The effect of the acid-sensitivity of 4-(N)-stearoyl gemcitabine-loaded micelles on drug resistance caused by RRM1 overexpression

Abstract

Chemoresistance is a major issue for most gemcitabine-related chemotherapies. The overexpression of ribonucleotide reductase subunit M1 (RRM1) plays a key role in gemcitabine resistance. In this study, we synthesized a new highly acid-sensitive amphiphilic micelle material by conjugating hydrophilic polyethylene glycol with a hydrophobic stearic acid derivative (C18) using a hydrazone bond, which was named as PHC-2. A lipophilic prodrug of gemcitabine, 4-(N)-stearoyl gemcitabine (GemC18), was loaded into micelles prepared with PHC-2, a previously synthesized less acid-sensitive PHC-1, and their acid-insensitive counterpart, PAC. GemC18 loaded in acid-sensitive micelles can overcome gemcitabine resistance, and GemC18 in the highly acid-sensitive PHC-2 micelles was more cytotoxic than in the less acid-sensitive PHC-1 micelles. Mechanistic studies revealed that upon cellular uptake and lysosomal delivery, GemC18 in the acid-sensitive micelles was released and hydrolyzed more efficiently. Furthermore, GemC18 loaded in the highly acid-sensitive PHC-2 micelles inhibited the expression of RRM1 and increased the level of gemcitabine triphosphate (dFdCTP) in gemcitabine resistant tumor cells. The strategy of delivering lipophilized nucleoside analogs using highly acid-sensitive micelles may represent a new platform technology to increase the antitumor activity of nucleoside analogs and to overcome tumor cell resistance to them.

Fig. 1

Characterization of PHC-2. (A) ¹H NMR spectrum of PHC-2 in CDCl₃. (B) ¹H NMR spectra of PHC-2 in CDCl₃ after incubation up to 24 h at pH 7.4, 6.8 or 5.5, 37°C. a, CHO from PEG-aldehyde, 9.79 ppm; b, CONHN=CH from PHC-2, 8.56 ppm. (C) Time- and pH-dependent degradation profiles of PHC-2 based on ¹H NMR spectra in B. (D) The plot of I₁/I₃ values versus the concentrations of PHC-2 in water (n = 3).

Fig. 2

In vitro release of GemC18 from PHC-2 micelles in PBS of different pH values (5.5, 6.8, or 7.4) at 37°C (n = 3).

Fig. 3

Cellular uptake and apoptosis studies. (A) The percentage of GemC18 internalized by TC-1-GR cells after incubation with GemC18, in a solution or in different micelles, for 2 h or 6 h (n = 3). a, P < 0.05, PHC-2 vs. PAC or PHC-1 for 6 h. (B) Representative cytograms of cell apoptosis analysis of TC-1-GR cellsafter treatment with GemHCl, GemC18, GemC18-loaded PAC, PHC-1 or PHC-2 micelles. Untreated cells were used as control. (C) The percentage of different TC-1-GR cell populations (nuclear debris, early apoptotic, late apoptotic and dead, and viable cells) after the treatment with GemHCl, GemC18, GemC18-loaded PAC, PHC-1 or PHC-2 micelles (n = 3).

Fig. 4

Mechanisms of the cellular uptake of GemC18 in solution or in various micelles. (A) A comparison of cellular uptake GemC18 in solution or in micelles at 4°C vs. 37°C (n = 3). a, P < 0.01 vs. the control. (B) The effect of various specific endocytosis inhibitors on the cellular uptake of GemC18 in three different PEG-C18 micelles (n = 3). b, P < 0.01 vs. the control.

Fig. 5

A comparison of the intracellular fate of PEG-C18 micelles with different acid-sensitivity. (A) Colocalization of DiO-loaded PEG-C18 micelles (green) and lysosomes (red). Cell nuclei are in blue. Bar, 20 μm. (B–C) The change of FRET effect in TC-1-GR cells. Cells were pre-incubated with DiI/DiO-loaded PEG-C18 micelles for 2 h, followed by six additional hours of incubation in fresh medium. Bar, 20 μm. Values in (C) are derived from the confocal micrographs in B (n = 7–9). a, P < 0.05 vs. PAC; b, P < 0.01 vs. PAC and PHC-1.

Fig. 6

The effect of NH₄Cl on the pro-apoptotic activity of GemC18 in solution or in micelles against TC-1-GR cells and the intracellular degradation of GemC18. The percentage of apoptotic cells after TC-1-GR cells were incubated in the presence (A) or absence (B) of NH₄Cl for 24 h. Cells were pre-incubated for 2 h with GemC18, GemC18-loaded PAC, PHC-1 or PHC-2 micelles. Cells pre-incubated with fresh medium were used as a negative control (n = 3). (C) The effect of NH₄Cl on the pro-apoptotic activity of the GemC18 in TC-1-GR cells, relative to the negative untreated control. (D) The percentage of GemC18 remaining in TC-1-GR cells 16 h after they were incubated in the presence or absence of NH₄Cl after internalization. Cells were pre-treated with GemC18, GemC18-loaded PAC, PHC-1 or PHC-2 micelles for 2 h to allow the internalization of GemC18 (n = 3). a, P < 0.01, 0 mM vs. 50 mM NH₄Cl.

Fig. 7

The effect of different formulations of GemHCl or GemC18 on RRM1 expression and dNTP and dFdCTP levels in TC-1-GR cells. (A) Immunoblotting analysis of RRM1 expression in TC-1-GR cells treated with GemHCl, GemC18, GemC18-loaded PAC, PHC-1, or PHC-2 micelles. Untreated cells were used as the control (Shown are typical data from more than 3 replicates). (B) Cellular dNTP level after treatment with GemHCl, GemC18, GemC18-loaded PAC, PHC-1, or PHC-2 micelles (n = 3). Untreated cells were used as the control. a, P < 0.05 vs. control; b, P < 0.05 vs. GemHCl; c, P < 0.01 vs. GemHCl; d, P < 0.05 vs. control. (C) Cellular dFdCTP level after treatment with GemHCl, GemC18, GemC18-loaded PAC, PHC-1, or PHC-2 micelles (n = 3). e, P < 0.01 vs. GemHCl and GemC18; f, P < 0.01 vs. others. (D) The ratio of dFdCTP to total dNTPs in TC-1-GR cells after treatment with GemHCl, GemC18, GemC18-loaded PAC, PHC-1, or PHC-2 micelles (n = 3). g, P < 0.01 vs. others; h, P < 0.05 vs. others.

Scheme 1

Schematic representation of micelle formation and the reaction process. (A) Chemical structures of the three PEG-C18 micelle materials, illustration of the preparation of GemC18-loaded micelles, and the expected acid-sensitive release profiles of GemC18 from the micelles. (B) The scheme of the synthesis of PHC-2 conjugate.