Theranostic Prodrugs Research Articles

Recent Advances in the Development of Targeted Activation Strategies for Organoboron Prodrugs

Elevated levels of reactive oxygen species (ROS) are a hallmark of varieties of diseases such as cancer, inflammation, and neurodegenerative disorders. Inspired by the discrepancy of ROS concentrations between pathological tissues and the normal counterparts, an increasing number of ROS‐responsive theranostic prodrugs are developed in past years, with particularly high proportions of organoboron‐based prodrugs that can respond to H2O2. Unfortunately, increasing studies have demonstrated that the intrinsic ROS (H2O2) levels in most pathological tissue are only slightly higher than normal tissues and are not adequate to activate organoboron prodrugs; in contrast, several organoboron compounds have been clinically approved in which boronic acid acts as electrophilic warhead. To this end, developing more robust and universal approaches for boronic acid‐prodrug activation becomes highly attractive. In this context, we discuss the recently reported activation strategies for boron‐caged prodrugs with a particular focus on their design principles and activation mechanisms. The perspectives on the future directions for this important research area are discussed as well.

A Rationally Designed Prodrug for the Fluorogenic Labeling of Albumin and Theranostic Effects on Drug-Induced Liver Injury.

The development of small-molecular fluorogenic tools for the chemo-selective labeling of proteins in live cells is important for the evaluation of intracellular redox homeostasis. Dynamic imaging of human serum albumin (HSA), an antioxidant protein under oxidative stress with concomitant release of antioxidant drugs to maintain redox homeostasis, affords potential opportunities for disease diagnosis and treatment. In this work, we developed a nonfluorogenic prodrug named TPA-NAC, by introducing N-acetyl-l-cysteine (NAC) into a conjugated acceptor skeleton. Through combined thiol and amino addition, coupling with HSA results in fluorescence turn-on and drug release. It was reasoned that the restricted intramolecular motion of the probe under an HSA microenvironment after covalent bonding inhibited the nonradiative transitions. Furthermore, the biocompatibility and photochemical properties of TPA-NAC enabled it to image exogenous and endogenous HSA in living cells in a wash-free manner. Additionally, the released drug evoked upregulation of superoxide dismutase (SOD), which synergistically eliminated reactive oxygen species in a drug-induced liver injury model. This study provides insights into the design of new theranostic fluorescent prodrugs for chemo-selective protein labeling and disease treatments.

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.

Allyl phenyl selenides as H2O2 acceptors to develop ROS-responsive theranostic prodrugs

Reactive oxygen species (ROS)-responsive prodrugs have received significant attention due to their capacity to target tumors to relieve the side effects caused by chemotherapy. Herein, a series of novel H2O2-activated theranostic prodrugs (CPTSe1-CPTSe7) were developed containing allyl phenyl selenide moieties as H2O2 acceptors. Compared with conventional boronate ester-based prodrug CPT-B, CPTSe1 was more stable in human plasma and showed a more complete release of camptothecin (CPT) in H2O2 inducing experiment. The selectively activated fluorescence signals of CPTSe1 in tumor cells make it useful for real-time monitoring of CPT release and H2O2 detection. Furthermore, excellent selectivity of CPTSe1 was achieved for tumor cells over normal cells. Our results provide a new platform for the development of H2O2-responsive theranostic prodrugs.

Hydrogen sulphide-triggered theranostic prodrugs based on the dynamic chemistry of tetrazines.

Dynamic nucleophilic aromatic substitution of tetrazines (SNTz) has been employed to build theranostic prodrugs that are activated by hydrogen sulfide. H2S is typically found in high concentrations in some kinds of cancer cells and it is able to trigger the disassembly of tetrazine prodrugs. In such a way, a dual release of drugs and/or fluorescent compounds can be selectively triggered.

Real-Time Multi-Photon Tracking and Bioimaging of Glycosylated Theranostic Prodrugs upon Specific Enzyme Triggered Release.

Real-time tracking of prodrug uptake, delivery and activation in vivo represents a major challenge for prodrug development. Herein, we demonstrate the use of novel glycosylated theranostics of the cancer pharmacophore Amonafide in highly-selective, enzymatic triggered release. We show that the use of endogenous enzymes for activated release of the therapeutic component can be observed, in real time, and monitored using one and two-photon bioimaging, offering unique insight into the prodrug pharmacokinetic profile. Furthermore, we demonstrate that the potent cytotoxicity of Amonafide is preserved using this targeted approach.

Theranostic Heterodimeric Prodrug with Dual-Channel Fluorescence Turn-On and Dual-Prodrug Activation for Synergistic Cancer Therapy.

Theranostic prodrugs that can precisely monitor drug activation with synergistic therapeutic effects are highly desirable for personalized medicine. In this study, a theranostic heterodimeric prodrug, CyNH-SS-DOX, with synchronous and independent dual-channel fluorescence turn-on and dual-prodrug activation for synergistic cancer therapy is developed. A hemicyanine fluorescent drug, CyNH2 , with good therapeutic effects found in this work, is conjugated to doxorubicin (DOX) through a disulfide linker to form CyNH-SS-DOX. Before activation, both the fluorescence of DOX and CyNH2 are in the off state and the toxicity is low. In the presence of intracellular glutathione, both the fluorescence of DOX and CyNH2 at different channels are turned on. Meanwhile, DOX and CyNH2 are activated in a synergistic anticancer effect. It is believed that CyNH-SS-DOX is promising for monitoring prodrug activation in dual-fluorescence channels and for enhancing therapeutic efficacy with few side effects.

Reactive Oxygen Species (ROS)-Responsive Prodrugs, Probes, and Theranostic Prodrugs: Applications in the ROS-Related Diseases.

Elevated levels of reactive oxygen species (ROS) have commonly been implicated in a variety of diseases, including cancer, inflammation, and neurodegenerative diseases. In light of significant differences in ROS levels between the nonpathogenic and pathological tissues, an increasing number of ROS-responsive prodrugs, probes, and theranostic prodrugs have been developed for the targeted treatment and precise diagnosis of ROS-related diseases. This review will summarize and provide insight into recent advances in ROS-responsive prodrugs, fluorescent probes, and theranostic prodrugs, with applications to different ROS-related diseases and various subcellular organelle-targetable and disease-targetable features. The ROS-responsive moieties, the self-immolative linkers, and the typical activation mechanism for the ROS-responsive release are also summarized and discussed.

Hydrogen Sulfide-Specific and NIR-Light-Controllable Synergistic Activation of Fluorescent Theranostic Prodrugs for Imaging-Guided Chemo-Photothermal Cancer Therapy

Theranostic prodrugs are promising for cancer medicine; however, the inability to activate these systems exclusively at the desired tumor location compromises the specificity and efficacy of cancer...

Nitroreductase-Activatable Theranostic Molecules with High PDT Efficiency under Mild Hypoxia Based on a TADF Fluorescein Derivative.

High specificity detection and site-specific therapy are still the main challenges for theranostic anticancer prodrugs. In this work, we reported two smart activatable theranostic molecules based on a thermally activated delayed fluorescence fluorescein derivative. Nitroreductase induced by a mild hypoxia microenvironment of a solid tumor was used to activate the fluorescence and photodynamic therapy (PDT) efficiency by employing the intramolecular photoinduced electron transfer mechanism. A high PDT efficiency under 10% oxygen concentration was achieved, which is better than that of porphyrin (PpIX), a traditional photosensitizer. Such an excellent PDT efficiency can be attributed to lysosome disruption because the theranostic molecule can specifically enter the lysosomes of cells. Importantly, the strategy of targeting the mild hypoxic cells in the edge of tumor tissue could heal the "Achilles' heel" of traditional PDT. We believe that this theranostic molecule has a high potential to be applied in clinical investigation as a theranostic anticancer prodrug.

Developing glutathione-activated catechol-type diphenylpolyenes as small molecule-based and mitochondria-targeted prooxidative anticancer theranostic prodrugs

Developing concise theranostic prodrugs is highly desirable for personalized and precision cancer therapy. Herein we used the glutathione (GSH)-mediated conversion of 2,4-dinitrobenzenesulfonates to phenols to protect a catechol moiety and developed stable pro-catechol-type diphenylpolyenes as small molecule-based prooxidative anticancer theranostic prodrugs. These molecules were synthesized via a modular route allowing creation of various pro-catechol-type diphenylpolyenes. As a typical representative, PDHH demonstrated three unique advantages: (1) capable of exploiting increased levels of GSH in cancer cells to in situ release a catechol moiety followed by its in situ oxidation to o-quinone, leading to preferential redox imbalance (including generation of H2O2 and depletion of GSH) and final selective killing of cancer cells over normal cells, and is also superior to 5-fluorouracil and doxorubicin, the widely used chemotherapy drugs, in terms of its ability to kill preferentially human colon cancer SW620 cells (IC50 = 4.3 μM) over human normal liver L02 cells (IC50 = 42.3 μM) with a favourable in vitro selectivity index of 9.8; (2) permitting a turn-on fluorescent monitoring for its release, targeting mitochondria and therapeutic efficacy without the need of introducing additional fluorophores after its activation by GSH in cancer cells; (3) efficiently targeting mitochondria without the need of introducing additional mitochondria-directed groups.

Aminopeptidase‐N‐activated Theranostic Prodrug for NIR Tracking of Local Tumor Chemotherapy

AbstractThe development of molecular theranostic prodrugs for in vivo cancer diagnosis and targeted chemotherapy is urgently required. Enzyme‐activated prodrugs display superior selectivity as a result of cancer‐specific enzymes which serve as cancer biomarkers. Herein, an aminopeptidase N (APN)‐activated theranostic prodrug Nile blue‐C6‐amide‐p‐fluorophenylalanyl‐l‐melphalanyl (NBFMel) is reported for fluorescence cancer diagnosis and local tumor treatment. NBFMel demonstrates negligible cytotoxicity and very weak fluorescence due to the photoinduced electron transfer (PET) between melphalan and Nile blue fluorophore. After activation caused by APN, the prodrug releases free melphalan that inhibits tumor cell growth. Simultaneously, the reaction blocks the PET process and switches on the fluorescence, which can be used for cancer diagnosis. NBFMel is successfully utilized to report the presence of tumor and for in situ tracking of drug release in tumor‐bearing mouse models. Moreover, NBFMel demonstrates efficient tumor inhibition when intravenously injected into mice. Therefore, the APN‐activated theranostic prodrug provides a new platform for in vivo cancer diagnosis and targeted anticancer chemotherapy.

Targeted Methotrexate Prodrug Conjugated With Heptamethine Cyanine Dye Improving Chemotherapy and Monitoring Itself Activating by Dual-Modal Imaging

Theranostic prodrug plays a vital role in reducing the side effects and evaluating the therapeutic efficiency of prodrug in vivo. In particular, small conjugate-based theranostic prodrugs have attracted much attention because of their clear and simple structures. In this work, we synthesized a novel tumor-targeting and glutathione-activated conjugate-based theranostic prodrug (Cy-SS-MTX). The prodrug was constructed by conjugating Cy (IR780) to methotrexate (MTX) via a disulphide bond. The Cy dye as targeting molecule bring prodrug to cancer cells and then the prodrug was activated by the high levels of glutathione in tumor. In cell experiments, the results showed the excellent ability of prodrug to target tumor. Meanwhile, the prodrug apparently improved the anti-tumor ability and hugely reduced toxicity of free MTX on normal cells. Furthermore, owing to intramolecular charge transfer between Cy and MTX, the Cy structure in the prodrug showed an absorption peak at 654 nm in UV-Vis spectroscopy. However, when the disulphide bond of prodrug was broken by glutathione, a new UV-Vis absorption peak at 802 nm of Cy structure in prodrug was arised. At the same time, the fluorescence (FL) emission peak at 750 nm (excitation at 640 nm) would turn into 808 nm (excitation at 745 nm). What’s more, the photoacoustic (PA) signal with excitation at 680 and 808 nm also changed. The experimental results in vivo showed that the prodrug has been successfully utilized for real-timely tracking MTX activation by FL and PA imaging upon near infrared laser excitation and cancer targeting therapy. Our studies further encourage application of small conjugate-based prodrug based on tumor-targeted heptamethine cyanine dye as reporter group for targeted therapy and real-timely tracking activation of drug.

Direct Real-Time Monitoring of Prodrug Activation by Chemiluminescence.

The majority of theranostic prodrugs reported so far relay information through a fluorogenic response generated upon release of the active chemotherapeutic agent. A chemiluminescence detection mode offers significant advantages over fluorescence, mainly due to the superior signal-to-noise ratio of chemiluminescence. Here we report the design and synthesis of the first theranostic prodrug monitored by a chemiluminescence diagnostic mode. As a representative model, we prepared a prodrug from the chemotherapeutic monomethyl auristatin E, which was modified for activation by β-galactosidase. The activation of the prodrug in the presence of β-galactosidase is accompanied by emission of a green photon. Light emission intensities, which increase with increasing concentration of the prodrug, were linearly correlated with a decrease in the viability of a human cell line that stably expresses β-galactosidase. We obtained sharp intravital chemiluminescent images of endogenous enzymatic activity in β-galactosidase-overexpressing tumor-bearing mice. The exceptional sensitivity achieved with the chemiluminescence diagnostic mode should allow the exploitation of theranostic prodrugs for personalized cancer treatment.

Dual-channel near-infrared fluorescent thiol-activatable theranostic prodrug for in vivo real-time tracking chemotherapy

Event Abstract Back to Event Dual-channel near-infrared fluorescent thiol-activatable theranostic prodrug for in vivo real-time tracking chemotherapy Jianbin Tang1, Minghzou Ye1, Xiaohang Wang2, Zhiqian Guo2, Youqing Shen1 and Weihong Zhu2 1 Zhejiang University, College of Chemical and Biological Engineering, China 2 East China University of Science and Technology, Key Laboratory for Advanced Materials and Institute of Fine Chemicals, China Introduction: Chemotherapy is one of the most important arsenals against cancer, but always results in undesired purgatorial side-effects such as hair loss, bone marrow suppression and organ damage. Tumor-activatable prodrugs that are selectively activated in tumors were developed to reduce off-target toxicity and improve the therapeutic index. Real-time monitoring when, where, and how the prodrugs are activated in vivo is critical for their development, but it remains as a great challenge since we lack capable theranostic prodrugs to track their behavior in vivo. The reported fluorescent theranostic prodrugs were plagued by their mono-channel fluorescence and short wavelength, which were insufficient for simultaneously tracking their biodistribution and activation in vivo[1],[2]. To solve the problem, we developed a novel theranostic prodrug (Cy-S-CPT) from cyanine dye and anticancer drug CPT, which were covalently connected with a disulfide bond linkage. The cleavage of the disulfide bond by GSH and the successive cyclization reaction produced the activated anti-cancer drug CPT and induced remarkable fluorescent shift from 825 to 650 nm, making a breakthrough with switchable dual-channel near-infrared (NIR) fluorescence to real-timely scrutinize the activation process of prodrugs with disulfide bond linker in vivo (Figure. 1). Figure. 1 Design and activation mechanism of dual-channel fluorescent theranostic prodrug Results and Discussion: The prodrug release process was tested at different conditions by using spectrometer, flow cytometer and confocal laser scanning microscopy, which verified the activation sensitivity and thiol-specificity in vitro. The in vivo fluorescent imaging of mice after the intravenous injection of the prodrug loaded in PEG-PLA nanoparticles was performed with different fluorescence channels (λex = 750 nm and λem = 825 nm vs λex = 530 nm, λem = 650 nm ) (Figure. 2B). Clearly, the NIR fluorescence of intact prodrug (represented in green) was quickly distributed in the whole body of mice soon after the injection, and then slowly faded out, demonstrating a quick distribution and gradual activation of Cy-S-CPT. Simultaneously, the red fluorescence of cyanine dye (represented in red) became stronger as time increased, and reached its peak at 4 h post injection, which indicated the highest prodrug activation level occurred at this time point. Intriguingly, even though red fluorescence dispersed broadly in the mice, indicating a comprehensive activation occurred in all organs, the tumor bestowed the strongest red fluorescence at 24 h post injection, which revealed a preferential prodrug accumulation and activation in tumor. Additionally, the in vivo antitumor studies performed on xenografted BCap-37 tumor model indicated that Cy-S-CPT loaded nanoparticles displayed significantly improved therapeutic efficacy than the clinical used drug CPT-11(Figure. 2B). Figure. 2 In vivo imaging (A) and antitumor activities (B) of the theranostic prodrug on BCap-37 tumor xenografted nude mice. Note: green signal represents the fluorescence from the intact prodrugs (λex = 750 nm, λem = 825 nm), indicating prodrug biodistribution; while red signal represents the fluorescence from CyA-K (λex = 530 nm, λem = 650 nm), indicating drug activation. Yellow represents the mix. Conclusion: A theranostic prodrug with a disulfide bond linkage possessing switchable NIR fluorescent from 825 to 650 nm was prepared and used to investigate GSH-responsive drug activation in vivo. The dual-channel fluorescent imaging indicated that although the prodrug with disulfide could be activated comprehensively in all the organs and tissues with a relatively high speed, tumor demonstrated the strongest prodrug activation at 24 h post injection. Moreover, Cy-S-CPT exhibited significant therapeutic efficacy which overwhelmed clinical used drug CPT-11. The theranostic prodrug with switchable dual-channel near-infrared (NIR) fluorescence provided a new strategy to real-time monitor the distribution and activation of prodrugs in vivo. National Basic Research Program (2014CB931900and 2013CB733700); NSFC for Creative Research Groups (21421004) and Distinguished Young Scholars (21325625), NSFC/China

NIR Fluorogenic Dye as a Modular Platform for Prodrug Assembly: Real‐Time in vivo Monitoring of Drug Release

The ability to monitor drug release in vivo provides essential pharmacological information. We developed a new modular approach for the preparation of theranostic prodrugs with a turn-ON near-infrared (NIR) fluorescence mode of action. The prodrugs release their chemotherapeutic cargo and an active cyanine fluorophore upon reaction with a specific analyte. The prodrug platform is based on the fluorogenic dye QCy7; upon removal of a triggering substrate, the dye fluoresces, and the free drug is released. The evaluated camptothecin prodrug was activated by endogenous hydrogen peroxide produced in tumor cells in vitro and in vivo. Drug release and in vitro cytotoxicity were correlated with the emitted fluorescence. The prodrug activation was effectively imaged in real time in mice bearing tumors. The modular design of the QCy7 fluorogenic platform should allow the preparation of numerous other prodrugs with various triggering substrates and chemotherapeutic agents. We anticipate that the development of real-time in vivo monitoring tools such as that described herein will pave the way for personalized therapy.

Photo-triggered fluorescent theranostic prodrugs as DNA alkylating agents for mechlorethamine release and spatiotemporal monitoring.

We describe a new theranostic strategy for selective delivery and spatiotemporal monitoring of mechlorethamine, a DNA alkylating agent. A photo-responsive prodrug is designed and composed of a photolabile o-nitrophenylethyl group, a DNA alkylating mechlorethamine drug and a coumarin fluorophore. Masking of the "N" in mechlorethamine in a positively charged state in the prodrug renders it inactive, non-toxic, selective and non-fluorescent. Indeed, the stable prodrug shows negligible cytotoxicity towards normal cells with and without UV activation and is completely non-fluorescent. However, upon photo-irradiation, the active mechlorethamine is released and induces efficient DNA cross-links, accompanied by a strong fluorescence enhancement (152 fold). Furthermore, DNA cross-linking activity from the release can be transformed into anticancer activity observed in in vitro studies of tumor cells. Importantly, the drug release progress and the movement can be conveniently monitored by fluorescence spectroscopy. The mechanistic study proves that the DNA cross-linking activity is mainly due to the release of DNA alkylating mechlorethamine. Altogether, the studies show the power of the theranostic strategy for efficient therapy in cancer treatment.

Imaging of doxorubicin release from theranostic macromolecular prodrugs via fluorescence resonance energy transfer.

Herein we present a FRET-based theranostic macromolecular prodrug (TMP) composed of (a) dendritic polyglycerol (PG) as polymeric nanocarrier, (b) doxorubicin (Dox) linked via a pH-sensitive hydrazone to (c) a tri-functional linker, and (d) an indodicarbocyanine dye (IDCC) attached in close proximity to Dox. The drug fluorescence is quenched via intramolecular FRET until the pH-sensitive hydrazone bond between the TMP and Dox is cleaved at acidic pH. By measuring its fluorescence, we characterized the TMP cleavage kinetics at different pH values in vitro. The intracellular release of Dox from the carrier was monitored in real time in intact cancer cells, giving more insight into the mode of action of a polymer drug conjugate.

Targeted theranostic prodrugs based on an aggregation-induced emission (AIE) luminogen for real-time dual-drug tracking.

A targeted theranostic delivery system containing two prodrugs with drug tracking and activation monitoring functions was developed for visualizing cancer cell ablation with synergistic anticancer effects.