaxel complex taken at an accelerating voltage of 5 kV. (a): Reprinted from [24].

Figure 5

Anticancer efficacy of a supramolecular complex against PTX-resistant melanoma B16F10 cells. (a) Survival of B16F10 melanoma cells treated with paclitaxel or the DDMC–paclitaxel complex for 48 h determined by using the MTT (WST8) method (blue solid line: DDMC/PTX, purple dashed line: DDMC, orange dashed line: PTX). (b) Tubulin polymerization and cell death (Cd) rates can be expressed by enzymatic kinetic parameters. Relationship between Cd and paclitaxel concentration, [E]₀, after 24 h and 48 h. After 24 h: Cd = 0.1062 [E]₀ + 0.0481; after 48 h: Cd = 0.1156 [E]₀ + 0.1082. (c) Apparatus constant (C₁) by cell culture time in WST8 vs time (h) for DDMC/PTX complex. Reprinted from [24].

Figure 6

Potential energy curve of paclitaxel (large) and DDMC/PTX complex (small) in the tumor inhibition reaction. E_a: activation energy of paclitaxel, E_a′: activation energy of the DDMC/PTX complex. Reprinted from [24].

Figure 7

In vivo analysis of the anticancer activity and survival rates of the DDMC/PTX complex in B16F10 melanoma cells. (a) The increase rate (V/V₀) of mean tumor volumes in the saline and DDMC/PTX4 (particle size: 50 nm) (p < 0.09). (b) The relationships between survival rate and time (hours) with the PTX, saline, DDMC/PTX4 (particle size: 50 nm) and DDMC/PTX5 (particle size: 290 nm) groups. (b): Reprinted from [62].

Figure 8

Two mice of both PTX (left) and DDMC/PTX4 (right) groups after 208 hours from I.P. In the mice of DDMC/PTX4 group necrosis was induced by TNF-α. The increase of TNF-α in an individual has been checked by the phoresis results after RT-PCR. Reprinted from [62].

Figure 9

α,β-tubulin dimer orientates with the active site (i.e., paclitaxel) of the DDMC/PTC complex. The DDMC/PTX complex (ca. 270 nm) will consists of more than 8.1·10³ DDMC molecules and 6.7·10⁶ paclitaxel molecules. Reprinted from [24].

Figure 10

Schematic representation of α,β-tubulin dimer polymerization showing the allosteric relationship between the DDMC/PTX complex and α,β-tubulin. Simplified structures are shown. Reprinted from [62].