Modularized supramolecular assemblies for hypoxia-activatable fluorescent visualization and image-guided theranostics

Rationale: Molecular imaging of microenvironment by hypoxia-activatable fluorescence probes has emerged as an attractive approach to tumor diagnosis and image-guided treatment. Difficulties remain in its translational applications due to hypoxia heterogeneity in tumor microenvironments, making it challenging to image hypoxia as a reliable proxy of tumor distribution. Methods: We report a modularized theranostics platform to fluorescently visualize hypoxia via light-modulated signal compensation to overcome tumor heterogeneity, thereby serving as a diagnostic tool for image-guided surgical resection and photodynamic therapy. Specifically, the platform integrating dual modules of fluorescence indicator and photodynamic moderator using supramolecular host-guest self-assembly, which operates cooperatively as a cascaded “AND” logic gate. First, tumor enrichment and specific fluorescence turn-on in hypoxic regions were accessible via tumor receptors and cascaded microenvironment signals as simultaneous inputs of the “AND” gate. Second, image guidance by a lighted fluorescence module and light-mediated endogenous oxygen consumption of a photodynamic module as dual inputs of “AND” gate collaboratively enabled light-modulated signal compensation in situ, indicating homogeneity of enhanced hypoxia-related fluorescence signals throughout a tumor. Results: In in vitro and in vivo analyses, the biocompatible platform demonstrated several strengths including a capacity for dual tumor targeting to progressively facilitate specific fluorescence turn-on, selective signal compensation, imaging-time window extension conducive to precise normalized image-guided treatment, and the functionality of tumor glutathione depletion to improve photodynamic efficacy. Conclusion: The hypoxia-activatable, image-guided theranostic platform demonstrated excellent potential for overcoming hypoxia heterogeneity in tumors.

The fluorescent probe protected by Boc group is hydrolyzed under acidic conditions, and the fluorescent probe CNP modified by the carboxylic acid base group is synthesized.Specifically, the yellow solid (35 mg) from the previous step was dissolved in DCM/TFA (1:1, v:v) solution, and stirred for 4 h at room temperature.After the reaction, the organic solvent is evaporated by the rotary evaporator under reduced pressure to obtain a yellow product (CNP).The product is used for the next reaction without further purified.

Stability of HTP-BM/CFN under different physiological conditions
It is important to test the stability of prepared nanoparticles under different physiological conditions, especially when considering their use in biomedical applications.We simulated in vivo serum conditions as well as tumor acidic conditions to assess the stability of the assemblies under different physiological conditions by measuring the changes in particle size.The specific operation was as follows: the samples were dissolved in RPMI-1640 medium containing 10 % fetal bovine serum and PBS with pH=6.5.The hydrodynamic sizes were measured at different time points (1, 12, 24, 36, and 48 h), respectively, as shown in the figure S14, there was no obvious change of the assemblies' particle sizes in serum solution within 48 h, which proved that the assemblies had a good stability, which is conducive to their long circulation in vivo, and provides an important reference for the effectiveness and safety of their application in organisms.However, the particle size of the assemblies changed under acidic physiological environment, which may be due to the fact that the assemblies were deconstructed under acidic environment, and the HTP in the core would be agglomerated, which caused the increase of the particle size.

HTP-BM/CF catabolism at normal pH and high GSH concentration
To assess the decomposition of the HTP-BM/CF under normal pH and high concentration GSH conditions, the HTP-BM/CF was incubated in the buffer solution with pH at 7.4 (normal pH) and high GSH concentration of 10 μM for 4 h .The HTP-BM/CF treated as below and the untreated HTP-BM/CF were both measured using gel permeation chromatography (GPC).As shown in the Figure S15 below, the untreated assembly at pH=7.4 had only one peak, which retention time were 8.7 min.
The peaks of HTP-BM/CF treated under normal pH and high concentration GSH appeared to shifted right and divided to two peaks in comparison to untreated HTP-BM/CF, indicating the molecular weight reduction of HTP-BM/CF.The two peaks of treated HTP-BM/CF may be assigned to CF (left) and the TP (right), in which the right peak is the decomposition product of HTP-BM inner core that was degraded by the high concentration GSH to generate TP.Based on this GPC result, it proves that HTP-BM/CF occurs decomposition under normal pH and high concentration GSH.

Release of self-assembled nanoparticles over time.
As requested, we tested the TP release of self-assembled HTP-BM/CF over time under in vitro (pH 7.4 without GSH) and in vivo (tumor environment (pH=6.5 and 10 μM GSH)) simulated conditions.As can be seen in the figure S16, the release of TP from the nano-assemblies in the PBS group has been stably kept at a low state, while under the condition of acidity plus high concentration of GSH, the self-assembled nanoparticles will gradually release porphyrin over time, and then the release rate will gradually slow down at a later stage to form a stable release phase, which also indicates that the stability of the assemblies is regulated by PH and GSH.

Figure S16 .
Figure S16.Release profiles of nanoparticles with time under different conditions.