Theranostic Properties of Crystalline Aluminum Phthalocyanine Nanoparticles as a Photosensitizer

Abstract

The study of phthalocyanines, known photosensitizers, for biomedical applications has been of high research interest for several decades. Of specific interest, nanophotosensitizers are crystalline aluminum phthalocyanine nanoparticles (AlPc NPs). In crystalline form, they are water-insoluble and atoxic, but upon contact with tumors, immune cells, or pathogenic microflora, they change their spectroscopic properties (acquire the ability to fluoresce and become phototoxic), which makes them upcoming agents for selective phototheranostics. Aqueous colloids of crystalline AlPc NPs with a hydrodynamic size of 104 ± 54 nm were obtained using ultrasonic dispersal and centrifugation. Intracellular accumulation and localization of AlPc were studied on HeLa and THP-1 cell cultures and macrophages (M0, M1, M2) by fluorescence microscopy. Crystallinity was assessed by XRD spectroscopy. Time-resolved spectroscopy was used to obtain characteristic fluorescence kinetics of AlPc NPs upon interaction with cell cultures. The photodynamic efficiency and fluorescence quantum yield of AlPc NPs in HeLa and THP-1 cells were evaluated. After entering the cells, AlPc NPs localized in lysosomes and fluorescence corresponding to individual AlPc molecules were observed, as well as destruction of lysosomes and a rapid decrease in fluorescence intensity during photodynamic action. The photodynamic efficiency of AlPc NPs in THP-1 cells was almost 1.8-fold that of the molecular form of AlPc (Photosens). A new mechanism for the occurrence of fluorescence and phototoxicity of AlPc NPs in interaction with cells is proposed.

Keywords: aluminum phthalocyanine; fluorescence lifetime; nanoparticles; photosensitizer; phototheranostic; phototoxicity.

Figure 1

(a) Aluminum phthalocyanine chloride (AlPcCl) used in the study; (b) SEM images of AlPc microparticles; (c) size distribution of the obtained particles in the colloid before and after centrifugation; (d) absorption spectra of Photosens and of crystalline AlPc NP colloids and the fluorescence spectrum of Photosens, as well as their visible and fluorescent images at 400 nm excitation (inset) (e) SEM images of obtained AlPc NPs; (f) absorption spectra for various concentrations of AlPc NP colloids: 10–250 mg/L (left), calibration curves for determining the concentration of AlPc NP colloids (right) by OD.

Figure 2

(a) AlPc NP absorption spectra at different pH; (b) AlPc NP fluorescence spectra (excitation at 633 nm) at different pH (due to the large difference in fluorescence intensity, the spectra are shown on a logarithmic scale); (c) AlPc NP fluorescence kinetics at different pH (d) calculated fluorescence lifetimes and amplitudes.

Figure 3

(a) Fluorescence spectra of Photosens in HeLa cells depending on the duration of incubation; (b) fluorescence spectra of AlPc NPs in HeLa cells depending on the duration of incubation; (c) fluorescence intensity of Photosens and AlPc NPs with addition of DMSO in HeLa cells.

Figure 4

Fluorescence images of HeLa cells (top row) after 24 h of incubation with AlPc NPs. Top row: green—Lysotracker fluorescence, red—AlPc NP fluorescence, from left to right—destruction of lysosomes by irradiation with a 633 nm laser. Bottom: fluorescence-intensity dynamics during laser irradiation.

Figure 5

Fluorescence images of M0, M1 and M2 macrophages after 24 h of incubation with AlPc NP colloids. AlPc NP fluorescence is red.

Figure 6

Fluorescence kinetics of AlPc NPs upon interaction with M0, M1 and M2 macrophages and calculated fluorescence lifetimes and amplitudes.

Figure 7

Dependence of hemoglobin oxygenation in the sample on the duration of laser irradiation at a wavelength of 675 nm with a power density of 200 mW/cm² for HeLa and THP-1 cells incubated with AlPc NPs and Photosens, as well as the calculated values of photodynamic efficiency coefficient (k).

Figure 8

Model of the fluorescence and phototoxicity mechanism of AlPc NPs captured by cells.