Quatsomes Loaded with Squaraine Dye as an Effective Photosensitizer for Photodynamic Therapy

Abstract

Photodynamic therapy is a non-invasive therapeutic strategy that combines external light with a photosensitizer (PS) to destroy abnormal cells. Despite the great progress in the development of new photosensitizers with improved efficacy, the PS's photosensitivity, high hydrophobicity, and tumor target avidity still represent the main challenges. Herein, newly synthesized brominated squaraine, exhibiting intense absorption in the red/near-infrared region, has been successfully incorporated into Quatsome (QS) nanovesicles at different loadings. The formulations under study have been characterized and interrogated in vitro for cytotoxicity, cellular uptake, and PDT efficiency in a breast cancer cell line. The nanoencapsulation of brominated squaraine into QS overcomes the non-water solubility limitation of the brominated squaraine without compromising its ability to generate ROS rapidly. In addition, PDT effectiveness is maximized due to the highly localized PS loadings in the QS. This strategy allows using a therapeutic squaraine concentration that is 100 times lower than the concentration of free squaraine usually employed in PDT. Taken together, our results reveal the benefits of the incorporation of brominated squaraine into QS to optimize their photoactive properties and support their applicability as photosensitizer agents for PDT.

Keywords: nanovesicles; photodynamic therapy; quatsomes; squaraine.

Scheme 1

Synthesis of Br-Sq-C12 dye. (i) anhydrous acetonitrile, iodododecane, MW, 60 min, 155 °C; (ii) squaric acid, toluene/n-butanol (1:1), MW, 30 min, 160 °C.

Figure 1

Br-Squaraine-C12-loaded QS components (a); graphic representation of the nanoparticles studied (b); schematic composition of the dye-loaded quatsomes and their mechanism of action in PDT (c).

Figure 2

Physicochemical characterization of Br-Sq-C12-loaded quatsomes. Macroscopic appearance of quatsomes after preparation, from left to right; QS_Blank, QS_Sq_160, and QS_Sq_200 (a). Size distribution obtained by DLS (b) and Cryo-TEM images of QS_Blank (c), QS_Sq_160 (d), and QS_Sq_200 (e).

Figure 3

Absorbance and fluorescence emission spectra of free Br-Sq-C12 in ethanol (a) and Br-Sq-C12-loaded QSs in water with a final dye concentration of 160 μM in sample QS_Sq_160 (b) and 200 μM in sample QS_Sq_200 (c).

Figure 4

DLS and ELS bar plot, showing hydrodynamic diameters (z-average) as bars and PDI as dots (a) and ζ-potential values (b). Absorbance spectra (c) and maximum absorbance values over time (d), measured at 1:10 dilution. Quantification of dye leaking 4 months after sample preparation (e).

Figure 5

Comparative generation of Reactive Oxygen Species (ROS) using Rose Bengal as the standard reference. The final concentration of the squaraine is 2.5 μM for both free and dye-loaded QS.

Figure 6

Cell viability assays based on a colorimetric method (MTT assay, Abs_490nm) on MCF-7 cells. Cells were treated O/N with QS_blank at two different concentrations of membrane components (Chol + Stk): 10 μg/mL (a) and 2 μg/mL (b). Data refer to one experiment representative of three. Statistical significance versus CNTRL (MCF-7 untreated): * p < 0.05, **** p < 0.0001 (t-test or Mann–Whitney test). (c) Cell viability assays on MCF-7 O/N treated with 10 μg/mL (brown line) and 2 μg/mL (beige line) of QS_blank, and 2 μg/mL QS loaded with 160 and 200 µM of Br-Sq-C12 (light blue and blue lines, respectively). Data are normalized on CNTRL at 24 h and represented as mean ± SEM of three independent experiments. Statistical significance versus 2 μg/mL QS blank: *** p < 0.001, **** p < 0.0001 (Ordinary one-way ANOVA with post-hoc Dunnett’s multiple comparisons test).

Figure 7

PDT evaluation on MCF-7 O/N treated with Br-Sq-C12 in its free form or incorporated into QS (2 μg/mL) at 24, 48, and 72 h after LED irradiation (640 nm, 7.2 J/cm²). Data are normalized on their own CNTRL (Br-Sq-12 samples on irradiated cells not treated; QS_Sq samples on irradiated cells treated with blank QS) at 24 h (dot line) and represented as mean ± SEM of three independent experiments. Statistical significance between Sq and QS_Sq: * p < 0.05 (paired t-test or Wilcoxon test).

Figure 8

Representative confocal fluorescence images of MCF-7 cells incubated O/N with either QS_Sq_200 (QS_Sq) (a) or free Br-Sq-C12 (Sq) (b) at the same final concentration (85 nM). Red signals refer to Calcein (λ_ex= 561 nm), and blue signals refer to squaraine (λ_ex= at 633 nm). For each sample, the overlay between the two signals in a 3D cellular volume reconstruction with orthogonal views is reported. Scale bar: 20 μm.