Transferrin-conjugated magnetic nanoparticles for the isolation of brain-derived blood exosomal MicroRNAs: A novel approach for Parkinson's disease diagnosis.

BACKGROUND: Brain-derived exosomes circulate in the bloodstream and other bodily fluids, serving as potential indicators of neurological disease progression. These exosomes present a promising avenue for the early and precise diagnosis of neurodegenerative conditions. Notably, miRNAs found in plasma extracellular vesicles (EVs) offer distinct diagnostic benefits due to their stability, abundance, and resistance to breakdown. RESULTS: In this study, we introduce a method using transferrin conjugated magnetic nanoparticles (TMNs) to isolate these exosomes from the plasma of patients with neurological disorders. This TMNs technique is both quick (<35 min) and cost-effective, requiring no high-priced ingredients or elaborate equipment for EV extraction. Our method successfully isolated EVs from 33 human plasma samples, including those from patients with Parkinson's disease (PD), Multiple Sclerosis (MS), and Dementia. Using quantitative polymerase chain reaction (PCR) analysis, we evaluated the potential of 8 exosomal miRNA profiles as biomarker candidates. Six exosomal miRNA biomarkers (miR-195-5p, miR-495-3p, miR-23b-3P, miR-30c-2-3p, miR-323a-3p, and miR-27a-3p) were consistently linked with all stages of PD. SIGNIFICANCE: The TMNs method provides a practical, cost-efficient way to isolate EVs from biological samples, paving the way for non-invasive neurological diagnoses. Furthermore, the identified miRNA biomarkers in these exosomes may emerge as innovative tools for precise diagnosis in neurological disorders including PD.

Molecularly imprinted polymer-based SERS sensing of transferrin in human serum.

Specific detection of glycoproteins such as transferrin (TRF) related to neurological diseases, hepatoma and other diseases always plays an important role in the field of disease diagnosis. We designed an antibody-free immunoassay sensing method based on molecularly imprinted polymers (MIPs) formed by the polymerization of multiple functional monomers for the sensitive and selective detection of TRF in human serum. In the sandwich surface-enhanced Raman spectroscopy (SERS) sensor, the TRF-oriented magnetic MIP nanoparticles (Fe3O4@SiO2-MIPs) served as capture units to specifically recognize TRF and 4-mercaptophenylboronic acid-functionalized gold nanorods (MPBA-Au NRs) served as SERS probes to label the targets. In order to achieve stronger interaction between the recognition cavities of the prepared MIPs and the different amino acid fragments that make up TRF, Fe3O4@SiO2-MIPs were obtained through polycondensation reactions between more silylating reagents, enhancing the specific recognition of the entire TRF protein and achieving high IF. This sensing method exhibited a good linear response to TRF within the TRF concentration range of 0.01 ng mL-1 to 1 mg mL-1 (R2 = 0.9974), and the LOD was 0.00407 ng mL-1 (S/N = 3). The good stability, reproducibility and specificity of the resulting MIP based SERS sensor were demonstrated. The determination of TRF in human serum confirmed the feasibility of the method in practical applications.

Transferrin Immobilized Graphene Oxide Nanocomposite for Targeted Cancer Chemodynamic Therapy via Increasing Intracellular Labile Fe2+ Concentration.

Recently, different alternative regulated cell death (RCD) pathways, viz., necroptosis, pyroptosis, ferroptosis, cuproptosis etc., have been explored as important targets for the development of cancer medications in recent years, as these can change the immunogenicity of the tumor microenvironment (TME) and will finally lead to the inhibition of cancer progression and metastasis. Here, we report the development of transferrin immobilized graphene oxide (Tfn@GOAPTES) nanocomposite as a therapeutic strategy toward cancer cell killing. The electrostatic immobilization of Tfn on the GOAPTES surface was confirmed by different spectroscopy and microscopy techniques. The Tfn immobilization was found to be ∼74 ± 4%, whereas the stability of the protein on the GO surface suggested a robust nature of the nanocomposite. The MTT assay suggested that Tfn@GOAPTES exhibited cytotoxicity toward HeLa cells via increased lipid peroxidation and DNA damage. Western blot studies resulted in decreased expression of acetylation on lysine 40 of α-tubulin and increased expression of LC3a/b for Tfn@GOAPTES treated HeLa cells, suggesting autophagy to be the main cause of the cell death mechanism. Overall, we predict that the present approach can be used as a therapeutic strategy for cancer cell killing via selective induction of a high concentration of intracellular iron.

A strategy for oral delivery of FGF21 for mitigating inflammation and multi-organ damage in sepsis.

Fibroblast growth factor 21 (FGF21) shows great therapeutic potential in metabolic, neurodegenerative and inflammatory diseases. However, current FGF21 administration predominantly relies on injection rather than oral ingestion due to its limited stability and activity post-gastrointestinal transit, thereby hindering its clinical utility. Milk-derived exosomes (mEx) have emerged as a promising vehicle for oral drug delivery due to their ability to maintain structural integrity in the gastrointestinal milieu. To address the challenge associated with oral delivery of FGF21, we encapsulated FGF21 within mEx (mEx@FGF21) to protect its activity post-oral administration. Additionally, we modified the surface of mEx@FGF21 by introducing transferrin (TF) to enhance intestinal absorption and transport, designated TF-mEx@FGF21. In vitro results demonstrated that the surface modification of TF promoted FGF21 internalization by intestinal epithelial cells. Orally administered TF-mEx@FGF21 showed promising therapeutic effects in septic mice. This study represents a practicable strategy for advancing the clinical application of oral FGF21 delivery.

Exploring the Specific Role of Iron Center in the Catalytic Activity of Human Serum Transferrin: CTAB-Induced Conformational Changes and Sequestration by Mixed Micelles.

Conformational changes play a seminal role in modulating the activity of proteins. This concept becomes all the more relevant in the context of metalloproteins, owing to the formation of specific conformation(s) induced by internal perturbations (like a change in pH, ligand binding, or receptor binding), which may carry out the binding and release of the metal ion/ions from the metal binding center of the protein. Herein, we investigated the conformational changes of an iron-binding protein, monoferric human serum transferrin (Fe-hTF), using several spectroscopic approaches. We could reversibly tune the cetyltrimethylammonium bromide (CTAB)-induced conformation of the protein, exploiting the concept of mixed micelles formed by three sequestrating agents: (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) hydrate (CHAPS) and two bile salts, namely, sodium cholate (NaC) and sodium deoxycholate (NaDC). The formation of mixed micelles between CTAB and these reagents (CHAPS/NaC/NaDC) results in the sequestration of CTAB molecules from the protein environment and aids the protein in reattaining its native-like structure. However, the guanidinium hydrochloride-induced denatured Fe-hTF did not acquire its native-like structure using these sequestrating agents, which substantiates the exclusive role of mixed micelles in the present study. Apart from this, we found that the conformation of transferrin (adopted in the presence of CTAB) displays pronounced esterase-like activity toward the para-nitrophenyl acetate (PNPA) substrate as compared to native transferrin. We also outlined the impact of the iron center and amino acids surrounding the iron center on the effective catalytic activity in the CTAB medium. We estimated ∼3 times higher specific catalytic efficiency for the iron-depleted Apo-hTF compared to the fully iron-saturated Fe2-hTF in the presence of CTAB.

A conserved asparagine residue stabilizes iron binding in Manduca sexta transferrin-1.

Transferrin 1 (Tsf1) is an insect-specific iron-binding protein that is abundant in hemolymph and other extracellular fluids. It binds iron tightly at neutral pH and releases iron under acidic conditions. Tsf1 influences the distribution of iron in the body and protects against infection. Elucidating the mechanisms by which Tsf1 achieves these functions will require an understanding of how Tsf1 binds and releases iron. Previously, crystallized Tsf1 from Manduca sexta was shown to have a novel type of iron coordination that involves four iron-binding ligands: two tyrosine residues (Tyr90 and Tyr204), a buried carbonate anion, and a solvent-exposed carbonate anion. The solvent-exposed carbonate anion was bound by a single amino acid residue, a highly conserved asparagine at position 121 (Asn121); thus, we predicted that Asn121 would be essential for high-affinity iron binding. To test this hypothesis, we analyzed the iron-binding and -release properties of five forms of recombinant Tsf1: wild-type, a Y90F/Y204F double mutant (negative control), and three Asn121 mutants (N121A, N121D and N121S). Each of the Asn121 mutants exhibited altered spectral properties, confirming that Asn121 contributes to iron coordination. The N121D and N121S mutations resulted in slightly lower affinity for iron, especially at acidic pH, while iron binding and release by the N121A mutant was indistinguishable from that of the wild-type protein. The surprisingly minor consequences of mutating Asn121, despite its high degree of conservation in diverse insect species, suggest that Asn121 may play a role that is essential in vivo but non-essential for high affinity iron binding in vitro.

Responsively Degradable Nanoarmor-Assisted Super Resistance and Stable Colonization of Probiotics for Enhanced Inflammation-Targeted Delivery.

Manipulation of the gut microbiota using oral microecological preparations has shown great promise in treating various inflammatory disorders. However, delivering these preparations while maintaining their disease-site specificity, stability, and therapeutic efficacy is highly challenging due to the dynamic changes associated with pathological microenvironments in the gastrointestinal tract. Herein, a superior armored probiotic with an inflammation-targeting capacity is developed to enhance the efficacy and timely action of bacterial therapy against inflammatory bowel disease (IBD). The coating strategy exhibits suitability for diverse probiotic strains and has negligible influence on bacterial viability. This study demonstrates that these armored probiotics have ultraresistance to extreme intraluminal conditions and stable mucoadhesive capacity. Notably, the HA-functionalized nanoarmor equips the probiotics with inflamed-site targetability through multiple interactions, thus enhancing their efficacy in IBD therapy. Moreover, timely "awakening" of ingested probiotics through the responsive transferrin-directed degradation of the nanoarmor at the site of inflammation is highly beneficial for bacterial therapy, which requires the bacterial cells to be fully functional. Given its easy preparation and favorable biocompatibility, the developed single-cell coating approach provides an effective strategy for the advanced delivery of probiotics for biomedical applications at the cellular level.

Cellulose nanocrystals from celery stalk as quercetin scaffolds: A novel perspective of human holo-transferrin adsorption and digestion behaviours.

In this study, cellulose nanocrystals (CNCs) were synthesized from celery stalks to be used as the platform for quercetin delivery. Additionally, CNCs and CNCs-quercetin were characterized using the results of scanning electron microscope (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, and zeta potential, while their interactions with human holo-transferrin (HTF) were also investigated. We examined their interaction under physiological conditions through the exertion of fluorescence, resonance light scattering, synchronized fluorescence spectroscopy, circular dichroism, three-dimensional fluorescence spectroscopy, and fluorescence resonance energy transfer techniques. The data from SEM and TEM exhibited the spherical shape of CNCs and CNCs-quercetin and also, a decrease was detected in the size of quercetin-loaded CNCs from 676 to 473 nm that indicated the intensified water solubility of quercetin. The success of cellulose acid hydrolysis was confirmed based on the XRD results. Apparently, the crystalline index of CNCs-quercetin was reduced by the interaction of CNCs with quercetin, which also resulted in the appearance of functional groups, as shown by FTIR. The interaction of CNCs-quercetin with HTF was also demonstrated by the induced quenching in the intensity of HTF fluorescence emission and Stern-Volmer data represent the occurrence of static quenching. Overall, the effectiveness of CNCs as quercetin vehicles suggests its potential suitability for dietary supplements and pharmaceutical products.

Real-time monitoring of Ti(IV) metal ion binding of transferrin using a solid-state nanopore.

Transferrin, a central player in iron transport, has been recognized not only for its role in binding iron but also for its interaction with other metals, including titanium. This study employs solid-state nanopores to investigate the binding of titanium ions [Ti(IV)] to transferrin in a single-molecule and label-free manner. We demonstrate the novel application of solid-state nanopores for single-molecule discrimination between apo-transferrin (metal-free) and Ti(IV)-transferrin. Despite their similar sizes, Ti(IV)-transferrin exhibits a reduced current drop, attributed to differences in translocation times and filter characteristics. Single-molecule analysis reveals Ti(IV)-transferrin's enhanced stability and faster translocations due to its distinct conformational flexibility compared to apo-transferrin. Furthermore, our study showcases solid-state nanopores as real-time monitors of biochemical reactions, tracking the gradual conversion of apo-transferrin to Ti(IV)-transferrin upon the addition of titanium citrate. This work offers insights into Ti(IV) binding to transferrin, promising applications for single-molecule analysis and expanding our comprehension of metal-protein interactions at the molecular level.

Holo/apo conversion two-dimensional urea PAGE for speciation of Fe3+-bound transferrin in serum.

This paper presents holo/apo conversion two-dimensional urea polyacrylamide gel electrophoresis (HAC-2D urea PAGE) as a novel method for speciating Fe3+-bound transferrin (Tf) species in biological samples, with a combination of metal ion contaminant sweeping (MICS) technique and Fe3+ detection PAGE. In the HAC-2D urea MICS-PAGE approach, HAC was performed to dissociate all the Fe3+ ions bound to Tf from the Fe-Tf species, during a two-step urea PAGE. Using this method, Fe2-Tf, FeN-Tf, and FeC-Tf (holo-Tf, Fe3+-bound Tf attached to N-lobe, and Fe3+-bound Tf attached C-lobe, respectively) were completely isolated based on the difference in the higher-order structure of Tf, visible as horizontally aligned spots off the diagonal. The Fe3+ ions bound to Tf in each gel fraction were determined using PAGE with a fluorescent probe. Without the MICS technique, which electrophoretically removes all contaminant Fe3+ ions from the gel medium to ensure accurate determination of the Fe3+ concentration, it becomes challenging to precisely measure the distribution of metalloprotein species owing to the contaminants. Finally, the distribution of each Fe-bound Tf in a standard human serum sample was successfully determined by complete separation from large amounts of coexisting proteins, and the free Fe3+ concentration in the serum was estimated.

Chitosan derivatives as dynamic coatings for transferrin glycoform separation in capillary electrophoresis.

Chitosan and its derivatives are interesting biopolymers for different field of analytical chemistry, especially in separation techniques. The present study was aimed at testing chitosan water soluble derivatives as dynamic coating agents for application to capillary electrophoresis. In particular, chitosan was modified following three different chemical reactions (nucleophilic substitution, reductive amination, and condensation) to introduce differences in charge and steric hindrance, and to assess the effect of these physico-chemical properties in capillary electrophoresis. The effects were tested on the capillary electrophoretic separation of the glycoforms of human transferrin, an important iron-transporting serum protein, one of which, namely disialo-transferrin (CDT), is a biomarker of alcohol abuse. Chitosan derivatives were characterized by using NMR and 1H NMR, HP-SEC-TDA, DLS, and rheology. The use of these compounds as dynamic coatings in the electrolyte running buffer in capillary electrophoresis was tested assessing the peak resolution of the main glycoforms of human transferrin and particularly of disialo-transferrin. The results showed distinct changes of the peak resolution produced by the different derivatives. The best results in terms of peak resolution were achieved using polyethylene glycol (PEG)-modified chitosan, which, in comparison to a reference analytical approach, provided an almost baseline resolution of disialo-transferrin from the adjacent peaks.

Understanding protein-nanoparticle interactions leading to protein corona formation: In vitro - in vivo correlation study.

Nanoparticles (NPs) in contact with biological fluids form a biomolecular corona through interactions with proteins, lipids, and sugars, acquiring new physicochemical properties. This work explores the interaction between selected proteins (hemoglobin and fetuin-A) that may alter NP circulation time and NPs of different surface charges (neutral, positive, and negative). The interaction with key proteins albumin and transferrin, the two of the most abundant proteins in plasma was also studied. Binding affinity was investigated using quartz crystal microbalance and fluorescence quenching, while circular dichroism assessed potential conformational changes. The data obtained from in vitro experiments were compared to in vivo protein corona data. The results indicate that electrostatic interactions primarily drive protein-NP interactions, and higher binding affinity does not necessarily translate into more significant structural changes. In vitro and single protein-NP studies provide valuable insights that can be correlated with in vivo observations, opening exciting possibilities for future protein corona studies.

Spectroscopy and molecular simulation on the interaction of Nano-Kaempferol prepared by oil-in-water with two carrier proteins: An investigation of protein-protein interaction.

In this work, the interaction of human serum albumin (HSA) and human holo-transferrin (HTF) with the prepared Nano-Kaempferol (Nano-KMP) through oil-in-water procedure was investigated in the form of binary and ternary systems by the utilization of different spectroscopy techniques along with molecular simulation and cancer cell experiments. According to fluorescence spectroscopy outcomes, Nano-KMP is capable of quenching both proteins as binary systems by a static mechanism, while in the form of (HSA-HTF) Nano-KMP as the ternary system, an unlinear Stern-Volmer plot was elucidated with the occurrence of both dynamic and static fluorescence quenching mechanisms in the binding interaction. In addition, the two acquired Ksv values in the ternary system signified the existence of two sets of binding sites with two different interaction behaviors. The binding constant values of HSA-Nano KMP, HTF-Nano-KMP, and (HSA-HTF) Nano-KMP complexes formation were (2.54 ± 0.03) × 104, (2.15 ± 0.02) × 104 and (1.43 ± 0.04) × 104M-1at the first set of binding sites and (4.68 ± 0.05) × 104 M-1 at the second set of binding sites, respectively. The data of thermodynamic parameters confirmed the major roles of hydrogen binding and van der Waals forces in the formation of HSA-Nano KMP and HTF-Nano KMP complexes. The thermodynamic parameter values of (HSA-HTF) Nano KMP revealed the dominance of hydrogen binding and van der Waals forces in the first set of binding sites and hydrophobic forces for the second set of binding sites. Resonance light scattering (RLS) analysis displayed the existence of a different interaction behavior for HSA-HTF complex in the presence of Nano-KMP as the ternary system. Moreover, circular dichroism (CD) technique affirmed the conformational changes of the secondary structure of proteins as binary and ternary systems. Molecular docking and molecular dynamics simulations (for 100 ns) were performed to investigate the mechanism of KMP binding to HSA, HTF, and HSA-HTF. Next to observing a concentration and time-dependent cytotoxicity, the down regulation of PI3K/AkT/mTOR pathway resulted in cell cycle arrest in SW480 cells.

Fabrication, in-silico, in-vitro, and in-vivo characterization of transferrin-targeted micelles containing cisplatin and gadolinium for improved theranostic applications in lung cancer therapy.

The targeted delivery of therapeutic and imaging agents is quite challenging in lung cancer therapy. Thus, lung cancer causes high mortality across the world. Herein, we developed TPGS-PF127 micelles containing cisplatin (CDDP) as a model anticancer drug and gadolinium (Gd) as a diagnostic agent by a slightly modified solvent casting method, further, the surface of the micelles was modified using TPGS-transferrin (TPGS-Tf) conjugate to improve targeted delivery of micelles to the lung cancer cells. Prior to this, the binding affinity of Tf over TfR (1E7U) and TfR (1E8W) was investigated with the help of in-silico studies. In-silico results showed good docking scores -7.8 and -7.2 kcal/mol of Tf -ligand towards 1E8W and 1E7U respectively promoting PI3K inhibition. Micelles have shown an average particle size range of 80-200 nm and have shown spherical morphology. The encapsulation efficiency of cisplatin (CDDP) in the CPT, CGPT, and CGPT-Tf micelles ranged from 75.63 % ± 1.58 % to 85.07 % ± 2.65 %. Furthermore, the encapsulation efficiency of gadolinium (Gd) in the CGPT and CGPT-Tf micelles was found to be 67.50 ± 0.32 % and 62.52 ± 0.52 %, respectively. CGPT-Tf micelles exhibited sustained release fashion for CDDP up to 48 h in physiological conditions. In the cytotoxicity study, CGPT-Tf micelles achieved higher cytotoxicity and caused a more antiproliferative effect in A549 cells compared to a commercial CDDP injection (Ciszest 50), after 24 h of treatment. Furthermore, the pharmacokinetic studies have proven the pharmacological effectiveness of developed CGPT-Tf micelles by achieving higher Cmax, Tmax, t1/2, and MRT of CDDP in systemic circulation compared to its counterparts and Ciszest 50. In lung theranostic observations, a higher internalization of Gd was noted in CGPT-TF compared to free Gd. The biochemical studies have proved the biocompatibility of developed micelles formulations by showing no sign of toxicity in the lungs. The developed micelles have great potential to be utilized in treating and diagnosing a wide variety of cancers.

Comprehensive insight into the molecular interaction of rutin with human transferrin: Implication of natural compounds in neurodegenerative diseases.

Neurodegeneration, a process of irreversible neuronal damage, is characterized by a damaged neuronal structure and function. The interplay between various proteins maintains homeostasis of essential metals in the brain, shielding neurons from degeneration; human transferrin (Htf) is essential in maintaining iron homeostasis. Any disruption in iron homeostasis results in the development of neurodegenerative diseases (NDs) and their pathology, mainly Alzheimer's disease (AD). Rutin is a known compound for its neuroprotective effects. In this work, we deciphered the binding of rutin with Htf in a bid to understand the interaction mechanism. The results of fluorescence and UV-vis spectroscopy demonstrated strong interaction between rutin and Htf. The enthalpy change (∆H°) and entropy change (∆S°) analysis demonstrated hydrophobic interactions as the prevalent forces. The binding mechanism of rutin was further assessed atomistically by molecular docking and extensive 200 ns molecular dynamic simulation (MD) studies; molecular docking showed binding of rutin within Htf's binding pocket. MD results suggested that binding of rutin to Htf does not cause significant structural switching or disruption of the protein's native packing. Overall, the study deciphers the binding of rutin with hTf, delineating the binding mechanism and providing a platform to use rutin in NDs therapeutics.

Affinity Maturated Transferrin Receptor Apical Domain Blocks Machupo Virus Glycoprotein Binding.

Transferrin receptor 1 (TfR) delivers iron across cellular membranes by shuttling the ion carrier protein transferrin. This ability to deliver large protein ligands inside cells is taken advantage of by pathogens to infiltrate human cells. Notably, the receptor's outermost ectodomain, the apical domain, is used as a point of attachment for several viruses including hemorrhagic arenaviruses. To better understand interactions with the receptor it would be advantageous to probe sequence determinants in the apical domain with viral spike proteins. Here, we carried out affinity maturation of our computationally designed apical domain from human TfR to identify underlying driving forces that lead to better binding. The improved variants were confirmed by in vitro surface plasmon resonance measurements with dissociation constants obtained in the lower nanomolar range. It was found that the strong binding affinities for the optimized variants matched the strength of interactions with the native receptor. The structure of the best variant was determined experimentally indicating that the conformational change in the hairpin binding motif at the protein-protein interface plays a crucial role. The experimental methodology can be straightforwardly applied to other arenavirus or pathogens that use the apical domain. It can further be useful to probe host-virus compatibility or therapeutic strategies based on the transferrin receptor decoys.

Nose to Brain Delivery of Transferrin conjugated PLGA nanoparticles for clonidine.

The present work focuses on the development of surface modified transferrin PLGA nanoparticles loaded with clonidine for nose to brain delivery. The CLD-Tf-PLGA-NPs were developed using double emulsification, followed by solvent evaporation and characterization. Particle size, PDI and Zeta potential of the nanoparticles was 199.5 ± 1.36 nm, 0.291, -17.4 ± 6.29 mV respectively with EE% 86.2 ± 2.12 %, and DL%, 7.8 ± 0.48 %. TEM, SEM and FTIR analysis were carried out to confirm the size and transferrin coating over the surface of nanoparticles. In-vitro drug release profile were studied in PBS (pH 7.4) and SNF (pH 5.5) for 72 h and highest release was observed in PBS 89.54 ± 3.17 %. Cellular assays were conducted on Neuro-2a cells to check the cytotoxicity and uptake of Tf-modified PLGA nanoparticles and the cell viability% was obtained to be 61.85 ± 4.48 % even at maximum concentration (40Cmax) with uptake of approximately 97 %. Histopathological studies were also performed to identify the cytotoxicity on nasal epithelium along with in-vivo biodistribution and pharmacodynamics studies to assess the concentration of drug in the mice brain and behavioural responses after intranasal delivery of surface modified nanoparticles. The results showed significant increase in concentration of drug in brain and behavioural improvements in mice (p < 0.05).

Interaction Between the Transferrin Protein and Plutonium (and Thorium), What's New?

Transferrin (Tf) is a glycoprotein that transports iron from the serum to the various organs. Several studies have highlighted that Tf can interact with metals other than Fe(III), including actinides that are chemical and radiological toxics. We propose here to report on the behavior of Th(IV) and Pu(IV) in comparison with Fe(III) upon Tf complexation. We considered UV-Vis and IR data of the M2 Tf complex (M=Fe, Th, Pu) and combined experimental EXAFS data with MD models. EXAFS data of the first M-O coordination sphere are consistent with the MD model considering 1 synergistic carbonate. Further EXAFS data analysis strongly suggests that contamination by Th/Pu colloids seems to occur upon Tf complexation, but it seems limited. SAXS data have also been recorded for all complexes and also after the addition of Deferoxamine-B (DFOB) in the medium. The Rg values are very close for apoTf, ThTf and PuTf, but slightly larger than for holoTf. Data suggest that the structure of the protein is more ellipsoidal than spherical, with a flattened oblate form. From this data, the following order of conformation size might be considered:holoTf

Macromolecular Crowding Promotes Re-entrant Liquid-Liquid Phase Separation of Human Serum Transferrin and Prevents Surface-Induced Fibrillation.

Protein aggregation and inactivation upon surface immobilization are major limiting factors for analytical applications in biotechnology-related fields. Protein immobilization on solid surfaces often requires multi-step surface passivation, which is time-consuming and inefficient. Herein, we have discovered that biomolecular condensates of biologically active human serum transferrin (Tf) can effectively prevent surface-induced fibrillation and preserve the native-like conformation of phase-separated Tf over a period of 30 days. It has been observed that macromolecular crowding promotes homotypic liquid-liquid phase separation (LLPS) of Tf through enthalpically driven multivalent hydrophobic interactions possibly via the involvement of its low-complexity domain (residues 3-20) containing hydrophobic amino acids. The present LLPS of Tf is a rare example of salt-mediated re-entrant phase separation in a broad range of salt concentrations (0-3 M) solely via the involvement of hydrophobic interactions. Notably, no liquid-to-solid-like phase transition has been observed over a period of 30 days, suggesting the intact conformational integrity of phase-separated Tf, as revealed from single droplet Raman, circular dichroism, and Fourier transform infrared spectroscopy measurements. More importantly, we discovered that the phase-separated condensates of Tf completely inhibit the surface-induced fibrillation of Tf, illustrating the protective role of these liquid-like condensates against denaturation and aggregation of biomolecules. The cell mimicking compact aqueous compartments of biomolecular condensates with a substantial amount of interfacial water preserve the structure and functionality of Tf. Our present study highlights an important functional aspect of biologically active protein condensates and may have wide-ranging implications in cell physiology and biotechnological applications.

FerryCalin: an Engineered Lipocalin Protein Directed against the Transferrin Receptor with Potential for Brain Drug Delivery.

The transferrin receptor (TfR) mediates transcytosis across the blood-brain barrier (BBB), which offers a promising approach for the non-invasive delivery of therapeutics into the brain parenchyma. Employing the recombinant homodimeric murine TfR ectodomain, prepared in a biochemically functional state, we have selected a cognate Anticalin via phage display and bacterial cell surface display from a random library based on the human lipocalin 2 (Lcn2). After affinity maturation, several engineered lipocalin variants were identified that bind murine TfR in a non-competitive manner with the natural ligand (transferrin ⋅ Fe3+ ), among those an Anticalin - dubbed FerryCalin - exhibiting a dissociation constant (KD ) of 3.8 nM. Epitope analysis using the SPOT technique revealed a sequential epitope in a surface region of TfR remote from the transferrin-binding site. Due to the fast kon rate and short complex half-life, as evidenced by real-time surface plasmon resonance (SPR) measurements, FerryCalin, or one of its related mutants, shows characteristics as a potential vehicle for the brain delivery of biopharmaceuticals.