Surfactant-Stripped Pheophytin Micelles for Multimodal Tumor Imaging and Photodynamic Therapy

Abstract

Porphyrin-based nanomaterials can inherently integrate multiple contrast imaging functionalities with phototherapeutic capabilities. We dispersed pheophytin (Pheo) into Pluronic F127 and carried out low-temperature surfactant-stripping to remove the bulk surfactant. Surfactant-stripped Pheo (ss-Pheo) micelles exhibited a similar size, but higher near-infrared fluorescence, compared to two other nanomaterials also with high porphyrin density (surfactant-stripped chlorophyll micelles and porphysomes). Singlet oxygen generation, which was higher for ss-Pheo, enabled photodynamic therapy (PDT). ss-Pheo provided contrast for photoacoustic and fluorescence imaging, and following seamless labeling with ⁶⁴Cu, was used for positron emission tomography. ss-Pheo had a long blood circulation and favorable accumulation in an orthotopic murine mammary tumor model. Trimodal tumor imaging was demonstrated, and PDT resulted in delayed tumor growth.

Keywords: fluorescence; micelles; pheophytin; photoacoustic; photodynamic therapy; positron emission tomography.

Figure 1.

Structure and representation of high-density porphyrin nanoparticles. (A) Pyro-lipid self-assembles to form porphysome nanovesicles with a porphyrin bilayer. (B) Chlorophyll (Chlr) or pheophytin (Pheo) can be formed into surfactant-stripped micelles with Pluronic F127 triblock copolymers. (C) Retention of Pheo and F127 during the surfactant-stripping process to generate ss-Pheo. Data show mean ± standard deviation for n = 3.

Figure 2.

Characterization of porphyrin nanomaterials. (A) Hydrodynamic diameter and (B) polydispersity index of ss-Pheo, ss-Chlr, and Porphs as determined by dynamic light scattering. (C) Q-band normalized absorption spectra in water. (D) Fluorescence emission spectra when nanoparticles were adjusted to have an absorbance of 0.05 at the excitation wavelength. Spectra are normalized to ss-Pheo emission maximum. (E) Relative fluorescence quantum yield. (F) Singlet Oxygen Sensor Green (SOSG) indicator dye signal increases as ss-Pheo, ss-Chlr, or porphysomes were irradiated at 665 nm (when adjusted to have an equal absorbance at 665 of 0.05). (G) Initial rate of SOSG increase during the first 5 s of irradiation. Values show mean ± standard deviation for n = 3.

Figure 3.

Surfactant-stripping enables concentrated micelle dispersions. (A) Absorbance of concentrated ss-Pheo compared to conventional formulation methods. (B) Hemolysis of Pheo samples at indicated concentrations following incubation with human red blood cells for 1 h at 37 °C. Values show mean ± standard deviation for n = 3.

Figure 4.

In vitro imaging of porphyrin nanoparticles using fluorescence and PAT. (A) Signal intensity in fluorescence counts for three photosensitizers as a function of tissue depth. (B) PAT signal intensity against tissue depth.

Figure 5.

Cell death induced by PDT with ss-Pheo. (A) Cells were incubated with ss-Pheo at indicated concentrations for 4 h and then subjected to light treatment with a 665 nm LED box. Cell viability was assessed 24 h after laser treatment using the XTT assay. (B) Following the indicated treatment, 4T1 cells were stained with Annexin V and PI and subjected to flow cytometry. Values show mean ± standard deviation for n = 3.

Figure 6.

Pheo concentration in serum in mice following intravenous administration of ss-Pheo (500 μg Pheo dose). Data shows mean ± standard deviation for n = 3 mice.

Figure 7.

Biodistribution of ss-Pheo and ⁶⁴Cu-labeled ss-Pheo (500 μg Pheo) 24 h after intravenous administration in mice bearing orthotopic 4T1 tumors. (A) Pheo concentration in tumor and different organs as assess by fluorometric analysis of tissue homogenates. (B) ⁶⁴Cu-labeled ss-Pheo (%ID/g) in tumor and different components of the body including cells, tissues, and organs. Data shows mean ± standard deviation for n = 3 mice.

Figure 8.

Trimodal imaging of ss-Pheo in vivo in mice bearing orthotopic 4T1 tumors. ss-Pheo (500 μg) was injected intravenously per mouse. (A) ⁶⁴Cu-labeled ss-Pheo PET scan at indicated time points. The tumor location is indicated by the white circle. (B) NIR fluorescence imaging in tumor-bearing mice either injected with ss-Pheo or untreated. (C) PAT imaging with control and treated mice. Representative images for n = 3 mice per group.

Figure 9.

Tumor growth inhibition by ss-Pheo via PDT. Mice were intravenously administered 500 μg of ss-Pheo and 24 h later were treated with 300 J/cm² (150 mW/cm² fluence rate) with a 665 nm laser diode. (A) Tumor volume for mice bearing a 4T1 tumor following indicated treatments. The asterisk shows there was a significant difference between ss-Pheo+laser compared to other groups based on one-way ANOVA (p < 0.5). (B) Tumor doubling delay was found for each treatment condition including Pheo alone, laser alone, and Pheo+laser. (C) Body weight in treated groups. Data show mean ± standard deviation for n = 5 mice per group.