Figures & data
Figure 1 Cytotoxicity determined by MTT assay in A549 cells.
Notes: Cells were incubated with doxorubicin-loaded composite micelles, free doxorubicin, and blank composite micelles for 72 hours.
Abbreviations: DOX, free doxorubicin; MIX, composite doxorubicin-loaded micelles.
Figure 2 Intracellular doxorubicin in A549 cells.
Notes: Doxorubicin uptake into A549 tumor cells after incubation with composite doxorubicin-loaded micelles or free doxorubicin for 4 hours, then compounds removed for one hour and 2.5 hours. Accumulation 4 hours: A549 cells incubated in composite doxorubicin-loaded micelles or free doxorubicin for 4 hours; release one hour. A549 cells incubated in composite doxorubicin-loaded micelles or free DOX for 4 hours, then compounds removed for one hour; release 2.5 hours, A549 cells incubated in composite doxorubicin-loaded micelles or free doxorubicin for 4 hours, then compounds removed for 2.5 hours.
Abbreviations: DOX, free doxorubicin; MIX, composite doxorubicin-loaded micelles.
Figure 3 Confocal laser scanning microscopy of cellular incorporation and release of doxorubicin in A549 multicellular spheroids. (A1) A549 spheroid cells incubated in free doxorubicin for 4 hours. (A2) Free doxorubicin released from the spheroid for one hour. (B1) A549 spheroid cells incubated in composite doxorubicin-loaded micelles with the same doxorubicin densities for 4 hours. (B2) Composite doxorubicin-loaded micelles released from the spheroid for one hour. (B3) Composite doxorubicin-loaded micelles released from the spheroid for 2.5 hours.
Figure 4 Flow cytometric histograms of A549 spheroid cells incubated with composite doxorubicin-loaded micelles or free doxorubicin with the same doxorubicin densities for 2 hours and 3 hours. DOX for 2 hours, multicellular spheroids incubated in free doxorubicin for 2 hours; DOX for 3 hours, multicellular spheroids incubated in free doxorubicin for 3 hours; MIX for 2 hours, multicellular spheroids incubated in MIX for 2 hours; MIX for 3 hours, multicellular spheroids incubated in MIX for 3 hours.
Abbreviations: DOX, free doxorubicin; MIX, composite doxorubicin-loaded micelles.
Figure 5 Flow cytometric histograms of multicellular spheroids incubated with MIX or free doxorubicin that achieved the same doxorubicin incorporation after 3 hours of exposure, at 2 and 4 hours following drug withdrawal.
Notes: DOX for 2 hours, multicellular spheroids incubated in free doxorubicin for 3 hours, then compounds removed for 2 hours; DOX for 4 hours, multicellular spheroids incubated in free doxorubicin for 3 hours, then compounds removed for 4 hours; MIX for 2 hours, multicellular spheroids incubated in MIX for 3 hours, then compounds removed for 2 hours; MIX for 4 hours, multicellular spheroids incubated in MIX for 3 hours, then compounds removed for 4 hours.
Abbreviations: DOX, free doxorubicin; MIX, composite doxorubicin-loaded micelles.
Figure 6 Cell survival curves after treatments with radiation alone or combined with 2.5 μg/mL blank composite micelles (radiation alone + blank composite micelles) or doxorubicin (radiation alone + doxorubicin) or doxorubicin-loaded composite micelle (radiation alone + MIX, P < 0.05 for radiation alone + MIX versus radiation alone).
Abbreviations: DOX, free doxorubicin; MIX, composite doxorubicin-loaded micelles; RT, radiation alone; Gy, gray.
Figure 7 Tumor cell migration from spheroids following irradiation and combination treatment (radiation alone + blank composite micelles, radiation alone + doxorubicin, radiation alone + MIX). Mean area of tumor cell migration (mm2).
Notes: Each treatment group (sham, irradiation alone, blank composite micelle + irradiation, free doxorubicin + irradiation, composite doxorubicin-loaded micelle + irradiation) consists of 30 A549 spheroids in five independent experiments. Changes at day 4 were significant (P < 0.05).
Abbreviations: DOX, free doxorubicin; MIX, composite doxorubicin-loaded micelles; RT, radiation alone.
Figure 8 Spheroid proliferation following single mode and combinational treatment.
Notes: Each treatment group (sham, irradiation alone, blank composite micelle + irradiation, free doxorubicin + irradiation, composite doxorubicin-loaded micelle + irradiation) consists of 30 A549 spheroids in five independent experiments. Illustration of mean volume growth of spheroids following irradiation and irradiation alone + blank composite micelles, irradiation alone + doxorubicin, irradiation alone + MIX (composite doxorubicin-loaded micelles). A significant reduction in spheroid volume was observed following irradiation alone + MIX on day 9 (P < 0.05).
Abbreviations: DOX, free doxorubicin; MIX, composite doxorubicin-loaded micelles; RT, radiation alone.
