Switching the NIR upconversion of nanoparticles for the orthogonal activation of photoacoustic imaging and phototherapy

Abstract

Phototheranostics based on upconversion nanoparticles (UCNPs) offer the integration of imaging diagnostics and phototherapeutics. However, the programmable control of the photoactivation of imaging and therapy with minimum side effects is challenging due to the lack of ideal switchable UCNPs agents. Here we demonstrate a facile strategy to switch the near infrared emission at 800 nm from rationally designed UCNPs by modulating the irradiation laser into pulse output. We further synthesize a theranostic nanoagent by combining with a photosensitizer and a photoabsorbing agent assembled on the UCNPs. The orthogonal activation of in vivo photoacoustic imaging and photodynamic therapy can be achieved by altering the excitation modes from pulse to continuous-wave output upon a single 980 nm laser. No obvious harmful effects during photoexcitation was identified, suggesting their use for long-term imaging-guidance and phototherapy. This work provides an approach to the orthogonal activation of imaging diagnostics and photodynamic therapeutics.

Fig. 1. Mechanistic design towards NIR switchable upconversion.

a Schematic of energy transfer rate (W) depending on the energy mismatch (ΔE) between the relevant energy levels of sensitizer (S) and activator (A). Inset shows the non-resonant energy transfer from S to A. Note that W₀ is the rate when ΔE = 0, and β presents a positive parameter. b Comparison of the resonant (left) and non-resonant (right) energy transfer upconversion in sensitizer-activator coupled systems. c Schematic of the orthogonal emissive properties of the specially designed UCNPs, which show red/NIR emissions with 980 nm CW laser excitation and the NIR emission vanishes with short pulse 980 nm PW laser excitation. d Upconversion emission spectra of the UCNPs under CW 980 nm excitation and PW 980 nm excitation with a reduction of the pulse width. e Dependence of the NIR-to-red emission intensity ratio of the UCNPs on pump power density upon CW and PW (with width of 10 ns) 980 nm laser.

Fig. 2. Switchable NIR upconversion mechanism through non-steady-state excitation.

a Time-dependent upconversion emission profiles of Er³⁺ at 660 nm and Tm³⁺ at 800 nm for the UCNPs under PW 980 nm excitation with a duration time of 8 ms. b Time-dependent upconversion emission profiles of Er³⁺ at 660 nm and Tm³⁺ at 800 nm for the UCNPs doping with only Er³⁺ and Tm³⁺, respectively, under PW 980 nm excitation with a duration time of 8 ms. c NIR-to-red emission intensity ratios under different excitation frequencies for the UCNPs. d Schematic dynamic control of upconversion in Yb³⁺/Er³⁺/Tm³⁺ codoped system for 660 nm red and switchable 800 nm NIR emissions under 980 nm excitation with CW or PW laser. e Comparison of the time-dependent upconversion emission profiles of Er³⁺ at 660 nm for the UCNPs with and without codoping of Tm³⁺ under 980 nm excitation with a duration time of 8 ms.

Fig. 3. Design and characterization of the orthogonal UCNPs-DI nanoagent.

a Schematic showing the orthogonal regulation of UCNPs-DI for photoacoustic imaging-guided on-demand treatment. b TEM images and DLS of UCNPs and UCNPs-DI, respectively. Scale bar, 20 nm. c Absorption spectra of ICG, DPP, UCNPs-I, UCNPs-D, and UCNPs-DI. d Upconversion emission spectra of UCNPs and UCNPs-DI under 980 nm PW or CW irradiation. e PA intensity of UCNPs-DI as a function of concentration (R² = 0.97789), and the inset shows the point PA signal. Data are presented as mean ± SD (n = 3). f Temperature changes of UCNPs-DI under irradiation of 980 nm PW or CW laser at the same power density (0.5 W/cm²). g Normalized SOSG fluorescence intensity in the presence of UCNPs-DI under 980 nm PW or CW irradiation.

Fig. 4. Cytotoxicity of UCNPs-DI in the orthogonal excitation.

a MCF7 cells were incubated with different concentrations of UCNPs-DI for 4 h, then cells were irradiated with a 980 nm PW laser (10 ns, 0.5 W/cm²) or CW laser (0.5 W/cm²) for 3 min. The relative viabilities of MCF7 cells were determined by the standard MTT assay. b Relative viabilities of cells pretreated UCNPs-DI (200 μg/mL, 4 h) recorded under different laser power density. c Intracellular ¹O₂ generation detected in SOSG-stained MCF7 cells with different treatments. Scale bar, 50 μm. d Confocal fluorescence microscope images of calcein AM and PI co-stained MCF7 cells after various treatments indicated. Green and red colors represented live and dead cells, respectively. Scale bar, 100 μm. e Flow cytometric analysis of MCF7 cells death after different treatments. In c–e, the nanoagent was used to treat the cells for 4 h at the concentration of 200 μg/mL and the 980 nm PW (10 ns) or CW laser was employed to irradiate the cells for 3 min at the power density of 0.5 W/cm². Mean values and error bars are defined as mean and SD, respectively. All data in a and b are presented as mean ± SD (n  =  3). Statistical differences p values were calculated by Student’s two-sided t-test (^nsP > 0.05).

Fig. 5. In vivo photoacoustic imaging of UCNPs-DI.

a Schematic illustration of photoacoustic imaging of UCNPs-DI. b Real-time photoacoustic images of the tumors after systematic administration of UCNPs-DI in mice under irradiation of 980 nm PW laser. c Photoacoustic images of the tumors after systematic administration of UCNPs, DPP, ICG, UCNPs-D, UCNPs-I, and UCNPs-DI at 12 h intravenous post-injection under irradiation of 980 nm PW laser. d Quantitation of photoacoustic signals at the tumor sites of UCNPs-DI. e Quantitation of photoacoustic signals at the tumor sites of groups depictured in (c). Mean values and error bars are defined as mean and SD, respectively. All data in d and e are presented as mean ± SD (n = 3). Statistical differences p values were calculated by Student’s two-sided t-test.

Fig. 6. Orthogonally regulated tumor photo-treatment.

a Schematic illustration of the PDT treatment regimen. b Photographs of tumor tissues extracted from the mice that received a set of treatments at 21 days including PBS, PW, CW, UCNPs-DI, PW + ICG, CW + ICG, PW + UCNPs-I, CW + UCNPs-I, PW + UCNPs-DI, and CW + UCNPs-DI. Relative tumor volume growth curve (c), mice body weight (d), and tumor weight (e) of the mice treated with different treatments. f SOSG immunofluorescence sections treated in series of groups. Scale bar, 100 μm. g H&E staining of tumor slices for treatments. Scale bar, 100 μm. All quantitative data in (c–e) are expressed as mean ± SD (n = 6, biologically independent mice). P values were calculated by Student’s two-sided t-test (^nsP > 0.05).