Layer-by-Layer Nanoparticles for Calcium Overload in situ Enhanced Reactive Oxygen Oncotherapy

Abstract

Background: Challenges such as poor drug selectivity, non-target reactivity, and the development of drug resistance continue to pose significant obstacles in the clinical application of cancer therapeutic drugs. To overcome the limitations of drug resistance in chemotherapy, a viable treatment strategy involves designing multifunctional nano-platforms that exploit the unique physicochemical properties of tumor microenvironment (TME).

Methods: Herein, layer-by-layer nanoparticles with polyporous CuS as delivery vehicles, loaded with a sonosensitizer (tetra-(4-aminophenyl) porphyrin, TAPP) and sequentially functionalized with pH-responsive CaCO₃, targeting group hyaluronic acid (HA) were designed and synthesized for synergistic treatment involving chemodynamic therapy (CDT), sonodynamic therapy (SDT), photothermal therapy (PTT), and calcium overload. Upon cleavage in an acidic environment, CaCO₃ nanoparticles released TAPP and Ca²⁺, with TAPP generating ¹O₂ under ultrasound trigger. Exposed CuS produced highly cytotoxic ·OH in response to H₂O₂ and also exhibited a strong PTT effect.

Results: CuS@TAPP-CaCO₃/HA (CTCH) delivered an enhanced ability to release more Ca²⁺ under acidic conditions with a pH value of 6.5, which in situ causes damage to HeLa mitochondria. In vitro and in vivo experiments both demonstrated that mitochondrial dysfunction greatly amplified the damage caused by reactive oxygen species (ROS) to tumor, which strongly confirms the synergistic effect between calcium overload and reactive oxygen therapy.

Conclusion: Collectively, the development of CTCH presents a novel therapeutic strategy for tumor treatment by effectively responding to the acidic TME, thus holding significant clinical implications.

Keywords: calcium overload; mitochondria damage; pH-responsive; reactive oxygen oncotherapy; synergistic treatment.

Scheme 1

Schematic diagram of the preparation process of CTCH and the combination of PTT, CDT and SDT for synergistic antitumor therapy.

Figure 1

Characterizations of CTCH. (A) Low-magnification TEM image of CuS (scale bar: 100 nm); (B) TEM image of CTCH (scale bar: 100 nm); (C) Element mapping of CTCH (scale bar: 100 nm); (D) FTIR spectra of HA, CuS, and CTCH; (E) XPS spectra of CTCH; (F) Zeta potentiogram of CuS, CuS@TAPP and CTCH.

Figure 2

Evaluation of photothermal properties of CTCH. (A) Infrared imaging and (B) temperature-time plot of CTCH at different concentrations irradiated by 808 nm (1.0 W/cm²) laser for 5 min; (C) Infrared imaging and (D) temperature-time plot of CTCH (200 μg/mL) irradiated by 808 nm laser at different laser powers for 5 min; (E) Photothermal heating curves of CTCH solution (200 μg/mL) upon exposure to 808 nm laser irradiation (1.0 W/cm²).

Figure 3

The performance of CTCH. UV-visible absorption spectra of TMB after co-incubation of (A) CTCH, (B) different concentrations CTCH and (C) CTCH with H₂O₂; (D) Ca²⁺ release curve under different pH conditions.

Figure 4

In vitro therapy evaluation. (A) The fluorescence images of HeLa cells stained with DCFH-DA (scale bar: 100 μm); (B) The fluorescence images of HeLa cells stained with JC-10 (scale bar: 50 μm); (C) Fluorescence intensity statistics corresponding to (B); (D) Effects of different concentrations of CuS, TAPP and CTCH on HcerEpic cell survival rate; (E) The effects of different concentrations of CTCH on the survival rate of HcerEpic cells after ultrasonic or laser treatment; (F) The effects of different concentrations of CTCH on the survival rate of HeLa cells under different conditions; (G) The fluorescence images of HeLa cells stained by calcein AM (green) and PI (red) after different treatments (scale bar: 100 μm); (H) Fluorescence intensity statistics corresponding to (G).

Figure 5

In vivo accumulation in tumor and photothermal capability of CTCH. (A) In vivo fluorescence images of tumor-bearing mice after intravenous injection with CTCH at different time points; (B) Photothermal imaging of CTCH in vivo; (C) Photothermal statistics in vivo; (D) Pictures of each group recorded at day 0, 7, and 14 during the treatment period; (E) Representative tumor images from each group after treatment; (F) Tumor volume changes of each group during treatment (**P < 0.01); (G) The tumor weight in each group after treatment (**P < 0.01); (H) The body-weight change of nude mice during treatment.