Core‑shell type thermo‑nanoparticles loaded with temozolomide combined with photothermal therapy in melanoma cells

Abstract

A novel core‑shell type thermo‑nanoparticle (CSTNP) co‑loaded with temozolomide (TMZ) and the fluorescein new indocyanine green dye IR820 (termed IT‑CSTNPs) was designed and combined with a near‑infrared (NIR) laser to realize its photothermal conversion. The IT‑CSTNPs were prepared using a two‑step synthesis method and comprised a thermosensitive shell and a biodegradable core. IR820 and TMZ were entrapped in the shell and the core, respectively. Dynamic light scattering results demonstrated that the average hydrodynamic size of the IT‑CSTNPs was 196.4±3.1 nm with a ζ potential of ‑24.9±1.3 mV. The encapsulation efficiencies of TMZ and IR820 were 6.1 and 16.6%, respectively. Temperature increase curves under NIR laser irradiation indicated that the IT‑CSTNPs exhibited the desired photothermal conversion efficiency. The in vitro drug release curves revealed a suitable release capability of IT‑CSTNP under physiological conditions, whereas NIR laser irradiation accelerated the drug release. Inverted fluorescence microscopy and flow cytometry results revealed that the uptake of IT‑CSTNPs by A375 melanoma cells occurred in a concentration‑dependent manner. Confocal laser scanning microscopy results indicated that IT‑CSTNPs entered tumour cells via endocytosis and were located in intercellular lysosomes. In summary, the present study explored the photothermal conversion capability, cellular uptake, and intracellular localization of IT‑CSTNPs.

Figure 1.

Schematic for preparation of core-shell type thermo-nanoparticles co-loaded with TMZ and the fluorescein new indocyanine green dye IR820. TMZ, temozolomide; PLGA, polylactide-co-glycolic acid; DPPC, dipalmitoyl phosphatidylcholine; MSPC, monostearoylphosphatidylcholine.

Figure 2.

Characterization of core-shell type thermo-nanoparticles co-loaded with temozolomide and the fluorescein new indocyanine green dye IR820. (A) Hydrodynamic size distribution in water and in culture media (DMEM+10% FBS). (B) ζ potential. (C) Transmission electron microscopy image.

Figure 3.

Determination of the TMZ content. (A) Degradation curve of TMZ in PBS pH 7.4. (B) Ultraviolet absorption curve of TMZ in 0.1 M HCl. (C) Ultraviolet absorption spectrum and stability of TMZ. TMZ, temozolomide.

Figure 4.

Temperature increase curve measurement. (A) Temperature increase curves of different concentrations of IT-CSTNPs (0, 5, 10 and 20 µg/ml) under near-infrared laser irradiation. (B) Temperature curves of PBS, IR820 (20 µg/ml), and IT-CSTNPs (IR820 concentration, 20 µg/ml) under near-infrared laser irradiation. IT-CSTNPs, core-shell type thermo-nanoparticle co-loaded with temozolomide and IR820; NP, nanoparticle.

Figure 5.

In vitro drug release profile of IT-CSTNPs. (A) Release profile of IT-CSTNPs at different pH conditions. (B) Release profile of IT-CSTNPs under NIR irradiation. IT-CSTNPs, core-shell type thermo-nanoparticle co-loaded with temozolomide and IR820; NIR, near-infrared.

Figure 6.

Cellular uptake of IT-CSTNPs. Dose-dependent cellular uptake of IT-CSTNPs by A375 melanoma cells was measured using (A) inverted fluorescence microscopy (magnification, ×200) and (B) flow cytometry. IT-CSTNPs, core-shell type thermo-nanoparticle co-loaded with temozolomide and IR820; FITC, fluorescein isothiocyanate; NP, nanoparticle.

Figure 7.

Intracellular localization measurement of core-shell type thermo-nanoparticles co-loaded with temozolomide and the fluorescein new indocyanine green dye IR820 in A375 cells. (A) and (B) Represent different magnifications. Scale bar, 20 µm.