Modulating Early Stage Amyloid Aggregates by Dipeptide-Linked Perylenebisimides: Structure-Activity Relationship, Inhibition of Fibril Formation in Human CSF and Aß1-40.

Amyloid aggregation is observed in many neurodegenerative diseases, but the formation of final plaque seldom correlates to the disease severity. Early and intermediate structures such as soluble oligomers are considered as primary toxic species in protein misfolding diseases specifically linked to Aß in Alzheimer's disease (AD). Two peptide-linked perylenebisimide isomers (PAPAP and APPPA) were developed to study the structure-activity relationship with a toxic Aß oligomer in commercial Aß as well as in human cerebrospinal fluid (CSF), diminish and inhibit them, and prevent them from forming toxic amyloid fibrils from an early stage. Self-aggregation of perylenebisimides enables the formation of nano/micro-objects that are used to interact with the hydrophobic regions of the peptide and direct the peptide aggregation into an "off-pathway", preventing mature fibril formation. Remarkably, one of the Ala-Phe dipeptide-linked perylenebisimide isomers (APPPA) showed a high selectivity toward an Aß oligomer and could also cross the endothelial monolayer barrier (blood-brain barrier, BBB) more efficiently than the other derivative (PAPAP). Kinetic ThT studies and AFM imaging provided strong proof of both of the isomers being able to inhibit fibrillation of prefibrillar and oligomeric Aß in both the commercial Aß1-40 peptide as well as in the real human CSF sample. Further, a correlation has been built using pristine fluorescence of perylenebisimides, showing modulation and "oligo-blocking". The obtained data provides clear evidence that the mutual aggregation between the modulator and amyloid aggregate becomes predominant compared to their individual aggregation. These results reinforce the development of the structural platform design to diminish toxic oligomers, inhibit them, and prevent the formation of toxic amyloid fibrils at an early stage.

Multifunctional (3-in-1) cancer theranostics applications of hydroxyquinoline-appended polyfluorene nanoparticles.

The accumulation of fluorescent hydroxyquinoline-affixed polyfluorene (PF-HQ) nanoparticles and their utility for multi-color bio-imaging and drug delivery for cancer treatment are reported. The formation of nanoparticles (PF-HQ) containing hydrophobic pockets via three-dimensional growth of a polymeric backbone in a higher water fraction (THF : H2O = 1 : 9) was observed. The nanoparticles showed incredible dual-state optical and fluorescence properties, which were further explored in multi-color cell imaging in both cancer and normal cells. The cell viability assay in various normal cells confirmed the biocompatible nature of PF-HQ, which was further supported by an ex vivo (chick chorioallantoic membrane assay) model. This encouraged us to fabricate PF-HQ-based new drug delivery systems (DDS: PF-HQ-DOX) upon conjugation with the FDA-approved anti-cancer drug doxorubicin (DOX) by filling the hydrophobic pockets of the polymer nanoparticles. The enhanced anti-cancer activity of the DDS (PF-HQ-DOX) compared with that of free DOX was observed in mouse melanoma cancer cells (B16F10) and a subcutaneous mouse (C57BL6/J) melanoma tumor model upon administration of PF-HQ-DOX. Ex vivo biodistribution studies using a fluorescence quantification method demonstrated the enhanced accumulation of DOX in tumor tissues in the PF-HQ-DOX-treated group compared to that of the free drug, signifying the drug delivery efficacy of the delivery system by a passive targeting manner. Based on the above biological data (in vitro and in the pre-clinical model), these robust and versatile fluorescent hydroxyquinoline-affixed polyfluorene (PF-HQ) nanoparticles could be effectively utilized for multifunctional biomedical applications (as they are biocompatible and can be used for bio-imaging and as a drug delivery vehicle).

An efficient strategy to assemble water soluble histidine-perylene diimide and graphene oxide for the detection of PPi in physiological conditions and in vitro.

A strategy to develop water soluble, biocompatible nanocomposite probe for the detection of pyrophosphate (PPi) in physiological conditions and in in vitro live melanoma cancer cells (B16F10) is reported. The self-assembled nanocomposite probe comprised of amino acid (histidine) functionalized perylenediimide (PDI-HIS), copper ion and graphene oxide (GO) and that could be utilized as a highly effective sensing platform in biological conditions and cellular environment via fluorescence "turn-on" for PPi detection. This controlled fabrication of metal organic self-assembled spheres along with GO proved very valuable for the detection of PPi in unprecedented sensitivity over other competing ions. The PDI-HIS-Cu-GO (PCG) nanocomposite sensor provides a unique platform for the fluorogenic detection of PPi having a very low limit of detection (LOD) of 0.60×10-7M based on the strong affinity (1.0×106M-1) between the copper complex of PDI-HIS receptor and PPi. The intracellular detection of PPi using PCG also carried out in B16F10 cells where >10 times observed as compared to the PDI-HIS+Cu2+ complex. Thus early cancer detection via PPi recognition in physiological conditions and in live cells was possible using PCG. Furthermore, the fabrication of PDI-HIS and PCG with PVA hydrogel films and on thin layer chromatography plates demonstrated the practical utility for the detection of PPi anions by "off-on" response rapidly in a label free manner.

Modulation of Amyloid Aggregates into Nontoxic Coaggregates by Hydroxyquinoline Appended Polyfluorene.

Inhibitory modulation toward de novo protein aggregation is likely to be a vital and promising therapeutic strategy for understanding the molecular etiology of amyloid related diseases such as Alzheimer's disease (AD). The building up of toxic oligomeric and fibrillar amyloid aggregates in the brain plays host to a downstream of events, causing damage to axons, dendrites, synapses, signaling, transmission, and finally cell death. Herein, we introduce a novel conjugated polymer (CP), hydroxyquinoline appended polyfluorene (PF-HQ), which has a typical "amyloid like" surface motif and exhibits inhibitory modulation effect on amyloid ß (Aß) aggregation. We delineate inhibitory effects of PF-HQ based on Thioflavin T (ThT) fluorescence, atomic force microscopy (AFM), circular dichroism (CD), and Fourier transform infrared (FTIR) studies. The amyloid-like PF-HQ forms nano coaggregates by templating with toxic amyloid intermediates and displays improved inhibitory impacts toward Aß fibrillation and diminishes amyloid cytotoxicity. We have developed a CP based modulation strategy for the first time, which demonstrates beneficiary amyloid-like surface motif to interact efficiently with the protein, the pendant side groups to trap the toxic amyloid intermediates as well as optical signal to acquire the mechanistic insight.

Aggregation induced emission enhancement and growth of naphthalimide nanoribbons via J-aggregation: insight into disaggregation induced unfolding and detection of ferritin at the nanomolar level.

A series of novel V-shaped naphthalimide derivatives are reported herein, designed through a strategy to achieve aggregation induced emission (AIE), giving rise to unexpected self-assembly properties. This includes an aggregation induced emission enhancement (AIEE) active small organic molecule viz. naphthalimide derivative functionalized with 8-hydroxyquinoline (α-NQ), that spontaneously forms highly fluorescent "nanoribbon" like structures with <100 nm diameter and hundreds of micron length in aqueous media irrespective of concentration and surface at room temperature. The classical head-to-tail π-π stacking, as revealed by the single crystal X-ray study, was found to be the main driving force for the J-type aggregation with unique self-aggregating behavior. A comparative mechanistic study confirms that the N-atom in the hydroxyquinoline moiety is responsible for directing the entire molecular behavior in solution as well as in its aggregated state. These α-NQ nanoribbons formed in aqueous media were found to be highly sensitive and selective towards the multi-functional nonheme protein ferritin (Ksv = 0.83 × 107 M-1) with a limit of detection 67.25 × 10-11 M (0.33 ng µL-1) under physiological conditions, which serves as a well-known inflammatory marker for various diseases. The fluorescent nanoribbons were also found to modify the α-helix content of ferritin, thereby inducing conformational changes in their secondary structure as confirmed through circular dichroism (CD) spectroscopy techniques. Collectively, these findings improve the fundamental understanding of the self-assembly of AIEE active molecules along with the photophysical properties of core substituted naphthalimide derivatives that report the highest sensitivity towards ferritin in the presence of a bright AIEEgen under physiological conditions.

Modulation of Amyloid-ß Fibrils into Mature Microrod-Shaped Structure by Histidine Functionalized Water-Soluble Perylene Diimide.

Alzheimer's disease (AD) is associated with different types of amyloid peptide aggregates including senile plaques, fibrils, protofibrils, and oligomers. Due to these difficulties, a powerful strategy is needed for the disaggregation of amyloid aggregates by modulating their self-aggregation behavior. Herein, we report a unique approach toward transforming the aggregated amyloidogenic peptides using an amino acid functionalized perylene diimide as a molecular modulator, which is a different nondestructive approach as compared to inhibiting the aggregation of peptides. The histidine functionalized perylenediimide (PDI-HIS) molecule could coassemble with amyloid ß (Aß) peptides via hydrogen bonding that leads to the enhancement in the π-π interactions between Aß and PDI-HIS moieties. The Thioflavin T (ThT) assay and various spectroscopic and microscopic techniques establish that the PDI-HIS molecules accelerate the Aß1-40 and the amyloid aggregates in CSF into micro size coassembled structures. These results give rise to a new and unique complementary approach for modulating the biological effects of the aggregates in amyloidogenic peptides.