Zwitterionic rhodamine-CPT prodrug nanoparticles with GSH/H2O2 responsiveness for cancer theranostics

Rationale: Fluorescently traceable prodrugs, which can monitor their biodistribution in vivo and track the kinetics of drug delivery in living cells, are promising for constructing theranostic medicines. However, due to their charge and hydrophobicity, most of the fluorescently traceable prodrugs exhibit high protein binding and non-specific tissue retention affecting in vivo distribution and toxicity, with high background signals. Methods: Herein, the zwitterionic rhodamine (RhB) and camptothecin (CPT) were bridged with a disulfide bond to construct a tumorous heterogeneity-activatable prodrug (RhB-SS-CPT). The interaction of zwitterionic RhB-SS-CPT with proteins was detected by UV and fluorescence spectroscopy, and further demonstrated by molecular docking studies. Then, intracellular tracking and cytotoxicity of RhB-SS-CPT were determined in tumor and normal cells. Finally, the in vivo biodistribution, pharmacokinetics, and anticancer efficacy of RhB-SS-CPT were evaluated in a mouse animal model. Results: The tumorous heterogeneity-activatable RhB-SS-CPT prodrug can self-assemble into stable nanoparticles in water based on its amphiphilic structure. Particularly, the zwitterionic prodrug nanoparticles reduce the non-specific binding to generate a low background signal for better identification of cancerous lesions, achieve rapid internalization into cancer cells, selectively release bioactive CPT as a cytotoxic agent in response to high levels of GSH and H2O2, and exhibit high fluorescence that contributes to the visual chemotherapy modality. In addition, the RhB-SS-CPT prodrug nanoparticles show longer circulation time and better antitumor activity than free CPT in vivo. Interestingly, the zwitterionic nature allows RhB-SS-CPT to be excreted through the renal route, with fewer side effects. Conclusions: Zwitterionic features and responsive linkers are important considerations for constructing potent prodrugs, which provide some useful insights to design the next-generation of theranostic prodrugs for cancer.

(Shanghai, China). Hoechst 33342, MitoTracker® Green FM, LysoTracker Green® DND-26, Calcein (AM) and ethidium homodimer-1 (EthD-1) were purchased from Invitrogen (Carlsbad, CA). All other reagents and solvents were used without further purification. 1 H and 13 C nuclear magnetic resonance spectra were recorded using a Bruker Ascend 400 FT-NMR. Mass analysis was measured at Brooke MicroFlex LRF in positive ion mode. Malvern Zetasizer Nano ZS90 was used to measure the particle size distribution at room temperature. HITACHI U-2910 UV-Vis absorption spectrometer and Hitachi F-2700 spectrometer were used to record absorption spectra and fluorescence spectrum, respectively. Confocal laser scanning microscopy (Nikon C2+) was used to observe the fluorescence of cells. H&E staining images and Live-Dead images were measured by using Olympus IX73 microscope.
The mixture continued to react at 35 °C for 3 hours. The solvent and oxalyl chloride were removed by concentration under vacuum to obtain a yellow oil as compound 1, which was used for the next step without further purification.

In Vitro CPT release
The release profiles of CPT from prodrug nanoparticles were studied in PBS (pH 7.4, containing 30% ethanol) [5]. RhB-SS-CPT (15 μM) were incubated with different concentrations of H2O2 or GSH in 2 mL release media at 37 °C. At predetermined time points, the concentration of the released CPT from prodrug nanoparticles was determined by high performance liquid chromatography (HPLC) (n=3 for each group).
The HPLC analysis of drug release was achieved on Agilent InfinityLab Poroshell 120 EC-C18 4 μm (4.6×150 mm) Column with a mobile phase of CH3OH: H2O=60:40, a column temperature at 20 ℃, a flow rate at 1 mL/min, and a UV detection wavelength at 364 nm. To further investigate the mechanism of H2O2-triggered or GSH-triggered CPT release, ESI-MS was used to monitor the molecular weight of RhB-SS-CPT nanoparticles after treated with H2O2 or GSH (10 mM) for 6 h.

In Vitro cytotoxicity assay
HeLa and L929 cells were cultured in a conventional medium supplemented with 10%

In Vivo anticancer efficacy
Firstly, Kunming mice were used to evaluate the pharmacokinetic profiles of RhB-SS-CPT nanoparticles. The animals were randomly divided into free CPT and RhB-SS-CPT groups, and were intravenously administrated with free CPT or RhB-SS-CPT at a dose of 4 mg/kg equivalent to CPT (n = 3 for each group). At predetermined time points, blood samples were collected, and 10 μL of blood was lysed by 50 μL RIPA Lysis buffer.