Doxorubicin loaded iron oxide nanoparticles overcome multidrug resistance in cancer in vitro

Abstract

Multidrug resistance (MDR) is characterized by the overexpression of ATP-binding cassette (ABC) transporters that actively pump a broad class of hydrophobic chemotherapeutic drugs out of cancer cells. MDR is a major mechanism of treatment resistance in a variety of human tumors, and clinically applicable strategies to circumvent MDR remain to be characterized. Here we describe the fabrication and characterization of a drug-loaded iron oxide nanoparticle designed to circumvent MDR. Doxorubicin (DOX), an anthracycline antibiotic commonly used in cancer chemotherapy and substrate for ABC-mediated drug efflux, was covalently bound to polyethylenimine via a pH sensitive hydrazone linkage and conjugated to an iron oxide nanoparticle coated with amine terminated polyethylene glycol. Drug loading, physiochemical properties and pH lability of the DOX-hydrazone linkage were evaluated in vitro. Nanoparticle uptake, retention, and dose-dependent effects on viability were compared in wild-type and DOX-resistant ABC transporter over-expressing rat glioma C6 cells. We found that DOX release from nanoparticles was greatest at acidic pH, indicative of cleavage of the hydrazone linkage. DOX-conjugated nanoparticles were readily taken up by wild-type and drug-resistant cells. In contrast to free drug, DOX-conjugated nanoparticles persisted in drug-resistant cells, indicating that they were not subject to drug efflux. Greater retention of DOX-conjugated nanoparticles was accompanied by reduction of viability relative to cells treated with free drug. Our results suggest that DOX-conjugated nanoparticles could improve the efficacy of chemotherapy by circumventing MDR.

Fig. 1

Synthesis schematic. a) Polyethylenimine (PEI) was activated with a hydrazine group through subsequent modifications with Traut’s reagent and BMPH to form PEI-BMPH. b) Doxorubicin (DOX) was attached to PEI-BMPH through a hydrazone bond. c) Amine terminated PEG coated iron oxide nanoparticles (NP-PEG) were activated with SIA to render a free iodoacetate group and subsequently reacted to PEI-DOX through a thioether linkage to form NP-DOX. Each NP-DOX had 216 ± 99 PEI, and 5 ± 2 DOX per PEI.

Fig. 2

Colloidal stability of NP-DOX. NP-DOX displayed no appreciable change in size during incubation at 37° C for 5 days in DMEM with 10% FBS.

Fig. 3

Drug release profiles showing the pH dependent release of DOX from NP-DOX. The pH tested correspond to that of blood (pH 7.5), tumor microenvironment (pH 6.5), and endosomes/lysosomes (pH 5.5 and 4.5).

Fig. 4

Accumulation of free DOX or NP-DOX in wild-type and drug-resistant C6 cells. Cells were treated for 4 hr with 1000 ng/mL DOX or equimolar concentration of DOX on NP-DOX, then intracellular DOX was determined by the fluorescence of cell lysate and normalized to cell number using Alamar Blue at a) 4 hr and b) 24 hr after initial drug exposure. N.S. indicates no significance, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001 as determined by Student’s t-test.

Fig. 5

Fluorescence visualization of DOX intracellular accumulation. Both a) drug sensitive C6 and b) drug resistant C6-ADR cells were treated with 1000 ng/mL free DOX or equivalent DOX concentration of NP-DOX for 4 hrs then allowed to grow for an additional 20 hrs. Scale bars correspond to 20 μm.

Fig. 6

Drug response curves. a) C6 and b) C6-ADR were treated with free DOX or NP-DOX and cell viability was analyzed 24, 48, and 72 hours post-treatment. DOX concentrations are in ng/mL.

Fig. 7

NP-DOX circumvents MDR-mediated resistance. a) Fold increase in viability of drug-resistant cells relative to drug-sensitive cells (C6-ADR C6) treated with 1000 ng/mL free DOX or equivalent dose of NP-DOX. b) Resistance factors for 72 hr time point. The resistance factor is the ratio of IC₅₀ of C6-ADR to the IC₅₀ of C6. N.S. indicates no significance, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001 as determined by Student’s t-test.