Nanoparticle-Based Platform for Activatable Fluorescence Imaging and Photothermal Ablation of Endometriosis

Abstract

Endometriosis is a painful disorder where endometrium-like tissue forms lesions outside of the uterine cavity. Intraoperative identification and removal of these lesions are difficult. This study presents a nanoplatform that concurrently delineates and ablates endometriosis tissues using real-time near-infrared (NIR) fluorescence and photothermal therapy (PTT). The nanoplatform consists of a dye, silicon naphthalocyanine (SiNc), capable of both NIR fluorescence imaging and PTT, and a polymeric nanoparticle as a SiNc carrier to endometriosis tissue following systemic administration. To achieve high contrast during fluorescence imaging of endometriotic lesions, nanoparticles are constructed to be non-fluorescent prior to internalization by endometriosis cells. In vitro studies confirm that these nanoparticles activate the fluorescence signal following internalization in macaque endometrial stromal cells and ablate them by increasing cellular temperature to 53 ^° C upon interaction with NIR light. To demonstrate in vivo efficiency of the nanoparticles, biopsies of endometrium and endometriosis from rhesus macaques are transplanted into immunodeficient mice. Imaging with the intraoperative Fluobeam 800 system reveals that 24 h following intravenous injection, nanoparticles efficiently accumulate in, and demarcate, endometriotic grafts with fluorescence. Finally, the nanoparticles increase the temperature of endometriotic grafts up to 47 °C upon exposure to NIR light, completely eradicating them after a single treatment.

Keywords: endometriosis; fluorescence imaging; nanoparticles; photothermal therapy.

Figure 1.

Schematic illustration of “always on” and “activatable” SiNc-loaded PEG-PCL nanoparticles (SiNc-NP). Green and orange colors represent hydrophilic PEG outer shell and hydrophobic PCL core of nanoparticles, respectively. In contrast to ‘always on” SiNc-NP (top panel), “activatable” SiNc-NP (lower panel) contain a higher amount of SiNc molecules (dark red spheres) inside the hydrophobic cores, causing fluorescence self-quenching. “Activatable” SiNc-NP generate fluorescence after internalization into endometriotic cells and subsequent relaxation of densely-packed, self-quenched SiNc molecules in the intracellular environment.

Figure 2.

A) Representative dynamic light scattering profile and B) cryo-TEM image of SiNc-loaded PEG-PCL nanoparticles (SiNc-NP). Scale bar is 25 μm in (B). C) Fluorescence spectra (excitation wavelength = 750 nm) of SiNc-NP with low (0.5% SiNc/1 mg PEG-PCL, “always on” SiNc-NP) and high (6% SiNc/1 mg PEG-PCL, “activatable” SiNc-NP) SiNc loading. Inset: fluorescence images of “always on” and “activatable” SiNc-NP solutions acquired using the Pearl Impulse Small Animal Imaging System. Scale bar: The blue and red colors reflect the lowest and highest fluorescence intensity, respectively.

Figure 3.

Representative fluorescence microscopy images of macaque endometriotic stromal cells incubated with “always on” SiNc-NP (top panel) and “activatable” SiNc-NP (low panel) for 1, 2, 4, and 24 h. The red and blue colors reflect the fluorescence signal generated by SiNc and NucBlue (nuclei staining), respectively.

Figure 4.

Viability of macaque endometriotic stromal cells after the following treatments: 1) cells incubated with medium; 2) cells exposed to 780 nm NIR light (0.9 W cm⁻²) for 15 min; 3) cells incubated with empty PEG-PCL nanoparticles (without SiNc) for 48 h;4) cells incubated with empty PEG-PCL nanoparticles for 48 h and exposed to 780 nm NIR light (0.9 W cm⁻²) for 15 min; 5) cells incubated with “activatable” SiNc-NP (SiNc=30μg mL⁻¹) for 48 h; 6) cells incubated with “activatable” SiNc-NP (SiNc = 30 μg mL⁻¹) for 48 h and exposed to 780 nm NIR light (0.9 W cm⁻²) for 15 min. *p < 0.05 when compared with the control (bar 1).

Figure 5.

Photomicrographs showing immunohistochemical staining (brown color) for A) ESR1, B) PGR, C) KI-67, and D) Factor 8 in endometriotic grafts collected from non-treated mice. All images captured at 20× magnification; “s” indicates stroma and “gl” indicates glands.

Figure 6.

A) Photograph (top) and NIR fluorescence image (bottom), recorded with Fluobeam 800, of a mouse bearing endometriotic grafts 24 h after intravenous injection of “activatable” SiNc-NP. B,C) NIR fluorescence images of a mouse bearing endometriotic graft (B) and resected tissues (C) recorded with Pearl Impulse Small Animal Imaging System 24 h after intravenous injection of “activatable” SiNc-NP. D,E) Representative fluorescence microscopy images of sections of endometriotic grafts collected 24 h after intravenous injection of SiNc-NP. Red color indicates NIR fluorescence generated by SiNc-NP. Yellow color represents blood vessels stained with the fluorescently labeled anti-CD31 antibody. Scale bars are 50 μm.

Figure 7.

A) Representative temperature profile inside of endometriotic graft during photothermal therapy (PTT) mediated by intravenously injected SiNc-NP in combination with 780 nm light (0.9 W cm⁻², red curve). Mice were injected with SiNc-NP 24 h prior to NIR light treatment. The black curve represents a temperature profile inside of endometriotic graft upon exposure to 780 nm light only (without SiNc-NP injection). B) Representative growth profiles of subcutaneous endometriotic grafts after the following treatments: 1) saline, mice injected with saline; 2) SiNc-NP, mice injected intravenously with SiNc-NP (3 mg SiNc kg⁻¹); 3) light, mice injected with saline and exposed to 780 nm light (0.9 W cm⁻²) for 15 min at 24 h post-injection; 4) SiNc-NP + light, mice injected intravenously with SiNc-NP (3 mg SiNc kg⁻¹) and exposed to 780 nm light (0.9 W cm⁻²) for 15 min at 24 h post-injection. C) Changes in body weights of mice injected intravenously with SiNc-NP (3 mg SiNc kg⁻¹) and exposed to 780 nm light for 15 min at 24 h post-injection.

Figure 8.

Effect of PTT on graft histology and steroid receptor staining. A,C) In control mice not receiving PTT, the grafts displayed enlarged endometriotic glands and stroma. E,G) In these mice, the grafts stained strongly for both ESR1 and PGR. B,D) Endometriotic glands and stroma were lost after PTT and graft sites were replaced with murine connective tissue. F,H) Minimal ESR1 and PGR staining nuclei were observed after PTT therapy. The inset in G shows irrelevant IgG control (anti-Br(d) U) showing staining specificity for both ESR1 and PGR. “gl” indicates glands; “s” indicates stroma; “sc” indicates stromal connective tissue with minimal ESR1 and PGR staining. 5X, 5× plan apochromatic objective; 20X, 20× plan apchromatic objective.