Hyaluronic acid-modified, IR780-conjugated and doxorubicin-loaded reduced graphene oxide for targeted cancer chemo/photothermal/photodynamic therapy

Abstract

We used reduced graphene oxide (rGO), which has two times higher photothermal conversion efficiency than graphene oxide (GO), as a photothermal agent for cancer photothermal therapy (PTT). By conjugating a photosensitizer IR780 to rGO, the IR780-rGO could be endowed with reactive oxygen species (ROSs) generation ability for concurrent photodynamic therapy (PDT). The IR780-rGO was coated with hyaluronic acid (HA) by electrostatic interaction to facilitate its intracellular uptake by U87 glioblastoma cells. The IR780-rGO/HA was loaded with doxorubicin (DOX) for chemotherapy (CT), to develop a pH-responsive drug delivery nano-platform for targeted multimodal cancer CT/PTT/PDT. We fully characterized the properties of all nanocomposites during the synthesis steps. The high loading efficiency of DOX on IR780-rGO-HA provides 3 mg/mg drug loading, while IR780-rGO-HA/DOX shows 3 times higher drug release at endosomal pH value (pH 5) than at pH 7.4. The mechanism for PTT/PDT was confirmed from the ability of IR780-rGO-HA to induce time-dependent temperature rise, synthesis of heat shock protein 70 (HSP70) and generation of intracellular ROSs, after exposure to 808 nm near infrared (NIR) laser light. The nano-vehicle IR780-rGO-HA shows high biocompatibility toward 3T3 fibroblast and U87 cancer cell lines, as well as enhanced intracellular uptake by U87 through active targeting. This translates into increased cytotoxicity of IR780-rGO-HA/DOX, by lowering the drug half-maximal inhibitory concentration (IC₅₀) from 0.7 to 0.46 μg/mL. This IC₅₀ is further decreased to 0.1 μg/mL by irradiation with NIR laser for 3 min at 1.5 W/cm². The elevated cancer cell killing mechanism was supported from flow cytometry analysis, where the highest cell apoptosis/necrosis rate was observed in combination CT/PTT/PDT. Using xenograft tumor model created by subcutaneous implantation of U87 cells in nude mice, IR780-rGO-HA/DOX delivered through intravenous (IV) injection and followed with 808 nm laser treatment for 5 min at 1.5 W/cm² results in the lowest tumor growth rate, with negligible change of tumor volume from its original value at the end 20-day observation period. The therapeutic efficacy was supported from inhibited cell proliferation rate, increased cell apoptosis rate, and increased production of HSP70 from immunohistochemical staining of tumor tissue slices. The safety of the NIR-assisted multimodal cancer treatment could be confirmed from non-significant change of body weight and hematological parameters of blood sample. Taken together, we conclude that IV delivery of IR780-rGO-HA/DOX plus NIR laser treatment is an effective nanomedicine approach for combination cancer therapy.

Keywords: Doxorubicin; Photodynamic therapy; Photosensitizer; Photothermal agent; Photothermal therapy; Reduced graphene oxide.