In vivo Multi-scale Photoacoustic Imaging Guided Photothermal Therapy of Cervical Cancer based on Customized Laser System and Targeted Nanoparticles

Abstract

Background: Effective treatment strategy for cervical carcinoma is subject to the limitation of its anatomical location and histological characteristics. Comprehensive imaging before cervical carcinoma treatment is of great significance for the patients. Current imaging methods cannot meet the requirements of high resolution, deep imaging depth and non-invasive imaging at the same time. Fortunately, Photoacoustic imaging (PAI) is a novel imaging method that combines rich optical contrast, high ultrasonic spatial resolution, and deep penetration depth in a single modality. Moreover, PAI-guided photothermal therapy (PTT) by aid of targeting nanoparticles is an emerging and effective cancer treatment in recent years.

Methods: Here, strong near-infrared region (NIR) absorption-conjugated polymer PIIGDTS (PD) nanoparticles with folic acid (FA) modification (namely, PD-FA) that targeted at Hela cell were specifically designed as cervical tumor imaging contrast agents and photothermal agents.

Results: The obtained PD-FA nanoparticles exhibited admirable photoacoustic contrast-enhancing ability and desirable PTT behavior with the photothermal conversion efficiency as high as 62.6% in vitro. Furthermore, the PAI performance and PTT efficiency were tested in HeLa tumor-bearing nude mice after injection of PD-FA nanoparticles. In vivo multi-scale, PAI provided B-san and 3D dimension imaging for intuitive and comprehensive information of Hela tumor. Moreover, the Hela tumor can be completely eliminated within 18 days after PTT, with no toxicity and side effects.

Conclusion: In summary, PD-FA injection combined with PAI and PTT systems provides a novel powerful tool for early diagnosis and precise treatment of cervical cancer.

Keywords: PIIGDTS nanoparticle; cervical cancer; multi-scale photoacoustic imaging; photothermal therapy.

Figure 1

Schematic illustration of the photoacoustic imaging (PAI) guided photothermal therapy (PTT) with the assistance of PD-FA nanoparticles for the imaging and therapy of cervical carcinoma.

Figure 2

Characterization and cytotoxicity of the as-prepared PD-FA nanoparticles. (A) UV-Vis absorption spectra of the PD-FA nanoparticles at different concentration dissolved in water. (B) Transmission Electron Micrograph (TEM) image of PD-FA nanoparticles with an average size of 9 ± 3 nm. The scale bar is 50 nm. (C) Hydrodynamic diameter of the as-prepared PD-FA nanoparticles dispersed in PBS measured by dynamic light scattering (DLS). (D) Cell viabilities of cos-7 cells incubated with the as-prepared PD-FA nanoparticles at various concentrations ranging from 0 μg mL⁻¹ to 80 μg mL⁻¹.

Figure 3

Photothermal properties of PD-FA nanoparticles in vitro. (A) A series of photothermal images of PD-FA nanoparticles solution (80 μg mL⁻¹) under the irradiation of 845 nm laser at the power density of 1.5 W cm⁻². (B) The temperature variation of the solution containing different concentrations of the PD-FA nanoparticles (0, 5, 10, 20, 40, 80 μg mL⁻¹) under the irradiation of 845 nm laser at the power density of 1.5 W cm⁻². (C) The temperature curves of PD-FA nanoparticles at the concentration of 80 μg mL⁻¹ under 845 nm laser irradiation at different power densities. (D) Temperature variations of the PD-FA nanoparticles under 845 nm laser irradiation at a power density of 1.5 W cm⁻² for five light on/off cycles laser irradiation. (E) Photothermal effect of PD-FA nanoparticles in water when irradiated with a 845 nm laser (1.5 W cm⁻²). The laser was switched off after irradiation for 10 min.

Figure 4

In vitro photoacoustic property of as-prepared PD-FA nanoparticles. (A) Photoacoustic spectrum of PD-FA nanoparticles at different wavelengths. (B) The photostability of photoacoustic signal under the irradiation of different laser powers for 20 min. (C) Photoacoustic images of PD-FA nanoparticles nuder excitation at 845 nm at different concentrations. (D) Photograph of sample in agar phantom. (E) Intensity profiles along the dashed line from (C). (F) The Photoacoustic amplitudes at 845 nm as a function of concentrations of PD-FA nanoparticles.

Figure 5

Cell uptaking and photothermal abilities incubated with HeLa cells of PD-FA nanoparticles and PD nanoparticles. (A) Confocal fluorescence images of HeLa cancer cells co-cultured with PD-FA nanoparticles and PD nanoparticles for 2, 4, 8 h. Scale bar is 20 μm. (B) Fluorescence images of Calcein AM (green, live cells) and propidium iodide (red, dead cells) contained HeLa cells after different treatments including; (a) PD-FA nanoparticles + Laser; (b) PD-FA nanoparticles + HeLa pre-treated by free FA+ Laser; (c) PD nanoparticles + Laser; (d) PD-FA nanoparticles, (e) Laser only; (f) Control (845 nm laser irradiation at a power density of 1 W cm⁻² for 10 min). Scale bar is 50 μm. (C) The cell viabilities of the HeLa cervical cancer cells after treatment with PD-FA nanoparticles plus laser, PD nanoparticles plus Laser and pre-treated by free FA before PD-FA nanoparticles plus laser.

Figure 6

PAI in tumor-mimicking phantom. (A) Dual-modal PAI and ultrasound imaging of medical coupling gel with different concentration of PD-FA nanoparticles. (B) Quantization of the photoacoustic signal of tumor phantom with different concentration of PD-FA nanoparticles in (A).

Figure 7

In vivo PAI of PD-FA nanoparticles. (A) Photoacoustic images of tumor site in HeLa tumor-bearing nude mice after intravenous injection of PD nanoparticles or PD-FA nanoparticles for 2, 4, 8, 12, 24 and 36 h. Scale bar is 1 mm. (B) Photoacoustic intensities of the tumor site at different time points obtained from (A). (C) In vivo 3D photoacoustic images of tumor tissues with injection of PD nanoparticles or PD-FA nanoparticles at 8h.

Figure 8

In vivo PTT of Hela tumor-bearing nude mice. (A) Thermal graphic images of HeLa tumor-bearing nude mice exposed to NIR laser for 5 min (845 nm, 1 W cm⁻²) after injection of PD-FA nanoparticles, PD nanoparticles or PBS. (B) Representative photos of HeLa tumor-bearing mice at different days after different treatments. Green circle indicated the tumor sites, red circle and red arrow indicated the recrudescence of tumor. (C) Tumor volume changes of mice after different treatments. (D) Body weight recording of mice after different treatment. (E) Survival rates of mice under different treatment after 30 days. (F) H&E staining images of tumor sections collected from HeLa tumor-bearing mice with different treatments. The scale bar is 200 μm.

Figure 9

H&E staining of major organs, including heart, liver, spleen, lung, and kidney of mice from each group after different treatment. The scale bar is 200 μm.