Perfluorocarbon-polyepinephrine core-shell nanoparticles as a near-infrared light activatable theranostic platform for bimodal imaging-guided photothermal/chemodynamic synergistic cancer therapy

Abstract

Background: Activatable multifunctional nanoparticles present considerable advantages in cancer treatment by integrating both diagnostic and therapeutic functionalities into a single platform. These nanoparticles can be precisely engineered to selectively target cancer cells, thereby reducing the risk of damage to healthy tissues. Once localized at the target site, they can be activated by external stimuli such as light, pH changes, or specific enzymes, enabling precise control over the release of therapeutic agents or the initiation of therapeutic effects. Furthermore, these nanoparticles can be designed to incorporate multiple therapeutic modalities, including chemotherapy, photothermal therapy (PTT), and chemodynamic therapy (CDT). This comprehensive approach facilitates real-time monitoring of treatment efficacy and allows for dynamic adjustments to therapy, resulting in more personalized and effective cancer treatments. Methods: This study reports the synthesis of perfluorocarbon (PFC)-encapsulated fluorescent polyepinephrine (PEPP) nanoshells chelated with Fe²⁺ (PFC@PEPP-Fe) and explores their potential for bimodal imaging and synergistic combination therapy in cancer treatment. The cellular uptake, cytotoxicity, and in vitro therapeutic efficacy of PFC@PEPP-Fe were assessed using 4T1 breast cancer cells. In vivo bimodal imaging using fluorescence (FL) and ultrasound (US) was conducted after injection into 4T1 tumor-bearing balb/c nude mice. The synergistic anticancer effects of PFC@PEPP-Fe, combining CDT and PTT, were evaluated following 808 nm laser irradiation (1 W/cm²) for 5 min, with treatment outcomes monitored over a 14 days period. Results: Both in vitro and in vivo studies demonstrated that PFC@PEPP-Fe enables effective bimodal imaging and exhibits substantial anticancer efficacy through the synergistic effects of PTT and CDT. Near-infrared (NIR) laser irradiation increased the temperature, enhancing the release of O₂ and the production of H₂O₂, which in turn amplified the CDT effect. The combination of PFC@PEPP-Fe administration and NIR laser significantly reduced tumor volume, slowed tumor growth, and improved survival in 4T1 tumor-bearing mice, confirming the strong anticancer activity due to the PTT/CDT synergy. Conclusions: As a multifunctional theranostic nanoparticle, PFC@PEPP-Fe not only enables cancer cell-specific US/FL bimodal imaging through the generation of microbubbles from its PFC core and fluorescent PEPP shells but also facilitates synergistic chemodynamic and photothermal therapeutic actions under NIR laser irradiation, which induces the self-supply of H₂O₂ and O₂ within cancer cells.

Keywords: Fenton reaction; bimodal imaging; cancer treatment; perfluorocarbon; polyepinephrine; theranostics.

Scheme 1

Scheme illustrating the working system of PFC@PEPP-Fe generating cancer-specific “OFF-ON” signals for activatable bimodal FL and US imaging at (A) physiological pH (below 37 ℃) and (B) endosomal pH (above 37 ℃). (C) Schematic illustration demonstrates the cancer treatment process of PFC@PEPP-Fe through combination of enhanced-CDT and PTT following cancer cell uptake facilitated by the EPR effect.

Figure 1

(A) Overall synthetic method of PFC@PEPP-Fe. Structure characterization of PFC@PEPP and PFC@PEPP-Fe using (B) DLS, (C) TEM, (D) TEM-associated EDS mapping, (E) FTIR, (F) XPS. (G) XPS narrow spectrum of PFC@PEPP-Fe includes specific peaks for Fe 2p.

Figure 2

(A) Proposed synthetic mechanism and chemical structure of PFC@PEPP. (B) FL change of PFC@PEPP treated with different volumes of EDA. (C) Theoretically calculated molecular orbitals of PFC@PEPP using density functional theory calculations at the B3LYP/3-31G(d, p) level. (D) FL spectra of PFC@PEPP with/without Fe²⁺ ions. (E) Stern-Volmer plot of PFC@PEPP with different concentrations of FeCl₂. (F) Schematic illustration of the FL “OFF-ON” working process of PFC@PEPP-Fe at different pH values. (G) FL change of PFC@PEPP-Fe in buffers with a pH of 7.4 and 5.4. (H) ICP-MS analysis of PFC@PEPP-Fe in pH 7.4 and pH 5.4 buffers at different time points.

Figure 3

(A) Time-dependent FL microscopic images of 4T1 cells incubated with pre-dissolved PFC@PEPP-Fe solutions at pH 7.4 and 5.4, and the corresponding 3D surface-plot analysis of the FL signal. (B) Time-dependent quantitative FL intensity of 4T1 cells incubated with pre-dissolved PFC@PEPP-Fe solutions with pH = 7.4 and 5.4 (n = 3). (C) Optical microscopy images of PFC@PEPP-Fe with/without US exposure at 37 ℃ (US frequency: 10 MHz). (D) US images of PBS buffer and aqueous solutions of PFC@PEPP and PFC@PEPP-Fe at different temperatures and (E) their quantitative gray value intensities (n = 3).

Figure 4

(A) Temperature variations of 1 mL of DI water and 1 mL of each aqueous solution of PFC@PEPP and PFC@PEPP-Fe under NIR laser irradiation (1 W cm^-2). (B) Photothermal conversion stability of 1 mL aqueous solution of PFC@PEPP-Fe over three NIR laser irradiation (1 W cm^-2) cycles. (C) Linear fit of the time vs. -ln(θ) plot of cooling-process data. (D) Schematics of the burst release of O₂ from PFC@PEPP-Fe upon NIR laser irradiation. (E) IR thermal camera and (F) optical microscopy images of PFC@PEPP-Fe under continuous NIR laser irradiation for 10 min. (G) US imaging of PBS buffer and aqueous solutions of PEPP, PFC@PEPP, and PFC@PEPP-Fe at different temperatures. (H) Changes in the O₂ concentration in aqueous solutions of PFC@PEPP and PFC@PEPP-Fe after NIR laser irradiation for 15 min.

Figure 5

(A) Proposed therapeutic mechanism of PFC@PEPP-Fe for cancer treatment via dual-mode imaging-guided synergistic combination of PTT and enhanced-CDT. (B) FL images of 4T1 cells stained with H₂O₂ assay kit after treatment with various sample groups. (C) FL intensities of HTA in 4T1 cells treated with the various samples (n = 3). (D) Intracellular FL images of 4T1 cells treated with chemicals containing different concentrations of ROS (top) and the corresponding 3D surface-plot analyses of the FL signal (bottom). (E) FL images of 4T1 cells stained using a live/dead kit after treatment with PFC@PEPP + NIR. (F) Cell viability assessment of 4T1 cells treated with different concentrations of the various samples (n = 3). (G) Flow cytometry analysis of 4T1 cells treated with different sample groups.

Figure 6

(A) US images of the tumor site (white and blue dotted line) in 4T1 tumor-bearing balb/c nude mice intravenously injected with PBS and PFC@PEPP-Fe, and (B) their quantitative intensities (n = 3). (C) FL images of 4T1 tumor-bearing balb/c nude mice intravenously injected with PBS and PFC@PEPP-Fe at 0 and 240 min. (D) FL image of the excised major organs and tumor of the mice after treatment with PFC@PEPP-Fe, and (E) their quantitative FL intensities (n = 3).

Figure 7

(A) IR thermal camera images of balb/c nude mice injected intravenously with PFC@PEPP-Fe under continuous NIR laser irradiation (1.0 W cm^-2) for 10 min. (B) Schematics of the therapeutic schedule for 4T1 tumor-bearing balb/c nude mice. (C) Average body weight and (D) relative tumor volume of 4T1 tumor-bearing balb/c nude mice treated with only PBS, PFC@PEPP, PFC@PEPP-Fe, and PFC@PEPP + NIR laser irradiation. (E) Survival rate of the 4T1 tumor-bearing mice in the different treatment groups. (F) Camera images of excised tumors from the different groups after 14 days of treatment (n = 5). Histological evaluation of tumor tissues from the different treatment groups by (G) H&E and (H) TUNEL staining (scale bar = 200 μm). Histological evaluation of tumor tissues from the different treatment groups by (I) Ki-67 and (J) CRT immunohistochemical staining (scale bar = 100 μm).