Cerenkov Radiation Induced Photodynamic Therapy Using Chlorin e6-Loaded Hollow Mesoporous Silica Nanoparticles

Abstract

Traditional photodynamic therapy (PDT) requires external light to activate photosensitizers for therapeutic purposes. However, the limited tissue penetration of light is still a major challenge for this method. To overcome this limitation, we report an optimized system that uses Cerenkov radiation for PDT by using radionuclides to activate a well-known photosensitizer (chlorin e6, Ce6). By taking advantage of hollow mesoporous silica nanoparticles (HMSNs) that can intrinsically radiolabel an oxophilic zirconium-89 (⁸⁹Zr, t_1/2 = 78.4 h) radionuclide, as well as possess great drug loading capacity, Ce6 can be activated by Cerenkov radiation from ⁸⁹Zr in the same nanoconstruct. In vitro cell viability experiments demonstrated dose-dependent cell deconstruction as a function of the concentration of Ce6 and ⁸⁹Zr. In vivo studies show inhibition of tumor growth when mice were subcutaneously injected with [⁸⁹Zr]HMSN-Ce6, and histological analysis of the tumor section showed damage to tumor tissues, implying that reactive oxygen species mediated the destruction. This study offers a way to use an internal radiation source to achieve deep-seated tumor therapy without using any external light source for future applications.

Keywords: Cerenkov radiation; chlorin e6; hollow mesoporous silica nanoparticles; photodynamic therapy; positron emission tomography.

Figure 1

Physicochemical characterization of HMSNs. (a) TEM image shows an average particle size ~110 nm. (Scale bar: 200 nm) Outset schematic depicts the structure of HMSN showing a hollow cavity with a mesoporous shell. (b) DLS measurements depicting monodispersed hydrodynamic size distribution of HMSN-NH₂ (polydispersity index, PDI = 0.09).

Figure 2

(a) Schematic represents the synthesis of [⁸⁹Zr]HMSN-Ce6 nanoconstructs. Ce6 was first loaded inside the hollow structure of HMSN-NH₂ followed by direct chelator-free labeling with oxophilic ⁸⁹Zr to form [⁸⁹Zr]HMSN-Ce6. (b) Digital photos of HMSN-Ce6 show nanoparticles in PBS before (left) and after centrifugation (right). (c) UV-vis-NIR spectra of Ce6 and HMSN-Ce6 in PBS (red). (d) Reduction of UV absorbance of Ce6 loaded in HMSN-Ce6 at day 1, 2, 3, 7 and 14, indicating that Ce6 slowly releases from HMSN over time at 25 °C.

Figure 3

(a) In vitro stability test of [⁸⁹Zr] HMSN-Ce6 after incubation in mouse serum, at 37 °C for different periods of time. (b) Luminescence imaging of PBS solution containing ⁸⁹Zr (tube 1), HMSN-Ce6 (tube 2) and [⁸⁹Zr]HMSN-Ce6 (tube 3). Images were acquired using an IVIS spectrum in vivo imaging system (emission filter 690 – 710 nm, excitation was blocked).

Figure 4

In vitro Cerenkov radiation induced photodynamic therapy. (a) Cell viability of 4T1 cells treated with various concentrations of Ce6 in HMSN-Ce6 and [⁸⁹Zr]HMSN-Ce6 nanoconstructs. (b) Cell viability of 4T1 cells treated with various amounts of ⁸⁹Zr in [⁸⁹Zr]HMSN-Ce6 nanoconstructs. (c) Cell viability of 4T1 cells treated with ⁸⁹Zr, HMSNs, HMSN-Ce6 and [⁸⁹Zr]HMSN-Ce6, the concentrations of Ce6 and HMSNs were kept the same. (d) Confocal images of γ-H₂AX stained 4T1 cells treated with PBS (control), HMSN-Ce6, free ⁸⁹Zr and [⁸⁹Zr]HMSN-Ce6 nanoconstructs., scale bar = 20 μm. Error bars represent SD of at least three replicates. P values: ***P < 0.001, ** P<0.01, and *P<0.5.

Figure 5

In vivo PET imaging of [⁸⁹Zr]HMSN-Ce6. (a) Maximum intensity projection PET images of 4T1 tumor-bearing mice taken at various time points (2, 7 and 14 day) post injection (p.i.) of [⁸⁹Zr]HMSN-Ce6. (b) Region-of-interest quantification of [⁸⁹Zr]HMSN-Ce6 uptake in the 4T1 tumor, liver, and bone at various time points p.i. The unit is the percentage of injected dose per gram of tissue (%ID/g). Error bars represent SD of three replicates.

Figure 6

In vivo Cerenkov radiation induced photodynamic therapy. (a) Tumor growth curves of different groups of mice after various treatments indicated; control group with no injection (black), mice injected with [⁸⁹Zr]HMSN (blue), HMSN-Ce6 (green) and [⁸⁹Zr]HMSN-Ce6 (red), n=4. Error bars were based on SD. (b) Representative photographs of tumors from different groups taken at the day 16 p.i. (c) Representative photographs of mice from different groups taken at the day 14 p.i. (d) H&E stained tumor slices collected from different groups of mice day 7 after various treatments. Statistical analysis was calculated by student’s T-test (*** p < 0.001, **p < 0.01, or *p < 0.05).