Unambiguous Detection of Elevated Levels of Hypochlorous Acid in Double Transgenic AD Mouse Brain

Alzheimer’s disease (AD) is one of the most prevalent forms of dementia. The current diagnosis methods based on the behavior and cognitive decline or imaging of core biomarkers, namely, amyloid-β (Aβ) plaques and neurofibrillary tangles (NFTs), in the brain offer poor to moderate success. Detection and imaging of biomarkers that cause additional traits of pathophysiological aberrations in the brain are invaluable to monitor early disease onset and progression of AD pathology. The pathological hallmark of AD is associated with generation of excessive reactive oxygen species (ROS) in the brain, which aggravate oxidative stress and inflammation. ROS production involves elevated levels of hypochlorous acid (HOCl) and can serve as one of the potential biomarkers for the diagnosis of AD. We report the design, synthesis, and characterization of switchable coumarin-morpholine (CM) conjugates as off-on fluorescence probes for the specific detection of HOCl produced and proximally localized with amyloid plaques. The nonfluorescent thioamide probe CM2 undergoes regioselective transformation to fluorescent amide probe CM1 in the presence of HOCl (∼90-fold fluorescence enhancement and 0.32 quantum yield) with high selectivity and sensitivity (detection limit: 0.17 μM). The excellent cellular uptake and blood-brain barrier (BBB) crossing ability of CM2 allowed unambiguous and differential detection, imaging, and quantification of HOCl in cellular milieu and in the wild type (WT) and AD mouse brains. This study demonstrates the elevated level of HOCl in the AD mouse brain and the potential to expand the repertoire of biomarkers for the diagnosis of AD

Unambiguous Detection of Elevated Levels of Hypochlorous Acid in Double Transgenic AD Mouse Brain

Alzheimer’s disease (AD) is one of the most prevalent forms of dementia. The current diagnosis methods based on the behavior and cognitive decline or imaging of core biomarkers, namely, amyloid-β (Aβ) plaques and neurofibrillary tangles (NFTs), in the brain offer poor to moderate success. Detection and imaging of biomarkers that cause additional traits of pathophysiological aberrations in the brain are invaluable to monitor early disease onset and progression of AD pathology. The pathological hallmark of AD is associated with generation of excessive reactive oxygen species (ROS) in the brain, which aggravate oxidative stress and inflammation. ROS production involves elevated levels of hypochlorous acid (HOCl) and can serve as one of the potential biomarkers for the diagnosis of AD. We report the design, synthesis, and characterization of switchable coumarin-morpholine (CM) conjugates as off-on fluorescence probes for the specific detection of HOCl produced and proximally localized with amyloid plaques. The nonfluorescent thioamide probe CM2 undergoes regioselective transformation to fluorescent amide probe CM1 in the presence of HOCl (∼90-fold fluorescence enhancement and 0.32 quantum yield) with high selectivity and sensitivity (detection limit: 0.17 μM). The excellent cellular uptake and blood-brain barrier (BBB) crossing ability of CM2 allowed unambiguous and differential detection, imaging, and quantification of HOCl in cellular milieu and in the wild type (WT) and AD mouse brains. This study demonstrates the elevated level of HOCl in the AD mouse brain and the potential to expand the repertoire of biomarkers for the diagnosis of AD

Cyclic Dipeptide-Guided Aggregation-Induced Emission of Naphthalimide and Its Application for the Detection of Phenolic Drugs

The development of novel aggregation-induced emission-based fluorophoric systems (AIEgens) has gained prominent importance in recent years, owing to their wide range of applications. Herein, we demonstrate the design, syntheses, and molecular architectonics of cyclic dipeptide tethered naphthalimides (CDP-NIs) to evaluate their AIEgenic properties and applications. CDPs are versatile molecular auxiliaries that form robust intermolecular hydrogen bonding and are tethered to naphthalic anhydride with the ACQ (aggregation-caused quenching) feature. The introduction of a CDP auxiliary was anticipated to promote the molecular assembly of the resulting naphthalimide product to form AIE-active aggregates through intermolecular hydrogen bonding in aqueous media. The systematic photophysical studies of CDP-naphthalimide (CDP-NI) conjugates led to the identification of two AIEgenic fluorophores. The AIEgenic property of the lead candidate 4a was employed for the detection of phenolic drugs in aqueous media. In particular, modulation of the AIEgenic property of 4a offered the sensitive detection of drugs such as doxorubicin and rifampicin (LOD = 18 nM/9.7 ppb and 202 nM/164 ppb, respectively)

Glucose-Responsive Self-Regulated Injectable Silk Fibroin Hydrogel for Controlled Insulin Delivery

Stimuli-responsive drug delivery systems are gaining importance in personalized medicine to deliver therapeutic doses in response to disease-specific stimulation. Pancreas-mimicking glucose-responsive insulin delivery systems offer improved therapeutic outcomes in the treatment of type 1 and advanced stage of type 2 diabetic conditions. Herein, we present a glucose-responsive smart hydrogel platform based on phenylboronic acid-functionalized natural silk fibroin protein for regulated insulin delivery. The modified protein was synergistically self-assembled and cross-linked through β-sheet and phenylboronate ester formation. The dynamic nature of the bonding confers smooth injectability through the needle. The cross-linked hydrogel structures firmly hold the glucose-sensing element and insulin in its pores and contribute to long-term sensing and drug storage. Under hyperglycemic conditions, the hydrogen peroxide generated from the sensing element induces hydrogel matrix degradation by oxidative cleavage, enabling insulin release. In vivo studies in a type 1 diabetic Wistar rat model revealed that the controlled insulin release from the hydrogel restored diabetic glucose level to physiological conditions for 36 h. This work establishes the functional modification of silk fibroin into a glucose-responsive hydrogel platform for regulated and functional insulin delivery application

Intrinsically Disordered Ku Protein-Derived Cell-Penetrating Peptides

Efficient delivery of bioactive ingredients into cells is a major challenge. Cell-penetrating peptides (CPPs) have emerged as promising vehicles for this purpose. We have developed novel CPPs derived from the flexible and disordered tail extensions of DNA-binding Ku proteins. Ku-P4, the lead CPP identified in this study, is biocompatible and displays high internalization efficacy. Biophysical studies show that the proline residue is crucial for preserving the intrinsically disordered state and biocompatibility. DNA binding studies showed effective DNA condensation to form a positively charged polyplex. The polyplex exhibited effective penetration through the cell membrane and delivered the plasmid DNA inside the cell. These novel CPPs have the potential to enhance the cellular uptake and therapeutic efficacy of peptide-drug or gene conjugates

Hybrid Multifunctional Modulators Inhibit Multifaceted Aβ Toxicity and Prevent Mitochondrial Damage

Amyloid beta (Aβ) aggregation is the key trait responsible for the pathological devastation caused by Alzheimer’s disease (AD). Among the various pathways of multifaceted toxicity exhibited by Aβ aggregates in neuronal cells, generation of reactive oxygen species (ROS) by Aβ-Cu^II complex and mitochondrial damage are prominent. Aβ interferes with mitochondrial transport channels, causing mitochondrial dysfunction. Herein, we present nontoxic hybrid multifunctional modulators (HMMs, TGR86–88) developed by integrating the structural and functional features of the metal chelating aggregation modulator, clioquinol (Clq), and the antioxidant epigallocatechin gallate (EGCG). Detailed biophysical and docking studies show that TGR86 interacts with Aβ and efficiently modulates both metal-dependent and metal-independent Aβ aggregation. TGR86 complexes with Cu^II, arrests its redox cycle, and thereby prevents the generation of ROS. The antioxidant nature of the HMMs effectively prevents DNA damage and protein oxidation. TGR86 rescued PC12 cells from Aβ-induced neurotoxicity by preventing the generation of ROS and foiling the interaction of toxic Aβ species with mitochondria, thereby averting its damage. These key attributes make TGR86 a potential candidate to develop therapeutics for the multifactorial Aβ toxicity in Alzheimer’s disease

Rationally Designed Molecules Synergistically Modulate Multifaceted Aβ Toxicity, Microglial Activation, and Neuroinflammation

Synergistic modulation of multifaceted toxicity is the key to tackle multifactorial Alzheimer’s disease (AD). The etiology of AD includes amyloid β (Aβ) amyloidosis, metal ion dyshomeostasis, reactive oxygen species (ROS), oxidative stress, mitochondrial damage, and neuroinflammation. We rationally designed multifunctional modulators by integrating pharmacophores for metal chelation, antioxidant and anti-inflammatory properties, and modulation of Aβ42 aggregation on the naphthalene monoimide (NMI) scaffold. The in vitro and cellular studies of NMIs revealed that M3 synergistically modulates metal-independent and -dependent amyloid toxicity, scavenges ROS, alleviates oxidative stress, and emulates Nrf2-mediated stress response in neuronal cells. M3 effectively reduced structural and functional damage of mitochondria, reduced Cyt c levels, and rescued cells from apoptosis. The biological atomic force microscopy and Western blot analysis revealed the ability of M3 to suppress microglial activation and neuroinflammation through inhibition of the NF-κβ pathway. The synergistic action of M3 is in agreement with our design strategy to develop a multifunctional therapeutic candidate by integrating multiple pharmacophores with distinct structural and functional elements to ameliorate the multifaceted toxicity of AD