Indocyanine Green-Loaded Quenched Nanoliposomes as Activatable Theranostics for Cancer

Abstract

Photodynamic therapy (PDT) and photothermal therapy (PTT) are considered to be one of the most effective methods for treating cancer due to their noninvasive nature, high effectiveness, and fewer side effects compared to standard therapeutic modalities for cancer. However, conventional always-on types of PDT and PTT agents have basic drawbacks in their in vivo applications, which include the unwanted generation of strong fluorescence signals and phototoxicity in normal tissues, including blood vessels, when exposed to light, resulting in poor imaging contrast and unwanted phototoxicity. Here, we propose indocyanine green-loaded quenched nanoliposomes (Q-ICG-NLs) as an activatable nanotheranostics. Q-ICG-NLs showed significant quenching in near-infrared fluorescence emission and singlet oxygen generation upon light irradiation. The photothermal effect of Q-ICG-NLs was 1.3 times greater than free indocyanine green. Its fluorescence and singlet oxygen generation were largely restored when taken up into cancer cells, enabling the selective detection and phototherapy of cancer cells. These results suggest that Q-ICG-NLs can be effectively used for selective near-infrared fluorescence imaging and the subsequent image-guided PDT and PTT of cancers.

Keywords: activatable; indocyanine green; nanoliposome; phototherapy.

Figure 1

A schematic illustration of Q-ICG-NLs demonstrates their application in activatable NIR fluorescence imaging, photodynamic therapy (PDT), and photothermal therapy (PTT) for cancer treatment. In normal tissues or the extracellular environment, the near-infrared (NIR) fluorescence and singlet oxygen generation of ICG molecules are mostly quenched. However, after Q-ICG-NLs accumulate in tumors via the enhanced permeation and retention (EPR) effect and are internalized by cancer cells, ICG molecules are released from the nanoliposomes, restoring strong fluorescence and enhancing phototoxicity. Furthermore, the imaging-guided PTT induces heat stress can amplify phototoxicity to the cancer cells [17].

Figure 2

(A) Hydrodynamic size distribution of Q-ICG-NLs. (B) Cryo-TEM image of Q-ICG-NLs showing their round-shaped unilamellar structure. (C) Stability analysis of Q-ICG-NLs over a 7-day period by monitoring changes in the hydrodynamic size.

Figure 3

(A) Representative UV/Vis absorption and (B) fluorescence spectra of Q-ICG-NLs and free ICG in deionized water. Fluorescence images of microtubes containing free ICG and Q-ICG-NLs are shown in the inset of (B). (C) Measurement of singlet oxygen generation (SOG) using Q-ICG-NLs and Q-ICG-NLs containing 1% SDS during 808 nm laser irradiation (n = 4).

Figure 4

Observation of photothermal effects. (A) Schematic illustration of the experimental setup for photothermal effect analysis and temperature imaging, demonstrating a heat map within the disposable cuvette. Time-dependent temperature changes in (B) Q-ICG-NLs and (C) free ICG in aqueous solution during 808 nm laser irradiation. Photographs taken before and after light irradiation are shown in the upper images.

Figure 5

Evaluation of the fluorescence recovery of Q-ICG-NLs after intracellular uptake. (A) Confocal fluorescence images of Calu-3 cells treated with Q-ICG-NLs and free ICG (λ_ex. = 633 nm, λ_em. = 700 ± 50 nm). Scale bar = 50 μm. (B) Quantitative analysis of NIR fluorescence signals in Calu-3 cells treated with Q-ICG-NLs and free ICG (n = 4, * p < 0.05).

Figure 6

(A) Cell viability of Calu-3 cells treated with free ICG or Q-ICG-NLs at various ICG-equivalent concentrations (n = 4). (B) In vitro phototoxicity test (n = 4). Calu-3 cells were treated with free ICG or Q-ICG-NLs for 16 h at various ICG-equivalent concentrations, washed twice, and then irradiated with an 808 nm CW laser (0.5 W/cm² for 10 min). After 24 h, cell viability was measured. * p < 0.01.