Fluorescent Prodrug Research Articles

Fingolimod phosphoramidate prodrugs: Synthesis, photophysical characterisation and lipid bilayer interaction of fluorescent tagged Prodrug

FTY720 (Fingolimod, Gilenya, 1), a structural analog of sphingosine (2) was the first orally administered drug approved for the treatment of multiple sclerosis (MS). However, the phosphate derivative of Fingolimod, namely Fingolimod-1-phosphate (FTY720-1P, 3) is the biologically active molecule in MS as well as in various lysosomal storage diseases such as mucolipidosis IV and Nieman-Pick. In all cases, Fingolimod requires to be phosphorylated, resembling the natural sphingosine phosphate (4), to accomplish its therapeutic/biological effects. Here, we report the synthesis of a small library of prodrugs that could provide the efficient delivery of 3, the bioactive species. Moreover, to gain insight about the biological behaviour of these novel candidates within human body such as cell and brain blood barrier penetration we have prepared and spectroscopically characterised a fluorescent tagged version of one of the new prodrugs. The behavior of our prodrugs with respect to biological membranes was evaluated by studying the interaction between the fluorescent prodrug 18 and DOPC/DPPC liposome models.

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.

A fluorescent prodrug to fight drug-resistant lung cancer cells via autophagy-driven ferroptosis

Lung cancer is the second most common malignant tumor worldwide. The problem of drug resistance in lung cancer leads to treatment failure and recurrence in more than 90% of cancer patients. Thus, developing chemical probes that can observe drug-resistant lung cancer cells in real-time and eliminate them is of great interest. However, such tools that can simultaneously probe drug-resistant lung cancer cells and therapeutic modes of action have not yet been developed. In this work, we reported a fluorescent prodrug that can be activated in cancer cell lysosomes to release amonafide (ANF) and aniline mustard, where the fluorescent signal of ANF (λex/λem = 405/570 −620 nm) can be tracked by super-resolution imaging in live cancer cells. With imaging studies, we found that this new fluorescent prodrug uniquely displays an autophagy-driven ferroptosis-inducing effect, accompanied by the accumulation of intracellular ferrous ions and lipid peroxidation. More importantly, the fluorescent prodrug exhibits potential anti-proliferative activity, especially against drug-resistant A549R lung cancer cells (IC50, ∼ 3 μM), better than the positive control drug cisplatin (IC50, ∼ 27 μM). This work indicates that the development of fluorescent prodrugs as autophagy-driven ferroptosis inducers may represent a viable approach for targeting drug-resistant cancer cells.

Cancer Cell-Specific Fluorescent Prodrug Delivery Platforms.

Targeting cancer cells with high specificity is one of the most essential yet challenging goals of tumor therapy. Because different surface receptors, transporters, and integrins are overexpressed specifically on tumor cells, using these tumor cell-specific properties to improve drug targeting efficacy holds particular promise. Targeted fluorescent prodrugs not only improve intracellular accumulation and bioavailability but also report their own localization and activation through real-time changes in fluorescence. In this review, efforts are highlighted to develop innovative targeted fluorescent prodrugs that efficiently accumulate in tumor cells in different organs, including lung cancer, liver cancer, cervical cancer, breast cancer, glioma, and colorectal cancer. The latest progress and advances in chemical design and synthetic considerations in fluorescence prodrug conjugates and how their therapeutic efficacy and fluorescence can be activated by tumor-specific stimuli are reviewed. Additionally, novel perspectives are provided on strategies behind engineered nanoparticle platforms self-assembled from targeted fluorescence prodrugs, and how fluorescence readouts can be used to monitor the position and action of the nanoparticle-mediated delivery of therapeutic agents in preclinical models. Finally, future opportunities for fluorescent prodrug-based strategies and solutions to the challenges of accelerating clinical translation for the treatment of organ-specific tumors are proposed.

A dual-amplified ROS-responsive nanosystem with self-accelerating drug release for synergistic chemotherapy.

In this work, we have developed a tumor-specific self-accelerating prodrug activation nanosystem consisting of self-amplifying degradable polyprodrug PEG-TA-CA-DOX and encapsulated fluorescent prodrug BCyNH2, equipped with a reactive oxygen species dual-cycle amplification effect. Furthermore, activated CyNH2 is a therapeutic agent with potential to synergistically improve chemotherapy.

Construction of hyperbranched polysiloxane-based multifunctional fluorescent prodrug for preferential cellular uptake and dual-responsive drug release.

Hyperbranched polymers hold great promise in nanomedicine for their controlled chemical structures, sizes, multiple terminal groups and enhanced stability than linear amphiphilic polymer assemblies. However, the rational design of hyperbranched polymer-based nanomedicine with low toxic materials, selective cellular uptake, controlled drug release, as well as real-time drug release tracking remains challenging. In this work, a hyperbranched multifunctional prodrug HBPSi-SS-HCPT is constructed basing on the nonconventional aggregation-induced emission (AIE) featured hyperbranched polysiloxanes (HBPSi). The HBPSi is a biocompatible AIE macromolecule devoid of conjugates, showing a high quantum yield of 17.88% and low cytotoxicity. By covalently grafting the anticancer drug, 10-hydroxycamptothecin (HCPT), to the HBPSi through 3,3'-dithiodipropionic acid, HBPSi-SS-HCPT is obtained. The HBPSis demonstrate obvious AIE features and it turned to aggregation-caused quenching (ACQ) after grafting HCPT owing to the FRET behavior between HBPSi and HCPT in HBPSi-SS-HCPT. In addition to on-demand HCPT release in response to changes in environmental pH and glutathione, a series of in vitro and in vivo studies revealed that HBPSi-SS-HCPT exhibits enhanced accumulation in tumor tissues through the enhanced permeation and retention (EPR) effect and preferential cancer cell uptake by charge reversal, thus resulting in apoptotic cell death subsequently. This newly developed multifunctional HBPSi-SS-HCPT prodrug provides a biocompatible strategy for controlled drug delivery, preferential cancer cell uptake, on-demand drug release and enhanced antitumor efficacy.

Research advances in integrated theranostic probes for tumor fluorescence visualization and treatment.

At present, cancer is obviously a major threat to human health worldwide. Accurate diagnosis and treatment are in great demand and have become an effective method to alleviate the development of cancer and improve the survival rate of patients. A large number of theranostic probes that combine diagnosis and treatment methods have been developed as promising tools for tumor precision medicine. Among them, fluorescent theranostic probes have developed rapidly in the frontier research field of precision medicine with their real time, low toxicity, and high-resolution merit. Therefore, this review focuses on recent advances in the development of fluorescent theranostic probes, as well as their applications for cancer diagnosis and treatment. Initially, small-molecule fluorescent theranostic probes mainly including tumor microenvironment-responsive fluorescent prodrugs and phototherapeutic probes were introduced. Subsequently, nanocomposite probes are expounded based on four types of nano-fluorescent particles combining different therapies (chemotherapy, photothermal therapy, photodynamic therapy, gene therapy, etc.). Then, the capsule-type "all in one" probes, which occupy an important position in theranostic probes, are summarized according to the surface carrier type. This review aims to present a comprehensive guide for researchers in the field of tumor-related theranostic probe design and development.

Kinetic stability-driven cytotoxicity of small-molecule prodrug nanoassemblies.

Nanoassemblies (NAs) of small-molecule lipophilic prodrugs have been widely investigated for efficient drug delivery in cancer therapy, but their kinetic stability has not attracted sufficient attention in the past studies. Herein, we reported that kinetic stability has a great influence on the drug release from the NAs of lipophilic prodrugs in physiologically relevant media. Based on the co-assembled FRET nanosystems of two lipophilic fluorescent prodrugs, we demonstrated that NAs constructed by lipophilic prodrugs containing shorter alkyl chains or those with higher unsaturated degrees displayed poorer kinetic stability, which further resulted in remarkably faster drug release in mouse plasma and various tissue homogenates. More importantly, these kinetically unstable NAs also induced rapid intracellular drug release, resulting in much more potent cytotoxicity. These findings highlight the crucial role of kinetic stability in determining the drug release from the NAs of lipophilic prodrugs, which would effectively guide their rational designs for cancer therapy.

Nanomized tumor-microenvironment-active NIR fluorescent prodrug for ensuring synchronous occurrences of drug release and fluorescence tracing.

Improving the bioavailability and tumor-targeting ability of a prodrug, as well as monitoring its active ingredient release in vivo, is still a challenge in cancer diagnosis and therapy. Herein, a specific nanomized tumor-microenvironment-active near-infrared (NIR) fluorescent DCM-S-GEM/PEG prodrug was developed as a potent monitoring platform, wherein we conjugated antitumor drug gemcitabine (GEM) and NIR fluorescent chromophore dicyanomethylene-4H-pyran (DCM) via glutathione (GSH)-activatable disulfide linker and encapsulated DCM-S-GEM into an amphiphilic polymer DSPE-mPEG by self-assembly. The nanomized DCM-S-GEM/PEG prodrug exhibits excellent photostability and high biocompatibility, significantly improving the therapeutic efficacy toward lung tumor cells with fewer side-effects toward normal cells. Furthermore, when compared with the standalone DCM-S-GEM prodrug, the micellization with diblock DSPE-mPEG avoids fast metabolism, facilitates the accumulation of drugs in lung tumor tissues, displays longer tumor retention, and realizes precise drug release in lung tumors. The nanomized DCM-S-GEM/PEG prodrug can be developed as a promising tool to monitor prodrug delivery and activation processes in vivo.

Near‐Infrared Fluorescent Theranostic Cisplatin Prodrug with Transcatheter Intra‐Arterial Therapy: Application to Rabbit Hepatocellular Carcinoma

AbstractTranscatheter intra‐arterial therapy (TIT) has become valuable in the battle against primary and secondary hepatic malignancies. However, the lack of a mechanism to visualize real‐time drug release and avoid fast metabolic clearance of chemotherapeutic agents is the primary barrier to TIT for hepatocellular carcinoma. Here, a specific near‐infrared (NIR) fluorescent prodrug platform, that is, DSPE‐mPEG/DCM‐S‐Pt micelles (DCM‐S‐Pt@PEG), to assist TIT in direct administration to large mammals like rabbits, is presented. DCM‐S‐Pt@PEG consists of a NIR fluorophore for tracing drug release, a nonspecific antitumor drug cisplatin for hepatocellular carcinoma treatment, a glutathione‐activatable disulfide linker, and a DSPE‐mPEG nano‐micelle carrier for controllable drug release. DCM‐S‐Pt@PEG can make the drug accumulation in tumor tissues enhance the therapeutic effect by: i) specific delivery of prodrug to hepatic tumor tissues through the femoral artery by TIT, ii) sustained drug‐release from the biodegradable lipid DSPE‐mPEG micelle to minimize metabolic clearance, and iii) cancer biomarker–activated drug release. It provides a promising strategy to assist TIT in treating unresectable devastating hepatocellular carcinoma administration in the rabbit model, rather than common mouse model.

Fluorescent contrast agents for tumor surgery.

Cancer is a leading cause of cases of mortality worldwide. The most effective method to cure solid tumors is surgery. Every year, >50% of cancer patients receive surgery to remove solid tumors. Surgery may increase the cure rate of most solid tumors by 4–11 fold. Surgery has many challenges, including identifying small lesions, locating metastases and confirming complete tumor removal. Fluorescence guidance describes a new approach to improve surgical accuracy. Near-infrared fluorescence imaging allows for real-time early diagnosis and intraoperative imaging of lesion tissue. The results of previous preclinical studies in the field of near-infrared fluorescence imaging are promising. This review provides examples introducing the three kinds of fluorescent dyes: The passive fluorescent dye indocyanine green, which has been approved by the Food and Drug Administration for clinical use in the USA, the fluorescent prodrug 5-aminolevulinic acid, a porphyrin precursor in the heme synthesis, and biomarker-targeted fluorescent dyes, which allow conjugation to different target sites.

Co-assembling FRET nanomedicine with self-indicating drug release

Two lipophilic fluorescent prodrugs co-assembled into nanoaggregates with the ability to release FRET-indicated drugs, which was used to investigate the role of reduction-responsive linkers on drug release kinetics in the cytoplasm and physiologically relevant media in a visualized, noninvasive manner.

Chemical Modulation of in Vivo Macrophage Function with Subpopulation-Specific Fluorescent Prodrug Conjugates.

Immunomodulatory agents represent one of the most promising strategies for enhancing tissue regeneration without the side effects of traditional drug-based therapies. Tissue repair depends largely on macrophages, making them ideal targets for proregenerative therapies. However, given the multiple roles of macrophages in tissue homeostasis, small molecule drugs must be only active in very specific subpopulations. In this work, we have developed the first prodrug–fluorophore conjugates able to discriminate closely related subpopulations of macrophages (i.e., proinflammatory M1 vs anti-inflammatory M2 macrophages), and employed them to deplete M1 macrophages in vivo without affecting other cell populations. Selective intracellular activation and drug release enabled simultaneous fluorescence cell tracking and ablation of M1 macrophages in vivo, with the concomitant rescue of a proregenerative phenotype. Ex vivo assays in human monocyte-derived macrophages validate the translational potential of this novel platform to develop chemical immunomodulatory agents as targeted therapies for immune-related diseases.

GSH-Activated NIR Fluorescent Prodrug for Podophyllotoxin Delivery.

Theranostic prodrug therapy enables the targeted delivery of anticancer drugs with minimized adverse effects and real-time in situ monitoring of activation of the prodrugs. In this work, we report the synthesis and biological assessment of the near-infrared (NIR) prodrug DCM-S-PPT and its amphiphilic copolymer (mPEG-DSPE)-encapsulated nanoparticles. DCM-S-PPT is composed of podophyllotoxin (PPT) as the anticancer moiety and a dicyanomethylene-4H-pyran (DCM) derivative as the NIR fluorescent reporter, which are linked by a thiol-specific cleavable disulfide bond. In vitro experiments indicated that DCM-S-PPT has low cytotoxicity and that glutathione (GSH) can activate DCM-S-PPT resulting in PPT release and a concomitant significant enhancement in NIR fluorescence at 665 nm. After being intravenously injected into tumor-bearing nude mice, DCM-S-PPT exhibited excellent tumor-activated performance. Furthermore, we have demonstrated that mPEG-DSPE as a nanocarrier loaded with DCM-S-PPT (mPEG-DSPE/DCM-S-PPT) showed even greater tumor-targeting performance than DCM-S-PPT on account of the enhanced permeability and retention effect. Its tumor-targeting ability and specific drug release in tumors make DCM-S-PPT a promising prodrug that could provide a significant strategy for theranostic drug delivery systems.

A dual fluorescent prodrug for in vivo tumor imaging and therapy

A dual fluorescent prodrug for in vivo tumor imaging and therapy Minghzou Ye, Xiaohang Wang, Jianbin Tang, Zhiqian Guo, He Tian, Weihong Zhu, Youqing Shen, Key Laboratory of Biomass Chemical Engineering of Ministry of Education and Center for Bionanoengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, Zhejiang, China., Institute of Fine Chemicals, East China University of Science and Technology, Shanghai, P.R. China ⁎Corresponding authors. E-mail addresses: yemzh@163.com (M. Ye), jianbin@zju.edu.cn (J. Tang)

Mesoporous silica nanoparticle-based cisplatin prodrug delivery and anticancer effect under reductive cellular environment.

This work demonstrates the judicious application of a prodrug delivery strategy to achieve a highly improved anticancer drug effect of cisplatin using mesoporous silica nanoparticles. Our effort primarily addressed several pressing needs to overcome various impediments such as toxicity concerns, rapid inactivation, and low drug efficiency of cisplatin prodrug. The developed delivery system utilizes fluorescent mesoporous silica nanoparticles as a template to host the cisplatin prodrug through a reducible linkage. The inactive oxidized Pt(iv) complex installed on the surface of the mesoporous silica nanoparticles in the prodrug conferred stability to cisplatin; however, in the reductive environment of cancer cell lines the active cisplatin form was regenerated. Prodrug-conjugated nanoparticles showed 63 times lower IC50 value than that of cisplatin in HeLa cell line. The delivery system not only demonstrates enhanced cellular uptake but also shows a high drug effect which should diminish the associated side effects. Furthermore, a new, easy and inexpensive fluorescent based Pt quantification method has been adopted instead of the commonly used ICP-based quantification method and this strategy of quantification could be elaborated to monitor fluorescent prodrug nanoparticles during real-time diagnosis.

Abstract 1682: Assessment of pharmacokinetics and pharmacodynamics correlation of a novel fluorescent anticancer prodrug

Abstract Prodrug therapies provide high promise in improving current cancer treatments. A novel anticancer prodrug 6-chloro-9-nitro-5-oxo-5H-benzo[a]phenoxazine (CNOB) and a bacterial nitroreductase (ChrR6) that can efficiently activate reductive prodrugs have recently been discovered by our group. This CNOB/ChrR6 regimen is a superior anticancer treatment compared to the prodrugs currently in clinical use (Mitomycin C) or in clinical trials. Both CNOB and its activated compound 9-amino-6-chloro-5H-benzo[a]phenoxazine-5-one (MCHB) are fluorescent with distinctive emission wavelengths at 550 nm and 625 nm, respectively. This feature provides a unique opportunity to evaluate prodrug activation and pharmacokinetics (PK). Moreover, it likely constitutes a reliable tool to predict pharmacodymamic (PD) outcomes based on PK evaluations. Our objectives are to appraise the correlation between the PK and PD parameters, especially based on the quantitative fluorescence measurements, which will provide an innovative approach for drug development and therapeutic monitoring. Murine mammary cancer (4T1) cells expressing luciferase gene (4T1-luc) and 4T1-luc transfected with humanized ChrR6 (4T1-luc/hChrR6) were utilized. In vitro cell viability from CNOB (15 μM) treatment was determined by both the MTS and SRB assays. Fluorescence of CNOB and MCHB in cell culture medium was measured using a Tecan plate reader. Both types of cells were implanted in the mammary fat pad of nude mice to generate tumor xenografts. CNOB was administered intravenously (3.3 mg/kg), and fluorescence images were taken at various times (1, 2, 4, 6, 12 and 24 h) via IVIS® Spectrum, followed by sacrifice for tissue drug analysis using high performance liquid chromatography (HPLC). Correlation and statistical analysis were performed via GraphPad Prism®. In vitro results demonstrate significant (p < 0.01) time-dependent responses both in MCHB fluorescence generation and cell viability reduction. MCHB fluorescence (PK) is highly correlated (ρ = 0.96) with the reduction of cell viability (PD) only in cells expressing HChrR6. We found a direct correlation between tumor MCHB levels determined via fluorescence imaging and HPLC analysis from in vivo studies. The imaging results show significantly higher (p < 0.05) MCHB fluorescence levels in 4T1-luc/hChrR6 tumors as compared to 4T1-luc tumors, with calculated MCHB area under the concentration curve (AUC) of 582.0 ± 246.2 and 50.7 ± 23.7 μg·h/L (n = 5), respectively. We are currently assessing other PD parameters, such as cell apoptosis and/or tumor regression, as well as more in depth PK analysis in order to establish the PK-PD correlation in vivo. In conclusion, the unique fluorescent prodrug described here provides a reliable marker for PK determination of the therapeutic compound (MCHB) in tumors, which can be further utilized for the prediction of cancer treatment outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1682.