Aggregate Species Research Articles

Exploring the Aβ1-42 fibrillogenesis timeline by atomic force microscopy and surface enhanced Raman spectroscopy.

Introduction: Alzheimer's disease (AD) is a progressive debilitating neurological disorder representing the most common neurodegenerative disease worldwide. Although the exact pathogenic mechanisms of AD remain unresolved, the presence of extracellular amyloid-β peptide 1-42 (Aβ1-42) plaques in the parenchymal and cortical brain is considered one of the hallmarks of the disease. Methods: In this work, we investigated the Aβ1-42 fibrillogenesis timeline up to 48h of incubation, providing morphological and chemo-structural characterization of the main assemblies formed during the aggregation process of Aβ1-42, by atomic force microscopy (AFM) and surface enhanced Raman spectroscopy (SERS), respectively. Results: AFM topography evidenced the presence of characteristic protofibrils at early-stages of aggregation, which form peculiar macromolecular networks over time. SERS allowed to track the progressive variation in the secondary structure of the aggregation species involved in the fibrillogenesis and to determine when the β-sheet starts to prevail over the random coil conformation in the aggregation process. Discussion: Our research highlights the significance of investigating the early phases of fibrillogenesis to better understand the molecular pathophysiology of AD and identify potential therapeutic targets that may prevent or slow down the aggregation process.

Structural characterization of amyloid aggregates with spatially resolved infrared spectroscopy.

The self-assembly of proteins and peptides into ordered structures called amyloid fibrils is a hallmark of numerous diseases, impacting the brain, heart, and other organs. The structure of amyloid aggregates is central to their function and thus has been extensively studied. However, the structural heterogeneities between aggregates as they evolve throughout the aggregation pathway are still not well understood. Conventional biophysical spectroscopic methods are bulk techniques and only report on the average structural parameters. Understanding the structure of individual aggregate species in a heterogeneous ensemble necessitates spatial resolution on the length scale of the aggregates. Recent technological advances have led to augmentation of infrared (IR) spectroscopy with imaging modalities, wherein the photothermal response of the sample upon vibrational excitation is leveraged to provide spatial resolution beyond the diffraction limit. These combined approaches are ideally suited to map out the structural heterogeneity of amyloid ensembles. AFM-IR, which integrates IR spectroscopy with atomic force microscopy enables identification of the structural facets the oligomers and fibrils at individual aggregate level with nanoscale resolution. These capabilities can be extended to chemical mapping in diseased tissue specimens with submicron resolution using optical photothermal microscopy, which combines IR spectroscopy with optical imaging. This book chapter provides the basic premise of these novel techniques and provides the typical methodology for using these approaches for amyloid structure determination. Detailed procedures pertaining to sample preparation and data acquisition and analysis are discussed and the aggregation of the amyloid β peptide is provided as a case study to provide the reader the experimental parameters necessary to use these techniques to complement their research efforts.

Inhibitors of amyloid fibril formation.

Many diseases are caused by misfolded and denatured proteins, leading to neurodegenerative diseases. In recent decades researchers have developed a variety of compounds, including polymeric inhibitors and natural compounds, antibodies, and chaperones, to inhibit protein aggregation, decrease the toxic effects of amyloid fibrils, and facilitate refolding proteins. The causes and mechanisms of amyloid formation are still unclear, and there are no effective treatments for Amyloid diseases. This section describes research and achievements in the field of inhibiting amyloid accumulation and also discusses the importance of various strategies in facilitating the removal of aggregates species (refolding) in the treatment of neurological diseases such as chemical methods like as, small molecules, metal chelators, polymeric inhibitors, and nanomaterials, as well as the use of biomolecules (peptide and, protein, nucleic acid, and saccharide) as amyloid inhibitors, are also highlighted.

Unveiling the therapeutic potential of Lobaria extract and its depsides/depsidones in combatting Aβ42 peptides aggregation and neurotoxicity in Alzheimer's disease.

Background: The development of effective inhibitors that can inhibit amyloid β (Aβ) peptides aggregation and promote neurite outgrowth is crucial for the possible treatment of Alzheimer's disease (AD). Lobaria (Schreb.) Hoffm., a traditional Chinese medicine used in Himalaya region for inflammatory diseases, contains depsides/depsidones (DEPs) such as gyrophoric acid, norstictic acid, and stictic acid known for their anti-cancer and anti-inflammation properties. Methods: Lobaria extracts were analyzed using HPLC to identify DEPs and establish standards. The inhibitory effects of Lobaria on Aβ42 fibrillization and depolymerization were assessed using various approaches with biophysical and cellular methods. The neuroprotective activity of Lobaria extracts and its DEPs aganist Aβ-mediated cytotoxicity was also evaluated. Results: Norstictic and stictic acid were found in the water extract, while norstictic, stictic, and gyrophoric acid were detected in the ethanol extract of Lobaria. Both extracts, and their DEPs effectively inhibited Aβ42 fibrillation and disaggregate mature Aβ42 fibrils. Notably, the ethanol extract showed superior inhibitory effect compared to the water extract, with gyrophoric acid being the most effective DEPs. Additionally, herbal extract-treated Aβ42 aggregation species significantly protected neuronal cells from Aβ42-induced cell damage and promoted neurite outgrowth. Conclusion: This study is the first to investigate the effect of Lobaria on Aβ42 and neuronal cell in AD. Given that Lobaria is commonly used in ethnic medicine and food with good safety records, our findings propose that Lobaria extracts and DEPs have potential as neuroprotective and therapeutic agents for AD patients.

Deep eutectic solvent-assisted preparation of resveratrol-based carbon dots with high biocompatibility for in situ screening inhibitors on amyloid protein aggregation and scavenging reactive oxygen species

It is important to monitor the aggregation process of amyloid protein for fundamental researches, diagnosis and drug development of the related diseases, like neurodegenerative and metabolic diseases. Resveratrol (Res) has attracted great attention due to its high medical values. However, the application of Res is seriously restricted, because of its poor bioavailability caused by low water solubility and poor absorption. To address the problem, for the first time, this study put forward a novel strategy to nano-derivatize Res to carbon dots, i.e. Res-DES-CDs, based on the ternary deep eutectic solvent (DES). DES played the roles to assist the solubility and carbonization of Res, and improve biocompatibility. Res-DES-CDs possessed the highest water compatibility as reported so far for Res related derivatives. Meanwhile, Res-DES-CDs retained some functionalities of Res, thus exhibiting the turn-on fluorescence probing ability towards amyloid fibrils without disturbing amyloid aggregation; it was applicable to in situ screen aggregation inhibitors in real time. In addition, Res-DES-CDs possessed better ability of scavenging reactive oxygen species than Res, but with lower cytotoxicity and higher cell uptake rate, which held a promise to alleviate oxidative stress in relative diseases. The proposed strategy could be generalized to enhance water solubility and biocompatibility of compounds of poor solubility.

Passive Immunotherapies Targeting Amyloid-β in Alzheimer's Disease: A Quantitative Systems Pharmacology Perspective.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by amyloid-β (Aβ) protein accumulation in the brain. Passive immunotherapies using monoclonal antibodies for targeting Aβ have shown promise for AD treatment. Indeed, recent US FDA approval of aducanumab and lecanemab, alongside positive donanemab Phase III results demonstrated clinical efficacy after decades of failed clinical trials for AD. However, the pharmacological basis distinguishing clinically effective from ineffective therapies remains unclear, impeding development of potent therapeutics. This study aimed to provide a quantitative perspective for effectively targeting Aβ with antibodies. We first reviewed the contradicting results associated with amyloid hypothesis and the pharmacological basis of Aβ immunotherapy. Subsequently, we developed a quantitative systems pharmacology (QSP) model that describes the non-linear progression of Aβ pathology and the pharmacologic actions of the Aβ-targeting antibodies. Using the QSP model, we analyzed various scenarios for effective passive immunotherapy for AD. The model revealed that binding exclusively to the Aβ monomer has minimal effect on Aβ aggregation and plaque reduction, therefore antibody affinity towards Aβ monomer is unwanted, as it could become a distractive mechanism for plaque reduction. Neither early intervention, high brain penetration, nor increased dose alone could yield significant improvement of clinical efficacy for antibodies targeting solely monomers. Antibodies that bind all Aβ species but lack effector function exhibited moderate effects in plaque reduction. Our model highlights the importance of binding aggregate Aβ species and incorporating effector functions for efficient and early plaque reduction, guiding the development of more effective therapies for this devastating disease. Significance Statement Despite previous unsuccessful attempts spanning several decades, passive immunotherapies utilizing monoclonal antibodies for targeting amyloid-beta (Aβ) have demonstrated promise with two recent FDA approvals. However, the pharmacological basis that differentiates clinically effective therapies from ineffective ones remains elusive. Our study offers a Quantitative Systems Pharmacology (QSP) perspective, emphasizing the significance of selectively targeting specific Aβ species and importance of antibody effector functions. This perspective sheds light on the development of more effective therapies for this devastating disease.

Sorption of U(VI) on dolomite in a dilute carbonate solution: behavior investigation and mechanism probe

Abstract The U(VI) sorption behavior on dolomite was systematically investigated in a dilute carbonate solution using batch technique. The distribution coefficients (K d) increased with the increasing time and temperature, but decreased with the increase of initial U(VI) concentration and ionic strength. As the initial pH increased from 2.0 to 10.5, the K d values first increased and then decreased, mainly due to the transition from electrostatic attraction to repulsion caused by the changes in U(VI) species. The sorption process was well-described by pseudo-second-order kinetic and Freundlich isotherm models, and was spontaneous, endothermic, heterogeneous, and pH-dependent. In the presence of HA/FA, at low pH, more HAs/FAs were adsorbed on dolomite surface, providing more sorption sites for the positively charged U(VI) species, and promoting the U(VI) sorption. At high pH, more HAs/FAs were dispersed in solution, encapsulating the negatively charged U(VI) species in aggregates, and inhibiting the U(VI) sorption. The promoting and inhibiting effects of HA on the sorption process are obviously stronger than FA. XPS spectra indicated that the surface ‒OH and CO3 2− on the hydroxylated dolomite and the –OH groups in HA/FA may be involved in U(VI) sorption. The results reported here provide valuable references for further understanding U(VI) migration in geological media.

Soil bacterial habitat generalists strengthen depth-dependent organic carbon accrual in coastal reclaimed lands after afforestation

The microbially driven soil organic carbon (SOC) turnover and accrual at the aggregate scale are crucial to reveal potential SOC sequestration and stabilization. However, information about how afforestation in reclaimed lands influences the bacterial contribution to SOC within soil aggregates remains unclear. Here, we investigated the bacterial taxonomic and functional composition and habitat associations of species (i.e., generalists and specialists) in different aggregates. Soil aggregates were isolated using an optimal soil moisture method. Bacterial taxonomic and functional composition were determined using high-throughput sequencing and PICRUSt2, respectively. We evaluated the effect of bacterial traits on SOC accrual within aggregates along the soil profile in unreclaimed tideland and two poplar plantations at different stages of reclamation (i.e., reclaimed 24 and 64 years prior to data collection) in East China. The bacterial traits and SOC concentration did not differ significantly among different-sized aggregates and that bacterial alpha diversity and SOC within aggregates increased with reclamation time, but this was only pronounced in surface soil. The relative abundance of generalists increased, specialists decreased in the soil profile with reclamation time, and the magnitude of variation declined with soil depth. The random forest model and variation partitioning analysis confirmed that generalists and specialists dominated the variation in SOC. Moreover, some subgroups from Proteobacteria, Acidobacteria, Actinobacteria, and Chloroflexi phyla were rich in generalists and may play crucial roles in SOC accrual. These findings suggest that the bacterial traits and SOC in coastal coarse-textured soil were not affected by aggregate sizes and might rely on other aggregate attributes (e.g., stability). Furthermore, SOC accrual was strongly affected by an increase in habitat generalists. We provide new insights into the variation pattern in SOC and bacterial traits at the aggregate scale and illustrate that bacterial generalists have the greatest impact on SOC accrual in coastal reclaimed lands after afforestation.

Biosensing strategies for amyloid‐like protein aggregates

AbstractProtein aggregate species play a pivotal role in the pathology of various degenerative diseases. Their dynamic changes are closely correlated with disease progression, making them promising candidates as diagnostic biomarkers. Given the prevalence of degenerative diseases, growing attention is drawn to develop pragmatic and accessible protein aggregate species detection technology. However, the performance of current detection methods is far from satisfying the requirements of extensive clinical use. In this review, we focus on the design strategies, merits, and potential shortcomings of each class of detection methods. The review is organized into three major parts: native protein sensing, seed amplification, and intricate program, which embody three different but interconnected methodologies. To the best of our knowledge, no systematic review has encompassed the entire workflow, from the molecular level to the apparatus organization. This review emphasizes the feasibility of the methods instead of theoretical detection limitations. We conclude that high selectivity does play a pivotal role, while signal compilation, multilateral profiling, and other patient‐oriented strategies (i.e. less invasiveness and assay speed) are also important.

First 3-D structural evidence of a native-like intertwined dimer in the acylphosphatase family

Acylphosphatase (AcP, EC 3.6.1.7) is a small model protein conformed by a ferredoxin-like fold, profoundly studied to get insights into protein folding and aggregation processes. Numerous studies focused on the aggregation and/or amyloidogenic properties of AcPs suggest the importance of edge-β-strands in the process. In this work, we present the first crystallographic structure of Escherichia coli AcP (EcoAcP), showing notable differences with the only available NMR structure for this enzyme. EcoAcP is crystalised as an intertwined dimer formed by replacing a single C-terminal β-strand between two protomers, suggesting a flexible character of the C-terminal edge of EcoAcP. Despite numerous works where AcP from different sources have been used as a model system for protein aggregation, our domain-swapped EcoAcP structure is the first 3-D structural evidence of native-like aggregated species for any AcP reported to date, providing clues on molecular determinants unleashing aggregation.

Identification of hybrid amyloid strains assembled from amyloid-β and human islet amyloid polypeptide

Cross-fibrillation of amyloid-β (Aβ) peptides and human islet amyloid polypeptides (hIAPP) has revealed a close correlation between Alzheimer’s disease and type 2 diabetes (T2D). Importantly, different amyloid strains are likely to lead to the clinical pathological heterogeneity of degenerative diseases due to toxicity. However, given the complicated cross-interactions between different amyloid peptides, it is still challenging to identify the polymorphism of the hybrid amyloid strains and reveal mechanistic insights into aggregation, but highly anticipated due to their significance. In this study, we investigated the cross-fibrillation of Aβ peptides and different hIAPP species (monomers, oligomers, and fibrils) using combined experimental and simulation approaches. Cross-seeding and propagation of different amyloid peptides monitored by experimental techniques proved that the three species of hIAPP aggregates have successively enhanced Aβ fibrillation, especially for hIAPP fibrils. Moreover, the polymorphism of these morphologically similar hybrid amyloid strains could be distinguished by testing their mechanical properties using quantitative nanomechanical mapping, where the assemblies of Aβ-hIAPP fibrils exhibited the high Young’s modulus. Furthermore, the enhanced internal molecular interactions and β-sheet structural transformation were proved by exploring the conformational ensembles of Aβ-hIAPP heterodimer and Aβ-hIAPP decamer using molecular dynamic simulations. Our findings pave the way for identifying different hybrid amyloid strains by quantitative nanomechanical mapping and molecular dynamic simulations, which is important not only for the precise classification of neurodegenerative disease subtypes but also for future molecular diagnosis and therapeutic treatment of multiple interrelated degenerative diseases.

Selective logging destabilizes the functioning and composition of forest ecosystems at multiple spatial scales

Selective logging is one of the most prevalent land uses of forests worldwide, affecting biodiversity and ecosystem functioning. However, the effect of selective logging on the dual nature of temporal stability, and the scale dependence of this effect, remain to be elucidated. By conducting several decade‐long experiments in temperate forest ecosystems, we tested the effects of selective logging on aggregate and compositional stability at multiple spatial scales. As expected, forest ecosystem stability at larger spatial scales was enhanced both by the stability of local scales (i.e. α stability) and asynchronous dynamics among local communities (i.e. spatial asynchrony). We found that the negative effects of selective logging on both facets of forest stability propagated from local to larger spatial scales due to reduced α stability and the biological insurance effects of α diversity. However, both spatial aggregate and compositional asynchrony were not affected by selective logging. Interestingly, despite the selective logging, α diversity still provided biological insurance effects for maintaining aggregate and compositional stability. Our results imply that selective logging may destabilize the aggregate ecosystem functioning and species composition of forest ecosystems at local and larger spatial scales. To our knowledge, this study provides the first evidence of the scale dependence of aggregate and compositional stability of forest ecosystems in response to selective logging. Our findings suggest that forest management should avoid excessive selective logging and strive to protect forest diversity to safeguard the sustainability of the functioning and composition of natural forest ecosystems at multiple spatial scales.Keywords: aggregate stability, compositional stability, diversity, ecosystem functioning, spatial asynchrony, stability

Estimation of the Compositional Proportions of Different Micelle Species in Water: Application of One-dimensional Aggregate Theory to Small Angle Neutron Scattering Analyzed Data

Abstract The one-dimensional aggregation theory was applied to the small angle neutron scattering (SANS) analyzed data of surfactant solutions. Plots of the hydrophobic bonding energy E to form an aggregate against the number-average aggregation number n were found to be powerful to estimate the compositional ratios of different aggregate species co-existing in a gross sample solution.

Super-resolution imaging unveils the self-replication of tau aggregates upon seeding

Tau is a soluble protein interacting with tubulin to stabilize microtubules. However, under pathological conditions, it becomes hyperphosphorylated and aggregates, a process that can be induced by treating cells with exogenously added tau fibrils. Here, we employ single-molecule localization microscopy to resolve the aggregate species formed in early stages of seeded tau aggregation. We report that entry of sufficient tau assemblies into the cytosol induces the self-replication of small tau aggregates, with a doubling time of 5h inside HEK cells and 1day in murine primary neurons, which then grow into fibrils. Seeding occurs in the vicinity of the microtubule cytoskeleton, is accelerated by the proteasome, and results in release of small assemblies into the media. In the absence of seeding, cells still spontaneously form small aggregates at lower levels. Overall, our work provides a quantitative picture of the early stages of templated seeded tau aggregation in cells.

SEVI Inhibits Aβ Amyloid Aggregation by Capping the β-Sheet Elongation Edges.

Inhibiting the aggregation of amyloid peptides with endogenous peptides has broad interest due to their intrinsically high biocompatibility and low immunogenicity. Here, we investigated the inhibition mechanism of the prostatic acidic phosphatase fragment SEVI (semen-derived enhancer of viral infection) against Aβ42 fibrillization using atomistic discrete molecular dynamic simulations. Our result revealed that SEVI was intrinsically disordered with dynamic formation of residual helices. With a high positive net charge, the self-aggregation tendency of SEVI was weak. Aβ42 had a strong aggregation propensity by readily self-assembling into β-sheet-rich aggregates. SEVI preferred to interact with Aβ42, rather than SEVI themselves. In the heteroaggregates, Aβ42 mainly adopted β-sheets buried inside and capped by SEVI in the outer layer. SEVI could bind to various Aβ aggregation species─including monomers, dimers, and proto-fibrils─by capping the exposed β-sheet elongation edges. The aggregation processes Aβ42 from the formation of oligomers to conformational nucleation into fibrils and fibril growth should be inhibited as their β-sheet elongation edges are being occupied by the highly charged SEVI. Overall, our computational study uncovered the molecular mechanism of experimentally observed inhibition of SEVI against Aβ42 aggregation, providing novel insights into the development of therapeutic strategies against Alzheimer's disease.

The effects of environmental variables on hotspots of Antarctic krill (Euphausia superba) in the Bransfield Strait during autumn

Antarctic krill (Euphausia superba), a typical aggregation species, is the keystone species in the Antarctic ecosystem and supports an important fishery in the Southern Ocean. The unique topographic conditions of this region provide an important habitat for krill, and it has therefore become a critical krill fishing ground. To investigate the factors affecting krill hotspot patterns in the central basin of the Bransfield Strait in autumn 2021, an area with a high level of krill biomass and a persistent fishing ground, we used Getis-Ord Gi* approach to perform a hotspot analysis of the spatial and temporal variability of krill distribution. From this analysis we examined the relationship between krill distribution in hotspot and coldspot areas and environment variables such as geospatial features and eddy kinetic energy. We found that environmental variables generally showed opposing trends for krill distribution at hot- and coldspot areas. Moreover, geospatial features, including the distance to 200m and 1000m isobaths and depth, may be the dominant factor determining krill habitat characteristics, and moderate levels of eddy kinetic energy were conducive to krill aggregation.

Thiophene-Based Ligands: Design, Synthesis and Their Utilization for Optical Assignment of Polymorphic-Disease-Associated Protein Aggregates.

The development of ligands for detecting protein aggregates is of great interest, as these aggregated proteinaceous species are the pathological hallmarks of several devastating diseases, including Alzheimer's disease. In this regard, thiophene-based ligands have emerged as powerful tools for fluorescent assessment of these pathological entities. The intrinsic conformationally sensitive photophysical properties of poly- and oligothiophenes have allowed optical assignment of disease-associated protein aggregates in tissue sections, as well as real-time in vivo imaging of protein deposits. Herein, we recount the chemical evolution of different generations of thiophene-based ligands, and exemplify their use for the optical distinction of polymorphic protein aggregates. Furthermore, the chemical determinants for achieving a superior fluorescent thiophene-based ligand, as well as the next generation of thiophene-based ligands targeting distinct aggregated species are described. Finally, the directions for future research into the chemical design of thiophene-based ligands that can aid in resolving the scientific challenges around protein aggregation diseases are discussed.

Increased reactive oxygen species lead to overactivation of platelets in essential thrombocythemia

IntroductionPlatelet function, rather than platelet count, plays a crucial role in thrombosis in essential thrombocythemia (ET). However, little is known about the abnormal function of platelets in ET. Here, we investigated the functional characteristics of platelets in ET hemostasis to explore the causes of ET platelet dysfunction and new therapeutic strategies for ET. Materials and methodsWe analyzed platelet aggregation, activation, apoptosis, and reactive oxygen species (ROS) in ET patients and JAK2V617F-positive ET-like mice. The effects of ROS on platelet function and the underlying mechanism were investigated by inhibiting ROS using N-acetylcysteine (NAC). ResultsPlatelet aggregation, activation, apoptosis, ROS, and clot retraction were elevated in ET. No significant differences were observed between ET patients with JAK2V617F or CALR mutations. Increased ROS activated the JAK-STAT pathway, which may further influence platelet function. Inhibition of platelet ROS by NAC reduced platelet aggregation, activation, and apoptosis, and prolonged bleeding time. Furthermore, NAC treatment reduced platelet count in ET-like mice by inhibiting platelet production from megakaryocytes. ConclusionsElevated ROS in ET platelets resulted in enhanced platelet activation, function and increased risk of thrombosis. NAC offers a potential therapeutic strategy for reducing platelet count.

Mitochondria-Directing Fluorogenic Probe: An Efficient Amyloid Marker for Imaging Lipid Metabolite-Induced Protein Aggregation in Live Cells and Caenorhabditis elegans.

The design and development of optical probes for sensing neurotoxic amyloid fibrils are active and important areas of research and are undergoing continuous advancements. In this paper, we have synthesized a red emissive styryl chromone-based fluorophore (SC1) for fluorescence-based detection of amyloid fibrils. SC1 records exceptional modulation in its photophysical properties in the presence of amyloid fibrils, which has been attributed to the extreme sensitivity of its photophysical properties toward the immediate microenvironment of the probe in the fibrillar matrix. SC1 also shows very high selectivity toward the amyloid-aggregated form of the protein as compared to its native form. The probe is also able to monitor the kinetic progression of the fibrillation process, with comparable efficiency as that of the most popular amyloid probe, Thioflavin-T. Moreover, the performance of SC1 is least sensitive to the ionic strength of the medium, which is an advantage over Thioflavin-T. In addition, the molecular level interaction forces between the probe and the fibrillar matrix have been interrogated by molecular docking calculations which suggest the binding of the probe to the exterior channel of the fibrils. The probe has also been demonstrated to sense protein aggregates from the Aβ-40 protein, which is known to be responsible for Alzheimer's disease. Moreover, SC1 exhibited excellent biocompatibility and exclusive accumulation at mitochondria which allowed us to successfully demonstrate the applicability of this probe to detect mitochondrial-aggregated protein induced by an oxidative stress indicator molecule 4-hydroxy-2-nonenal (4-HNE) in A549 cell lines as well as in a simple animal model like Caenorhabditis elegans. Overall, the styryl chromone-based probe presents a potentially exciting alternative for the sensing of neurotoxic protein aggregation species both in vitro as well as in vivo.

Controlled lipid self-assembly for scalable manufacturing of next-generation immune stimulating complexes

Immune stimulating complexes (ISCOMs) are safe and effective saponin-based adjuvants formed by the self-assembly of saponin, cholesterol, and phospholipids in water to form cage-like 30–40 nm diameter particles. Inclusion of the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) in ISCOM particles yields a promising next-generation adjuvant termed Saponin-MPLA NanoParticles (SMNP). In this work, we detail protocols to produce ISCOMs or SMNP via a tangential flow filtration (TFF) process suitable for scalable synthesis and Good Manufacturing Practice (GMP) production of clinical-grade adjuvants. SMNP or ISCOM components were solubilized in micelles of the surfactant MEGA-10, then diluted below the critical micelle concentration (CMC) of the surfactant to drive ISCOM self-assembly. Assembly of ISCOM/SMNP particles using the purified saponin QS-21 used in clinical-grade saponin adjuvants was found to require controlled stepwise dilution of the initial micellar solution, to prevent formation of undesirable kinetically-trapped aggregate species. An optimized protocol gave yields of ∼77% based on the initial feed of QS-21 and the final SMNP particle composition mirrored the feed ratios of the components. Further, samples were highly homogeneous with comparable quality to that of material prepared at lab scale by dialysis and purified via size-exclusion chromatography. This protocol may be useful for clinical preparation of ISCOM-based vaccine adjuvants and therapeutics.