Polyprodrug nanomedicine for chemiexcitation-triggered self-augmented cancer chemotherapy and gas therapy.

Carbon monoxide (CO) has emerged as a potential antitumor agent by inducing the dysfunction of mitochondria and the apoptosis of cancer cells. However, it remains challenging to deliver appropriate amount of CO into tumor to ensure efficient tumor growth suppression with minimum side effects. Herein we developed a CO prodrug-loaded nanomedicine based on the self-assembly of camptothecin (CPT) polyprodrug amphiphiles. The polyprodrug nanoparticles readily dissociate upon exposure to endogenous H2O2 in the tumor, resulting in rapid release of CPT and generation of high-energy intermediate dioxetanedione. The latter can transfer the energy to neighboring CO prodrugs to activate CO production by chemiexcitation, while CPT promotes the generation of H2O2 in tumors, which in turn facilitates cascade CPT and CO release. As a result, the polyprodrug nanoparticles display remarkable tumor suppression in both subcutaneous and orthotopic breast tumor-bearing mice owing to the self-augmented CPT release and CO generation. In addition, no obvious systemic toxicity was observed in mice treated with the metal-free CO prodrug-loaded nanomedicine, suggesting the good biocompatibility of the polyprodrug nanoparticles. Our work provides new insights into the design and construction of polyprodrug nanomedicines for synergistic chemo/gas therapy.

Investigation of the Chemiluminescent Reaction of a Fluorinated Analog of Marine Coelenterazine.

Bioluminescence (BL) and chemiluminescence (CL) are remarkable processes in which light is emitted due to (bio)chemical reactions. These reactions have attracted significant attention for various applications, such as biosensing, bioimaging, and biomedicine. Some of the most relevant and well-studied BL/CL systems are that of marine imidazopyrazine-based compounds, among which Coelenterazine is a prime example. Understanding the mechanisms behind efficient chemiexcitation is essential for the optimization and development of practical applications for these systems. Here, the CL of a fluorinated Coelenterazine analog was studied using experimental and theoretical approaches to obtain insight into these processes. Experimental analysis revealed that CL is more efficient under basic conditions than under acidic ones, which could be attributed to the higher relative chemiexcitation efficiency of an anionic dioxetanone intermediate over a corresponding neutral species. However, theoretical calculations indicated that the reactions of both species are similarly associated with both electron and charge transfer processes, which are typically used to explain efficiency chemiexcitation. So, neither process appears to be able to explain the relative chemiexcitation efficiencies observed. In conclusion, this study provides further insight into the mechanisms behind the chemiexcitation of imidazopyrazinone-based systems.

The Role of Acetyl Zingerone and Its Derivatives in Inhibiting UV-Induced, Incident, and Delayed Cyclobutane Pyrimidine Dimers.

Cyclobutane pyrimidine dimers (CPDs) are ultraviolet radiation (UV)-induced carcinogenic DNA photoproducts that lead to UV signature mutations in melanoma. Previously, we discovered that, in addition to their incident formation (iCPDs), UV exposure induces melanin chemiexcitation (MeCh), where UV generates peroxynitrite (ONOO-), which oxidizes melanin into melanin-carbonyls (MCs) in their excited triplet state. Chronic MeCh and energy transfer by MCs to DNA generates CPDs for several hours after UV exposure ends (dark CPD, dCPDs). We hypothesized that MeCh and the resulting dCPDs can be inhibited using MeCh inhibitors, and MC and ONOO- scavengers. Here, we investigated the efficacy of Acetyl Zingerone (AZ), a plant-based phenolic alkanone, and its chemical analogs in inhibiting iCPDs and dCPDs in skin fibroblasts, keratinocytes, and isogenic pigmented and albino melanocytes. While AZ and its methoxy analog, 3-(4-Methoxy-benzyl)-Pentane-2,4-dione (MBPD) completely inhibited the dCPDs, MBPD also inhibited ~50% of iCPDs. This suggests the inhibition of ~80% of total CPDs at any time point post UV exposure by MBPD, which is markedly significant. MBPD downregulated melanin synthesis, which is indispensable for dCPD generation, but this did not occur with AZ. Meanwhile, AZ and MBPD both upregulated the expression of nucleotide excision repair (NER) pathways genes including Xpa, Xpc, and Mitf. AZ and its analogs were non-toxic to the skin cells and did not act as photosensitizers. We propose that AZ and MBPD represent "next-generation skin care additives" that are safe and effective for use not only in sunscreens but also in other specialized clinical applications owing to their extremely high efficacy in blocking both iCPDs and dCPDs.

Tuning the Intramolecular Chemiexcitation of Neutral Dioxetanones by Interaction with Ionic Species.

The intramolecular chemiexcitation of high-energy peroxide intermediates, such as dioxetanones, is an essential step in different chemi- and bioluminescent reactions. Here, we employed the Time-Dependent Density Functional Theory (TD-DFT) methodology to evaluate if and how external stimuli tune the intramolecular chemiexcitation of model dioxetanones. More specifically, we evaluated whether the strategic placement of ionic species near a neutral dioxetanone model could tune its thermolysis and chemiexcitation profile. We found that these ionic species allow for the "dark" catalysis of the thermolysis reaction by reducing the activation barrier to values low enough to be compatible with efficient chemi- and bioluminescent reactions. Furthermore, while the inclusion of these species negatively affected the chemiexcitation profile compared with neutral dioxetanones, these profiles appear to be at least as efficient as anionic dioxetanones. Thus, our results demonstrated that the intramolecular chemiexcitation of neutral dioxetanones can be tuned by external stimuli in such a way that their activation barriers are decreased. Thus, these results could help to reconcile findings that neutral dioxetanones could be responsible for efficient chemi-/bioluminescence, while being typically associated with high activation parameters.

Chemiexcitation-Triggered Prodrug Activation for Targeted Carbon Monoxide Delivery.

Photolysis-based prodrug strategy can address some critical drug delivery issues, which otherwise are very challenging to tackle with traditional prodrug strategy. However, the need for external light irradiation significantly hampers its in vivo application due to the poor light accessibility of deep tissue. Herein, we propose a new strategy of chemiexcitation-triggered prodrug activation, wherein a photoresponsive prodrug is excited for drug payload release by chemiexcitation instead of photoirradiation. As such, the bond-cleavage power of photolysis can be employed to address some critical drug delivery issues while obviating the need for external light irradiation. We have established the proof of concept by the successful development of a chemiexcitation responsive carbon monoxide delivery platform, which exhibited specific CO release at the tumor site and pronounced tumor suppression effects. We anticipate that such a concept of chemiexcitation-triggered prodrug activation can be leveraged for the targeted delivery of other small molecule-based drug payloads.

Triplet-Energy Quenching Functions of Antioxidant Molecules.

UV-like DNA damage is created in the dark by chemiexcitation, in which UV-activated enzymes generate reactive oxygen and nitrogen species that create a dioxetane on melanin. Thermal cleavage creates an electronically excited triplet-state carbonyl whose high energy transfers to DNA. Screening natural compounds for the ability to quench this energy identified polyenes, polyphenols, mycosporine-like amino acids, and related compounds better known as antioxidants. To eliminate false positives such as ROS and RNS scavengers, we then used the generator of triplet-state acetone, tetramethyl-1,2-dioxetane (TMD), to excite the triplet-energy reporter 9,10-dibromoanthracene-2-sulfonate (DBAS). Quenching measured as reduction in DBAS luminescence revealed three clusters of 50% inhibitory concentration, ~50 µM, 200-500 µM, and >600 µM, with the former including sorbate, ferulic acid, and resveratrol. Representative triplet-state quenchers prevented chemiexcitation-induced "dark" cyclobutane pyrimidine dimers (dCPD) in DNA and in UVA-irradiated melanocytes. We conclude that (i) the delocalized pi electron cloud that stabilizes the electron-donating activity of many common antioxidants allows the same molecule to prevent an electronically excited species from transferring its triplet-state energy to targets such as DNA and (ii) the most effective class of triplet-state quenchers appear to operate by energy diversion instead of electron donation and dissipate that energy by isomerization.

Target-Oriented Synthesis of Marine Coelenterazine Derivatives with Anticancer Activity by Applying the Heavy-Atom Effect.

Photodynamic therapy (PDT) is an anticancer therapeutic modality with remarkable advantages over more conventional approaches. However, PDT is greatly limited by its dependence on external light sources. Given this, PDT would benefit from new systems capable of a light-free and intracellular photodynamic effect. Herein, we evaluated the heavy-atom effect as a strategy to provide anticancer activity to derivatives of coelenterazine, a chemiluminescent single-molecule widespread in marine organisms. Our results indicate that the use of the heavy-atom effect allows these molecules to generate readily available triplet states in a chemiluminescent reaction triggered by a cancer marker. Cytotoxicity assays in different cancer cell lines showed a heavy-atom-dependent anticancer activity, which increased in the substituent order of hydroxyl < chlorine < bromine. Furthermore, it was found that the magnitude of this anticancer activity is also dependent on the tumor type, being more relevant toward breast and prostate cancer. The compounds also showed moderate activity toward neuroblastoma, while showing limited activity toward colon cancer. In conclusion, the present results indicate that the application of the heavy-atom effect to marine coelenterazine could be a promising approach for the future development of new and optimized self-activating and tumor-selective sensitizers for light-free PDT.

Role of Melanin Chemiexcitation in Melanoma Progression and Drug Resistance.

Melanoma is the deadliest type of skin cancer. Human melanomas often show hyperactivity of nitric oxide synthase (NOS) and NADPH oxidase (NOX), which, respectively, generate nitric oxide (NO · ) and superoxide (O2 ·- ). The NO · and O2 - react instantly with each other to generate peroxynitrite (ONOO-) which is the driver of melanin chemiexcitation. Melanoma precursors, the melanocytes, are specialized skin cells that synthesize melanin, a potent shield against sunlight's ultraviolet (UV) radiation. However, melanin chemiexcitation paradoxically demonstrates the melanomagenic properties of melanin. In a loop, the NOS activity regulates melanin synthesis, and melanin is utilized by the chemiexcitation pathway to generate carcinogenic melanin-carbonyls in an excited triplet state. These carbonyl compounds induce UV-specific DNA damage without UV. Additionally, the carbonyl compounds are highly reactive and can make melanomagenic adducts with proteins, DNA and other biomolecules. Here we review the role of the melanin chemiexcitation pathway in melanoma initiation, progression, and drug resistance. We conclude by hypothesizing a non-classical, positive loop in melanoma where melanin chemiexcitation generates carcinogenic reactive carbonyl species (RCS) and DNA damage in normal melanocytes. In parallel, NOS and NOX regulate melanin synthesis generating raw material for chemiexcitation, and the resulting RCS and reactive nitrogen species (RNS) regulate cellular proteome and transcriptome in favor of melanoma progression, metastasis, and resistance against targeted therapies.

Chemiexcitation and Its Implications for Disease.

Quantum mechanics rarely extends to molecular medicine. Recently, the pigment melanin was found to be susceptible to chemiexcitation, in which an electron is chemically excited to a high-energy molecular orbital. In invertebrates, chemiexcitation causes bioluminescence; in mammals, a higher-energy process involving melanin transfers energy to DNA without photons, creating the lethal and mutagenic cyclobutane pyrimidine dimer that can cause melanoma. This process is initiated by NO and O2- radicals, the formation of which can be triggered by ultraviolet light or inflammation. Several chronic diseases share two properties: inflammation generates these radicals across the tissue, and the diseased cells lie near melanin. We propose that chemiexcitation may be an upstream event in numerous human diseases.

Chemical excitation of electrons: A dark path to melanoma.

Sunlight's ultraviolet wavelengths induce cyclobutane pyrimidine dimers (CPDs), which then cause mutations that lead to melanoma or to cancers of skin keratinocytes. In pigmented melanocytes, we found that CPDs arise both instantaneously and for hours after UV exposure ends. Remarkably, the CPDs arising in the dark originate by a novel pathway that resembles bioluminescence but does not end in light: First, UV activates the enzymes nitric oxide synthase (NOS) and NADPH oxidase (NOX), which generate the radicals nitric oxide (NO) and superoxide (O2(-)); these combine to form the powerful oxidant peroxynitrite (ONOO(-)). A fragment of the skin pigment melanin is then oxidized, exciting an electron to an energy level so high that it is rarely seen in biology. This process of chemically exciting electrons, termed "chemiexcitation", is used by fireflies to generate light but it had never been seen in mammalian cells. In melanocytes, the energy transfers radiationlessly to DNA, inducing CPDs. Chemiexcitation is a new source of genome instability, and it calls attention to endogenous mechanisms of genome maintenance that prevent electronic excitation or dissipate the energy of excited states. Chemiexcitation may also trigger pathogenesis in internal tissues because the same chemistry should arise wherever superoxide and nitric oxide arise near cells that contain melanin.

UV-induced Melanin Chemiexcitation: A New Mode of Melanoma Pathogenesis.

Mutations in sunlight-induced melanoma arise from cyclobutane pyrimidine dimers (CPDs), DNA photoproducts usually created picoseconds after an ultraviolet (UV) photon is absorbed at thymine or cytosine. Surprisingly, we found that, in melanocytes, CPDs were generated for hours after UVA or UVB exposure. These "dark CPDs" constituted the majority of CPDs in cultured human and murine melanocytes and in mouse skin, and they were most prominent in skin containing pheomelanin, the melanin responsible for blonde and red hair. The mechanism was also a surprise. Dark cyclobutane pyrimidine dimers (CPDs) arise when ultraviolet (UV)-induced superoxide and nitric oxide combine to form peroxynitrite, one of the few biological molecules capable of exciting an electron. This process, termed "chemiexcitation," is the source of bioluminescence in lower organisms. Excitation occurred in fragments of melanin, creating a quantum triplet state that had the energy of a UV photon but which induced CPDs by radiationless energy transfer to DNA. UVA and peroxynitrite also solubilized melanin and permeabilized the nuclear membrane, allowing melanin to enter. Melanin is evidently carcinogenic as well as protective. Chemiexcitation may also trigger pathogenesis in internal tissues because the same chemistry should arise wherever superoxide and nitric oxide arise near cells that contain melanin.