Green Tea Catechin-Based Complex Micelles Combined with Doxorubicin to Overcome Cardiotoxicity and Multidrug Resistance

Abstract

Chemotherapy for cancer treatment has been demonstrated to cause some side effects on healthy tissues and multidrug resistance of the tumor cells, which greatly limits therapeutic efficacy. To address these limitations and achieve better therapeutic efficacy, combination therapy based on nanoparticle platforms provides a promising approach through delivering different agents simultaneously to the same destination with synergistic effect. In this study, a novel green tea catechin-based polyion complex (PIC) micelle loaded with doxorubicin (DOX) and (-)-Epigallocatechin-3-O-gallate (EGCG) was constructed through electrostatic interaction and phenylboronic acid-catechol interaction between poly(ethylene glycol)-block-poly(lysine-co-lysine-phenylboronic acid) (PEG-PLys/PBA) and EGCG. DOX was co-loaded in the PIC micelles through π-π stacking interaction with EGCG. The phenylboronic acid-catechol interaction endowed the PIC micelles with high stability under physiological condition. Moreover, acid cleavability of phenylboronic acid-catechol interaction in the micelle core has significant benefits for delivering EGCG and DOX to same destination with synergistic effects. In addition, benefiting from the oxygen free radicals scavenging activity of EGCG, combination therapy with EGCG and DOX in the micelle core could protect the cardiomyocytes from DOX-mediated cardiotoxicity according to the histopathologic analysis of hearts. Attributed to modulation of EGCG on P-glycoprotein (P-gp) activity, this kind of PIC micelles could effectively reverse multidrug resistance of cancer cells. These results suggested that EGCG based PIC micelles could effectively overcome DOX induced cardiotoxicity and multidrug resistance.

Keywords: cardiotoxicity; catechin; combination therapy; multidrug resistance..

Figure 1

(A) Schematic illustrating the formation of the core cross-linked micelle co-loaded with DOX. (B) Proposed mechanism to overcome multidrug resistance. (C) Mechanism to reduce cardiotoxicity.

Figure 2

(A) Hydrodynamic diameter distribution of CLM and TEM images of CLM at pH 7.4 in 10 mM phosphate buffer solution (inset). (B) Stability of CLM and NCLM. Size variation of CLM and NCLM in PBS buffer (10 mM, pH 7.4) at 37 °C.

Figure 3

(A) Illustration of pH responsiveness of CLM. (B) UV-vis spectra of FPBA, EGCG, mixture of EGCG and FPBA at pH 7.4 and mixture of EGCG and FPBA at pH < 5.0. (C) UV-vis spectra of CLM over time under different pH condition.

Figure 4

In vitro release profiles of DOX from (A) CLM-DOX and (B) NP-DOX under different pH conditions.

Figure 5

Superoxide scavenging activity of CLM. (A) Illustration of the role of CLM in NBT assay. (B) Effect of CLM on the inhibition of the NBT reduction under different micelle concentration. (C) Superoxide scavenging activity of CLM at 3 min under different micelle concentration.

Figure 6

Inverted fluorescent microscopy images of H9C2 cells treated with free DOX, NP-DOX and EGCG based CLM-DOX after 4 h incubation at equivalent DOX concentration of 10 μg/mL. From left to right, the images show DOX fluorescence (red), cell nuclei stained by DAPI (blue), and the merge of the two images. Scale bars are 25 μm.

Figure 7

(A) ROS content in H9C2 cells treated with PBS, free DOX, NP-DOX and EGCG based CLM-DOX after 2 h incubation. (B) Cytotoxicity of free DOX, NP-DOX and EGCG based CLM-DOX on H9C2 cells.

Figure 8

Histopathologic analysis of hearts after H&E staining, which were harvested from mice treated with free DOX, NP-DOX, CLM-DOX and saline. Scale bars are 50 μm.

Figure 9

Inverted fluorescent microscopy image of MCF-7/Adr cells after incubation with free DOX, NP-DOX and EGCG based CLM-DOX. From left to right, cell nucleus stained by DAPI (blue), the images show DOX fluorescence (red), and the merge of the two images.

Figure 10

(A) Cytotoxicity of different micelle against resistant human breast cancer cells MCF-7/Adr. (B) Dose-response curves of DOX in CLM-DOX and NP-DOX against MCF-7/Adr. (Mean ±standard deviation, n=3).

Figure 11

ex vivo fluorescence (from DOX) imaging of the heart and normal tissues harvested from BALB/c mice at 1 h, 6 h and 24 h post-injection. The numeric label for each organ is as follows: 1, heart; 2, liver; 3, spleen; 4, lung; 5, kidney.