Fibril-Droplet Relationship Through Liquid-Liquid Phase Separation; A BSA-MOF Case

Fibril-Droplet Relationship Through Liquid-Liquid Phase Separation; A BSA-MOF Case | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Fibril-Droplet Relationship Through Liquid-Liquid Phase Separation; A BSA-MOF Case Amirhossein Latifi, Elnaz Hosseini, Hossein Daneshgar, Mohammad Edrisi, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3953695/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract By providing a suitable platform that accelerates early-stage occurrences needed for triggering protein aggregation, liquid-liquid phase separation (LLPS) has the potential to promote this phenomenon. Among different proteins which their condensation propensity has been investigated, bovine serum albumin (BSA) has attracted attentions because of its globular and stable structure. BSA undergoes phase separation and phase transition in the presence of polyethylene glycol as a molecular crowder. The control of hydrophobicity, hydrogen bonding, and electrostatic forces as the main forces that conduct LLPS were provided by functionalizing a UiO-66 metal-organic framework (MOF) with -NH2 and -COOH functional groups. This work evaluated how the functionalized UiO-66 with -NH2 modulates the LLPS of BSA. Successful synthesis and functionalization of UiO-66 were confirmed using various physical and chemical analyses. Optical and fluorescence microscopy images correlated BSA LLPS droplet size with spectroscopic measurements of resultant BSA fibrils. UiO-66-NH2 was found to cause significant conformational changes in BSA, resulting in a decrease in its LLPS and aggregation rate, as demonstrated by various biophysical methods. This study suggested that more hydrophobic surrounding micro-environments caused by UiO-66-NH2 inhibited BSA LLPS, leading to decreased droplet size and number. The direct correlation between droplet size and fibril length also confirmed the role of LLPS as an important alternative pathway enabling fibril formation even in globular proteins. Fibril droplet BSA MOF UiO-66 LLPS Hydrophobicity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Liquid-liquid phase separation (LLPS) is a crucial biological process in cells [ 1 ] that drives intracellular compartmentalization [ 2 ], organizes biochemical reactions, signaling cascades [ 3 ], and enables proteins to gather in condensed droplet-like regions as water is expelled through volume exclusion [ 4 ]. This region coexists with a less dense phase. The concentrated protein content in the condensed phase can facilitate amyloid aggregation not only for disordered aggregation-prone proteins [ 5 ] like tau[ 6 ], alpha-synuclein[ 7 ], and TDP-43[ 8 ] but also for ordered globular proteins such as lysozyme and serum albumin [ 4 , 9 ]. Thus, LLPS can promote amyloid fibril formation[ 9 ], which is implicated in cancer, Alzheimer’s, and Parkinson's diseases[ 10 , 11 ], diabetes, and associated complications [ 12 ]. Bovine serum albumin (BSA) is a model globular protein to understand the main factors behind LLPS with a well-defined tertiary structure with 583 amino acids constrained by 17 disulfide bridges into a predominantly helical, stable conformation [ 13 , 14 ]. Elucidation of the governing protein-protein and protein-solvent interactions enabling spontaneous BSA demixing into highly concentrated liquid droplets, which paves the way for amyloid fibrillogenesis, requires control over solution conditions modulating protein conformation and inter and intramolecular interactions. Metal-organic frameworks (MOFs) comprise metal ion coordination complexes interconnected into crystalline lattices containing nanoporous channels and cavities capable of sequestering molecules. UiO-66 is one such MOF constructed from Zr 6 O 4 (OH) 4 clusters linked by terephthalate organic ligands, forming a structure with both octahedral and tetrahedral pores accessible through small triangular windows [ 15 ]. With a high surface area exceeding 1200 m 2 /g and pore volumes around 0.5 cm3/g arising from its high porosity, UiO-66 is ideally suited for biosensing [ 16 ], encapsulating and transporting cargo [ 17 , 18 ]. Recent progresses in implication of MOFs and specifically UiO-66 family in the diagnosis, treatment and detection of neurodegenerative diseases such as Alzheimer disease [ 19 , 20 ] in their early stages by targeting LLPS prone proteins and peptides[ 21 ], makes them an adequate candidate for modulating this process. Appending functional groups (e.g. −NH 2 , −COOH) onto the UiO-66 scaffold can modulate loading capacity and release kinetics to optimize delivery applications [ 22 , 23 ]. For instance, amine groups boost storage via increased protein affinity while carboxyl moieties confer higher hydrophilicity and electrostatic attraction to cationic molecules. Capitalizing on such functionalization furnishes means to fine-tune UiO-66 properties and govern encapsulated cargo interactions [ 24 ]. In this study, synthesized MOFs functionalized with and without -NH 2 and -COOH have been used for investigating LLPS and fibrillation of BSA. Using UiO-66 MOFs with different functional groups allowed us to investigate the role of hydrophobic/hydrophilic and electrostatic interactions in the phase separation process. Also, MOFs effect on phase separation determined droplets of different sizes, paving the way for investigating the role of LLPS as an alternative pathway for amyloid fibrillation. Experimental section Material Fatty acid-free BSA catalog number 1.12018 was obtained from Sigma Aldrich, and 50 mM phosphate buffer was prepared at pH 7 using NaH 2 PO 4 and Na 2 HPO 4 . Polyethylene glycol (PEG) (MW 1450 g/mol), Thioflavin T (ThT), 1-anilino-8-naphthalene sulfonate (ANS), NaCl, NaH 2 PO 4 , and Na 2 HPO 4 were acquired from Merck, and all components for MOF synthesis, including ZrCl 4 , 1,2,4,5-benzene tetracarboxylic acid (BDC), Acetic acid, Dimethylformamide (DMF) purchased from Merck, FITC conjugated BSA catalog number: A23015 purchased from Thermo Fisher. Preparing BSA and Concentration determination A proper amount of BSA protein was dissolved in 50 mM phosphate buffer (pH 7.4) with a target concentration of 5 mM, and the concentration was confirmed by UV-Vis spectrophotometer (UV-3100 Shimadzu) measurement at 280 nm assuming the extinction coefficient as 43824 M − 1 cm − 1 . BSA concentration was fixed at 450 µM for all samples. MOF synthesis UiO-66 and UiO-66-NH 2 UiO-66 and UiO-66-NH 2 were synthesized according to the previous reports [ 15 , 25 , 26 ]. Briefly, ZrCl 4 (166.6 mg, 0.72 mmol) was dissolved in 20 ml DMF and stirred for 20 min at 40°C. Then, for UiO-66 BDC (82 mg, 1 mmol) and for UiO-66-NH 2 , BDC-NH 2 (181.1 mg, 1 mmol) was added, and the resultant mixture was sonicated in an ultrasonic bath for 20 min. After that, the resulting mixture was heated at 120°C for 24 hours. In the next step, methanol was added, and the solution was centrifuged at 7000 rpm for 5 min. This process was repeated three times, and the remaining solid pellet was dried under a vacuum. UiO-66-(COOH) 2 To synthesize this MOF, first, ZrCl 4 (0.304 g) was dissolved in 15 ml deionized water and stirred by using a thermo stirrer [ 27 ]. BDC (0.318 g) separately was dissolved in 15 ml deionized water and 10 ml acetic acid, again mixed using a thermo stirrer. The second mixture was sonicated for 10 minutes in an ultrasonic bath. Then, the two solutions were combined and heated at 110°C for 24 hours. After cooling down to room temperature, the product was centrifuged at 7000 rpm for 5 min, and the supernatant was decanted. The remaining solid was washed three times with methanol, centrifuging, and decanting after each wash. Finally, the product dried at room temperature for 24 hours. MOFs stock preparation UiO-66, UiO-66-(COOH) 2, and UiO-66-NH 2 stocks were prepared by mixing 2 mg of each sample with 1 ml phosphate buffer (pH 7.4) and, for appropriate dispersion, sonicated (UHP-400 Ultrasonic Homogenizer) for at least 30 min with probe sonication. NaCl stock preparation 1 M NaCl stock was prepared by dissolving 58.44 mg of NaCl in 1 ml 50 mM phosphate buffer (pH 7.4). Polyethylene glycol stock preparation A polyethylene glycol (PEG) stock solution was prepared by dissolving 800 mg PEG 4000 Da in 1 ml phosphate buffer (pH 7.4). Samples preparation Depending on the final volume of each experiment, the proper volume of protein, salt, MOFs, and PEG have been used to reach 450 µM, 300 mg/ml, 0.28 mg/ml, and 200 mg/ml as the final concentration, respectively. The addition order was: 1) buffer, 2) salt, 3) MOF, 4) protein. PEG was added whenever LLPS induction was needed. This protocol ensured controlled protein-MOF interaction before undergoing LLPS. To further promote interactions, samples were incubated overnight at 8°C, 300 rpm in a thermoshaker (Eppendorf Thermomixer C) before each assay. Droplet formation experiments To investigate the ionic strength impact on the propensity of BSA to undergo LLPS, solutions with varying sodium chloride (NaCl) concentrations (50–300 mM) were prepared. The extent of LLPS was assessed by measuring droplet diameters from optical microscopy (Zeiss Axioskop 2 FS plus) images and using ImageJ software. This enabled quantitative comparisons of phase separation intensity across different NaCl concentrations. Protein conformational changes To probe BSA tertiary structural changes, extrinsic fluorescence spectroscopy utilizing 1-anilino-8-naphthalene sulfonate (ANS), and intrinsic fluorescence spectroscopy were employed. ANS fluorescence was recorded at 340 nm excitation and 480 nm emission. Intrinsic fluorescence used 280 nm excitation and 340 nm emission using an Agilent BioTek Synergy H4 Hybrid Microplate Reader. Furthermore, Far UV circular dichroism (CD) spectroscopy was recorded at room temperature from 190–260 nm using a Jasco J-810 to investigate alterations in the secondary structure content upon treatments. Samples were diluted to reduce interference from turbidity caused by droplet formation by the ratio of 1:25 and 1:80 for fluorescence and CD spectroscopy, respectively. Protein fluorescence labeling To visually confirm condensed protein within liquid droplets, BSA was fluorescently labeled with fluorescein isothiocyanate (FITC). Its excitation and emission maximums were 491 nm and 516 nm here, respectively. A 10:990 ratio of FITC-BSA to unlabeled BSA was used for observation. This low FITC level provided sufficient fluorescence to detect droplets while minimizing impacts on phase separation. MOF characterization The morphology of the samples was visualized using Field Emission Scanning Electron Microscopy (FESEM) with a TESCAN MIRA3 SEM. Crystallinity was determined by X-ray diffraction (XRD) analysis conducted on an Inel EQUINOX3000 X-ray diffractometer using copper K-alpha radiation (wavelength = 1.5418 Angstroms) over a range of 5 to 80 theta. To analyze the sample chemical composition, Fourier transform infrared (FT-IR) spectroscopy was performed on a BRUKER Equinox 55 spectrometer with a spectral range of 400–4000 cm − 1 using potassium bromide plates. Physicochemical properties The contact angle and zeta potential have been operated as indices of hydrophobicity and surface charge of MOFs, respectively. The contact angle measurement was performed by a Wilhelmy plate method using a force tensiometer (Kruss K100C). For this purpose, MOFs with 2 mg/ml concentration has been dispersed in 2 ml of the buffer. Zeta potential measurements were performed using the Laser Doppler electrophoresis technique on a HORIBA SZ100 Z instrument. The surface charge of the MOFs was determined by running the measurements across an applied voltage spectrum spanning from − 200 to + 200 mV. Results FESEM results Electron microscopy images indicated that synthesized MOFs were semi-spherical, and adding NH 2 and COOH functional groups did not affect the MOF's morphology (Fig. 1 , A, B, and C). The EDS element analysis results confirmed the presence of essential functional group elements (Fig. 1 , D, E, and F). Based on Fig. 1 , adding functional groups didn't change UiO-66 clusters' morphology and/or size significantly in this study, which is consistent with previous reports[ 23 ]. In this case, other functional groups' physicochemical properties may determine the differences observed in droplet and fibril dimensions. XRD patterns X-ray diffraction (XRD) analysis determined the crystal structure of UiO-66 (Fig. 1 G). UiO-66 matched literature patterns with peaks at 7.4, 8.5, 14.8, 18.5, 25.4, and 31.1, confirming successful synthesis [ 17 ]. UiO-66-(COOH) 2 showed a similar XRD pattern to UiO-66 [ 28 , 29 ]. The NH 2 -grafted framework also matched the original UiO-66, indicating that NH 2 groups did not modify crystal structure [ 23 , 25 ]. FTIR spectrum The Fourier transform infrared (FTIR) spectra of the synthesized MOF samples (Fig. 1 H) reveal distinct bands attributable to specific chemical structures and functional groups. For UiO-66, the band at 1568 cm − 1 indicates coordination of -COOH with Zr 4+ . Bands at 1506 cm − 1 and 1395 cm − 1 correspond to vibrations of the benzene ring and carboxylate group of the organic linker [ 17 ]. Additional bands at 1019 cm − 1 , 760–780 cm − 1 , and 660 cm − 1 arise from C-H vibrations. UiO-66-NH 2 and UiO-66-(COOH) 2 exhibit similar spectra, with extra bands due to grafted functional groups. The band at 1620 cm − 1 in UiO-66-NH 2 originates from the N-H bending of aromatic amines. The 1252 cm − 1 band indicates C-N stretching[ 23 , 25 ]. For UiO-66-(COOH) 2 , the band at 1712 cm − 1 confirms the presence of uncoordinated -COOH groups. The FTIR data confirms the complete, uncorrupted synthesis of the MOF samples with full retention of chemical structures. [ 28 , 29 ] BSA – MOF interaction In order to investigate the strength of interaction between MOFs and BSA an experiment was design as follow. BSA was incubated overnight with varying concentrations of UiO-66, UiO-66-NH 2 , and UiO-66-(COOH) 2 MOFs. Subsequently, the samples were centrifuged at 18,000 rpm for 5 minutes. The rationale was that if robust interactions such as bond formation occurred between any of the MOFs and BSA, then after centrifugation BSA would co-sediment with the MOFs due to the high density of the MOF metal clusters. In this case, increasing MOF concentration would increase co-sedimentation, which could be quantified by measuring BSA concentration in the supernatant using UV-Vis spectroscopy. The concentrations used for each of MOFs UiO-66, UiO-66-(COOH) 2 , UiO-66-NH 2 was as mentioned in Table 1 . Table 1 Different concentrations of MOFs in MOF-BSA interaction experiment. Mg.ml − 1 0.04 0.12 mg.ml − 1 mg.ml − 1 0.2 0.28 mg.ml − 1 mg.ml − 1 0.36 Mg.ml − 1 0.44 In the next step, intrinsic fluorescence emission and ANS-binding assays were performed to evaluate structural changes in the protein. Intrinsic tryptophan fluorescence was measured at 340 nm, corresponding to the emission peak wavelength of tryptophan residues in BSA. This allowed detection of changes in the local environment of aromatic amino acids like tryptophan. Additionally, ANS binding assays were carried out, with fluorescence recorded at 440 nm. Since hydrophobic dye ANS binds to exposed hydrophobic regions on protein surfaces, changes in ANS fluorescence indicate alterations of surface hydrophobicity. The results of UV-vis spectroscopy, intrinsic fluorescence, and ANS-binding assays after incubating BSA with UiO-66, UiO-66-(COOH) 2 , and UiO-66-NH 2 followed by centrifugation to remove MOFs are shown in Fig. 2 . The UV-vis absorption profiles indicate no decrease compared to BSA alone. Rather, in most cases, a slight increase in UV-vis absorbance is observed upon addition of MOFs. The intrinsic fluorescence results reveal decreased emission intensities for BSA incubated with the NH 2 -functionalized MOF at all concentrations tested. A similar effect is seen for some concentrations of non-functionalized UiO-66. In contrast, the COOH-functionalized MOF generally increases or does not affect intrinsic fluorescence, except at the highest 0.44 mg/mL concentration. Finally, ANS fluorescence is enhanced in the presence of UiO-66-NH 2 , reflecting greater exposure of hydrophobic regions. However, the differences in ANS emission are not significant. These results demonstrate that MOFs neither form strong bonds with BSA proteins and nor cause significant permanent conformational change in protein structure. The BSA phase separates into liquid droplets. Increasing NaCl concentration from 50 to 300 mM led to larger BSA droplets (Fig. 3 A), with 300 mM NaCl resulting in significantly larger droplet diameters (Fig. 3 B). Since the total BSA concentration was held constant, this suggests that more BSA protein partitioned from solution into droplets at 300 mM NaCl to form the larger condensed phases observed. FITC-labeled BSA has been used to confirm the protein nature of droplets (Fig. 3 C). Fluorescence microscopy using the FITC-BSA showed intense fluorescence within the liquid droplets, verifying that condensed protein accumulation had occurred. Furthermore, larger droplets exhibited higher fluorescence intensity compared to smaller droplets. Kinetics of droplet size evolution To examine the dynamic nature of liquid-liquid phase separated proteins, it was necessary to track droplet size over time and visualize growth [ 30 ]. Optical microscopy visualized droplets at 0, 2, 6, and 24 hours after inducing phase separation (Fig. 4 ). Over 24 hours, the number of droplets decreased while the size of remaining droplets increased, consistent with the coalescence and Ostwald ripening phenomenon [ 31 , 32 ]. By 24 hours, only a sparse population of enlarged droplets remained. Relating LLPS droplet size to subsequent fibril length provided insights into their relationship under different conditions [ 33 ]. To have a better quantitative comparison about the impact of UiO-66, UiO-66-NH 2 and UiO-66-(COOH) 2 on the size of droplets, their diameters were measured by ImageJ software plotted in Fig. 4 B. The result was in consistent with fluorescence microscopy results. Conformational changes due to protein interaction with MOFs and LLPS Intrinsic and ANS fluorescence probed BSA tertiary structure and surface hydrophobicity changes after phase separation (Fig. 6 A and B). Also, UiO-66-NH 2 slightly decreased ANS emission and increased intrinsic fluorescence, but these changes were not significant which implies that MOFs did not cause any significant conformational change. Circular dichroism (Fig. 6 C) monitored BSA secondary structure changes after separation. The signal appearance remained constant despite a slight intensity decrease, likely due to the presence of PEG. [ 34 ] Fibril formation kinetics After phase separation (7 days of incubation at room temperature), Thioflavin T (ThT) stained samples were observed by fluorescence microscopy (Fig. 6 A). Fibril length measurement demonstrated that UiO-66-NH 2 significantly decreased BSA fibril length in comparison with other synthesized MOFs and the sample without MOF (Fig. 6 B). Fibrils from the smallest droplets induced by UiO-66-NH 2 were significantly shorter than those of the control. UiO-66-NH 2 had lower ThT emission than the control, probably correlating with its smaller and fewer droplets. However, non-functionalized UiO-66 had a more significant ThT increase versus control, indicating fluorescence depends on both droplet size and number. Zeta potential and contact angels’ results In this case, the functional groups' physico-chemical properties determined the differences in droplet and fibril dimensions (Table 2 ). Measuring zeta potential and contact angle revealed that UiO-66-NH 2 had the highest contact angle and, thus, the greatest hydrophobicity, and UiO-66-(COOH) 2 had the highest zeta potential[ 35 – 38 ]. The zeta potential results at pH = 7.4, as shown in Table 2 , indicate that the potential of frameworks with a carboxyl functional group is more negative. As seen in Table 1 , despite the significant difference in the potential of the carboxyl group, adding the NH 2 functional group did not cause a significant change compared to the state without the functional group. Table 1 also indicates that the contact angle for UiO-66, UiO-66-NH 2 , and UiO-66-(COOH) 2 were obtained as 26.19, 42.98, and 24.9, respectively. Table 2 Results of zeta potential and contact angle measurements. MOFs Zeta potential (mV) Contact angle (rad) UiO-66 -18.53 26 UiO-66-(COOH) 2 -27.2 25 UiO-66NH 2 -19.46 43 Discussion Comparison of NaCl concentrations reveals more extensive liquid-liquid phase separation (LLPS) at higher ionic strengths, aligning with BSA's isoelectric point (around 4.7) where repulsive forces typically hinder aggregation. Higher salt likely screens this repulsion, allowing attractive hydrophobic and van der Waals forces to dominate, promoting BSA association into expanding droplets rather than new droplet formation [ 39 , 40 ]. Turning to UiO-66 and its functionalized forms, no strong bonds were observed with BSA protein. Notably, NH2-functionalized UiO-66 successfully prevented LLPS-induced droplet formation. Conversely, COOH groups resulted in larger droplets compared to the unfunctionalized version. The correlation of increased fluorescence with larger droplets suggests higher protein content yielding more fluorescence emission [ 41 ]. Introducing the NH2 group significantly reduced both the number and size of droplets (Fig. 4 C), indicating its role as an LLPS inhibitor. Interestingly, droplet size also decreased in the absence of functional groups, but with an increased number of droplets [ 42 ]. Figure 4 D clearly shows the decrease in droplet number with UiO-66-NH2. This decrease implies that UiO-66-NH2 slows down BSA aggregation, further confirmed by a reduced apparent aggregation constant in its presence (slopes in Fig. 6 C). Importantly, fibrils formed with UiO-66-NH2 (inducing the smallest droplets) were significantly shorter than the control. This aligns with previous studies on the role of functional groups in amyloid β fibril size, where NH2 groups shortened the resultant fibrils [45]. Notably, Figs. 4 B and 6 B exhibit a similar size pattern (Control > UiO-66-(COOH)2 > UiO-66 > UiO-66-NH2), suggesting a correlation between droplet and fibril size. Furthermore, the number of fibrils, at least for UiO-66-NH2, seems correlated with the number of droplets. This suggests that smaller BSA precursor droplets yield shorter BSA fibrils, implying that droplets provide a platform for further interactions triggering BSA aggregation at room temperature.Finally physico-chemical investigations reveald that increasing hydrophobicity decreases proteins' propensity to aggregate into droplets while raising environmental charge increases protein aggregation and phase separation. Based on the sticker-spacer model [ 39 ], the results indicate that hydrophobic interactions play a major role in BSA phase separation. The model prioritizes intermolecular associations over intramolecular hydrophobic interactions under conditions promoting aggregation. Therefore, these findings agree that exposed hydrophobic regions on BSA drive intermolecular interactions and subsequent phase separation [36, 46]. As seen here, increasing salt concentration that disrupts electrostatic interactions by raising ionic strength allowed favorable hydrophobic interactions to overcome the electrostatic interactions, which promote protein solubility, leading to phase separation [ 42 ]. Similarly, the increased hydrophobicity of UiO-66-NH 2 likely disturbed the crucial hydrophobic interactions for phase separation. The effect of increasing hydrophobicity on inhibiting LLPS of HSA has also been reported in the work of Patel et al. [ 41 ] Decreased ANS fluorescence in the presence of UiO-66-NH 2 might have occurred due to exposed hydrophobic regions of BSA binding to MOF surfaces, blocking ANS or causing structural changes reducing ANS binding [48]. In conclusion, reducing interactions favoring phase separation may result in reduced protein aggregation and fibril formation, explaining the observed decrease in fibril size and the direct relationship between droplet and fibril dimensions. These findings highlight the significance of phase separation droplets as potentially important alternative pathways influencing protein aggregation. Declarations Conflicts of Interest: The authors declare that they have no competing interests. Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Author Contribution Ali Akbar Moosavi-Movahedi and Amirhossein Latifi wrote the main manuscript text, Elnaz Hosseini and Payam Arghavani prepared figures, Mohammad Edrisi and Hossein Daneshgar wrote the part related to MOF synthesis part based on the information provided by Mojtaba Bagherzadeh, Reza Yousefi reviewed the article as a scientific editor, and all authors reviewed the manuscript. Acknowledgment: The support of the University of Tehran, National Natural Science Foundation (INSF), UNESCO Chair on Interdisciplinary Research in Diabetes is greatly acknowledged. 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Nat Commun 12. https://doi.org/10.1038/s41467-021-24111-x Zwicker D, Seyboldt R, Weber CA et al (2017) Growth and division of active droplets provides a model for protocells. Nat Phys 13:408–413. https://doi.org/10.1038/nphys3984 Mukherjee S, Sakunthala A, Gadhe L et al (2023) Liquid-liquid Phase Separation of α-Synuclein: A New Mechanistic Insight for α-Synuclein Aggregation Associated with Parkinson’s Disease Pathogenesis. J Mol Biol 435 Black KA, Priftis D, Perry SL et al (2014) Protein encapsulation via polypeptide complex coacervation. ACS Macro Lett 3:1088–1091. https://doi.org/10.1021/mz500529v Yusof NF, Raffi AA, Yahaya NZS et al (2023) Surface Modification of UiO-66 on Hollow Fibre Membrane for Membrane Distillation. Membr (Basel) 13. https://doi.org/10.3390/membranes13030253 Lawrence MC, Katz MJ (2022) Analysis of the Water Adsorption Isotherms in UiO-Based Metal–Organic Frameworks. J Phys Chem C 126:1107–1114. https://doi.org/10.1021/acs.jpcc.1c05190 Ibrahim AH, El-Mehalmey WA, Haikal RR et al (2019) Tuning the Chemical Environment within the UiO-66-NH2 Nanocages for Charge-Dependent Contaminant Uptake and Selectivity. Inorg Chem. https://doi.org/10.1021/acs.inorgchem.9b01611 Li TT, Liu YM, Wang T et al (2018) Regulation of the surface area and surface charge property of MOFs by multivariate strategy: Synthesis, characterization, selective dye adsorption and separation. Microporous Mesoporous Mater 272:101–108. https://doi.org/10.1016/j.micromeso.2018.06.023 Michels JJ, Brzezinski M, Scheidt T et al (2022) Role of Solvent Compatibility in the Phase Behavior of Binary Solutions of Weakly Associating Multivalent Polymers. Biomacromolecules 23:349–364. https://doi.org/10.1021/acs.biomac.1c01301 Ji Y, Li F, Qiao Y (2022) Modulating liquid-liquid phase separation of FUS: mechanisms and strategies. J Mater Chem B 10:8616–8628 Patel CK, Singh S, Saini B, Mukherjee TK (2022) Macromolecular Crowding-Induced Unusual Liquid-Liquid Phase Separation of Human Serum Albumin via Soft Protein-Protein Interactions. J Phys Chem Lett 13:3636–3644. https://doi.org/10.1021/acs.jpclett.2c00307 Brady JP, Farber PJ, Sekhar A et al (2017) Structural and hydrodynamic properties of an intrinsically disordered region of a germ cell-specific protein on phase separation. Proc Natl Acad Sci U S A 114:E8194–E8203. https://doi.org/10.1073/pnas.1706197114 Togashi DM, Ryder AG (2008) A fluorescence analysis of ANS bound to bovine serum albumin: Binding properties revisited by using energy transfer. J Fluoresc 18:519–526. https://doi.org/10.1007/s10895-007-0294-x Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3953695","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":273615173,"identity":"763f4b2d-8caf-4607-a4aa-f7ead8bd5fb8","order_by":0,"name":"Amirhossein Latifi","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Amirhossein","middleName":"","lastName":"Latifi","suffix":""},{"id":273615174,"identity":"7231e1fb-0e47-453e-9aa4-6fb093f3a86a","order_by":1,"name":"Elnaz Hosseini","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Elnaz","middleName":"","lastName":"Hosseini","suffix":""},{"id":273615175,"identity":"b65fbc6b-fc83-48d5-b324-890d29dbb45b","order_by":2,"name":"Hossein Daneshgar","email":"","orcid":"","institution":"Sharif University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Hossein","middleName":"","lastName":"Daneshgar","suffix":""},{"id":273615176,"identity":"b3d6ec70-8099-44e8-b1d2-0f3ea44bba24","order_by":3,"name":"Mohammad Edrisi","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"","lastName":"Edrisi","suffix":""},{"id":273615177,"identity":"03db2a9e-dd6c-43bd-bd21-b3032653544f","order_by":4,"name":"Payam Arghavani","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Payam","middleName":"","lastName":"Arghavani","suffix":""},{"id":273615178,"identity":"702bfb07-d7ab-4086-8bdc-080e2cf7f85d","order_by":5,"name":"Mojtaba Bagherzadeh","email":"","orcid":"","institution":"Sharif University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Mojtaba","middleName":"","lastName":"Bagherzadeh","suffix":""},{"id":273615179,"identity":"a6e68767-42ca-45d8-97fa-16fac3bf02e1","order_by":6,"name":"Reza Yousefi","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Reza","middleName":"","lastName":"Yousefi","suffix":""},{"id":273615180,"identity":"9b4fd81a-b800-42eb-9f2c-7a3ac0d2409c","order_by":7,"name":"Ali Akbar Moosavi-Movahedi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYPACCTk2CR4Yh7GBKC3GEC0JxGthSGxAaCEAdNubD3/42WaR3ifde4CZ9weDPH8Dc9sHfFrMzhxLk+xtk8htkzmXwMyTwGA44wBj8wy8Wm7kmDHwnAFqkcgxAGlh3MDA2IzXYUAtxh//nJFIZ4NqsSdGi4E0T4VEAkxLImEtQL9Iy1RIGIL8cnBOmkTyjMOEtBxvPvzxjUGdvPzs3oMP3tjY2Pa3tz/GqwUFHADGKQMDM/EaRsEoGAWjYBTgAAAXkD9cZZk9JAAAAABJRU5ErkJggg==","orcid":"","institution":"University of Tehran","correspondingAuthor":true,"prefix":"","firstName":"Ali","middleName":"Akbar","lastName":"Moosavi-Movahedi","suffix":""}],"badges":[],"createdAt":"2024-02-13 14:20:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3953695/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3953695/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51442829,"identity":"373b50f5-7aba-49bc-b4d7-b1b3fd42e539","added_by":"auto","created_at":"2024-02-21 17:54:16","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":203387,"visible":true,"origin":"","legend":"\u003cp\u003eMOFs characterization. (A) Morphology and size determination of metal-organic frameworks and EDS element analysis of UiO-66 (A, E), UiO-66-NH\u003csub\u003e2\u003c/sub\u003e (B, E), and UiO-66-(COOH)\u003csub\u003e2\u003c/sub\u003e (C, F) using field emission scanning electron microscope. (G) XRD patterns. (H) FTIR analysis.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3953695/v1/174a3e50bf1cdf585c9c027d.jpg"},{"id":51441837,"identity":"4aeb31e1-6855-40d4-b549-bd98cf651591","added_by":"auto","created_at":"2024-02-21 17:46:16","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":178887,"visible":true,"origin":"","legend":"\u003cp\u003eComparison chart of the effects of different concentrations of metal-organic frameworks on the tertiary structure of protein using the results of intrinsic and ANS fluorescence emission and UV-visible absorption.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3953695/v1/3a9652585e112479be9e30a2.jpg"},{"id":51441836,"identity":"72ee6368-3a48-4634-82d2-71937652a031","added_by":"auto","created_at":"2024-02-21 17:46:16","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":127683,"visible":true,"origin":"","legend":"\u003cp\u003e(A) A representative microscope image of 450 μM of BSA in the presence of different concentrations of NaCl, (B) a comparison of the size of droplets under the influence of different salt concentrations, (C) representative fluorescence microscopic images of FITC-BSA in Control (MOF-free droplets) and in the presence of UiO-66, UiO-66-NH\u003csub\u003e2\u003c/sub\u003e, UiO-66-(COOH)\u003csub\u003e2\u003c/sub\u003e. In all images, the scale is 5μ.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3953695/v1/0a7e03d791bdece0d683cb00.jpg"},{"id":51441842,"identity":"c4e88673-d7d3-4eeb-8bea-d8cbd501b38e","added_by":"auto","created_at":"2024-02-21 17:46:16","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":218322,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Optical microscopy images of droplet evolution within 24 hours in the absence and presence of synthesized MOFs. The scale is 5μ. (B) obtained diameter of about 90 droplets at time 0h in panel (A) by ImageJ. (C) Comparison of the number of droplets at time 0h.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3953695/v1/f305f946e20edd9bb6f03420.jpg"},{"id":51441844,"identity":"5487fc35-673f-4ca3-9a2b-fd9613f9541a","added_by":"auto","created_at":"2024-02-21 17:46:16","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":213650,"visible":true,"origin":"","legend":"\u003cp\u003eStructural evaluations by (A) intrinsic fluorescence (inset, peak signal comparison), (B) ANS fluorescence (inset, peak signal comparison), and (C) CD spectra.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3953695/v1/242833d5d6dd0ee12e39bf0d.jpg"},{"id":51441843,"identity":"17f7d6a7-42cf-492a-b23b-678eb6a0614c","added_by":"auto","created_at":"2024-02-21 17:46:16","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":483647,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Microscopy images of fibrils in the absence and presence of synthesized MOFs. Obtained length (B) and number (C) of fibrils in panel (A) by ImageJ. (D) BSA aggregation kinetics through droplet formation.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3953695/v1/1215a4ff11cb349378e51d50.jpg"},{"id":51445573,"identity":"bcaa6f99-2bb8-47b2-ba79-8ed22be18c25","added_by":"auto","created_at":"2024-02-21 18:10:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1025804,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3953695/v1/95dc0cde-f4bb-4c27-b825-d58b242d3293.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Fibril-Droplet Relationship Through Liquid-Liquid Phase Separation; A BSA-MOF Case","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLiquid-liquid phase separation (LLPS) is a crucial biological process in cells [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] that drives intracellular compartmentalization [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], organizes biochemical reactions, signaling cascades [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and enables proteins to gather in condensed droplet-like regions as water is expelled through volume exclusion [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This region coexists with a less dense phase. The concentrated protein content in the condensed phase can facilitate amyloid aggregation not only for disordered aggregation-prone proteins [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] like tau[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], alpha-synuclein[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and TDP-43[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] but also for ordered globular proteins such as lysozyme and serum albumin [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Thus, LLPS can promote amyloid fibril formation[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], which is implicated in cancer, Alzheimer\u0026rsquo;s, and Parkinson's diseases[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], diabetes, and associated complications [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBovine serum albumin (BSA) is a model globular protein to understand the main factors behind LLPS with a well-defined tertiary structure with 583 amino acids constrained by 17 disulfide bridges into a predominantly helical, stable conformation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Elucidation of the governing protein-protein and protein-solvent interactions enabling spontaneous BSA demixing into highly concentrated liquid droplets, which paves the way for amyloid fibrillogenesis, requires control over solution conditions modulating protein conformation and inter and intramolecular interactions.\u003c/p\u003e \u003cp\u003eMetal-organic frameworks (MOFs) comprise metal ion coordination complexes interconnected into crystalline lattices containing nanoporous channels and cavities capable of sequestering molecules. UiO-66 is one such MOF constructed from Zr\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e(OH)\u003csub\u003e4\u003c/sub\u003e clusters linked by terephthalate organic ligands, forming a structure with both octahedral and tetrahedral pores accessible through small triangular windows [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. With a high surface area exceeding 1200 m\u003csup\u003e2\u003c/sup\u003e/g and pore volumes around 0.5 cm3/g arising from its high porosity, UiO-66 is ideally suited for biosensing [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], encapsulating and transporting cargo [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent progresses in implication of MOFs and specifically UiO-66 family in the diagnosis, treatment and detection of neurodegenerative diseases such as Alzheimer disease [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] in their early stages by targeting LLPS prone proteins and peptides[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], makes them an adequate candidate for modulating this process. Appending functional groups (e.g. \u0026minus;NH\u003csub\u003e2\u003c/sub\u003e, \u0026minus;COOH) onto the UiO-66 scaffold can modulate loading capacity and release kinetics to optimize delivery applications [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. For instance, amine groups boost storage via increased protein affinity while carboxyl moieties confer higher hydrophilicity and electrostatic attraction to cationic molecules. Capitalizing on such functionalization furnishes means to fine-tune UiO-66 properties and govern encapsulated cargo interactions [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, synthesized MOFs functionalized with and without -NH\u003csub\u003e2\u003c/sub\u003e and -COOH have been used for investigating LLPS and fibrillation of BSA. Using UiO-66 MOFs with different functional groups allowed us to investigate the role of hydrophobic/hydrophilic and electrostatic interactions in the phase separation process. Also, MOFs effect on phase separation determined droplets of different sizes, paving the way for investigating the role of LLPS as an alternative pathway for amyloid fibrillation.\u003c/p\u003e"},{"header":"Experimental section","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterial\u003c/h2\u003e \u003cp\u003eFatty acid-free BSA catalog number 1.12018 was obtained from Sigma Aldrich, and 50 mM phosphate buffer was prepared at pH 7 using NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e and Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e. Polyethylene glycol (PEG) (MW 1450 g/mol), Thioflavin T (ThT), 1-anilino-8-naphthalene sulfonate (ANS), NaCl, NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, and Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e were acquired from Merck, and all components for MOF synthesis, including ZrCl\u003csub\u003e4\u003c/sub\u003e, 1,2,4,5-benzene tetracarboxylic acid (BDC), Acetic acid, Dimethylformamide (DMF) purchased from Merck, FITC conjugated BSA catalog number: A23015 purchased from Thermo Fisher.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePreparing BSA and Concentration determination\u003c/h2\u003e \u003cp\u003eA proper amount of BSA protein was dissolved in 50 mM phosphate buffer (pH 7.4) with a target concentration of 5 mM, and the concentration was confirmed by UV-Vis spectrophotometer (UV-3100 Shimadzu) measurement at 280 nm assuming the extinction coefficient as 43824 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003ecm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. BSA concentration was fixed at 450 \u0026micro;M for all samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMOF synthesis\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eUiO-66 and UiO-66-NH\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003eUiO-66 and UiO-66-NH\u003csub\u003e2\u003c/sub\u003e were synthesized according to the previous reports [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Briefly, ZrCl\u003csub\u003e4\u003c/sub\u003e (166.6 mg, 0.72 mmol) was dissolved in 20 ml DMF and stirred for 20 min at 40\u0026deg;C. Then, for UiO-66 BDC (82 mg, 1 mmol) and for UiO-66-NH\u003csub\u003e2\u003c/sub\u003e, BDC-NH\u003csub\u003e2\u003c/sub\u003e (181.1 mg, 1 mmol) was added, and the resultant mixture was sonicated in an ultrasonic bath for 20 min. After that, the resulting mixture was heated at 120\u0026deg;C for 24 hours. In the next step, methanol was added, and the solution was centrifuged at 7000 rpm for 5 min. This process was repeated three times, and the remaining solid pellet was dried under a vacuum.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eUiO-66-(COOH)\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003eTo synthesize this MOF, first, ZrCl\u003csub\u003e4\u003c/sub\u003e (0.304 g) was dissolved in 15 ml deionized water and stirred by using a thermo stirrer [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. BDC (0.318 g) separately was dissolved in 15 ml deionized water and 10 ml acetic acid, again mixed using a thermo stirrer. The second mixture was sonicated for 10 minutes in an ultrasonic bath. Then, the two solutions were combined and heated at 110\u0026deg;C for 24 hours. After cooling down to room temperature, the product was centrifuged at 7000 rpm for 5 min, and the supernatant was decanted. The remaining solid was washed three times with methanol, centrifuging, and decanting after each wash. Finally, the product dried at room temperature for 24 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMOFs stock preparation\u003c/h2\u003e \u003cp\u003eUiO-66, UiO-66-(COOH)\u003csub\u003e2,\u003c/sub\u003e and UiO-66-NH\u003csub\u003e2\u003c/sub\u003e stocks were prepared by mixing 2 mg of each sample with 1 ml phosphate buffer (pH 7.4) and, for appropriate dispersion, sonicated (UHP-400 Ultrasonic Homogenizer) for at least 30 min with probe sonication.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eNaCl stock preparation\u003c/h2\u003e \u003cp\u003e1 M NaCl stock was prepared by dissolving 58.44 mg of NaCl in 1 ml 50 mM phosphate buffer (pH 7.4).\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003ePolyethylene glycol stock preparation\u003c/h2\u003e \u003cp\u003eA polyethylene glycol (PEG) stock solution was prepared by dissolving 800 mg PEG 4000 Da in 1 ml phosphate buffer (pH 7.4).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSamples preparation\u003c/h2\u003e \u003cp\u003eDepending on the final volume of each experiment, the proper volume of protein, salt, MOFs, and PEG have been used to reach 450 \u0026micro;M, 300 mg/ml, 0.28 mg/ml, and 200 mg/ml as the final concentration, respectively. The addition order was: 1) buffer, 2) salt, 3) MOF, 4) protein. PEG was added whenever LLPS induction was needed. This protocol ensured controlled protein-MOF interaction before undergoing LLPS. To further promote interactions, samples were incubated overnight at 8\u0026deg;C, 300 rpm in a thermoshaker (Eppendorf Thermomixer C) before each assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDroplet formation experiments\u003c/h2\u003e \u003cp\u003eTo investigate the ionic strength impact on the propensity of BSA to undergo LLPS, solutions with varying sodium chloride (NaCl) concentrations (50\u0026ndash;300 mM) were prepared. The extent of LLPS was assessed by measuring droplet diameters from optical microscopy (Zeiss Axioskop 2 FS plus) images and using ImageJ software. This enabled quantitative comparisons of phase separation intensity across different NaCl concentrations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eProtein conformational changes\u003c/h2\u003e \u003cp\u003eTo probe BSA tertiary structural changes, extrinsic fluorescence spectroscopy utilizing 1-anilino-8-naphthalene sulfonate (ANS), and intrinsic fluorescence spectroscopy were employed. ANS fluorescence was recorded at 340 nm excitation and 480 nm emission. Intrinsic fluorescence used 280 nm excitation and 340 nm emission using an Agilent BioTek Synergy H4 Hybrid Microplate Reader. Furthermore, Far UV circular dichroism (CD) spectroscopy was recorded at room temperature from 190\u0026ndash;260 nm using a Jasco J-810 to investigate alterations in the secondary structure content upon treatments. Samples were diluted to reduce interference from turbidity caused by droplet formation by the ratio of 1:25 and 1:80 for fluorescence and CD spectroscopy, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eProtein fluorescence labeling\u003c/h2\u003e \u003cp\u003eTo visually confirm condensed protein within liquid droplets, BSA was fluorescently labeled with fluorescein isothiocyanate (FITC). Its excitation and emission maximums were 491 nm and 516 nm here, respectively. A 10:990 ratio of FITC-BSA to unlabeled BSA was used for observation. This low FITC level provided sufficient fluorescence to detect droplets while minimizing impacts on phase separation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMOF characterization\u003c/h2\u003e \u003cp\u003eThe morphology of the samples was visualized using Field Emission Scanning Electron Microscopy (FESEM) with a TESCAN MIRA3 SEM. Crystallinity was determined by X-ray diffraction (XRD) analysis conducted on an Inel EQUINOX3000 X-ray diffractometer using copper K-alpha radiation (wavelength\u0026thinsp;=\u0026thinsp;1.5418 Angstroms) over a range of 5 to 80 theta. To analyze the sample chemical composition, Fourier transform infrared (FT-IR) spectroscopy was performed on a BRUKER Equinox 55 spectrometer with a spectral range of 400\u0026ndash;4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e using potassium bromide plates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePhysicochemical properties\u003c/h2\u003e \u003cp\u003eThe contact angle and zeta potential have been operated as indices of hydrophobicity and surface charge of MOFs, respectively. The contact angle measurement was performed by a Wilhelmy plate method using a force tensiometer (Kruss K100C). For this purpose, MOFs with 2 mg/ml concentration has been dispersed in 2 ml of the buffer. Zeta potential measurements were performed using the Laser Doppler electrophoresis technique on a HORIBA SZ100 Z instrument. The surface charge of the MOFs was determined by running the measurements across an applied voltage spectrum spanning from \u0026minus;\u0026thinsp;200 to +\u0026thinsp;200 mV.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eFESEM results\u003c/h2\u003e \u003cp\u003eElectron microscopy images indicated that synthesized MOFs were semi-spherical, and adding NH\u003csub\u003e2\u003c/sub\u003e and COOH functional groups did not affect the MOF's morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, A, B, and C). The EDS element analysis results confirmed the presence of essential functional group elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, D, E, and F). Based on Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, adding functional groups didn't change UiO-66 clusters' morphology and/or size significantly in this study, which is consistent with previous reports[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In this case, other functional groups' physicochemical properties may determine the differences observed in droplet and fibril dimensions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eXRD patterns\u003c/h2\u003e \u003cp\u003eX-ray diffraction (XRD) analysis determined the crystal structure of UiO-66 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). UiO-66 matched literature patterns with peaks at 7.4, 8.5, 14.8, 18.5, 25.4, and 31.1, confirming successful synthesis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. UiO-66-(COOH)\u003csub\u003e2\u003c/sub\u003e showed a similar XRD pattern to UiO-66 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The NH\u003csub\u003e2\u003c/sub\u003e-grafted framework also matched the original UiO-66, indicating that NH\u003csub\u003e2\u003c/sub\u003e groups did not modify crystal structure [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eFTIR spectrum\u003c/h2\u003e \u003cp\u003eThe Fourier transform infrared (FTIR) spectra of the synthesized MOF samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH) reveal distinct bands attributable to specific chemical structures and functional groups. For UiO-66, the band at 1568 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicates coordination of -COOH with Zr\u003csup\u003e4+\u003c/sup\u003e. Bands at 1506 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1395 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e correspond to vibrations of the benzene ring and carboxylate group of the organic linker [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Additional bands at 1019 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 760\u0026ndash;780 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 660 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e arise from C-H vibrations. UiO-66-NH\u003csub\u003e2\u003c/sub\u003e and UiO-66-(COOH)\u003csub\u003e2\u003c/sub\u003e exhibit similar spectra, with extra bands due to grafted functional groups. The band at 1620 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in UiO-66-NH\u003csub\u003e2\u003c/sub\u003e originates from the N-H bending of aromatic amines. The 1252 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e band indicates C-N stretching[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. For UiO-66-(COOH)\u003csub\u003e2\u003c/sub\u003e, the band at 1712 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e confirms the presence of uncoordinated -COOH groups. The FTIR data confirms the complete, uncorrupted synthesis of the MOF samples with full retention of chemical structures. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eBSA \u0026ndash; MOF interaction\u003c/h2\u003e \u003cp\u003eIn order to investigate the strength of interaction between MOFs and BSA an experiment was design as follow. BSA was incubated overnight with varying concentrations of UiO-66, UiO-66-NH\u003csub\u003e2\u003c/sub\u003e, and UiO-66-(COOH)\u003csub\u003e2\u003c/sub\u003e MOFs. Subsequently, the samples were centrifuged at 18,000 rpm for 5 minutes. The rationale was that if robust interactions such as bond formation occurred between any of the MOFs and BSA, then after centrifugation BSA would co-sediment with the MOFs due to the high density of the MOF metal clusters. In this case, increasing MOF concentration would increase co-sedimentation, which could be quantified by measuring BSA concentration in the supernatant using UV-Vis spectroscopy. The concentrations used for each of MOFs UiO-66, UiO-66-(COOH)\u003csub\u003e2\u003c/sub\u003e, UiO-66-NH\u003csub\u003e2\u003c/sub\u003e was as mentioned in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDifferent concentrations of MOFs in MOF-BSA interaction experiment.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMg.ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e 0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.12 mg.ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emg.ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e 0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.28 mg.ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emg.ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e 0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMg.ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e 0.44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn the next step, intrinsic fluorescence emission and ANS-binding assays were performed to evaluate structural changes in the protein. Intrinsic tryptophan fluorescence was measured at 340 nm, corresponding to the emission peak wavelength of tryptophan residues in BSA. This allowed detection of changes in the local environment of aromatic amino acids like tryptophan. Additionally, ANS binding assays were carried out, with fluorescence recorded at 440 nm. Since hydrophobic dye ANS binds to exposed hydrophobic regions on protein surfaces, changes in ANS fluorescence indicate alterations of surface hydrophobicity.\u003c/p\u003e \u003cp\u003eThe results of UV-vis spectroscopy, intrinsic fluorescence, and ANS-binding assays after incubating BSA with UiO-66, UiO-66-(COOH)\u003csub\u003e2\u003c/sub\u003e, and UiO-66-NH\u003csub\u003e2\u003c/sub\u003e followed by centrifugation to remove MOFs are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The UV-vis absorption profiles indicate no decrease compared to BSA alone. Rather, in most cases, a slight increase in UV-vis absorbance is observed upon addition of MOFs.\u003c/p\u003e \u003cp\u003eThe intrinsic fluorescence results reveal decreased emission intensities for BSA incubated with the NH\u003csub\u003e2\u003c/sub\u003e-functionalized MOF at all concentrations tested. A similar effect is seen for some concentrations of non-functionalized UiO-66. In contrast, the COOH-functionalized MOF generally increases or does not affect intrinsic fluorescence, except at the highest 0.44 mg/mL concentration.\u003c/p\u003e \u003cp\u003eFinally, ANS fluorescence is enhanced in the presence of UiO-66-NH\u003csub\u003e2\u003c/sub\u003e, reflecting greater exposure of hydrophobic regions. However, the differences in ANS emission are not significant. These results demonstrate that MOFs neither form strong bonds with BSA proteins and nor cause significant permanent conformational change in protein structure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe BSA phase separates into liquid droplets.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIncreasing NaCl concentration from 50 to 300 mM led to larger BSA droplets (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), with 300 mM NaCl resulting in significantly larger droplet diameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Since the total BSA concentration was held constant, this suggests that more BSA protein partitioned from solution into droplets at 300 mM NaCl to form the larger condensed phases observed.\u003c/p\u003e \u003cp\u003eFITC-labeled BSA has been used to confirm the protein nature of droplets (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Fluorescence microscopy using the FITC-BSA showed intense fluorescence within the liquid droplets, verifying that condensed protein accumulation had occurred. Furthermore, larger droplets exhibited higher fluorescence intensity compared to smaller droplets.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eKinetics of droplet size evolution\u003c/h2\u003e \u003cp\u003eTo examine the dynamic nature of liquid-liquid phase separated proteins, it was necessary to track droplet size over time and visualize growth [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOptical microscopy visualized droplets at 0, 2, 6, and 24 hours after inducing phase separation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Over 24 hours, the number of droplets decreased while the size of remaining droplets increased, consistent with the coalescence and Ostwald ripening phenomenon [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. By 24 hours, only a sparse population of enlarged droplets remained. Relating LLPS droplet size to subsequent fibril length provided insights into their relationship under different conditions [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo have a better quantitative comparison about the impact of UiO-66, UiO-66-NH\u003csub\u003e2\u003c/sub\u003e and UiO-66-(COOH)\u003csub\u003e2\u003c/sub\u003e on the size of droplets, their diameters were measured by ImageJ software plotted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB. The result was in consistent with fluorescence microscopy results.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eConformational changes due to protein interaction with MOFs and LLPS\u003c/h2\u003e \u003cp\u003eIntrinsic and ANS fluorescence probed BSA tertiary structure and surface hydrophobicity changes after phase separation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and B). Also, UiO-66-NH\u003csub\u003e2\u003c/sub\u003e slightly decreased ANS emission and increased intrinsic fluorescence, but these changes were not significant which implies that MOFs did not cause any significant conformational change. Circular dichroism (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) monitored BSA secondary structure changes after separation. The signal appearance remained constant despite a slight intensity decrease, likely due to the presence of PEG. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eFibril formation kinetics\u003c/h2\u003e \u003cp\u003eAfter phase separation (7 days of incubation at room temperature), Thioflavin T (ThT) stained samples were observed by fluorescence microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Fibril length measurement demonstrated that UiO-66-NH\u003csub\u003e2\u003c/sub\u003e significantly decreased BSA fibril length in comparison with other synthesized MOFs and the sample without MOF (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eFibrils from the smallest droplets induced by UiO-66-NH\u003csub\u003e2\u003c/sub\u003e were significantly shorter than those of the control. UiO-66-NH\u003csub\u003e2\u003c/sub\u003e had lower ThT emission than the control, probably correlating with its smaller and fewer droplets. However, non-functionalized UiO-66 had a more significant ThT increase versus control, indicating fluorescence depends on both droplet size and number.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eZeta potential and contact angels\u0026rsquo; results\u003c/h2\u003e \u003cp\u003eIn this case, the functional groups' physico-chemical properties determined the differences in droplet and fibril dimensions (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Measuring zeta potential and contact angle revealed that UiO-66-NH\u003csub\u003e2\u003c/sub\u003e had the highest contact angle and, thus, the greatest hydrophobicity, and UiO-66-(COOH)\u003csub\u003e2\u003c/sub\u003e had the highest zeta potential[\u003cspan additionalcitationids=\"CR36 CR37\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe zeta potential results at pH\u0026thinsp;=\u0026thinsp;7.4, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, indicate that the potential of frameworks with a carboxyl functional group is more negative. As seen in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, despite the significant difference in the potential of the carboxyl group, adding the NH\u003csub\u003e2\u003c/sub\u003e functional group did not cause a significant change compared to the state without the functional group. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e also indicates that the contact angle for UiO-66, UiO-66-NH\u003csub\u003e2\u003c/sub\u003e, and UiO-66-(COOH)\u003csub\u003e2\u003c/sub\u003e were obtained as 26.19, 42.98, and 24.9, respectively.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults of zeta potential and contact angle measurements.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMOFs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZeta potential\u003c/p\u003e \u003cp\u003e(mV)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eContact angle\u003c/p\u003e \u003cp\u003e(rad)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eUiO-66\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-18.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eUiO-66-(COOH)\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-27.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eUiO-66NH\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-19.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eComparison of NaCl concentrations reveals more extensive liquid-liquid phase separation (LLPS) at higher ionic strengths, aligning with BSA's isoelectric point (around 4.7) where repulsive forces typically hinder aggregation. Higher salt likely screens this repulsion, allowing attractive hydrophobic and van der Waals forces to dominate, promoting BSA association into expanding droplets rather than new droplet formation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTurning to UiO-66 and its functionalized forms, no strong bonds were observed with BSA protein. Notably, NH2-functionalized UiO-66 successfully prevented LLPS-induced droplet formation. Conversely, COOH groups resulted in larger droplets compared to the unfunctionalized version. The correlation of increased fluorescence with larger droplets suggests higher protein content yielding more fluorescence emission [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIntroducing the NH2 group significantly reduced both the number and size of droplets (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), indicating its role as an LLPS inhibitor. Interestingly, droplet size also decreased in the absence of functional groups, but with an increased number of droplets [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD clearly shows the decrease in droplet number with UiO-66-NH2. This decrease implies that UiO-66-NH2 slows down BSA aggregation, further confirmed by a reduced apparent aggregation constant in its presence (slopes in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eImportantly, fibrils formed with UiO-66-NH2 (inducing the smallest droplets) were significantly shorter than the control. This aligns with previous studies on the role of functional groups in amyloid β fibril size, where NH2 groups shortened the resultant fibrils [45]. Notably, Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB exhibit a similar size pattern (Control\u0026thinsp;\u0026gt;\u0026thinsp;UiO-66-(COOH)2\u0026thinsp;\u0026gt;\u0026thinsp;UiO-66\u0026thinsp;\u0026gt;\u0026thinsp;UiO-66-NH2), suggesting a correlation between droplet and fibril size. Furthermore, the number of fibrils, at least for UiO-66-NH2, seems correlated with the number of droplets. This suggests that smaller BSA precursor droplets yield shorter BSA fibrils, implying that droplets provide a platform for further interactions triggering BSA aggregation at room temperature.Finally physico-chemical investigations reveald that increasing hydrophobicity decreases proteins' propensity to aggregate into droplets while raising environmental charge increases protein aggregation and phase separation. Based on the sticker-spacer model [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], the results indicate that hydrophobic interactions play a major role in BSA phase separation. The model prioritizes intermolecular associations over intramolecular hydrophobic interactions under conditions promoting aggregation. Therefore, these findings agree that exposed hydrophobic regions on BSA drive intermolecular interactions and subsequent phase separation [36, 46]. As seen here, increasing salt concentration that disrupts electrostatic interactions by raising ionic strength allowed favorable hydrophobic interactions to overcome the electrostatic interactions, which promote protein solubility, leading to phase separation [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Similarly, the increased hydrophobicity of UiO-66-NH\u003csub\u003e2\u003c/sub\u003e likely disturbed the crucial hydrophobic interactions for phase separation. The effect of increasing hydrophobicity on inhibiting LLPS of HSA has also been reported in the work of Patel et al. [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] Decreased ANS fluorescence in the presence of UiO-66-NH\u003csub\u003e2\u003c/sub\u003e might have occurred due to exposed hydrophobic regions of BSA binding to MOF surfaces, blocking ANS or causing structural changes reducing ANS binding [48].\u003c/p\u003e \u003cp\u003eIn conclusion, reducing interactions favoring phase separation may result in reduced protein aggregation and fibril formation, explaining the observed decrease in fibril size and the direct relationship between droplet and fibril dimensions. These findings highlight the significance of phase separation droplets as potentially important alternative pathways influencing protein aggregation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of Interest:\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAli Akbar Moosavi-Movahedi and Amirhossein Latifi wrote the main manuscript text, Elnaz Hosseini and Payam Arghavani prepared figures, Mohammad Edrisi and Hossein Daneshgar wrote the part related to MOF synthesis part based on the information provided by Mojtaba Bagherzadeh, Reza Yousefi reviewed the article as a scientific editor, and all authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgment:\u003c/h2\u003e \u003cp\u003eThe support of the University of Tehran, National Natural Science Foundation (INSF), UNESCO Chair on Interdisciplinary Research in Diabetes is greatly acknowledged.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlberti S, Gladfelter A, Mittag T (2019) Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates. 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J Fluoresc 18:519\u0026ndash;526. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10895-007-0294-x\u003c/span\u003e\u003cspan address=\"10.1007/s10895-007-0294-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Fibril droplet, BSA, MOF, UiO-66, LLPS, Hydrophobicity","lastPublishedDoi":"10.21203/rs.3.rs-3953695/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3953695/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"By providing a suitable platform that accelerates early-stage occurrences needed for triggering protein aggregation, liquid-liquid phase separation (LLPS) has the potential to promote this phenomenon. Among different proteins which their condensation propensity has been investigated, bovine serum albumin (BSA) has attracted attentions because of its globular and stable structure. BSA undergoes phase separation and phase transition in the presence of polyethylene glycol as a molecular crowder. The control of hydrophobicity, hydrogen bonding, and electrostatic forces as the main forces that conduct LLPS were provided by functionalizing a UiO-66 metal-organic framework (MOF) with -NH2 and -COOH functional groups. This work evaluated how the functionalized UiO-66 with -NH2 modulates the LLPS of BSA. Successful synthesis and functionalization of UiO-66 were confirmed using various physical and chemical analyses. Optical and fluorescence microscopy images correlated BSA LLPS droplet size with spectroscopic measurements of resultant BSA fibrils. UiO-66-NH2 was found to cause significant conformational changes in BSA, resulting in a decrease in its LLPS and aggregation rate, as demonstrated by various biophysical methods. This study suggested that more hydrophobic surrounding micro-environments caused by UiO-66-NH2 inhibited BSA LLPS, leading to decreased droplet size and number. The direct correlation between droplet size and fibril length also confirmed the role of LLPS as an important alternative pathway enabling fibril formation even in globular proteins.","manuscriptTitle":"Fibril-Droplet Relationship Through Liquid-Liquid Phase Separation; A BSA-MOF Case","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-21 17:46:11","doi":"10.21203/rs.3.rs-3953695/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"128b3ec3-592f-407b-8893-a925d328cf0c","owner":[],"postedDate":"February 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-02-21T17:46:13+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-21 17:46:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3953695","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3953695","identity":"rs-3953695","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}