Breaking the depth dependency of phototherapy with Cerenkov radiation and low-radiance-responsive nanophotosensitizers

Abstract

The combination of light and photosensitizers for phototherapeutic interventions, such as photodynamic therapy, has transformed medicine and biology. However, the shallow penetration of light into tissues and the reliance on tissue oxygenation to generate cytotoxic radicals have limited the method to superficial or endoscope-accessible lesions. Here we report a way to overcome these limitations by using Cerenkov radiation from radionuclides to activate an oxygen-independent nanophotosensitizer, titanium dioxide (TiO2). We show that the administration of transferrin-coated TiO2 nanoparticles and clinically used radionuclides in mice and colocalization in tumours results in either complete tumour remission or an increase in their median survival. Histological analysis of tumour sections showed the selective destruction of cancerous cells and high numbers of tumour-infiltrating lymphocytes, which suggests that both free radicals and the activation of the immune system mediated the destruction. Our results offer a way to harness low-radiance-sensitive nanophotosensitizers to achieve depth-independent Cerenkov-radiation-mediated therapy.

Figure 1. Titanium dioxide and Titanocene photoagents for CRIT

a, Schematic of CR mediated excitation of TiO₂ nanoparticles to generate cytotoxic hydroxyl and superoxide radicals from water and dissolved oxygen, respectively, through electron-hole pair generation. CR is generated by PET radionuclides (not to scale). b, Schematic of CR mediated excitation of Tc to generate a cyclopentadienyl radical and a titanium-centred radical through photofragmentation (not to scale). In aerated media, the radicals transform into more potent peroxyl radicals. c, Schematic illustrating the development of TiO₂-PEG,TiO₂-Tf by coating TiO₂ with Tf and subsequent generation of TiO₂-Tf-Tc construct by simple addition of Tc, which docks into the iron binding site of Tf (not to scale). Below (left to right) are the TEM images of TiO₂-PEG, TiO₂ aggregates, TiO₂-Tf and TiO₂-Tf-Tc (right). Scale bar, 50 nm. CR and NPS generate disparate and regenerative cytotoxic free radicals for CRIT.

Figure 2. Cellular uptake of NPS

a, TEM image of a HT1080 tumour cell showing internalized and endo-lysosomal localization of the TiO₂-Tf constructs (arrows). Scale bar, 2 μm. Inset shows two lysosomal compartments with TiO₂-Tf. Scale bar, 400 nm. b, In cellulo uptake of TiO₂-AlexaTf and successful blocking with holo-Tf suggesting Tf receptor mediated internalization as the mechanism of uptake. Scale bar, 20 μm. c, Quantitation of successful blocking of TiO₂-AlexaTf internalization by saturating doses of holo-Tf in HT1080 cells. Values are means ± s.e.m. (experiments for each group were run in triplicates and replicated 2X). **P < 0.01. Tf receptor mediates endocytosis of Tf-coated NPS in tumour cells.

Figure 3. In vitro assessment of CRIT

a, Cell viability assay comparing the TiO₂-Tf, Tc-Tf and TiO₂-Tf-Tc constructs with and without exposure to ⁶⁴Cu and FDG on HT1080 cells. Values are means ± s.e.m. (experiments for each group were run in triplicates). ***P < 0.001. b, Cell viability assay using TiO₂-Tf-Tc with different activities of FDG demonstrating significant cell killing even at 0.2 mCi/0.1 ml. Values are means ± s.e.m. (experiments for each group were run in triplicates and replicated 3X). **P < 0.01, ***P < 0.001. c, Confocal microscopy image of merged bright-field and fluorescence images of Matrigel^™ suspended cells with extracellular TiO₂ and d, intracellular TiO₂, with 0.5 mCi/0.1 ml ⁶⁴Cu. Live/Dead^® cell viability stain was used to distinguish live cells (green) from dead cells (red). Scale bar, 50 μm. Experiments were replicated 3X. e, Examples of alkaline comet assay results. The images show undamaged and damaged DNA as a result of free radical damage and apoptosis. Image marked (i) is representative of undamaged DNA, from the controls, including untreated cells and either exposed to NPS or radionuclide alone. Notice there is negligible DNA in the tail (0.15%). In comparison, cells treated with the NPS and radionuclide, show considerable DNA damage as shown in (ii, iii, iv). Cells in the same treatment group exhibited variable DNA damage, such as 22.32%, 45.87% and 71.84% DNA in the tail. The fluorescence intensity is represented in pseudocolour. f, Cells undergoing CRIT demonstrated an overall higher percent of damaged DNA. 100 cells were counted from each group. Values are means ± s.e.m. **P < 0.01. Experiments were replicated 3X. g, TEM image of a normal HT1080 cell. Scale bar, 3 μm. h, TEM image showing a necrotic cell that was treated with TiO₂-Tf (arrows) and FDG. Notice loss of cell membrane integrity and highly vacuolated cytoplasm. Scale bar, 2 μm. i, TEM image showing an apoptotic cell that was treated with TiO₂-Tf (arrows) and FDG. Notice surface blebbing and condensed chromatin. Scale bar, 1.4 μm. j, TEM image of an apoptotic cell that was treated with Tc-Tf and FDG. Notice nuclear fragmentation and chromatin margination. Scale bar, 1.4 μm. k, Comparison between HT1080 cells not undergoing and undergoing CRIT with TiO₂-Tf and Tc-Tf show higher output of free radicals such as hydroxyl, superoxide and peroxyl species as measured using HPF and Mitosox fluorescent dyes. Values are means ± s.e.m. (experiments for each group were run in triplicates and replicated 3X). l, Lipid peroxidation assay using BODIPY 581/591 C11 reagent on HT1080 cells showing higher degree of lipid peroxidation in cells treated with Tc-Tf and FDG. Values are means ± s.e.m (experiments for each group were run in triplicates and replicated 3X). *P < 0.05. Intracellular localization of both radionuclide and NPS is critical for effective CRIT.

Figure 4. CRIT through intratumoural administration of TiO₂ and ⁶⁴Cu

a, In vivo CRIT through a one-time intratumoural administration of PEGylated TiO₂ and ⁶⁴Cu in HT1080 tumour bearing Athymic nu/nu mice. Toxicity through elemental Cu was eliminated by using non-radioactive CuCl₂, with and without TiO₂-PEG. Values are means ± s.e.m. (n = 4 mice per group). Experiments were replicated 2X. b, Representative photographs at day 1, 3 & 45 of HT1080 tumour bearing mice injected with a single dose of 2.5 μg/ml of TiO₂-PEG and 0.5 mCi/0.1 ml of ⁶⁴Cu intratumourally at day 1. Scale bar, 5 mm. Complete tumour elimination was achieved after PDT at day 45 (dotted circle). c, H&E stained HT1080 tumour section before PDT showing typical herringbone architecture of fibrosarcoma. Scale bar, 1 mm. (n = 4 histological sections per group). d, H&E stained HT1080 tumour section 3 d after commencement of PDT showing extensive necrotic centres and destruction of the tumour architecture. Scale bar, 1 mm. (n = 4 histological sections per group). CRIT can achieve tumour remission through intratumoural administration of both radionuclide and NPS.

Figure 5. In vivo biodistribution of NPS

a, In vivo biodistribution profile of AlexaTf in HT1080 tumour bearing Athymic nude mice at 24 h following tail vein injection (n = 5). Experiments were replicated 3X. b, Ex vivo fluorescence image of dissected organs from (a). Notice the high fluorescence from blood suggesting circulating AlexaTf. c, In vivo biodistribution profile of TiO₂-AlexaTf in HT1080 tumour bearing Athymic nude mice at 24 h following tail vein injection (n = 5). d, Ex vivo fluorescence image of dissected organs from (c). Fluorescence imaging was performed using an excitation and emission wavelength of 685nm and 720 nm, respectively. e, In vivo biodistribution of TiO₂-AlexaTf AlexaTf and TiO₂-PEG alone in HT1080 tumour bearing Athymic nu/nu mice over 24 h. Intrinsic fluorescence of TiO₂ nanoparticles was used for the ex vivo imaging of TiO₂-PEG. Values are means ± s.e.m. (n = 5 mice per group). f, TEM image of tumour sections showing localization of the TiO₂-Tf-Tc constructs (arrow) in tumour cells after i.v. administration. Scale bar, 500 nm g, TEM image of tumour sections of mice injected with TiO₂-PEG showing absence of TiO₂ in the tumour cells. Scale bar, 1μm. High tumour uptake and retention of Tf-coated NPS relative to non-tumour tissues demonstrate the feasibility of CRIT via i.v. administration of CR source following selective retention of the NPS in tumours.

Figure 6. Evaluation of CRIT through systemically administered photoagents and FDG

a, In vivo CRIT through a one-time systemic administration of the constructs and FDG in HT1080 tumour bearing Athymic nu/nu mice. Values are means ± s.e.m. (n = 6 mice per group). **P < 0.01, ***P < 0.001. Experiments were replicated 3X. b, Kaplan-Meier survival curves representing treatment with 0.87 mCi/0.1 ml FDG. *** P < 0.001. c, Survival curves representing treatment with 0.14 and 0. 43 mCi/0.1 ml FDG (n = 4 mice per group). Experiments were replicated 2X. **P < 0.01. d, In vivo CRIT in A549 tumour bearing Athymic nu/nu mice using TiO₂-Tf-Tc and FDG. Values are means ± s.e.m. (n = 4 mice per group). Experiments were replicated 2X. ***P < 0.001. e, FDG-PET images of untreated (left) mouse (15 d) with bilateral HT1080 tumours and after CRIT (30 d), imaged by administering 0.19 mCi/0.1 ml FDG i.v. Notice the right tumour in mouse undergoing CRIT displays a necrotic zone. f, Standard uptake value of FDG is considerably low in mouse that underwent CRIT. ***P < 0.001. g, Histological analysis of H&E stained HT1080 tumour sections from an untreated mouse are compared to mice that underwent CRIT. Normal tumour tissue is marked as T, necrotic tissue as N, and denuded areas suggesting macrophage assisted clearance is marked as *. Magnified images show tumour-infiltrating lymphocytes in the treated tumour sections. h, TEM image of tumour section extracted from untreated mice showing healthy cells. Scale bar, 3 μm i, TEM image of tumour section extracted from mice that underwent CRIT showing majority of cells are apoptotic. Scale bar, 3 μm. j, Magnified TEM image of (i) showing internalized NPS (arrows) in apoptotic tumour cells. Scale bar, 500 nm. k, TEM image of tumour section from necrotic region showing necrotic cells with internalized NPS (arrows). Scale bar, 2 μm. Disparate and regenerative cytotoxic free radicals from both Tc and TiO₂ in TiO₂ in TiO₂-Tf-Tc NPS utilized apoptosis and necrosis cell death pathways to enhance CRIT.