Azo-based small molecular hypoxia responsive theranostic for tumor-specific imaging and therapy
Graphical abstract
Introduction
Hypoxia represents a unique feature of solid tumors with limited oxygen supply, which results in the stabilization and upregulation of the hypoxia-inducible factor (HIF-1α/β) [1]. The latter controls several biological processes such as cell proliferation, angiogenesis, pH regulation, apoptosis, immortality, and migration [[2], [3], [4]]. On the cellular level, hypoxia occurs due to a reduction in oxygen diffusion rate with increasing distance from the vasculature system [5]. As a result, hypoxia spreads heterogeneously within the solid tumor and eventually results in the formation of metastatic tumor masses [6]. The low oxygen (pO2 ≤ 20 mmHg) pressure in hypoxia often directly affects the efficacy of anticancer modalities used today in cancer treatment programs [7]. In radiotherapy, low oxygen concentrations in hypoxic regions often result in radiation resistance due to an insufficient formation of reactive oxygen species (ROS). Thus, radiation treatment often fails to create lethal damages to cancerous DNAs [8]. Additionally, the hypoxia-driven HIF-1α/β factor and free oxygen radicals may induce an angiogenesis factor to overcome damages intended to be caused by radiation therapy [9]. Similarly, the efficacy of chemotherapeutics is reduced in hypoxic tumor regions. As hypoxia forms far away from blood vessels (≥150 μM), the tumor microenvironment is oxygen deficient. Thus, chemotherapeutics may also fail to reach a hypoxic tumor region [10]. Moreover, high extracellular acidity in hypoxia plays a significant role in blocking the cellular uptake of chemotherapeutics such as anthracycline analogs and others [11]. Thus, the development of more alternative treatments and targeted drug delivery strategies would be beneficial.
To attain the desired pharmaceutical properties of a chemotherapeutic, understanding the underlying mechanisms of hypoxia and relevant cellular parameters are crucial. A recent study has shown that in mammalian cells, several enzymes such as NADPH-nitroreductases, NADPH-cytochrome P450 reductase, cytochrome b5 reductase and xanthine oxidase, and DT-diaphorase are highly active, eventually propagating a reducing ambiance in the solid tumor microenvironment [12]. Therefore, nitroaromatic, azo-derivatives, and quinone moieties have been extensively used in recent years as hypoxia-sensitive markers. [[12], [13], [14], [15], [16], [17]] Such compounds have been shown to be spontaneously reduced by one or two-electron reductase enzymes in the hypoxic microenvironment. Accompanying several fluorophores in a tandem with the hypoxia- markers, have also been used for hypoxia imaging [[18], [19], [20], [21]]. Similar strategies have been exploited in tumor hypoxia therapy to reach maximum clinical benefits [[22], [23], [24]]. In past few years, fluorescence modulated theranostic systems has gained significant attention in cancer therapy and imaging, because of the high synthetic simplicity, targeting specificity, enhanced cellular uptake and low cytotoxicity to neighboring healthy cells [[25], [26], [27], [28], [29], [30], [31]]. Recently, Kim and Hong et al. have reported a theranostic system based on a nitroaryl derivative of SN-38 [30,32]. Meanwhile, Zhang et al. have developed a nitro-aromatic based gemcitabine prodrug, which released gemcitabine to the hypoxia region by sequentially triggering nitroreductase with photolytic cleavage [33]. Also, Kim et al. have developed an azobenzene derivative of DNA alkylating agent N, N′-bis(2-chloroethyl)-1,4-benzenediamine for selectively hypoxia treatment [34]. Current diagnostics have provided an incentive for us to explore an improved strategy to combine hypoxia responsive therapy and imaging techniques with maximum clinical benefits.
Our present study has been designed to develop a suitable theranostic prodrug, which may further allow us to quantify the extent of drug release in the hypoxia tumor region specifically with therapeutic intervention. Moreover, the low synthetic burden for accessing the final prodrug was also one of the goals from pharmacoeconomic point of view [35]. We herein reported a theranostic prodrug that meets the desired requirements mentioned above. Specifically, the prodrug AzP1 proves to be highly advantageous concerning synthetic aspects, photophysical property changes in the presence of liver microsomes selectively, the extent of drug release (SN-38) in cells, in vivo bio-distribution of prodrug and finally, in vivo therapeutic efficacy. As described in subsequent sections, the rat liver microsomes induced cleavage of the ‘azo’ bond in the prodrug, resulted in a fluorescence turn-on response signal thus providing information on the extent of drug release (SN-38).
Section snippets
Materials
All reagents were purchased from the commercial sources (Aldrich, Alfa, TCI etc) and were used as received without any further purification. For compound purification, flash column chromatography was performed (300–400 mesh silica gel). The 1H NMR and 13C NMR spectra were recorded on Bruker 500 MHz magnetic resonance spectrometer using deuterated solvents with trimethylsilane (TMS) as internal standard. Chemical shifts were presented in ppm and the coupling constants (J) in Hz. Mass
Results and discussion
The theranostic prodrug AzP1 contains the antineoplastic irinotecan analog SN-38, a topoisomerase I inhibitor with a hypoxia-sensitive moiety, aryl-azo benzyl alcohol. AzP1 releases the active form of SN-38 once it encounters a hypoxic microenvironment in the solid tumor tissue. Free SN-38 then exhibited a fluorescence emission at λem = 560 nm and thereby exhibit information about the distribution of SN-38 in the biological system including real-time drug release information (Scheme 1). The
Conclusion
We have developed an azo-reductase stimulating theranostic AzP1. Theranostic exhibited chemical inertness towards biological analytes and wide pH-range. Furthermore, the azo-bond (NN) in the theranostic prodrug AzP1 has been shown to be very susceptible towards reduction by NADPH-cytochrome P450 enriched rat-liver microsomes. AzP1 exhibited a ratiometric fluorescence enhancement at λem = 560 nm with a 13-fold increase compared to the emission at λem = 450 nm. The prodrug has been shown to
Author contributions
Y. Z., M. M. and A.S. contributed equally to this work.
Notes
The authors declare no competing financial interest.
Acknowledgments
SB thanked to DST-SERB, India for a research grant (ECR/2015/00035). This work was supported by the National Natural Science Foundation of China (Nos. 21462050 and 21672185), the Foundation of the Department of Science and Technology of Yunnan Province of China (Nos. 2013HB062, 2014HB008, 2016FB020), and the Program for Excellent Youth Talents (No. C6176102), Yunnan University, and Future Planning in Korea (CRI project no. 2018R1A3B1052702, J. S. K.)
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2023, Sensors and Actuators B: ChemicalCitation Excerpt :In 2019, Wang et al. reported a novel azo-replaced semi-cyanine fluorescent probe AZO-Cy that was used for monitoring the hypoxic environment of A549 cancer cells [24]. At the same year, our group reported an azo-derivative probe, AzP1, that can be activated by cytochrome P450 reductase in the hypoxic TME [25]. We then extended our study by evaluating the near-infrared multifunctional fluorescent azo-compound, YLOD [26].
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These authors contributed equally.