Dual-channel NIR activatable theranostic prodrug for in vivo spatiotemporal tracking thiol-triggered chemotherapy

Real-time tracking of where, when, and how prodrugs are established. A novel theranostic prodrug based on the disulfide linkage with two distinct switchable near-infrared (NIR) fluorescence can precisely extract the prodrug release profile in vivo through dual-channel fluorescent imaging for the first time.


Experimental section
All solvents were of analytical grade. 1 H and 13 C NMR in CDCl 3 were obtained by a Bruker AV-400 spectrometer with tetramethylsilane (TMS) as internal standard. High Resolution Mass Spectra (HRMS) were obtained by a Waters LCT Premier XE spectrometer.
Absorption spectra were measured on a Varian Cary 500 spectrophotometer at 37 °C.
Fluorescence spectra were recorded on a Varian Cary Eclipse fluorescence spectrophotometer (1 cm quartz cell) at 37 °C. Deionized water was used to prepare all aqueous solutions. was removed by rotary evaporation to obtain red oil product, and then the product was purified by column chromatography (silica gel column, dichloromethane: triethylamine = 100 : 1) to obtain a red powder (210 mg), yield 64 %. 1 Figure S6-8).  Figure S9-11).

Synthesis of CPT-C-OH
The control compound CPT-C was synthesized using the same procedure used in the synthesis of CPT-S-OH. 1 Figure S15-17).

Synthesis of Cy-C-CPT
The control compound CPT-C-CPT was synthesized using the same procedure used in the synthesis of CPT-S-CPT. 1  and maintained under standard conditions.

Encapsulation of Cy-S-CPT and Cy-C-CPT
The prodrug-loaded nanoparticles were prepared using the method reported by Sun 1 with slight modifications. Briefly, Cy-S-CPT or Cy-C-CPT (1 mg) and PEG5000-PLA3000 (2) A dynamic laser scattering spectrometer (DLS, nano series ZEN3600, Malvern Instruments Ltd., UK) was employed to measure the sizes and zeta potentials of PEG-PLA nanoparticles loaded with the prodrugs, while the nanoparticle concentrations were 10 mg/mL in the samples.

In vitro cytotoxicity assay
3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were used to assess the cytotoxicity of the prodrugs on BCap-37 and MDCK cell lines. Briefly, cells were seeded in 96 well plates at a density of 5000 cells per well and incubated overnight. Cells were exposed to serial dilutions of drugs and cultivated for another 48 h, then replaced by fresh medium containing 0.75 mg/mL MTT. After 3 h incubation, the yellow tetrazolium salt (MTT) was metabolized into dark blue formazan crystals, and the MTT medium S7 solution was carefully removed. Finally, DMSO was added into the wells and the plate was gently shaken to dissolve the precipitates. The absorbance in each well was determined at 562 nm with a microplate spectrophotometer (Molecular Devices, SpectraMax M2e, USA).
Cell viability was calculated as the ratio of absorbance of the wells incubated with drug to that of the wells incubated with culture medium.

Cellular uptake and intracellular localization observation
Tumor cells were cultured in glass-bottom petri dishes at a density of 50,000 cell/mL 24 h before treatment. Cells were exposed to the prodrugs at a final concentration of 10 μM for 2 h. Cell nuclei and lysosomes were respectively stained by DAPI and Lyso Tracker Green for 30 min. After washing with PBS for 3 times, cell images were observed using a confocal laser scanning microscope (CLSM, Nikon-A1 system, Japan). Fluorescence of CyA-K released from Cy-S-CPT or Cy-C-CPT was excited using a 488 nm laser, and the emission wavelength was read from 570-620 nm.

Flow cytometry measurement
Tumor cells were plated onto six-well plates at 50,000 cells/mL (2 mL medium per well), including heart, kidneys, spleen, lung, stomach, liver and tumors were excised, washed with 0.9% saline and imaged with the same parameter described for in vivo imaging.

Plasma pharmacokinetic study
Female ICR mice were randomly divided into 3 groups (n = 3), and intravenously injected with PEG-PLA/Cy-S-CPT, PEG-PLA/Cy-C-CPT and PEG-PLA/CyA-K at a CPTequivalent dose of 10 μmol/kg respectively. Blood samples were collected into heparinized tubes at different time intervals, centrifuged at 5000 rpm for 10 min at 4 °C, and 100 μL of its supernate plasma was mixed with 900 μL acetonitrile to precipitate all the proteins. After centrifuged, the organic layer was collected. One part of 50 μL acetonitrile solution was acidized using hydrochloric acid and tested by HPLC to determine the CPT concentration; and rest 800 μL acetonitrile solution were concentrated and determine both prodrug level and CyA-K level using HPLC. The concentrations of intact prodrugs in blood plasma were determined by using a Waters 2998 detector with the absorption wavelength of 750 nm; CPT concentration was monitored using the same detector with the absorption wavelength of 360 nm; and CyA-K concentration was determined by a Waters 2475 detector with excitation of 530 nm and emission wavelength read at 650 nm. Standard curves of every components were constructed previously, which were used to determine the exact plasma concentration.

Tumor section and fluorescence imaging analysis
The prodrugs loaded in nanoparticles were intravenously injected into the nude mice with

Histological examination of tumor tissues
The excised tumors were fixed using 4% paraformaldehyde buffer, embedded in paraffin and sectioned into 5-um thick slices. The sections were stained with hematoxylin and esion (H&E, Fisher Scientific, USA) for histological examinations, observed under inverted microscope.

Statistical analysis
Data were presented as means ± standard errors. All the statistical analyses were performed using Student's t-test. Differences were considered statistically significant at a level of p<0.05, and very significant when p<0.01.