Protein Aggregation Research Articles

Structural diversity of Alzheimer-related protein aggregations revealed using photothermal ratio-metric micro-spectroscopy

The crucial link between pathological protein aggregations and lipids in Alzheimer’s disease pathogenesis is increasingly recognized, yet its spatial dynamics remain challenging for labeling-based microscopy. Here, we demonstrate photothermal ratio-metric infrared spectro-microscopy (PRISM) to investigate the in situ structural and molecular compositions of pathological features in brain tissues at submicron resolution. By identifying the vibrational spectroscopic signatures of protein secondary structures and lipids, PRISM tracks the structural dynamics of pathological proteins, including amyloid and hyperphosphorylated Tau (pTau). Amyloid-associated lipid features in major brain regions were observed, notably the enrichment of lipid-dissociated plaques in the hippocampus. Spectroscopic profiling of pTau revealed significant heterogeneity in phosphorylation levels and a distinct lipid-pTau relationship that contrasts with the anticipated lipid-plaque correlation. Beyond in vitro studies, our findings provide direct visualization evidence of aggregate-lipid interactions across the brain, offering new insights into mechanistic and therapeutic research of neurodegenerative diseases.

New Insights into the Development of Donepezil-Based Hybrid and Natural Molecules as Multi-Target Drug Agents for Alzheimer’s Disease Treatment

Alzheimer’s disease (AD) involves a complex pathophysiology with multiple interconnected subpathologies, including protein aggregation, impaired neurotransmission, oxidative stress, and microglia-mediated neuroinflammation. Current treatments, which generally target a single subpathology, have failed to modify the disease’s progression, providing only temporary symptom relief. Multi-target drugs (MTDs) address several subpathologies, including impaired aggregation of pathological proteins. In this review, we cover hybrid molecules published between 2014 and 2024. We offer an overview of the strategies employed in drug design and approaches that have led to notable improvements and reduced hepatotoxicity. Our aim is to offer insights into the potential development of new Alzheimer’s disease drugs. This overview highlights the potential of multi-target drugs featuring heterocycles with N-benzylpiperidine fragments and natural compounds in improving Alzheimer’s disease treatment.

In situ editing of tumour cell membranes induces aggregation and capture of PD-L1 membrane proteins for enhanced cancer immunotherapy

Immune checkpoint blockade (ICB) therapy has emerged as a new therapeutic paradigm for a variety of advanced cancers, but wide clinical application is hindered by low response rate. Here we use a peptide-based, biomimetic, self-assembly strategy to generate a nanoparticle, TPM1, for binding PD-L1 on tumour cell surface. Upon binding with PD-L1, TPM1 transforms into fibrillar networks in situ to facilitate the aggregation of both bound and unbound PD-L1, thereby resulting in the blockade of the PD-1/PD-L1 pathway. Characterizations of TPM1 manifest a prolonged retention in tumour ( > 7 days) and anti-cancer effects associated with reinvigorating CD8+ T cells in multiple mice tumour models. Our results thus hint TPM1 as a potential strategy for enhancing the ICB efficacy.

Alternative production of pro-death Bax∆2 protein via ribosomal frameshift in Alzheimer’s disease

Pro-death Bax family member, Bax∆2, forms protein aggregates in Alzheimer’s disease neurons and causes stress granule-mediated neuronal death. Production of Bax∆2 originated from two events: alternative splicing of Bax exon 2 and a microsatellite mutation (a deletion from poly guanines, G8 to G7). Each event alone leads to a reading frameshift and premature termination. While Bax exon 2 alternative splicing is common in Alzheimer’s brains, the G7 mutation is not, which is inconsistent with the high Bax∆2 protein levels detected in clinical samples. Here, we report an alternative mechanism to produce Bax∆2 protein in the absence of the G7 mutation. Using dual-tag systems, we showed that a ribosomal frameshift (RFS) can compensate the lack of G7 mutation in translating Bax∆2 protein. Intriguingly, the microsatellite poly G repeat is neither essential nor the site for the RFS. However, disruption of the poly G sequence impaired the RFS, potentially due to alteration of the local RNA structure. Using immunoprecipitation-mass spectrometry, we pinpointed the RFS site at 15 base pairs upstream of the microsatellite. These results uncover an alternative mechanism for generating functional Bax∆2-subfamily isoforms, highlighting the production plasticity of Bax family isoforms and unveiling potential new therapeutic targets for Alzheimer’s disease.

SARS-CoV-2 and HSV-1 Induce Amyloid Aggregation in Human CSF Resulting in Drastic Soluble Protein Depletion.

The corona virus (SARS-CoV-2) pandemic and the resulting long-term neurological complications in patients, known as long COVID, have renewed interest in the correlation between viral infections and neurodegenerative brain disorders. While many viruses can reach the central nervous system (CNS) causing acute or chronic infections (such as herpes simplex virus 1, HSV-1), the lack of a clear mechanistic link between viruses and protein aggregation into amyloids, a characteristic of several neurodegenerative diseases, has rendered such a connection elusive. Recently, we showed that viruses can induce aggregation of purified amyloidogenic proteins via the direct physicochemical mechanism of heterogeneous nucleation (HEN). In the current study, we show that the incubation of HSV-1 and SARS-CoV-2 with human cerebrospinal fluid (CSF) leads to the amyloid aggregation of several proteins known to be involved in neurodegenerative diseases, such as APLP1 (amyloid β precursor like protein 1), ApoE, clusterin, α2-macroglobulin, PGK-1 (phosphoglycerate kinase 1), ceruloplasmin, nucleolin, 14-3-3, transthyretin, and vitronectin. Importantly, UV-inactivation of SARS-CoV-2 does not affect its ability to induce amyloid aggregation, as amyloid formation is dependent on viral surface catalysis via HEN and not its ability to replicate. Additionally, viral amyloid induction led to a dramatic drop in the soluble protein concentration in the CSF. Our results show that viruses can physically induce amyloid aggregation of proteins in human CSF and result in soluble protein depletion, thus providing a potential mechanism that may account for the association between persistent and latent/reactivating brain infections and neurodegenerative diseases.

Distinct Chemical Determinants are Essential for Achieving Ligands for Superior Optical Detection of Specific Amyloid-β Deposits in Alzheimer's Disease.

Aggregated forms of different proteins are common hallmarks for several neurodegenerative diseases, including Alzheimer's disease, and ligands that selectively detect specific protein aggregates are vital. Herein, we investigate the molecular requirements of thiophene-vinyl-benzothiazole based ligands to detect a specific type of Aβ deposits found in individuals with dominantly inherited Alzheimer's disease caused by the Arctic APP E693G mutation. The staining of these Aβ deposits was alternated when switching the terminal heterocyclic moiety attached to the thiophene-vinyl-benzothiazole scaffold. The most prevalent staining was observed for ligands having a terminal 3-methyl-1H-indazole moiety or a terminal 1,2-dimethoxybenzene moiety, verifying that specific molecular interactions between these ligands and the aggregates were necessary. The synthesis of additional thiophene-vinyl-benzothiazole ligands aided in pinpointing additional crucial chemical determinants, such as positioning of nitrogen atoms and methyl substituents, for achieving optimal staining of Aβ aggregates. When combining the optimized thiophene-vinyl-benzothiazole based ligands with a conventional ligand, CN-PiB, distinct staining patterns were observed for sporadic Alzheimer's disease versus dominantly inherited Alzheimer's disease caused by the Arctic APP E693G mutation. Our findings provide chemical insights for developing novel ligands that allow for a more precise assignment of Aβ deposits, and might also aid in creating novel agents for clinical imaging of distinct Aβ aggregates in AD.

Parametric influences on flow, heat transfer, and nutrient transport in the knee joint cavity, a numerical approach

A novel aspect of this study is the investigation of the impact of temperature-dependent Arrhenius reactions and metabolic heat generation on the overall heat transfer within a knee joint cavity. This is a unique contribution where the interaction between thermal effects and metabolic activity is shown to influence the transport of nutrients and the removal of waste products. For the first time, the study also aims to examine the influence of key non-dimensional numbers on the characteristics of mixed convection flow, providing new insights into the dominant physical mechanisms at play. An innovative feature of the model is the incorporation of a non-linear reaction term to address the balance between protein synthesis and degradation. By integrating both thermal and biochemical dynamics, this approach offers a comprehensive analysis of protein aggregation in the knee joint, with potential implications for understanding and treating joint diseases, particularly those related to the health of synovial fluid and cartilage, such as osteoarthritis. The findings have the potential to inform the development of improved therapeutic strategies for managing these conditions. In the context of knee joint cavity dynamics, a low Cauchy number Ca plays a crucial role in ensuring steady and laminar flow, which reduces inertia and ensures that the continuity equation is satisfied. This laminar nature of the flow is essential for maintaining stable synovial fluid movement within the cavity, which aids in the smooth transport of nutrients and waste products. Additionally, higher Peclet number Pe values indicate an increased dominance of convective transport over diffusion, thereby enhancing the supply of essential nutrients to cartilage and bone, while also facilitating the removal of waste products and inflammatory mediators. This efficient convective transport mechanism is vital for the overall health of the joint and for mitigating inflammation. Furthermore, the Zeldovich number β significantly influences the temperature sensitivity of the Arrhenius reaction term within the cavity, where higher values reflect increased reactivity to temperature changes. This temperature dependence impacts metabolic heat generation and protein dynamics, further influencing the biochemical processes that occur within the joint. Collectively, these non-dimensional parameters highlight key aspects of synovial fluid dynamics, nutrient transport, and biochemical reactions that are essential to the functioning and health of the knee joint.

Physicochemical Properties and Metal Ion-Binding Capacity of Thermal-Induced Antarctic Krill Protein Aggregates under Different pH Conditions.

Protein properties can be modified by thermal treatment at different pH values, resulting in the formation of protein aggregates with diverse morphologies and functionalities. This study investigated the morphology of aggregates of Antarctic krill protein (AKPS) formed by thermal treatment (90 °C, 15 min) at pH 2, 3, 4, and 6-12, characterized the different morphologies of AKPS, and determined the metal ion (Ca2+, Fe2+, and Zn2+)-binding capacities. Results showed that heat treatment with different pH values generated various AKPS with distinct morphology and metal ion-binding abilities. AKPS have formed fibrils at pH ≤ 3, particles at pH 4-7, and amorphous aggregates at pH ≥ 8. Fibrous AKPS (pH 2) exhibited a high solubility (80.36%) and strong reducing effect on iron. The binding capacities of Ca2+ and Fe2+ reached 36.08% and 66.75%. Particulate AKPS (pH 6 and 7) primarily only showed Zn2+-binding capacity similar to that of casein phosphopeptide (17.33%). Amorphous AKPS (pH 9-11) displayed the optimal capacity to bind Zn2+ and Fe2+ (26.90% and 74.94%). The alterations in morphology and functional characteristics of AKPS permit the design of various nanostructures for food-derived mineral supplements, thus developing their potential for application in functional foods.

Boron: An intriguing factor in retarding Alzheimer's progression

Alzheimer's disease (AD) is a neurodegenerative disorder that is the fifth most common cause of mortality worldwide and the second most common cause of death in developed countries. The etiology of AD remains poorly understood; however, it is correlated with the accumulation of proteins in the brain, ultimately leading to cellular damage. Multiple factors, including genetic and environmental factors such as chemicals or food, have been linked to protein aggregation and cell death in AD. Boron is a vital micronutrient that is necessary for plant growth and is abundantly present in various fruits and nuts. Prior research has emphasized the importance of boron as a neuroprotective agent and necessary component for the preservation of brain health and function. However, the precise function of boron in the brain remains poorly understood. This review elucidates the molecular role of boron in the brain by examining existing information about its impact on neurodegenerative diseases and may provide a deeper understanding of the etiology of AD and, ultimately, lead to the development of novel approaches for its treatment.

Redox regulation, protein S-nitrosylation, and synapse loss in Alzheimer's and related dementias.

Redox-mediated posttranslational modification, as exemplified by protein S-nitrosylation, modulates protein activity and function in both health and disease. Here, we review recent findings that show how normal aging, infection/inflammation, trauma, environmental toxins, and diseases associated with protein aggregation can each trigger excessive nitrosative stress, resulting in aberrant protein S-nitrosylation and hence dysfunctional protein networks. These redox reactions contribute to the etiology of multiple neurodegenerative disorders as well as systemic diseases. In the CNS, aberrant S-nitrosylation reactions of single proteins or, in many cases, interconnected networks of proteins lead to dysfunctional pathways affecting endoplasmic reticulum (ER) stress, inflammatory signaling, autophagy/mitophagy, the ubiquitin-proteasome system, transcriptional and enzymatic machinery, and mitochondrial metabolism. Aberrant protein S-nitrosylation and transnitrosylation (transfer of nitric oxide [NO]-related species from one protein to another) trigger protein aggregation, neuronal bioenergetic compromise, and microglial phagocytosis, all of which contribute to the synapse loss that underlies cognitive decline in Alzheimer's disease and related dementias.

Amyloid beta-induced signalling in leptomeningeal cells and its impact on astrocyte response.

The pathological signature of Alzheimer's disease (AD) includes the accumulation of toxic protein aggregates, mainly consisting of amyloid beta (Aβ). Recent strides in fundamental research underscore the pivotal role of waste clearance mechanisms in the brain suggesting it may be an early indication of early onset AD. This study delves into the involvement of leptomeningeal cells (LMCs), crucial components forming integral barriers within the clearance system, in the context of AD. We examined the inflammatory cytokine responses of LMCs in the presence of Aβ, alongside assessments of LMC growth response, viability, oxidative stress, and changes in vimentin expression. The LMCs showed no changes in growth, viability, oxidative stress, or vimentin expression in the presence of Aβ, indicating that LMCs are less susceptible to Aβ damage compared to other CNS cells. However, LMCs exhibited a unique pro-inflammatory response to Aβ when compared to an LPS inflammatory control, showing an mRNA expression of pro-inflammatory cytokines such IL-6, IL-10 and IL-33 but no changes in IL-1α and IL-1β. Furthermore, LMCs influenced the astrocyte response to Aβ, as conditioned media from Aβ-treated LMCs was observed to downregulate somatic S100β in astrocytes. We also investigated whether the JAK/STAT3 pathway was involved in the Aβ response of the LMCs, as this pathway has been shown to be activated in astrocytes and neurons in the presence of Aβ. JAK/STAT3 activation was assessed through phosphorylated STAT3, revealing that JAK/STAT3 was not active in the cells when in the presence of Aβ. However, when JAK1 and JAK2 were inhibited, cytokine protein levels of IL7, IL10, IL15 and IL33 levels, which had shown alteration when LMCs were treated with Aβ, returned to base levels. This indicates that although JAK1/STAT3 and JAK2/STAT3 are not the direct pathway for Aβ response in LMCs, JAK1 and JAK2 may still play a role in regulating cytokine levels, potentially through indirect means or crosstalk. Overall, our findings reveal that LMCs are resilient to Aβ toxicity and suggest that JAK1/STAT3 and JAK2/STAT3 does not play a central role in the inflammatory response, providing new insights into the cellular mechanisms underlying AD.

Starch and Protein Characteristics of Chestnut Flours and Their Applications in Gluten-Free Products.

In the context of valorizing underutilized crops, this study investigated the starch and protein characteristics of chestnut varieties (Balestrera and Rossera) and their potential for use at 15% and 25% in reformulating gluten-free baked snacks and fresh pasta. Chestnut varieties differ in protein and starch content (Balestrera > Rossera), as well as in amylose, dietary fiber, and lipids (Balestrera < Rossera). Differences in starch and amylose content affected starch gelatinization (Balestrera < Rossera), pasting and retrogradation properties, and water absorption capacity at 90 °C (Balestrera > Rossera). No differences in water and oil absorption capacity and mixing properties were observed, neither in the protein profile nor in the nature of protein aggregates. Both varieties exhibited a good aptitude for producing gluten-free baked snacks and fresh pasta, even at enrichment levels of 25%. Overall, this study could provide valuable information for the development of gluten-free products using sustainable, underutilized crops.

High-intensity ultrasound modification of techno-functional and structural properties of white finger millet protein fractions.

In this study, albumin, globulin, and glutelin were extracted from white finger millet, and their amino acid content, functional and structural properties were investigated. The protein concentration of albumin, globulin, and glutelin were 76.01%, 74.32%, and 69.55%, respectively. The results showed that all the fractions had a significant amount of essential amino acids. Aqueous protein dispersions (10%, w/v) were treated for 12min at different ultrasound power levels (100, 200, and 300W). The solubility, emulsifying, and foaming properties of albumin and glutelin were significantly (p<0.05) improved after ultrasound treatment (20kHz) which indicates that ultrasound could unfold protein aggregates. A decrease in particle size, increase in surface hydrophobicity, and zeta potential correlated with improved functional properties. Ultrasound treatment reduced the size of all proteins except for fractions at 300W and also sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a significant change in the molecular weight of albumin and glutelin at 300W. Scanning electron microscopy of treated protein fraction showed distinctive microstructure with irregular structure compared to untreated protein fraction. Although Fourier transform infrared spectroscopy spectra of proteins were similar after ultrasonication, a partial increase in the intensity of the Amide A band was observed. In conclusion, the ultrasound-treated protein fraction can be used as a high-value plant-based emulsifier.

Molecular mechanisms at the basis of the protective effect exerted by EPPS on neurodegeneration induced by prefibrillar amyloid oligomers

It has been shown recently, without an explanation of the possible molecular mechanisms involved, that 4-(2-hydroxyethyl)-1-piperazinepropanesulphonic (EPPS) acid effectively protects from the neurotoxicity induced by oligomers and plaques formed by the protein amyloid-β protein. Here we report the same protective effect, obtained in vitro (HT22-diff cell line) and ex vivo (hippocampal slices) models, against amyloid neurotoxicity induced by oligomers of salmon Calcitonin (sCT), which has been shown to be a good model for the study of neurodegenerative diseases. Based on biophysical studies focusing on the protein aggregation kinetic and the interaction of the aggregates with model membranes, we propose a possible molecular mechanism underlying the protective effects. Taken together, our results indicate that EPPS is able to counteract the direct association (primary aggregation) of harmless low-molecular weight aggregates (dimers and trimers) or their aggregation catalysed by surfaces present in the solution (secondary aggregation). Thus, EPPS stabilizes harmless aggregates and hinders the formation of toxic and metastable prefibrillar oligomers. Overall, our data demonstrate that EPPS is an excellent drug candidate for the treatment of neurodegeneration due to misfolded proteins, such as Alzheimer’s or Parkinson’s disease.

Decoding Nucleotide Repeat Expansion Diseases: Novel Insights from Drosophila melanogaster Studies.

Drosophila melanogaster usage has provided substantial insights into the pathogenesis of several nucleotide repeat expansion diseases (NREDs), a group of genetic diseases characterized by the abnormal expansion of DNA repeats. Leveraging the genetic simplicity and manipulability of Drosophila, researchers have successfully modeled close to 15 NREDs such as Huntington's disease (HD), several spinocerebellar ataxias (SCA), and myotonic dystrophies type 1 and 2 (DM1/DM2). These models have been instrumental in characterizing the principal associated molecular mechanisms: protein aggregation, RNA toxicity, and protein function loss, thus recapitulating key features of human disease. Used in chemical and genetic screenings, they also enable us to identify promising small molecules and genetic modifiers that mitigate the toxic effects of expanded repeats. This review summarizes the close to 150 studies performed in this area during the last seven years. The relevant highlights are the achievement of the first fly-based models for some NREDs, the incorporation of new technologies such as CRISPR for developing or evaluating transgenic flies containing repeat expanded motifs, and the evaluation of less understood toxic mechanisms in NREDs such as RAN translation. Overall, Drosophila melanogaster remains a powerful platform for research in NREDs.

Protein aggregation in plant mitochondria lacking Lon1 inhibits translation and induces unfolded protein responses.

Loss of Lon1 led to stunted plant growth and accumulation of nuclear-encoded mitochondrial proteins including Lon1 substrates. However, an in-depth label-free proteomics quantification of mitochondrial proteins in lon1 revealed that the majority of mitochondrial-encoded proteins decreased in abundance. Additionally, we found that lon1 mutants contained protein aggregates in the mitochondrial that were enriched in metabolic enzymes, ribosomal subunits and PPR-containing proteins of the translation apparatus. These mutants exhibited reduced general mitochondrial translation as well as deficiencies in RNA splicing and editing. These findings support the role of Lon1 in maintaining a functional translational apparatus for mitochondrial-encoded gene translation. Transcriptome analysis of lon1 revealed a mitochondrial unfolded protein response reminiscent of the mitochondrial retrograde signalling dependent on the transcription factor ANAC017. Notably, lon1 mutants exhibited transiently elevated ethylene production, and the shortened hypocotyl observed in lon1 mutants during skotomorphogenesis was partially alleviated by ethylene inhibitors. Furthermore, the short root phenotype was partially ameliorated by introducing a mutation in the ethylene receptor ETR1. Interestingly, the upregulation of only a select few target genes was linked to ETR1-mediated ethylene signalling. Together this provides multiple steps in the link between loss of Lon1 and signalling responses to restore mitochondrial protein homoeostasis in plants.

Impact of protein aggregation on the immunogenicity of a human monoclonal antibody following pulmonary administration in mice

Spray drying is a widely employed method for generating dry powder formulations for inhalation. Yet, it presents substantial challenges when applied to therapeutic proteins due to stability issues. The formation of protein aggregates during the atomization and the heating steps can diminish protein activity and raise immunogenicity concerns. Here, we assessed the impact of varying levels of protein aggregates generated during spray-drying on the fate and the immunogenicity of the human monoclonal antibody NIP228 following intratracheal administration in mice. Aggregate-free rhodamine labelled NIP228 was spray-dried with or without 1% polysorbate 80 surfactant, resulting in the generation of powder formulations with associated low and high protein aggregate levels, respectively. Confocal imaging highlighted the presence of aggregates in the lungs for both powders but not for the solution, while flow cytometry analysis designated alveolar macrophages as the main immune cells taking up rhod-NIP228 in the lungs with very little involvement of dendritic cells and interstitial macrophages following a single dose administration. Notably, repeated intratracheal administration of the three formulations in mice did not impact the magnitude of the anti-drug antibody response in sera or broncho-alveolar lavages. The pulmonary route appeared to evoke a more robust immune response when compared to subcutaneous administration. Overall, the level of NIP228 aggregation in this study did not appear to be the primary driver of NIP228 immunogenicity following delivery to the lungs in mice, shedding new light on the interplay between protein aggregation and immunogenicity in the context of the pulmonary delivery of therapeutic proteins.

Improvement on gel properties of chicken myofibrillar protein with electron beam irradiation: Based on protein structure, gel quality, water state

This study aimed to investigate the effects of electron beam (E-beam) irradiation at different doses (0–15 kGy) on the solubility, rheological properties, emulsification characteristics, and moisture distribution of chicken myofibrillar proteins (MPs). Irradiation treatment notably increased the solubility, surface hydrophobicity, emulsification properties, and apparent viscosity of MPs, based on conformational changes caused by irradiation-induced oxidative denaturation of proteins. However, high doses of irradiation (15 kGy) induced in excessive cross-linking and aggregation of proteins, reducing the solubility, emulsification properties, and shear stress. Degradation of myosin heavy and light chains in irradiated MPs increased the content of β-turns and random coils. Additionally, the initial relaxation times of T21 and T22 in irradiated protein gels were reduced, and the peak value of P21 was increased, which improved the water-capturing ability of protein gels. Altogether, these results findings suggest that electron beam irradiation can be applied as a potential technique for modifying muscle proteins.

Deciphering the mechanism underlying poor aqueous solubility of extracted quinoa proteins

This study aimed to decipher the mechanisms underlying poor solubility of quinoa proteins by investigating the form of quinoa proteins dispersed in water and how protein-protein interactions influenced the kinetic stability of proteins in the dispersions. Specifically, the relative solubility and the forms of quinoa proteins in 1–5 w/w% protein dispersions were determined by separating proteins via centrifugation and/or ultrafiltration. The kinetic stability of quinoa proteins in the supernatants over a 3-week storage period was characterized by determining the changes of concentration, composition and physicochemical properties of quinoa proteins and predicting protein-protein interactions. The results showed that quinoa proteins existed mainly as differently-sized protein aggregates in the dispersions, leading to low relative solubility. The coagulation of protein aggregates in the supernatants caused severe precipitation during the first week of storage whereas they were disassociated simultaneously. With further storage, the remaining proteins in the supernatants reached kinetic stability, which was contributed by stronger electrostatic repulsion and lower surface hydrophobicity. Moreover, 11S globulin and 2S albumin were precipitated and solubilized together during storage, which was ascribed to intermolecular interactions driven by multiple sites between 11S globulin and/or 2S albumin. This study lays a foundation for extensive utilization of quinoa proteins.

Surface-enhanced infrared spectroscopic study of extracellular vesicles using plasmonic gold nanoparticles

Extracellular vesicles (EVs), sub-micrometer lipid-bound particles released by most cells, are considered a novel area in both biology and medicine. Among characterization methods, infrared (IR) spectroscopy, especially attenuated total reflection (ATR), is a rapidly emerging label-free tool for molecular characterization of EVs. The relatively low number of vesicles in biological fluids (~1010 particle/mL), however, and the complex content of the EVs’ milieu (protein aggregates, lipoproteins, buffer molecules) might result in poor signal-to-noise ratio in the IR analysis of EVs. Exploiting the increment of the electromagnetic field at the surface of plasmonic nanomaterials, surface-enhanced infrared spectroscopy (SEIRS) provides an amplification of characteristic IR signals of EV samples. Negatively charged citrate-capped and positively charged cysteamine-capped gold nanoparticles with around 10nm diameter were synthesized and tested with blood-derived EVs. Both types of gold nanoparticles contributed to an enhancement of the EVs’ IR spectroscopic signature. Joint evaluation of UV-Vis and IR spectroscopic results, supported by FF-TEM images, revealed that proper interaction of gold nanoparticles with EVs is crucial, and an aggregation or clustering of gold nanoparticles is necessary to obtain the SEIRS effect. Positively charged gold nanoparticles resulted in higher enhancement, probably due to electrostatic interaction with EVs, commonly negatively charged.