Intrinsic nucleus-targeted ultra-small metal-organic framework for the type I sonodynamic treatment of orthotopic pancreatic carcinoma

Abstract

Background: Sonodynamic therapy (SDT) strategies exhibit a high tissue penetration depth and can achieve therapeutic efficacy by facilitating the intertumoral release of reactive oxygen species (ROS) with a short lifespan and limited diffusion capabilities. The majority of SDT systems developed to date are of the highly O₂-dependent type II variety, limiting their therapeutic utility in pancreatic cancer and other hypoxic solid tumor types.

Results: Herein, a nucleus-targeted ultra-small Ti-tetrakis(4-carboxyphenyl)porphyrin (TCPP) metal-organic framework (MOF) platform was synthesized and shown to be an effective mediator of SDT. This MOF was capable of generating large quantities of ROS in an oxygen-independent manner in response to low-intensity ultrasound (US) irradiation (0.5 W cm^-2), thereby facilitating both type I and type II SDT. This approach thus holds great promise for the treatment of highly hypoxic orthotopic pancreatic carcinoma solid tumors. This Ti-TCPP MOF was able to induce in vitro cellular apoptosis by directly destroying DNA and inducing S phase cell cycle arrest following US irradiation. The prolonged circulation, high intratumoral accumulation, and nucleus-targeting attributes of these MOF preparations significantly also served to significantly inhibit orthotopic pancreatic tumor growth and prolong the survival of tumor-bearing mice following Ti-TCPP + US treatment. Moreover, this Ti-TCPP MOF was almost completely cleared from mice within 7 days of treatment, and no apparent treatment-associated toxicity was observed.

Conclusion: The nucleus-targeted ultra-small Ti-TCPP MOF developed herein represents an effective approach to the enhanced SDT treatment of tumors in response to low-intensity US irradiation.

Keywords: Hypoxia; Intrinsic nucleus-targeted; Orthotopic pancreatic carcinoma; Type I sonodynamic therapy; Ultra-small metal–organic framework.

Scheme 1.

A schematic illustration of ultra-small Ti-TCPP MOF application in nuclear-targeted SDT

Fig. 1

a The method of ultra-small Ti-TCPP MOF synthesis. DLS curves (b), TEM images (c), zeta potentials (d), XRD pattern (e) and XPS spectra of Ti-TCPP MOF (f–i)

Fig. 2

Concentration-dependent ¹O₂ generation (a), O₂ˉ generation (b), H₂O₂ generation (c) and ·OH generation (d) after US irradiation under normoxic or hypoxic conditions. e Normalized intensity of photoacoustic signal versus the concentration of Ti-TCPP MOF solution

Fig. 3

Cellular uptake and nuclear localization of Ti-TCPP MOF in BxPC-3 cells. Flow cytometry (a), ICP analysis (b) and confocal images (c) of BxPC-3 cells after incubation with Ti-TCPP MOF at different time periods. Scale bar: 20 μm. (n = 3). (d) Bio-TEM images of BxPC-3 cells before and 6 h after incubation with Ti-TCPP MOF. Scale bar: 2 µm. Red arrows denote Ti-TCPP MOF. *P < 0.05, ***P < 0.001

Fig. 4

a BxPC-3 cell viability after incubation with different concentrations of Ti-TCPP MOF for 24 h (n = 3). b Viability of BxPC-3 cells incubated with Ti-TCPP MOF for 6 h, then subjected to US irradiation (0.5 W cm⁻², 1 MHz, 50% duty cycle, 1 min) and incubated for an additonal 18 h (n = 3). c, d Flow cytometry analysis of cells after various treatments (n = 3). e Live (green) and dead (red) cell staining after various treatments. Scale bar: 100 μm. *P < 0.05, ***P < 0.001

Fig. 5

a Western blotting analysis of Bax, Bcl-2, and Caspase 3 in BxPC-3 cells incubated under various treatment conditions. β-tubulin was used as an internal control. ImageJ was used to quantify protein levels. Data are means ± S.D. (n = 3). **P < 0.01. b Cell cycle progression was evaluated by staining cancer cells with PI and was assessed via flow cytometry after various treatments (n = 3). c Confocal images of cancer cells in which the nuclei were stained blue with Hoechst and the γ-H2AX foci bright green following nuclear-targeting Ti-TCPP MOF treatment and US irradiation. d A DNA ladder assay was used to evaluate DNA damage after nucleus-targeted SDT therapy. A Control, B US, C Ti-TCPP, D Ti-TCPP + US

Fig. 6

Evaluation of Ti-TCPP MOF in vivo biosafety. a Different concentrations of Ti-TCPP MOF were used in a hemolysis assay. The inset images are of samples following centrifugation after incubation of RBCs with Ti-TCPP MOF (400, 200, 100, 50, or 25 µg mL⁻¹) or water, respectively (n = 3). b Blood urea nitrogen (BUN) levels in healthy mice 14 days post-Ti-TCPP MOF injection (i.v.) (n = 3). c Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, and alkaline phosphatase (ALP) levels in healthy mice at 14 days post-Ti-TCPP MOF injection (i.v.) (n = 3). d H&E-stained images of major organs from healthy control mice 14 days after the i.v. injection of PBS and Ti-TCPP MOF (Scale Bar = 100 µm). Ti-TCPP MOF was injected at a dose of 20 mg/kg

Fig. 7

a In vivo fluorescence images at different time points post-injection of Ti-TCPP MOF, and of various organs and tumors at 12 h post-injection. 1 tumor, 2 heart, 3 liver, 4 spleen, 5 lung and 6 kidney. b In vivo photoacoustic images at different time points post-Ti-TCPP MOF injection. Grayscale images represent ultrasound images, and colored images represent photoacoustic images. c Circulating Ti-TCPP MOF levels after i.v. injection, as assessed via ICP-OES. Ti-TCPP MOF pharmacokinetics followed a two-compartment model (n = 3). d Ti-TCPP MOF biodistribution in BxPC-3 tumor-bearing mice at 8 h post-i.v. injection (n = 3). e Time-dependent distribution of Ti in the primary organs of healthy mice after the i.v. injection of Ti-TCPP MOF (n = 3)

Fig. 8

In vivo antitumor evaluation. a Schematic illustration of the therapy process. b Fluorescence images of tumor-bearing mice after various treatments. c Tumor fluorescence intensity changes and d survival rates of tumor-bearing mice after different treatments. Data are means ± SD (n = 5). e H&E, TUNEL, Ki67, and γ-H2AX staining of tumors 15 day after various treatments (Scale bar = 100 µm)