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"Harnessing Intramolecular Rotation to Enhance Two-photon Imaging of Aβ Plaques Through Minimizing Background Fluorescence" Angew. Chem. Int. Ed., 5648-5652, 58, 2019.
  • The aggregation of amyloid beta (Aβ) proteins in senile plaques is a critical event during the development of Alzheimer\'s disease, and the postmortem detection of Aβ‐rich proteinaceous deposits through fluorescent staining remains one of the most robust diagnostic tools. In animal models, fluorescence imaging can be employed to follow the progression of the disease, and among the different imaging methods, two‐photon microscopy (TPM) has emerged as one of the most powerful. To date, several near‐infrared‐emissive two‐photon dyes with a high affinity for Aβ fibrils have been developed, but there has often been a tradeoff between excellent two‐photon cross‐sections and large fluorescence signal‐to‐background ratios. In the current work, we introduced a twisted intramolecular charge state (TICT)‐based de‐excitation pathway, which results in a remarkable fluorescence increase of around 167‐fold in the presence of Aβ fibrils, while maintaining an excellent two‐photon cross section, thereby enabling high‐contrast ex vivo and in vivo TPM imaging. Overall, the results suggest that adopting TICT de‐excitation in two‐photon fluorophores may represent a general method to overcome the tradeoff between probe brightness and signal‐to‐background ratio.
"Chemiluminescent Probe for the In Vitro and In Vivo Imaging of Cancers Over-expressing NQO1" Angew. Chem. Int. Ed. 1739-1743, 58, 2019.
  • Activatable (turn‐on) probes that permit the rapid, sensitive, selective, and accurate identification of cancer‐associated biomarkers can help drive advances in cancer research. Herein, a NAD(P)H:quinone oxidoreductase‐1 (NQO1)‐specific chemiluminescent probe 1 is reported that allows the differentiation between cancer subtypes. Probe 1 incorporates an NQO1‐specific trimethyl‐locked quinone trigger moiety covalently tethered to a phenoxy‐dioxetane moiety through a para‐aminobenzyl alcohol linker. Bio‐reduction of the quinone to the corresponding hydroquinone results in a chemiluminescent signal. As inferred from a combination of in vitro cell culture analyses and in vivo mice studies, the probe is safe, cell permeable, and capable of producing a “turn‐on” luminescence response in an NQO1‐positive A549 lung cancer model. On this basis, probe 1 can be used to identify cancerous cells and tissues characterized by elevated NQO1 levels.
"Overcoming Drug Resistance by Targeting Cancer Bioenergetics with an Activatable Prodrug" Chem (Cell Press), 2370-2383, 4, 2018.
  • Nearly without exception, all known cancer chemotherapeutics elicit a resistance response over time. The resulting resistance is correlated with poor clinical outcomes. Here, we report an approach to overcoming resistance through reprogramming oncogene-directed alterations in mitochondrial metabolism before drug activation while simultaneously circumventing drug efflux pumps. Conjugate C1 increases cancer cell apoptosis and inhibits regrowth of drugresistant tumors, as inferred from efficacy studies carried out in human cancer cells and in Dox-resistant xenograft tumor models. It also displays minimal whole-animal toxicity. These benefits are ascribed to an ability to evade chemoresistance by switching cancer cell metabolism back to normal mitochondrial oxidative phosphorylation while helping target the active Dox to first the mitochondrion and then the nucleus.
"Rational Design of In Vivo Tau Tangle-Selective Near Infrared Fluorophores: Expanding the BODIPY Universe" J. Am. Chem. Soc., 2017, 139 (38), pp 13393–13403
  • The elucidation of the cause of Alzheimer’s disease remains one of the greatest questions in neurodegenerative research. The lack of highly reliable low-cost sensors to study the structural changes in key proteins during the progression of the disease is a contributing factor to this lack of insight. In the current work, we describe the rational design and synthesis of two fluorescent BODIPY-based probes, named Tau 1 and Tau 2. The probes were evaluated on the molecular surface formed by a fibril of the PHF6 (306VQIVYK311) tau fragment using molecular docking studies to provide a potential molecular model to rationalize the selectivity of the new probes as compared to a homologous Aβ-selective probe. The probes were synthesized in a few steps from commercially available starting products and could thus prove to be highly costeffective. We demonstrated the excellent photophysical properties of the dyes, such as a large Stokes shift and emission in the near-infrared window of the electromagnetic spectrum. The probes demonstrated a high selectivity for self-assembled microtubule-associated protein tau (Tau protein), in both solution and cell-based experiments. Moreover, the administration to an acute murine model of tauopathy clearly revealed the staining of self-assembled hyperphosphorylated tau protein in pathologically relevant hippocampal brain regions. Tau 1 demonstrated efficient blood−brain barrier penetrability and demonstrated a clear selectivity for tau tangles over Aβ plaques, as well as the capacity for in vivo imaging in a transgenic mouse model. The current work could open up avenues for the cost-effective monitoring of the tau protein aggregation state in animal models as well as tissue staining. Furthermore, these fluorophores could serve as the basis for the development of clinically relevant sensors, for example based on PET imaging.
"A mitochondria-targeted cryptocyanine-based photothermogenic photosensitizer" J. Am. Chem. Soc., 2017, 139 (29), pp 9972–9978
  • Cryptocyanine-based probes exhibit highly efficient photothermal conversion and represent a new class of photothermal agents for use in photothermal therapy (PTT). With the thermal susceptibility of mitochondria in mind, we have prepared a mitochondriatargeted, NIR-absorbing cryptocyanine probe (Mito-CCy) and evaluated its photophysical properties, photothermal conversion effici ncy, biological compatibility, cytotoxicity, and mitochondrial localization in HeLa cells. Upon subjecting 0.5 mL of a PBS buffer solution (10 mM, pH 7.4, containing 50% DMSO) of Mito-CCy (0.5 mM) to 730 nm laser irradiation at 2.3 W/cm2 , the temperature of the solution increased by 13.5 °C within 5 min. In contrast, the corresponding cryptocyanine (CCy) lacking the triarylphosphonium group gave rise to only an ∼3.4 °C increase in solution temperature under otherwise identical conditions. Mito-CCy also exhibited high cytotoxicity in HeLa cells when subject to photoirradiation. This light-induced cytotoxicity is attributed to the endogenous production of reactive oxygen species (ROS) induced under conditions of local heating. ROS are known to interfere with the mitochondrial defense system and to trigger apoptosis. By targeting the mitochondria, the present sensitizer-based photothermogenic approach is rendered more effective. As such, the system reported here represents the vanguard of what might be a new generation of organelle-targeted photothermal therapeutics.
"Overcoming the Limits of Hypoxia in Photodynamic Therapy: A Carbonic Anhydrase IX-Targeted Approach" J. Am. Chem. Soc., 2017, 139 (22), pp 7595–7602
  • A major challenge in photodynamic cancer therapy (PDT) is avoiding PDT-induced hypoxia, which can lead to cancer recurrence and progression through activation of various angiogenic factors and significantly reduce treatment outcomes. Reported here is an acetazolamide (AZ)-conjugated BODIPY photosensitizer (AZBPS) designed to mitigate the effects of PDT-based hypoxia by combining the benefits of anti-angiogenesis therapy with PDT. AZBPS showed specific affinity to aggressive cancer cells (MDA-MB231 cells) that overexpress carbonic anhydrase IX (CAIX). It displayed enhanced photocytotoxicity compared to a reference compound, BPS, which is an analogous PDT agent that lacks an acetazolamide unit. AZ-BPS also displayed an enhanced in vivo efficacy in a xenograft mouse tumor regrowth model relative to BPS, an effect attributed to inhibition of tumor angiogenesis by both PDT-induced ROS generation and CAIX knockdown. AZ-BPS was evaluated successfully in clinical samples collected from breast cancer patients. We thus believe that the combined approach described here represents an attractive therapeutic approach to targeting CAIX-overexpressing tumors.
"Liposomal Texaphyrin Theranostics for Metastatic Liver Cancer" J. Am. Chem. Soc., 2016, 138 (50), pp 16380–16387
  • Reported here is a new theranostic agent, 1, which consists of a Gd3+-texaphyrin core conjugated to a doxorubicin prodrug via a disulfide bond. Conjugate 1 was designed to undergo cleavage in the presence of glutathione (GSH), a species typically upregulated in cancer cells. As prepared, conjugate 1 displays no appreciable fluorescence. However, when exposed to excess GSH an increase in the fluorescence intensity at 592 nm is observed that is ascribed to release of free doxorubicin. To improve the solubility and enhance the tumor targeting of 1, it was loaded into folate-receptor-targeted liposomes to produce FL-1 (for folate liposome loaded with 1). As inferred from both fluorescence turn on studies and independent HPLC analyses, FL-1 was found to undergo selective uptake and cleavage to release free Dox in the KB and CT26 cell lines, which express folate receptors on the cell surface, relative to the HepG2 and NIH3T3 cell lines, which show low expression of those receptors. FL-1 was found to produce a greater antiproliferative effect in the case of the KB and CT26 cell lines as compared to that in the HepG2 and NIH3T3 cell lines. FL-1 was also found to provide enhanced magnetic resonance imaging in vivo under conditions of T1 contrast in the early stage of metastatic cancer progression. Finally, time-dependent tumor regrowth studies involving both subcutaneous and metastatic liver cancer mouse models revealed that FL-1 is capable of reducing the tumor burden in vivo
"Disulfide-Based Multifunctional Conjugates for Targeted Theranostic Drug Delivery" Acc. Chem. Res., 2015, 48 (11), pp 2935–2946
  • Theranostics, chemical entities designed to combine therapeutic effects and imaging capability within one molecular system, have received considerable attention in recent years. Much of this interest reflects the promise inherent in personalized medicine, including disease-targeted treatments for cancer patients. One important approach to realizing this latter promise involves the development of so-called theranostic conjugates, multicomponent constructs that selectively target cancer cells and deliver cytotoxic agents while producing a readily detectable signal that can be monitored both in vitro and in vivo. This requires the synthesis of relatively complex systems comprising imaging reporters, masked chemotherapeutic drugs, cleavable linkers, and cancer targeting ligands. Ideally, the cleavage process should take place within or near cancer cells and be activated by cellular components that are associated with cancer states or specifically expressed at a higher level in cancer cells. Among the cleavable linkers currently being explored for the construction of such localizing conjugates, disulfide bonds are particularly attractive. This is because disulfide bonds are stable in most blood pools but are efficiently cleaved by cellular thiols, including glutathione (GSH) and thioredoxin (Trx), which are generally found at elevated levels in tumors. When disulfide bonds are linked to fluorophores, changes in emission intensity or shifts in the emission maxima are typically seen upon cleavage as the result of perturbations to internal charge transfer (ICT) processes. In well-designed systems, this allows for facile imaging. In this Account, we summarize our recent studies involving disulfide-based fluorescent drug delivery conjugates, including preliminary tests of their biological utility in vitro and in vivo. To date, a variety of chemotherapeutic agents, such as doxorubicin, gemcitabine, and camptothecin, have been used to create disulfide-based conjugates, as have a number of fluorophores, including naphthalimide, coumarin, BODIPY, rhodol, and Cy7. The resulting theranostic core (drug-disulfide-fluorophore) can be further linked to any of several site-localizing entities, including galactose, folate, biotin, and the RGD (Arg-Gly-Asp) peptide sequence, to create systems with an intrinsic selectivity for cancer cells over normal cells. Site-specific cleavage by endogenous thiols serves to release the cytotoxic drug and produce an easy-to-monitor change in the fluorescence signature of the cell. On the basis of the results summarized in this Account, we propose that disulfide-based cancer-targeting theranostics may have a role to play in advancing drug discovery efforts, as well as improving our understanding of cellular uptake and drug release mechanisms.
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