Two Beats One: Osteosarcoma Therapy with Light-Activated and Chemo-Releasing Keratin Nanoformulation in a Preclinical Mouse Model

Abstract

Osteosarcoma treatment is moving towards more effective combination therapies. Nevertheless, these approaches present distinctive challenges that can complicate the clinical translation, such as increased toxicity and multi-drug resistance. Drug co-encapsulation within a nanoparticle formulation can overcome these challenges and improve the therapeutic index. We previously synthetized keratin nanoparticles functionalized with Chlorin-e6 (Ce6) and paclitaxel (PTX) to combine photo (PDT) and chemotherapy (PTX) regimens, and the inhibition of osteosarcoma cells growth in vitro was demonstrated. In the current study, we generated an orthotopic osteosarcoma murine model for the preclinical evaluation of our combination therapy. To achieve maximum reproducibility, we systematically established key parameters, such as the number of cells to generate the tumor, the nanoparticles dose, the design of the light-delivery device, the treatment schedule, and the irradiation settings. A 60% engrafting rate was obtained using 10 million OS cells inoculated intratibial, with the tumor model recapitulating the histological hallmarks of the human counterpart. By scheduling the treatment as two cycles of injections, a 32% tumor reduction was obtained with PTX mono-therapy and a 78% reduction with the combined PTX-PDT therapy. Our findings provide the in vivo proof of concept for the subsequent clinical development of a combination therapy to fight osteosarcoma.

Keywords: chemotherapy; drug delivery; keratin nanoparticles; musculoskeletal tumors; orthotopic osteosarcoma murine model; osteosarcoma; photodynamic therapy.

Figure 1

Physical characterizations of PTX-Ce6@ker nanoformulation. (A,B) Representative Transmission electron microscopy (TEM) micrographs of PTX-Ce6@ker nanoparticles performed at a final concentration of 0.1 mg/mL (5 μm and 500 nm scale bar, respectively). (C) The graph shows the diameter distribution of keratin-based nanoparticles determined by analysis of TEM images (n = 319).

Figure 2

Chemical characterizations of PTX-Ce6@ker nanoformulation. (A,B) The graphs show the absorption spectra reflecting ROS and ¹O₂ production, respectively, measured at different irradiation times, expressed as minutes for ROS (‘) and seconds (‘’) for ¹O₂. (C) Release kinetics of PTX from PTX-Ce6@ker nanoparticles, as determined by equilibrium dialysis at 37 °C in (PBS, pH 7.4)/ethanol (75:25, v/v) and HPLC-UV analysis. Korsmeyer–Peppas model: $y=0.1461x^{0.6406}$ (R² = 0.9962); Weibull model: $y=1-e^{-0.1621x^{0.7898}}$ (R² = 0.9987).

Figure 3

Preclinical Osteosarcoma mouse model set up. (A) Representative paraffin section stained with H&E showing the entire tibia right after the inoculation of Saos-2 cells (TIME 0). The magnification of the red squared area is shown in the right panel. The red hash marks (#) indicate the OS tumor cells, while the red asterisks (*) the bone marrow cells. (B) Representative X-ray imaging of four animals from the 5 × 10⁶ Saos-2 group at week 6 from initial inoculation, and of four animals from the 10 × 10⁶ Saos-2 group at week 8 from initial inoculation. The red hash marks (#) point to the detectable tumor mass. (C) Representative paraffin sections stained with H&E of 10 × 10⁶ Saos-2 inoculation groups at 8 weeks. The § indicates the necrotic area, the hash marks (#) indicate new bone matrix, the head-arrows indicate the bone trabeculae damage due to tumor growth, while the asterisks the chondroid matrix.

Figure 4

Device prototyped for PDT in the in vivo experimentation. Two 660 ± 2 nm wavelength LED lights were assembled on two distinct flexible arms to focus the light directly on the treated area. To converge the irradiation, two cylinders (4 cm × 1 cm) were added on top of the light source to focus the light area over the animal’s tibia, and two heat dissipators were mounted to prevent excessive thermal surcharge on the lights.

Figure 5

Treatment regime set up: nanoformulation dosage and treatment’s schedule. (A) Timeline of the treatment regimen. (B–D) Histological evaluation of tumor tissue after treatments. Representative brightfield images of paraffin sections stained with H&E from samples treated with DOSE I (B), DOSE II (C) and irradiated immediately after (0 h), and of DOSE III (D) irradiated at 0 h and after 48 h. (E) Representative confocal images of samples from mouse treated with DOSE III and euthanised immediately (0 h) or 48 h after treatment. The red signal corresponds to the Ce6 molecule, while nuclei are shown in white. (F) Representative figure of FLIM analysis showing the Ce6 signal distribution in the tissue area right after PTX-Ce6@ker inoculation (time 0 h) with the colour scale indicating the average lifetime. The blue regions with τ_av of around 1 ns are indicative of Ce6, while the red-coloured regions, characterized by a τ_av of > 2 ns, are indicative of autofluorescence and Hoechst staining in the nuclei.

Figure 6

Preclinical evaluation of the therapy efficacy. (A) Representative H&E stained paraffin sections from tibia explants of animals from control groups (D-PBS −/+ PDT) and treatment groups (PTX-Ce6@ker −/+ PDT). Magnification of the black squared area is shown on the right side of the main panels. (B,C) The graphs show the quantification of tumor area and tumor cell number, respectively, performed with QuPath software. Data are expressed as the mean ± SD and analyzed using the One-way ANOVA test, and Tukey’s multiple comparison test as a post-test. Results were statistically significant at p values < 0.05 (** p-values< 0.01; *** p-values < 0.001).

Figure 7

Preclinical evaluation of the therapy efficacy. (A) Representative paraffin sections from tibia explants of animals from control groups (D-PBS −/+ PDT) and treatment groups (PTX-Ce6@ker −/+ PDT) stained with the following markers: H&E, K_i-67, and TUNEL. (B) Representative paraffin sections from muscle explants surrounding the tumor, stained with H&E. (C,D) The graphs show the quantification of K_i-67 and TUNEL positive cells in four selected ROI/slices along the tumor tissue, respectively. (E) The graph shows the quantification of the immune cells in the muscle tissues after treatments. Data are expressed as the mean ± SD and analyzed using the one-way ANOVA test and Tukey’s multiple comparison test as a post-test. Results were statistically significant at p values < 0.05 (** p values < 0.01; *** p values < 0.001).