Upconversion superballs for programmable photoactivation of therapeutics

Abstract

Upconversion nanoparticles (UCNPs) are the preferred choice for deep-tissue photoactivation, owing to their unique capability of converting deep tissue-penetrating near-infrared light to UV/visible light for photoactivation. Programmed photoactivation of multiple molecules is critical for controlling many biological processes. However, syntheses of such UCNPs require epitaxial growth of multiple shells on the core nanocrystals and are highly complex/time-consuming. To overcome this bottleneck, we have modularly assembled two distinct UCNPs which can individually be excited by 980/808 nm light, but not both. These orthogonal photoactivable UCNPs superballs are used for programmed photoactivation of multiple therapeutic processes for enhanced efficacy. These include sequential activation of endosomal escape through photochemical-internalization for enhanced cellular uptake, followed by photocontrolled gene knockdown of superoxide dismutase-1 to increase sensitivity to reactive oxygen species and finally, photodynamic therapy under these favorable conditions. Such programmed activation translated to significantly higher therapeutic efficacy in vitro and in vivo in comparison to conventional, non-programmed activation.

Fig. 1

Orthogonal excitation superballs with mixed UCNPs A and B. TEM images of UCNPs A (a) and UCNPs B (b). 808 nm (c) laser and 980 nm laser (d) excited upconversion emission spectra of cyclohexane solutions at room temperature (25 °C) comprising A (red line) and B (black line) with same concentration. e TEM images of OP-SBs. f Schematic illustration of OP-SBs with luminescent photos taken by smart phone camera. g Normalized 808 nm laser (left) and 980 nm laser (right) excited upconversion emission spectra of superball aqueous suspension at room temperature (25 °C) with different mixing ratio between A and B. 808 and 980 nm laser power (1 W) are kept same throughout the measurement. h 808 nm laser (left) and 980 nm laser (right) excited upconversion emission spectra with increasing laser power of superball (A:B = 1:1) aqueous suspension at room temperature (25 °C). i Smartphone images of OP-SBs (A:B = 1:1) pattern under different 980 nm:808 nm laser power ratio. Scale bar: 50 nm for a, b, 200 nm for e. UCNPs A, NaYF₄: 60%Yb, 20%Gd, 2%Er@NaLuF₄: 25%Y; UCNPs B, NaYF₄: 30Yb, 0.5%Tm@NaYF₄: 10%Yb@NaNdF₄: 10%Yb

Fig. 2

Orthogonal photoactivation of multiple therapeutics. a Schematic showing the difference in emission profile between Control-SBs@azo-Psi and OP-SBs@azo-Psi with programmed activation using 980/808 nm laser. b Release of singlet oxygen from OP-SBs@azo-Psi in solution over time with 980 nm irradiation. c Step-wise release of singlet oxygen from OP-SBs@azo-Psi in solution with 980 nm irradiation. d Release of siRNA from OP-SBs@azo-Psi in solution over time with 808 nm irradiation. e Step-wise release of siRNA from OP-SBs@azo-Psi in solution with 808 nm irradiation. f Singlet oxygen production using 980 nm excitation of the same OP-SBs@azo-Psi at different time points. g Orthogonal activation of singlet oxygen production and siRNA release from OP-SBs@azo-Psi over time. h Non-orthogonal activation of singlet oxygen production and siRNA release from Control-SBs@azo-Psi over time. i Programmed activation of OP-SBs@azo-Psi with different durations of 980 and 808 nm NIR irradiation. Error bars represent the standard deviation of measurements from three (n = 3) distinct samples

Fig. 3

Programmed orthogonal photoactivation. a Schematic illustration of orthogonal excitation of photosensitizers and azobenzene-based caps for endosomal escape, siRNA release and photodynamic therapy. b, c Cells without and with siRNA loaded OP-SBs and irradiated with 808 nm NIR for siRNA release, scale bar is 50 μm. d Production of singlet oxygen in cells with NIR irradiation in comparison to non-irradiated cells (e), scale bar is 50 μm. f, g Uptake of OP-SBs@azo-Psi in 2D culture cells (scale bar is 50 μm) and 3D tumor spheroids (scale bar is 200 μm). h Comparison of HeLa and Cal27 cell killing by OP-SBs@azo-Psi based PDT without and with PCI. i Comparison of SOD1 gene expression without and with PCI. j, k Comparison of simultaneous (non-orthogonal) and subsequent (orthogonal) activation of endosomal escape, siRNA release and PDT and their effects on cell viability of HeLa and Cal27 2D culture and 3D tumor spheroids. Error bars represent the standard deviation of measurements from three (n = 3) distinct samples

Fig. 4

Photoactivation of therapeutic processes using OP-SBs in vivo. Serum levels of alkaline phosphatase (ALP) activity (a), alanine aminotransferase (ALT) activity (b) and blood urea nitrogen (BUN) (c), in mice injected intravenously with OP-SBs at doses of 0 (untreated controls), 25 and 50 mg/kg and sacrificed at 1 week and 1 month post injection. Error bars represent the standard deviation of measurements from three (n = 3) distinct samples. d, e Hematoxylin and eosin staining of tissue sections obtained from the mice major organs at 1 week and 1 month post injection of the OP-SBs, showing that the histological features between mice intravenously injected with OP-SBs of dose 0 (control), 25 and 50 mg/kg, scale bar is 100 μm. f Tumor regression studies of Cal27 tumors on Balb/c nude mice injected with different nanoformulations and irradiated with a 980 and/or 808 nm NIR laser. g Body weight of treated mice over the treatment period. h, i Distribution of OP-SBs@azo-Psi in tumor at 8 and 24 h after intratumoral injection, scale bar is 100 μm. Error bars represent the standard deviation of measurements from five (n = 5) distinct samples. *p < 0.05 in comparison to Group 1 (ANOVA), *^#p < 0.05 in comparison to Group 1 and 3 (ANOVA)