Multifunctional Liposomes Targeting Amyloid-ß Oligomers for Early Diagnosis and Therapy of Alzheimer's Disease.

Early detection and treatment are crucial for Alzheimer's disease (AD) management. Current diagnostic and therapeutic methods focus on late-stage amyloid fibrils and plaques, overlooking toxic soluble amyloid ß oligomers (AßOs) accumulating early in AD. A multifunctional liposome-based platform is designed for early diagnosis and therapy of AD, leveraging a novel self-assembled cyclic d,l-α-peptide (CP-2) that selectively targets AßOs. Biocompatible CP-2 conjugated liposomes (CP-2-LPs) effectively disrupt Aß aggregation and mitigate Aß-mediated toxicity in human neuroblastoma cells. In transgenic Caenorhabditis elegans AD models, CP-2-LPs significantly outperformed free CP-2 by improving cognitive and behavioral functions, extending lifespan, and reducing toxic AßO levels. Intravenous injection of fluorescently labeled CP-2-LPs reveals effective blood-brain barrier penetration, with significantly higher brain fluorescence in transgenic mice than WT, enabling precise diagnosis. These findings underscore CP-2-LPs as a valuable tool for early detection and targeted therapy in AD.

Noninvasive Treatment of Alzheimer's Disease with Scintillating Nanotubes.

Effective and accessible treatments for Alzheimer's disease (AD) are urgently needed. Soluble Aß oligomers are identified as neurotoxic species in AD and targeted in antibody-based drug development to mitigate cognitive decline. However, controversy exists concerning their efficacy and safety. In this study, an alternative strategy is proposed to inhibit the formation of Aß oligomers by selectively oxidizing specific amino acids in the Aß sequence, thereby preventing its aggregation. Targeted oxidation is achieved using biocompatible and blood-brain barrier-permeable multicomponent nanoscintillators that generate singlet oxygen upon X-ray interaction. Surface-modified scintillators interact selectively with Aß and, upon X-ray irradiation, inhibit the formation of neurotoxic aggregates both in vitro and in vivo. Feeding transgenic Caenorhabditis elegans expressing human Aß with the nanoscintillators and subsequent irradiation with soft X-ray reduces Aß oligomer levels, extends lifespan, and restores memory and behavioral deficits. These findings support the potential of X-ray-based therapy for AD and warrant further development.

Aza-Residue Modulation of Cyclic d,l-α-Peptide Nanotube Assembly with Enhanced Anti-Amyloidogenic Activity.

Transient soluble oligomers of amyloid-ß (Aß) are considered among the most toxic species in Alzheimer's disease (AD). Soluble Aß oligomers accumulate early prior to insoluble plaque formation and cognitive impairment. The cyclic d,l-α-peptide CP-2 (1) self-assembles into nanotubes and demonstrates promising anti-amyloidogenic activity likely by a mechanism involving engagement of soluble oligomers. Systematic replacement of the residues in peptide 1 with aza-amino acid counterparts was performed to explore the effects of hydrogen bonding on propensity to mitigate Aß aggregation and toxicity. Certain azapeptides exhibited improved ability to engage, alter the secondary structure, and inhibit aggregation of Aß. Moreover, certain azapeptides disassembled preformed Aß fibrils and protected cells from Aß-mediated toxicity. Substitution of the l-norleucine3 and d-serine6 residues in peptide 1 with aza-norleucine and aza-homoserine provided, respectively, nontoxic [azaNle3]-1 (4) and [azaHse6]-1 (7), that significantly abated symptoms in a transgenic Caenorhabditis elegans AD model by decreasing Aß oligomer levels.

Early diagnosis and treatment of Alzheimer's disease by targeting toxic soluble Aß oligomers.

Transient soluble oligomers of amyloid-ß (Aß) are toxic and accumulate early prior to insoluble plaque formation and cognitive impairment in Alzheimer's disease (AD). Synthetic cyclic D,L-α-peptides (e.g., 1) self-assemble into cross ß-sheet nanotubes, react with early Aß species (1-3 mers), and inhibit Aß aggregation and toxicity in stoichiometric concentrations, in vitro. Employing a semicarbazide as an aza-glycine residue with an extra hydrogen-bond donor to tune nanotube assembly and amyloid engagement, [azaGly6]-1 inhibited Aß aggregation and toxicity at substoichiometric concentrations. High-resolution NMR studies revealed dynamic interactions between [azaGly6]-1 and Aß42 residues F19 and F20, which are pivotal for early dimerization and aggregation. In an AD mouse model, brain positron emission tomography (PET) imaging using stable 64Cu-labeled (aza)peptide tracers gave unprecedented early amyloid detection in 44-d presymptomatic animals. No tracer accumulation was detected in the cortex and hippocampus of 44-d-old 5xFAD mice; instead, intense PET signal was observed in the thalamus, from where Aß oligomers may spread to other brain parts with disease progression. Compared with standard 11C-labeled Pittsburgh compound-B (11C-PIB), which binds specifically fibrillar Aß plaques, 64Cu-labeled (aza)peptide gave superior contrast and uptake in young mouse brain correlating with Aß oligomer levels. Effectively crossing the blood-brain barrier (BBB), peptide 1 and [azaGly6]-1 reduced Aß oligomer levels, prolonged lifespan of AD transgenic Caenorhabditis elegans, and abated memory and behavioral deficits in nematode and murine AD models. Cyclic (aza)peptides offer novel promise for early AD diagnosis and therapy.

Cu2+-Induced self-assembly and amyloid formation of a cyclic D,L-α-peptide: structure and function.

In a wide spectrum of neurodegenerative diseases, self-assembly of pathogenic proteins to cytotoxic intermediates is accelerated by the presence of metal ions such as Cu2+. Only low concentrations of these early transient oligomeric intermediates are present in a mixture of species during fibril formation, and hence information on the extent of structuring of these oligomers is still largely unknown. Here, we investigate dimers as the first intermediates in the Cu2+-driven aggregation of a cyclic D,L-α-peptide architecture. The unique structural and functional properties of this model system recapitulate the self-assembling properties of amyloidogenic proteins including ß-sheet conformation and cross-interaction with pathogenic amyloids. We show that a histidine-rich cyclic D,L-α-octapeptide binds Cu2+ with high affinity and selectivity to generate amyloid-like cross-ß-sheet structures. By taking advantage of backbone amide methylation to arrest the self-assembly at the dimeric stage, we obtain structural information and characterize the degree of local order for the dimer. We found that, while catalytic amounts of Cu2+ promote aggregation of the peptide to fibrillar structures, higher concentrations dose-dependently reduce fibrillization and lead to formation of spherical particles, showing self-assembly to different polymorphs. For the initial self-assembly step to the dimers, we found that Cu2+ is coordinated on average by two histidines, similar to self-assembled peptides, indicating that a similar binding interface is perpetuated during Cu2+-driven oligomerization. The dimer itself is found in heterogeneous conformations that undergo dynamic exchange, leading to the formation of different polymorphs at the initial stage of the aggregation process.

Glucose-Functionalized Liposomes for Reducing False Positives in Cancer Diagnosis.

Fluorodeoxyglucose-positron emission tomography (18F-FDG-PET) is a powerful tool for cancer detection, staging, and follow-up. However, 18F-FDG-PET imaging has high rates of false positives, as it cannot distinguish between tumor and inflammation regions that both feature increased glucose metabolic activity. In the present study, we engineered liposomes coated with glucose and the chelator dodecane tetraacetic acid (DOTA) complexed with copper, to serve as a diagnostic technology for differentiating between cancer and inflammation. This liposome technology is based on FDA-approved materials and enables complexation with metal cations and radionuclides. We found that these liposomes were preferentially uptaken by cancer cell lines with high metabolic activity, mediated via glucose transporter-1. In vivo, these liposomes were avidly uptaken by tumors, as compared to liposomes without glucose coating. Moreover, in a combined tumor-inflammation mouse model, these liposomes accumulated in the tumor tissue and not in the inflammation region. Thus, this technology shows high specificity for tumors while evading inflammation and has potential for rapid translation to the clinic and integration with existing PET imaging systems, for effective reduction of false positives in cancer diagnosis.

Bifunctional Carbon Dots-Magnetic and Fluorescent Hybrid Nanoparticles for Diagnostic Applications.

There is a huge demand for materials capable of simple detection or separation after conjugation with specific biologic substances when applied as a diagnostic tools. Taking into account the photoluminescence properties of C-dots and the highly magnetic properties of Fe(0), a new hybrid composite of these components was synthesized via ultrasound irradiation. The material was fully characterized by various physicochemical techniques. The main goal of the current study was to obtain a highly magnetic and intense fluorescent hybrid material. The goal was achieved. In addition, magnetic particles tended to agglomerate. The new hybrid can be suspended in ethanol, which is an additional feature of the current research. The dispersion of the hybrid nanoparticles in ethanol was achieved by utilizing the interaction of iron particles with C-dots which were decorated with functional groups on their surface. The newly formed hybrid material has potential applications in diagnostic by conjugating with specific antibodies or with any other biologic compounds. Such application may be useful in detection of various diseases such as: cancer, tuberculosis, etc.

Neuroprotective Effect of Nerve Growth Factor Loaded in Porous Silicon Nanostructures in an Alzheimer's Disease Model and Potential Delivery to the Brain.

Nerve growth factor (NGF) plays a vital role in reducing the loss of cholinergic neurons in Alzheimer's disease (AD). However, its delivery to the brain remains a challenge. Herein, NGF is loaded into degradable oxidized porous silicon (PSiO2 ) carriers, which are designed to carry and continuously release the protein over a 1 month period. The released NGF exhibits a substantial neuroprotective effect in differentiated rat pheochromocytoma PC12 cells against amyloid-beta (Aß)-induced cytotoxicity, which is associated with Alzheimer's disease. Next, two potential localized administration routes of the porous carriers into murine brain are investigated: implantation of PSiO2 chips above the dura mater, and biolistic bombardment of PSiO2 microparticles through an opening in the skull using a pneumatic gene gun. The PSiO2 -implanted mice are monitored for a period of 8 weeks and no inflammation or adverse effects are observed. Subsequently, a successful biolistic delivery of these highly porous microparticles into a live-mouse brain is demonstrated for the first time. The bombarded microparticles are observed to penetrate the brain and reach a depth of 150 µm. These results pave the way for using degradable PSiO2 carriers as potential localized delivery systems for NGF to the brain.

Photoactive chlorin e6 is a multifunctional modulator of amyloid-ß aggregation and toxicity via specific interactions with its histidine residues.

The self-assembly of Aß to ß-sheet-rich neurotoxic oligomers is a main pathological event leading to Alzheimer's disease (AD). Selective targeting of Aß oligomers without affecting other functional proteins is therefore an attractive approach to prevent the disease and its progression. In this study, we report that photodynamic treatment of Aß in the presence of catalytic amounts of chlorin e6 can selectively damage Aß and inhibit its aggregation and toxicity. Chlorin e6 also reversed the amyloid aggregation process in the dark by binding its soluble and low molecular weight oligomers, as shown by thioflavin T (ThT) fluorescence and photoinduced cross-linking of unmodified protein (PICUP) methods. Using HSQC NMR spectroscopy, ThT assays, amino acid analysis, SDS/PAGE, and EPR spectroscopy, we show that catalytic amounts of photoexcited chlorin e6 selectively damage the Aß histidine residues H6, H13, and H14, and induce Aß cross-linking by generating singlet oxygen. In contrast, photoexcited chlorin e6 was unable to cross-link ubiquitin and α-synuclein, demonstrating its high selectivity for Aß. By binding to the Aß histidine residues, catalytic amounts of chlorin e6 can also inhibit the Cu2+-induced aggregation and toxicity in darkness, while at stoichiometric amounts it acts as a chelator to reduce the amount of free Cu2+. This study demonstrates the great potential of chlorin e6 as a multifunctional agent for treatment of AD, and shows that the three N-terminal Aß histidine residues are a suitable target for Aß-specific drugs.

Distinct Effects of O-GlcNAcylation and Phosphorylation of a Tau-Derived Amyloid Peptide on Aggregation of the Native Peptide.

Protein phosphorylation and O-GlcNAcylation are very common nucleoplasmic post-translational modifications. Mono-addition of either the phosphate or the O-GlcNAc group were shown to inhibit the self-aggregation of amyloidogenic proteins and peptides, which is the hallmark of various protein misfolding diseases. However, their comparable effect upon co-incubation with a native non-modified amyloid scaffold has not been reported. O-linked glycans and phosphate variants of the tau protein-derived VQIVYK hexapeptide motif were generated as a simplified amyloid scaffold model and demonstrate that, while self-aggregation can be attenuated by either a single glycan or a phosphate unit, only co-incubation with the O-GlcNAc variant inhibits aggregation of the native peptide. These results shed light on the role of post-translational modifications in protein aggregation and suggest a novel therapeutic approach to protein misfolding diseases.