IL-1 Family Cytokines Use Distinct Molecular Mechanisms to Signal through Their Shared Co-receptor.

Within the interleukin 1 (IL-1) cytokine family, IL-1 receptor accessory protein (IL-1RAcP) is the co-receptor for eight receptor-cytokine pairs, including those involving cytokines IL-1ß and IL-33. Unlike IL-1ß, IL-33 does not have a signaling complex that includes both its cognate receptor, ST2, and the shared co-receptor IL-1RAcP, which we now present here. Although the IL-1ß and IL-33 complexes shared structural features and engaged identical molecular surfaces of IL-1RAcP, these cytokines had starkly different strategies for co-receptor engagement and signal activation. Our data suggest that IL-1ß binds to IL-1RI to properly present the cytokine to IL-1RAcP, whereas IL-33 binds to ST2 in order to conformationally constrain the cognate receptor in an IL-1RAcP-receptive state. These findings indicate that members of the IL-1 family of cytokines use distinct molecular mechanisms to signal through their shared co-receptor, and they provide the foundation from which to design new therapies to target IL-33 signaling.

Wheat-Based Glues in Conservation and Cultural Heritage: (Dis)solving the Proteome of Flour and Starch Pastes and Their Adhering Properties.

Plant-based adhesives, such as those made from wheat, have been prominently used for books and paper-based objects and are also used as conservation adhesives. Starch paste originates from starch granules, whereas flour paste encompasses the entire wheat endosperm proteome, offering strong adhesive properties due to gluten proteins. From a conservation perspective, understanding the precise nature of the adhesive is vital as the longevity, resilience, and reaction to environmental changes can differ substantially between starch- and flour-based pastes. We devised a proteomics method to discern the protein content of these pastes. Protocols involved extracting soluble proteins using 0.5 M NaCl and 30 mM Tris-HCl solutions and then targeting insoluble proteins, such as gliadins and glutenins, with a buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris, and 75 mM DTT. Flour paste's proteome is diverse (1942 proteins across 759 groups), contrasting with starch paste's predominant starch-associated protein makeup (218 proteins in 58 groups). Transformation into pastes reduces proteomes' complexity. Testing on historical bookbindings confirmed the use of flour-based glue, which is rich in gluten and serpins. High levels of deamidation were detected, particularly for glutamine residues, which can impact the solubility and stability of the glue over time. The mass spectrometry proteomics data have been deposited to the ProteomeXchange, Consortium (http://proteomecentral.proteomexchange.org) via the MassIVE partner repository with the data set identifier MSV000093372 (ftp://MSV000093372@massive.ucsd.edu).

Identification of the functional PD-L1 interface region responsible for PD-1 binding and initiation of PD-1 signaling.

The PD-1/PD-L1 checkpoint pathway is important for regulating immune responses and can be targeted by immunomodulatory drugs to treat a variety of immune disorders. However, the precise protein-protein interactions required for the initiation of PD-1/PD-L1 signaling are currently unknown. Previously, we designed a series of first-generation PD-1 targeting peptides based on the native interface region of programmed death ligand 1 (PD-L1) that effectively reduced PD-1/PD-L1 binding. In this work, we further characterized the previously identified lead peptide, MN1.1, to identify key PD-1 binding residues and design an optimized peptide, MN1.4. We show MN1.4 is significantly more stable than MN1.1 in serum and retains the ability to block PD-1/PD-L1 complex formation. We further characterized the immunomodulatory effects of MN1.4 treatment by measuring markers of T cell activation in a co-culture model with ovarian cancer cells and peripheral blood mononuclear cells. We found MN1.4 treatment reduced cytokine secretion and suppressed T cell responses in a similar manner as recombinant PD-L1. Therefore, the PD-L1 interface region used to design MN1.4 appeared sufficient to initiate PD-1 signaling and likely represents the minimum necessary region of PD-L1 required for PD-1 recognition. We propose a peptide agonist for PD-1, such as MN1.4, could have several applications for treating autoimmune disorders caused by PD-1 deficiencies such as type 1 diabetes, inflammatory arthritis, or autoimmune side effects arising from monoclonal antibody-based cancer immunotherapies.

Effect of Cholesterol on the Structure and Composition of Glyco-DIBMA Lipid Particles.

Different amphiphilic co-polymers have been introduced to produce polymer-lipid particles with nanodisc structure composed of an inner lipid bilayer and polymer chains self-assembled as an outer belt. These particles can be used to stabilize membrane proteins in solution and enable their characterization by means of biophysical methods, including small-angle X-ray scattering (SAXS). Some of these co-polymers have also been used to directly extract membrane proteins together with their associated lipids from native membranes. Styrene/maleic acid and diisobutylene/maleic acid are among the most commonly used co-polymers for producing polymer-lipid particles, named SMALPs and DIBMALPs, respectively. Recently, a new co-polymer, named Glyco-DIBMA, was produced by partial amidation of DIBMA with the amino sugar N-methyl-d-glucosamine. Polymer-lipid particles produced with Glyco-DIBMA, named Glyco-DIBMALPs, exhibit improved structural properties and stability compared to those of SMALPs and DIBMALPs while retaining the capability of directly extracting membrane proteins from native membranes. Here, we characterize the structure and lipid composition of Glyco-DIBMALPs produced with either 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Glyco-DIBMALPs were also prepared with mixtures of either POPC or DMPC and cholesterol at different mole fractions. We estimated the lipid content in the Glyco-DIBMALPs and determined the particle structure and morphology by SAXS. We show that the Glyco-DIBMALPs are nanodisc-like particles whose size and shape depend on the polymer/lipid ratio. This is relevant for designing nanodisc particles with a tunable diameter according to the size of the membrane protein to be incorporated. We also report that the addition of >20 mol % cholesterol strongly perturbed the formation of Glyco-DIBMALPs. Altogether, we describe a detailed characterization of the Glyco-DIBMALPs, which provides relevant inputs for future application of these particles in the biophysical investigation of membrane proteins.

Structural Organization of Cardiolipin-Containing Vesicles as Models of the Bacterial Cytoplasmic Membrane.

The bacterial cytoplasmic membrane is the innermost bacterial membrane and is mainly composed of three different phospholipid species, i.e., phosphoethanolamine (PE), phosphoglycerol (PG), and cardiolipin (CL). In particular, PG and CL are responsible for the negative charge of the membrane and are often the targets of cationic antimicrobial agents. The growing resistance of bacteria toward the available antibiotics requires the development of new and more efficient antibacterial drugs. In this context, studying the physicochemical properties of the bacterial cytoplasmic membrane is pivotal for understanding drug-membrane interactions at the molecular level as well as for designing drug-testing platforms. Here, we discuss the preparation and characterization of PE/PG/CL vesicle suspensions, which contain all of the main lipid components of the bacterial cytoplasmic membrane. The vesicle suspensions were characterized by means of small-angle neutron scattering, dynamic light scattering, and electron paramagnetic spectroscopy. By combining solution scattering and spectroscopy techniques, we propose a detailed description of the impact of different CL concentrations on the structure and dynamics of the PE/PG bilayer. CL induces the formation of thicker bilayers, which exhibit higher curvature and are overall more fluid. The experimental results contribute to shed light on the structure and dynamics of relevant model systems of the bacterial cytoplasmic membrane.

Structural and Biophysical Properties of Supercharged and Circularized Nanodiscs.

Nanodiscs based on membrane scaffold proteins (MSPs) and phospholipids are used as membrane mimics to stabilize membrane proteins in solution for structural and functional studies. Combining small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and time-resolved small-angle neutron scattering (TR-SANS), we characterized the structure and lipid bilayer properties of five different nanodiscs made with dimyristoylphosphatidylcholine and different MSPs varying in size, charge, and circularization. Our SAXS modeling showed that the structural parameters of the embedded lipids are all similar, irrespective of the MSP properties. DSC showed that the lipid packing is not homogeneous in the nanodiscs and that a 20 Å wide boundary layer of lipids with perturbed packing is located close to the MSP, while the packing of central lipids is tighter than in large unilamellar vesicles. Finally, TR-SANS showed that lipid exchange rates in nanodiscs decrease with increasing nanodisc size and are lower for the nanodiscs made with supercharged MSPs compared to conventional nanodiscs. Altogether, the results provide a thorough biophysical understanding of the nanodisc as a model membrane system, which is important in order to carry out and interpret experiments on membrane proteins embedded in such systems.

Protein painting, an optimized MS-based technique, reveals functionally relevant interfaces of the PD-1/PD-L1 complex and the YAP2/ZO-1 complex.

Protein-protein interactions lie at the heart of many biological processes and therefore represent promising drug targets. Despite this opportunity, identification of protein-protein interfaces remains challenging. We have previously developed a method that relies on coating protein surfaces with small-molecule dyes to discriminate between solvent-accessible protein surfaces and hidden interface regions. Dye-bound, solvent-accessible protein regions resist trypsin digestion, whereas hidden interface regions are revealed by denaturation and sequenced by MS. The small-molecule dyes bind promiscuously and with high affinity, but their binding mechanism is unknown. Here, we report on the optimization of a novel dye probe used in protein painting, Fast Blue B + naphthionic acid, and show that its affinity for proteins strongly depends on hydrophobic moieties that we call here "hydrophobic clamps." We demonstrate the utility of this probe by sequencing the protein-protein interaction regions between the Hippo pathway protein Yes-associated protein 2 (YAP2) and tight junction protein 1 (TJP1 or ZO-1), uncovering interactions via the known binding domain as well as ZO-1's MAGUK domain and YAP's N-terminal proline-rich domain. Additionally, we demonstrate how residues predicted by protein painting are present exclusively in the complex interface and how these residues may guide the development of peptide inhibitors using a case study of programmed cell death protein 1 (PD-1) and programmed cell death 1 ligand 1 (PD-L1). Inhibitors designed around the PD-1/PD-L1 interface regions identified via protein painting effectively disrupted complex formation, with the most potent inhibitor having an IC50 of 5 µm.

Peptide Disc Mediated Control of Membrane Protein Orientation in Supported Lipid Bilayers for Surface-Sensitive Investigations.

In vitro characterization of membrane proteins requires experimental approaches providing mimics of the microenvironment that proteins encounter in native membranes. In this context, supported lipid bilayers provide a suitable platform to investigate membrane proteins by a broad range of surface-sensitive techniques such as neutron reflectometry (NR), quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance (SPR), atomic force microscopy (AFM), and fluorescence microscopy. Nevertheless, the successful incorporation of membrane proteins in lipid bilayers with sufficiently high concentration and controlled orientation relative to the bilayer remains challenging. We propose the unconventional use of peptide discs made by phospholipids and amphipathic 18A peptides to mediate the formation of supported phospholipid bilayers with two different types of membrane proteins, CorA and tissue factor (TF). The membrane proteins are reconstituted in peptide discs, deposited on a solid surface, and the peptide molecules are then removed with extensive buffer washes. This leaves a lipid bilayer with a relatively high density of membrane proteins on the support surface. As a very important feature, the strategy allows membrane proteins with one large extramembrane domain to be oriented in the bilayer, thus mimicking the in vivo situation. The method is highly versatile, and we show its general applicability by characterizing with the above-mentioned surface-sensitive techniques two different membrane proteins, which were efficiently loaded in the supported bilayers with ∼0.6% mol/mol (protein/lipid) concentration corresponding to 35% v/v for CorA and 8% v/v for TF. Altogether, the peptide disc mediated formation of supported lipid bilayers with membrane proteins represents an attractive strategy for producing samples for structural and functional investigations of membrane proteins and for preparation of suitable platforms for drug testing or biosensor development.

Proteomic analysis reveals pathogen-derived biomarkers of acute babesiosis in erythrocytes, plasma, and urine of infected hamsters.

Babesiosis among humans is on the rise in North America. Current diagnostic assays for the screening of babesiosis require blood collection by venipuncture, which is an invasive method. Urine on the other hand is a desirable biospecimen for biomarker analysis of Babesia microti infections because it can be collected periodically and non-invasively. Our group uses a new class of biomarker harvesting nanocage technology, which, when combined with mass spectrometry (MS), can determine the presence of parasite proteins shed in different bodily fluids of mammalian hosts, including urine. Using the hamster model of babesiosis, our nanoparticle-MS approach identified several B. microti proteins in erythrocytes, plasma, and urine samples. Surface and secreted antigens previously shown to elicit host immune responses against the parasite were particularly abundant in erythrocytes and plasma compared to other proteins. Two of these antigens, BmSA1 and BMR1_03g00947, showed different localization patterns by immunofluorescence of infected erythrocytes. Hamster urine samples from parasitemic animals harbored lower numbers of B. microti proteins compared to erythrocytes and plasma, with glycolytic enzymes, cytoskeletal components, and chaperones being the most frequently detected proteins. By applying novel nanoparticle-MS methods, a high level of analytical sensitivity can be achieved to detect multiple B. microti proteins in blood and urine. This is generally difficult to obtain with other techniques due to the masking of parasite biomarkers by the complex biomolecular matrix of bodily fluids from the host.

RPPA: Origins, Transition to a Validated Clinical Research Tool, and Next Generations of the Technology.

RPPA technology has graduated from a research tool to an essential component of clinical drug discovery research and personalized medicine. Next generations of RPPA technology will be a single clinical instrument that integrates all the steps of the workflow.

Affinity enrichment for mass spectrometry: improving the yield of low abundance biomarkers.

INTRODUCTION: Mass spectrometry (MS) is the premier tool for discovering novel disease-associated protein biomarkers. Unfortunately, when applied to complex body fluid samples, MS has poor sensitivity for the detection of low abundance biomarkers (≪10 ng/mL), derived directly from the diseased tissue cells or pathogens. Areas covered: Herein we discuss the strengths and drawbacks of technologies used to concentrate low abundance analytes in body fluids, with the aim to improve the effective sensitivity for MS discovery. Solvent removal by dry-down or dialysis, and immune-depletion of high abundance serum or plasma proteins, is shown to have disadvantages compared to positive selection of the candidate biomarkers by affinity enrichment. A theoretical analysis of affinity enrichment reveals that the yield for low abundance biomarkers is a direct function of the binding affinity (Association/Dissociation rates) used for biomarker capture. In addition, a high affinity capture pre processing step can effectively dissociate the candidate biomarker from partitioning with high abundance proteins such as albumin. Expert commentary: Properly designed high affinity capture materials can enrich the yield of low abundance (0.1-10 picograms/mL) candidate biomarkers for MS detection. Affinity capture and concentration, as an upfront step in sample preparation for MS, combined with MS advances in software and hardware that improve the resolution of the chromatographic separation can yield a transformative new class of low abundance biomarkers predicting disease risk or disease latency.

Localization of Cholesterol within Supported Lipid Bilayers Made of a Natural Extract of Tailor-Deuterated Phosphatidylcholine.

Cholesterol is an essential component of mammalian membranes and is known to induce a series of physicochemical changes in the lipid bilayer. Such changes include the formation of liquid-ordered phases with an increased thickness and a configurational order as compared to liquid-disordered phases. For saturated lipid membranes, cholesterol molecules localize close to the lipid head group-tail interface. However, the presence of polyunsaturated lipids was recently shown to promote relocation of cholesterol toward the inner interface between the two bilayer leaflets. Here, neutron reflection is used to study the location of cholesterol (both non-deuterated and per-deuterated versions are used) within supported lipid bilayers composed of a natural mixture of phosphatidylcholine (PC). The lipids were produced in a genetically modified strain of Escherichia coli and grown under specific deuterated conditions to give an overall neutron scattering length density (which depends on the level of deuteration) of the lipids matching that of D2O. The combination of solvent contrast variation method with specific deuteration shows that cholesterol is located closer to the lipid head group-tail interface in this natural PC extract rather than in the center of the core of the bilayer as seen for very thin or polyunsaturated membranes.

Toward detection of toxoplasmosis from urine in mice using hydro-gel nanoparticles concentration and parallel reaction monitoring mass spectrometry.

Diagnosis of clinical toxoplasmosis remains a challenge, thus limiting the availability of human clinical samples. Though murine models are an approximation of human response, their definitive infection status and tissue availability make them critical to the diagnostic development process. Hydrogel mesh nanoparticles were used to concentrate antigen to detectable levels for mass spectrometry. Seven Toxoplasma gondii isolates were used to develop a panel of potential peptide sequences for detection by parallel reaction monitoring (PRM) mass spectrometry. Nanoparticles were incubated with decreasing concentrations of tachyzoite lysate to explore the limits of detection of PRM. Mice whose toxoplasmosis infection status was confirmed by quantitative real-time PCR had urine tested by PRM after hydrogel mesh concentration for known T. gondii peptides. Peptides from GRA1, GRA12, ROP4, ROP5, SAG1, and SAG2A proteins were detected by PRM after nanoparticle concentration of urine, confirming detection of T. gondii antigen in the urine of an infected mouse.

Identification of novel candidate circulating biomarkers for malignant soft tissue sarcomas: Correlation with metastatic progression.

Soft tissue sarcomas (STS) are a heterogeneous group of rare tumors for which identification and validation of biological markers may improve clinical management. The fraction of low-molecular-weight (LMW) circulating proteins and fragments of proteins is a rich source of new potential biomarkers. To identify circulating biomarkers useful for STS early diagnosis and prognosis, we analyzed 53 high-grade STS sera using hydrogel core-shell nanoparticles that selectively entrap LMW proteins by size exclusion and affinity chromatography, protect them from degradation and amplify their concentration for mass spectrometry detection. Twenty-two analytes mostly involved in inflammatory and immunological response, showed a progressive increase from benign to malignant STS with a relative difference in abundance, more than 50% when compared to healthy control. 16 of these were higher in metastatic compared to non-metastatic tumors. Cox's regression analysis revealed a statistical significant association between the abundance of lactotransferrin (LTF) and complement factor H-related 5 (CFHR5) and risk of metastasis. In particular, CFHR5 was associated with the risk of metastasis. The role of circulating proteins involved in metastatic progression will be crucial for a better understanding of STS biology and patient management.

Class I Hydrophobin Vmh2 Adopts Atypical Mechanisms to Self-Assemble into Functional Amyloid Fibrils.

Hydrophobins are fungal proteins whose functions are mainly based on their capability to self-assemble into amphiphilic films at hydrophobic-hydrophilic interfaces (HHI). It is widely accepted that class I hydrophobins form amyloid-like structures, named rodlets, which are hundreds of nanometers long, packed into ordered lateral assemblies and do not exhibit an overall helical structure. We studied the self-assembly of the Class I hydrophobin Vmh2 from Pleurotus ostreatus in aqueous solutions by dynamic light scattering (DLS), thioflavin T (ThT), fluorescence assay, circular dichroism (CD), cryogenic trasmission electron microscopy (cryo-TEM), and TEM. Vmh2 does not form fibrillar aggregates at HHI. It exhibits spherical and fibrillar assemblies whose ratio depends on the protein concentration when freshly solubilized at pH ≥ 7. Moreover, it spontaneously self-assembles into isolated, micrometer long, and twisted amyloid fibrils, observed for the first time in fungal hydrophobins. This process is promoted by acidic pH, temperature, and Ca(2+) ions. A model of self-assembly into amyloid-like structures has been proposed.

Functionalized SPIONs: the surfactant nature modulates the self-assembly and cluster formation.

SuperParamagnetic Iron Oxide Nanoparticles (SPIONs) represent a suitable system for several applications especially in nanomedicine. Great efforts have been made to design stable and biocompatible functionalized SPIONs suitable for diagnostics and drug delivery. In particular, zwitterionic-surfactant functionalized SPIONs, obtained through a coating strategy based on hydrophobic interaction, are promising systems for biomedical applications. The size of functionalized SPIONs has emerged as a crucial parameter determining their fate in living organisms. However, not all the proposed functionalization strategies lead to monodispersed systems and SPION clustering often occurs. In this study, we report a systematic investigation on different surfactant-functionalized SPIONs in order to explore the possibility of tuning the particle size by choosing an appropriate amphiphilic molecule. By combining Small-Angle Neutron Scattering (SANS) and Dynamic Light Scattering (DLS) analysis, we have provided a detailed description of the functionalized SPION structure. Furthermore, we have also related the surfactant aggregation properties, i.e. the Critical Micelle Concentration (CMC), to their efficiency in coating the SPION surface. A lack in the formation of a compact shell leads to a clusters formation. On this basis, the present study contributes to furnishing decisive information to define synthetic strategies able to tune functionalized-SPION design.

Application of Nanotrap technology for high sensitivity measurement of urinary outer surface protein A carboxyl-terminus domain in early stage Lyme borreliosis.

OBJECTIVES: Prompt antibiotic treatment of early stage Lyme borreliosis (LB) prevents progression to severe multisystem disease. There is a clinical need to improve the diagnostic specificity of early stage Lyme assays in the period prior to the mounting of a robust serology response. Using a novel analyte harvesting nanotechnology, Nanotrap particles, we evaluated urinary Borrelia Outer surface protein A (OspA) C-terminus peptide in early stage LB before and after treatment, and in patients suspected of late stage disseminated LB. METHOD: We employed Nanotrap particles to concentrate urinary OspA and used a highly specific anti-OspA monoclonal antibody (mAb) as a detector of the C-terminus peptides. We mapped the mAb epitope to a narrow specific OspA C-terminal domain OspA236-239 conserved across infectious Borrelia species but with no homology to human proteins and no cross-reactivity with relevant viral and non-Borrelia bacterial proteins. 268 urine samples from patients being evaluated for all categories of LB were collected in a LB endemic area. The urinary OspA assay, blinded to outcome, utilized Nanotrap particle pre-processing, western blotting to evaluate the OspA molecular size, and OspA peptide competition for confirmation. RESULTS: OspA test characteristics: sensitivity 1.7 pg/mL (lowest limit of detection), % coefficient of variation (CV) = 8 %, dynamic range 1.7-30 pg/mL. Pre-treatment, 24/24 newly diagnosed patients with an erythema migrans (EM) rash were positive for urinary OspA while false positives for asymptomatic patients were 0/117 (Chi squared p < 10(-6)). For 10 patients who exhibited persistence of the EM rash during the course of antibiotic therapy, 10/10 were positive for urinary OspA. Urinary OspA of 8/8 patients switched from detectable to undetectable following symptom resolution post-treatment. Specificity of the urinary OspA test for the clinical symptoms was 40/40. Specificity of the urinary OspA antigen test for later serology outcome was 87.5 % (21 urinary OspA positive/24 serology positive, Chi squared p = 4.072e(-15)). 41 of 100 patients under surveillance for persistent LB in an endemic area were positive for urinary OspA protein. CONCLUSIONS: OspA urinary shedding was strongly linked to concurrent active symptoms (e.g. EM rash and arthritis), while resolution of these symptoms after therapy correlated with urinary conversion to OspA negative.

Unlocking the secrets to protein-protein interface drug targets using structural mass spectrometry techniques.

Protein-protein interactions (PPIs) drive all biologic systems at the subcellular and extracellular level. Changes in the specificity and affinity of these interactions can lead to cellular malfunctions and disease. Consequently, the binding interfaces between interacting protein partners are important drug targets for the next generation of therapies that block such interactions. Unfortunately, protein-protein contact points have proven to be very difficult pharmacological targets because they are hidden within complex 3D interfaces. For the vast majority of characterized binary PPIs, the specific amino acid sequence of their close contact regions remains unknown. There has been an important need for an experimental technology that can rapidly reveal the functionally important contact points of native protein complexes in solution. In this review, experimental techniques employing mass spectrometry to explore protein interaction binding sites are discussed. Hydrogen-deuterium exchange, hydroxyl radical footprinting, crosslinking and the newest technology protein painting are compared and contrasted.

Developing functionalized Fe3O4-Au nanoparticles: a physico-chemical insight.

Nanotechnology for biomedicine has recently attracted increasing interest from the scientific community. In particular, among the different nanodevices suitable for this application, multifunctionalizable hybrid nanoparticles are one of the most investigated research topics. Here we present a detailed physico-chemical characterization of hybrid magneto-plasmonic iron oxide-gold nanoparticles (NPs) with core-shell structure. In particular, we underline all the synthetic difficulties concerning the preparation of these systems. Based on all our results, after different tests of a commonly reported protocol for the synthesis of the core-shell system, we believe that several issues are still open in the synthetic preparation of these particular NPs. Indeed, at least for the conditions that we adopted, core-shell morphology nanoparticles cannot be produced. However, independent of the core structure, we describe here an optimized and efficient functionalization protocol to obtain stable nanoparticle aqueous suspensions, which can be easily exported to other kinds of metal and metal-oxide NPs and used to develop biocompatible systems. Furthermore, reliable information that could be useful for researchers working in this field is extensively discussed.

Tris buffer modulates polydopamine growth, aggregation, and paramagnetic properties.

Despite the growing technological interest of polydopamine (dopamine melanin)-based coatings for a broad variety of applications, the factors governing particle size, shape, and electronic properties of this bioinspired multifunctional material have remained little understood. Herein, we report a detailed characterization of polydopamine growth, particle morphology, and paramagnetic properties as a function of dopamine concentration and nature of the buffer (pH 8.5). Dynamic Light Scattering data revealed an increase in the hydrodynamic radii (Rh) of melanin particles with increasing dopamine concentration in all buffers examined, especially in phosphate buffer. Conversely, a marked inhibition of particle growth was apparent in Tris buffer, with Rh remaining as low as <100 nm during polymerization of 0.5 mM dopamine. Small angle neutron scattering data suggested formation of bidimensional structures in phosphate or bicarbonate buffers, while apparently three-dimensional fractal objects prevailed in Tris buffer. Finally, electron paramagnetic resonance spectra revealed a broader signal amplitude with a peculiar power saturation decay profile for polydopamine samples prepared in Tris buffer, denoting more homogeneous paramagnetic centers with respect to similar samples obtained in phosphate and bicarbonate buffers. Overall, these results disclose Tris buffer as an efficient modulator of polydopamine buildup and properties for the rational control and fine-tuning of melanin aggregate size, morphology, and free radical behavior.