Dual-targeted and pH-sensitive Doxorubicin Prodrug-Microbubble Complex with Ultrasound for Tumor Treatment

Abstract

In this study, we investigated the potential of a dual-targeted pH-sensitive doxorubicin prodrug-microbubble complex (DPMC) in ultrasound (US)-assisted antitumor therapy. The doxorubicin prodrug (DP) consists of a succinylated-heparin carrier conjugated with doxorubicin (DOX) via hydrazone linkage and decorated with dual targeting ligands, folate and cRGD peptide. Combination of microbubble (MB) and DP, generated via avidin-biotin binding, promoted intracellular accumulation and improved therapeutic efficiency assisted by US cavitation and sonoporation. Aggregates of prepared DP were observed with an inhomogeneous size distribution (average diameters: 149.6±29.8 nm and 1036.2±38.8 nm, PDI: 1.0) while DPMC exhibited a uniform distribution (average diameter: 5.804±2.1 μm), facilitating its usage for drug delivery. Notably, upon US exposure, DPMC was disrupted and aggregated DP dispersed into homogeneous small-sized nanoparticles (average diameter: 128.6±42.3 nm, PDI: 0.21). DPMC could target to angiogenic endothelial cells in tumor region via α_vβ₃-mediated recognition and subsequently facilitate its specific binding to tumor cells mediated via recognition of folate receptor (FR) after US exposure. In vitro experiments showed higher tumor specificity and killing ability of DPMC with US than free DOX and DP for breast cancer MCF-7 cells. Furthermore, significant accumulation and specificity for tumor tissues of DPMC with US were detected using in vivo fluorescence and ultrasound molecular imaging, indicating its potential to integrate tumor imaging and therapy. In particular, through inducing apoptosis, inhibiting cell proliferation and antagonizing angiogenesis, DPMC with US produced higher tumor inhibition rates than DOX or DPMC without US in MCF-7 xenograft tumor-bearing mice while inducing no obvious body weight loss. Our strategy provides an effective platform for the delivery of large-sized or aggregated particles to tumor sites, thereby extending their therapeutic applications in vivo.

Keywords: Doxorubicin prodrug; Dual-targeted; Microbubble complex; Ultrasound.; pH-sensitive.

Figure 1

Schematic illustration of US combined with DPMC to deliver DOX into nuclei and induce cytotoxicity. Using US cavitation and sonoporation, DPMCs were disrupted, facilitating drugs penetration into the tumor sites through temporary gaps in the endothelium. DPs were targeted to tumor cells, followed by release of pH-triggered DOX into nuclei.

Figure 2

Characterization of DP and DPMC. (A) TEM images and DLS graphs of DP pre-destruction with US (left) and DPMC post-destruction with US (right). (B) Confocal laser scanning microscopy images and corresponding bright field images of DPMC pre-destruction (left) and post-destruction (right) by US. DPMCs were visualized using the fluorescence of bound DOX. Data are presented as means ± standard deviation (n=3).

Figure 3

Flow cytometry and corresponding histogram profiles of MCF-7 cells incubated with H-F-DOX, H-RGD-DOX and H-F-RGD-DOX (A) and DOX, DP and DPMC with US (C). In vitro release of DOX from DP and DPMC with or without US after incubation at 37^oC in phosphate buffer (pH 5.0 and 7.4) (B). Cytotoxicity of MCF-7 cells incubated with MB with US, DOX, DP and DPMC with US (D). Values represent means ± SD (n=3).

Figure 4

Confocal microscope images of MCF-7 cells treated with free DOX (DOX), pH-insensitive prodrug (pH-insensitive), DP (pH-sensitive) and DPMC with US (pH-sensitive+US) for 4 h. Scale bar represents 20 μm (A) . Confocal microscope images of MCF-7 cells and A549 cells incubated with small-sized nanoparticles from DPMC disrupted by US for 5 mins. Scale bar represents 10 μm (B).

Figure 5

Schematic illustration of the image and analysis protocol (A). After monitoring for 4 min, targeted MBs were considered to adhere to the tumor site firmly. Blank MBs or DPMC were destroyed by the Flash mode of the ultrasound system. The process was monitored, starting from injection to at least 30 s after Flash destruction. The video intensity (VI) from adherent MBs (Targeted VI) was assessed by calculating the average pre- and post-destruction VI, and subtracting average post- from pre-destruction intensity. Ultrasound molecular images representing intensity from adherent blank MBs or DPMC displayed as color maps overlaid on B-mode images (B). Yellow line represents the region of interest (ROI). Scale bar represents 1 cm. Image signals are quantitatively presented (*** p<0.001) (C).

Figure 6

In vivo fluorescence images of tumor-bearing mice administered Cy 5.5-labeled DPMC with US, Cy 5.5-labeled DPMC and Cy 5.5 at different time-points (A). Quantification of the in vivo tumor fluorescence accumulation of different formulations expressed as fluorescence per mm² of tumor(B). Data are presented as mean values ± SD (n=5).

Figure 7

In vivo tumor growth inhibition of DPMC with or without US. Comparison of the tumor inhibition effect of DPMC with US versus DPMC without US, DOX and saline in a breast tumor model (n=5). DPMC with US achieved significant tumor inhibition (***p<0.001, compared to saline; ** p< 0.01, compared to DPMC without US) (A). During the treatment period, mice administered DPMC with US showed no significant body loss compared to those given saline, but differences were observed in relation to the DOX treatment group (* p< 0.05) (B). At the end of the experiment, tumor tissues were collected from each sacrificed animal after 20 days of treatment, photographed (D) and weighed (*** p<0.001, compared to saline and DPMC without US; * p< 0.05, compared to DOX) (C).

Figure 8

Histological analysis of tumors from mice treated with different formulations. Images (A) and corresponding quantification (B) of Caspase-3, Ki67 and CD34 staining of tumors treated with saline, DOX, DPMC without US and DPMC with US. Scale bar represents 100 μm.