Journal of Colloid and Interface Science

Crowding agents, such as polyethylene glycol (PEG, are often used to mimic the cellular cytoplasm in protein assembly studies. Despite the perception that crowding agents have an inert nature, recent work has shown they are not bystanders while proteins interact. Here, we explore the diverse effects of PEG on the phase separation and maturation of proteins. We use two proteins, the FG domain of Nup98 and bovine serum albumin (BSA), which represent an intrinsically disordered protein and a protein with well-established secondary structure, respectively. PEG expedites the maturation of Nup98, enhancing denser protein packing and fortifying hydrophobic interactions which hasten beta-sheet formation and subsequent droplet gelation. In contrast for BSA, PEG appears to enhance droplet stability and limits available solvent for the protein solubilization, without inducing significant changes to the secondary structure, pointing towards a significantly different behavior of the crowding agent. Interestingly, we detect almost no presence of PEG in Nup droplets whereas PEG is detectable within BSA droplets. Our findings demonstrate a nuanced interplay between crowding agents and proteins. PEG can accelerate protein maturation in LLPS systems but its partitioning and effect on protein structure in droplets is protein specific. This suggests that crowding phenomena are specific to each protein-crowding agent pair.

Knowledge of surface characteristics is a major step in the evaluation of bacterial cells for potential use as Pickering emulsion stabilizers. Here, the cell surface characteristics of 31 strains of the ex-Lactobacillus genus were studied with the aim of evaluating their intrinsic abilities to serve as Pickering stabilizers of oil-in-water emulsions. About 77.42% of the tested strains demonstrated relatively highly negative zeta potential (-43.76 mV [&le;] zeta potential [&le;] -19.23 mV), while [~]58% of the strains demonstrated high cell surface hydrophobicity (microbial adhesion to hexadecane or MATH [&ge;] 30%). By combining these findings, four different cell surface features were defined (I, II, II and IV). Strains mainly demonstrated the type I surface feature ([~]45%), with most expressing strongly negative zeta potential and high surface hydrophobicity (zeta potential < -15 mV and MATH [&ge;] 30%, respectively). It appeared that the abundance of negative charge on the surfaces of ex-Lactobacillus cells positively influences surface hydrophobicity. Assessment of intrinsic Pickering stabilization potential using 12 selected strains indicated that four strains showed profound droplet size stability. At least one strain was observed to have natural propensity to form relativley compact and small emulsion droplets (63{+/-}3 {micro}m), leading to enhanced firmness and storage stability of the Pickering emulsions.

The coacervation and structural rearrangement of the protein alpha-synuclein (Syn) into cytotoxic oligomers and amyloid fibrils are considered pathological hallmarks of Parkinsons disease. While aggregation is recognized as the key element of amyloid diseases, liquid-liquid phase separation (LLPS) and its interplay with aggregation have gained increasing interest. Previous work showed that factors promoting or inhibiting amyloid formation have similar effects on phase separation. Here, we provide a detailed scanning of a wide range of parameters including protein, salt and crowding concentrations at multiple pH values, revealing different salt dependencies of aggregation and phase separation. The influence of salt on aggregation under crowded conditions follows a non-monotonic pattern, showing increased effects at medium salt concentrations. This behavior can be elucidated through a combination of electrostatic screening and salting-out effects on the intramolecular interactions between the N-terminal and C-terminal regions of Syn. By contrast, we find a monotonic salt dependence of phase separation due to the intermolecular interaction. Furthermore, we observe the time evolution of the two distinct assembly states, with macroscopic fibrillar-like bundles initially forming at medium salt concentration but subsequently converting into droplets after prolonged incubation. The droplet state is therefore capable of inhibiting aggregation or even dissolving the aggregates through a variety of heterotypic interactions, thus preventing Syn from its dynamically arrested state.

In this letter, the pull-off forces of adsorbed films of four Bap1-inspired peptides in various solvents were investigated on negatively charged mica substrates using the surface forces apparatus (SFA), complemented with dynamic light scattering (DLS) for characterizing the aggregation behavior of peptides in solution. Bap1-inspired peptides consisted of the 57 amino acid wild-type sequence (WT); a scrambled version of the WT used to investigate the impact of the primary amino acid sequence in pull-off forces (Scr); a ten amino acid sequence rich in hydrophobic content (CP) of the WT sequence, and an eight amino acid sequence (Sh1) that corresponds to the pseudo-repeating sequence in the 57 AA. SFA results showed remarkable pull-off forces for CP, particularly in the presence of salts: measured pull-off forces were 26.0 {+/-} 7.0 mN/m for no dwell-time and up to 42.0 {+/-} 8.8 mN/m when surfaces were left in contact for 30 minutes. DLS observations indicate that salts favor large peptide aggregation for all constructs (Hz > 1 {micro}m), as compared to milliQ (Hz {approx} 100-500 nm) water and DMSO (Hz {approx} 100 nm), resulting in heterogeneous peptide film thicknesses. This letter concludes with a comparison to the pull-off forces of mussel foot protein-inspired peptides reported in the literature.

Renewable and biodegradable plastics derived from soy protein isolate (SPI) offer a promising alternative to conventional petroleum-based plastics, particularly for film-grade bioplastics applications such as plastic bags. However, even with reinforcement from cellulose nanocrystals (CNCs), their mechanical properties including stiffness lag behind those of petroleum-based plastics. To identify pathways for improving CNC-reinforced SPI composites, we studied stiffening mechanisms by interpreting experimental data using homogenization models that accounted for CNC agglomeration and the formation of CNC/SPI interphases. To model effects of surface modification of CNCs with polydopamine (polyDOPA), we incorporated two key mechanisms: enhanced CNC dispersion and modified CNC-SPI interfacial interactions. Models accounted for interphases surrounding CNCs, arising from physicochemical interactions with the polyDOPA-modified CNC surfaces. Consistent wih experimental observations of polyDOPA modification enhancing mechanical properties through both increased spatial distribution of CNCs and matrix-filler interactions, results demonstrated that improved dispersion and interfacial bonding contribute to increased composite stiffness. Results highlight the potential of biodegradable CNC/SPI bio-nanocomposites as sustainable plastic alternatives, and suggest pathways for further enhancing their mechanical properties.

Reference materials (RM)-assisted Rayleigh-Gans-Debye approximation (rm-RGDA) has been developed and used to in situ determine the size and thickness of the adlayer on the particles in solution. The particle size determined by rm-RGDA is quite close to that measured by electron microscopy but significantly smaller than that measured by DLS. The BSA adlayer absorbed on PS50, PS100 and SiO2 NPs is 3.3, 0.9 and 1.2 nm, respectively, and close to those observed by SEM, which is 4.6, 1.3 and 3.8 nm, respectively. The FTIR analysis results show that the BSA absorbed on larger particles or hydroxyl-abundant surface, e.g. PS100 and SiO2 NPs can lose its secondary structure, e.g. -helix, to a great extent and that absorbed on a more curve surface, e.g. smaller PS50 particles can largely preserve its secondary structure as its free state. The measurement results show the curvature of the NPs is closely related to the structure change of the adsorbed protein. This method provide a facile and new approach to measure the size and its adlayer change of the hybrid and core-shell structured nanoparticles in a wide range of wavelength. SIGNIFICANCEQuantitative study on the adsorption of the protein on colloidal nanoparticles is an important approach to understand the biophysical effect, compared with other ex situ methods such as TEM and SEM, where the specimen are undergone pre-processing and no longer the original state in measurement. It is, therefore, a big challenge. In order to cope with this challenge, UV-vis based RGDA has been developed and applied to in situ measure the size of the dispersed colloidal nanoparticles and their protein adlayer thickness, where the protein adlayer thickness on the colloidal nanoparticles can be easily determined. We believe this method provide a facile and sensitive way to in situ measure the dimension change of hybrid colloidal nanoparticles.

Optimizing the production of lipid nanoparticle (LNP) therapeutics is necessary for drug delivery efficiency, stability, and scalability. A small but growing body of literature has begun to recognize that LNP properties (e.g., size, shape, and internal structure) depend on the flow conditions during mixing for antisolvent precipitation, in which LNPs are formulated. Here, we use different mixers, varying flow patterns (e.g., laminar or turbulent mixing) and flow rate ratios (FRR), i.e., 3:1 and 1:1, to prepare a standard LNP formulation. We then characterize the resulting formulations using small angle x-ray scattering (SAXS) to provide insights into particle shape/morphology, internal organization (L and HII phases) of yeast RNA (yRNA), and structural differences/similarities that arise from the different mixing methods. The effect of ethanol removal on the LNPs structure, formulated from each mixing technique, is also discussed. We observed the 3:1 FRR mixers outperform the 1:1 configurations in certain desired LNP physiochemical properties. The differences observed in the LNPs produced across the two configurations are discussed. Furthermore, we use computational fluid dynamics to explain the turbulent mixing schemes among the 3:1 and 1:1 mixers.

Nanoparticles are promising alternatives to antibiotics since nanoparticles are easy to manufacture, non-toxic, and do not promote resistance. Nanoparticles act via physical disruption of the bacterial membrane and/or the generation of high concentrations of reactive-oxygen species locally. Potential for physical disruption of the bacterial membrane may be quantified by free energy methods, such as the extended Derjuan-Landau-Verwey-Overbeek theory, which predicts the initial surface-material interactions. The generation of reactive-oxygen species may be quantified using enthalpies of formation to predict minimum inhibitory concentrations. Neither of these two quantitative structure-activity values describes the dynamic, in situ behavioral changes in the bacterias struggle to survive. In this paper, borrowing parameters from logistic, oscillatory, and diauxic growth models, we use principal component analysis and agglomerative hierarchical clustering to classify survival modes across nanoparticle types and concentrations. We compare the growth parameters of 170 experimental interactions between nanoparticles and bacteria. The bacteria studied include Escherichia coli, Staphylococcus aureus, Methicillin-Resistant Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Helicobacter pylori, and were tested across multiple concentrations of liposomal drug delivery systems, amphiphilic peptide, and silver and selenium nanoparticles. Clustering reveals specific pairs of bacteria and nanoparticles where the nanoparticle induced growth dynamics could potentially spread the infection through the development of resistance and tolerance. This rapid screening also shows that bacteria generated nanoparticles do not induce growth modes indicative of the development of resistance. This methodology can be used to rapidly screen for novel therapeutics that do not induce resistance before using more robust intracellular content screening. This methodology can also be used as a quality check on batch manufactured nanoparticles.

Efficient drug delivery using nanoparticles (NPs) critically depends on their ability to diffuse through biological tissues to reach target cells at therapeutic concentrations. The extracellular matrix (ECM) poses a key barrier to such transport, which directly influences bio-distribution, cellular uptake, and overall therapeutic efficacy. A key regulator of this transport is hyaluronic acid/hyaluronan (HA), a major ECM polysaccharide that forms a hydrated, viscoelastic network. Increased/reduced hyaluronan concentration can elevate/decrease ECM bulk and effective viscosity. Increase in effective viscosity at the nanometer/micrometer length scales can hinder NP mobility through steric obstruction and hydrodynamic drag. There is a large variability in the HA molecular weights and concentrations, especially across age, tissue/organ, and pathological conditions. This work aims to study the diffusion of different NP types in the mixtures of HA polymers with variable molecular weights using the dynamic light scattering technique (DLS). Furthermore, we perform coarse-grained molecular dynamics (CG-MD) simulations for a model system to complement our findings from the dynamic light scattering experiments. We observe NP undergo anomalous diffusion, which is strongly dependent on the ratio of particle size/HA network mesh size, especially for higher molecular weight mixtures. This is strongly influenced by the effective viscosity, which is defined at the local environment experienced by the NPs. Our work highlights developing a simplified predictive framework coupled with simulations for a target-specific extracellular matrix environment.

AbstractVirus-like particles (VLPs) are emerging as nano-scaffolds in a variety of biomedical applications including the delivery of vaccine antigens to mucosal surfaces. These soft, colloidal, and proteinaceous structures (capsids) are nevertheless susceptible to mucosal environmental factors which limit their usefulness. We addressed this issue by crosslinking multiple capsid surface reactive residues using polyethylene glycol tethers. Surface crosslinking enhanced the colloidal stability and mechanical strength of VLPs against low pH, proteases, and mechanical agitation, while it did not interfere with function as vaccine. Chemical crosslinking thus offers a viable means to enhance the resilience of VLPs in mucosal applications.

It is well-established that -synuclein aggregation may proceed through an initial lipid-dependent aggregate formation and, if at acidic pH, a subsequent aggregate-dependent proliferation. It has also been recently reported that the aggregation of -synuclein may also take place through an alternative pathway, which takes place within dense liquid condensates produced through liquid-liquid phase separation. The microscopic mechanism of this process, however, remains to be clarified. Here, we developed a fluorescence-based assay to perform a kinetic analysis of the aggregation process of -synuclein within liquid condensates, and applied it to determine the corresponding mechanism of aggregation. Our analysis shows that at pH 7.4 the aggregation process of -synuclein within dense condensates starts with spontaneous primary nucleation followed by rapid aggregate-dependent proliferation. Taken together, these results reveal a highly efficient pathway for the appearance and proliferation of -synuclein aggregates at physiological pH.

Lipids, and cationic lipids in particular, are of interest as delivery vectors for hydrophobic drugs such as the cancer therapeutic paclitaxel, and the structures of lipid assemblies affect their efficacy. We investigated the effect of incorporating the multivalent cationic lipid MVL5 (+5e) and poly(ethylene glycol)-lipids (PEG-lipids), alone and in combination, on the structure of fluid-phase lipid assemblies of the charge-neutral lipid 1,2-dioleoyl-sn-glycero-phosphocholine (DOPC). This allowed us to elucidate lipid-liposome structure correlations in sonicated formulations with high charge density, which are not accessible with univalent lipids such as the well-studied DOTAP (+1e). Cryogenic TEM allowed us to determine the structure of the lipid assemblies, revealing diverse combinations of vesicles and disc-shaped, worm-like, and spherical micelles. Remarkably, MVL5 forms an essentially pure phase of disc micelles at 50 mol% MVL5. At higher (75 mol%) content of MVL5, short and intermediate-length worm-like micellar rods were observed and, in ternary mixtures with PEG-lipid, longer and highly flexible worm-like micelles formed. Independent of their length, the worm-like micelles coexisted with spherical micelles. In stark contrast, DOTAP forms mixtures of vesicles, disc micelles and spherical micelles at all studied compositions, even when combined with PEG-lipids. The observed similarities and differences in the effects of charge (multivalent versus univalent) and high curvature (multivalent charge versus PEG-lipid) on assembly structure provide insights into parameters that control the size of fluid lipid nanodiscs, relevant for future applications.

Formation of extracellular polymeric substances (EPS) is a crucial step for bacterial biofilm growth. Dependence of EPS composition on the growth substrate and the conditioning of the latter is thus of primary importance. Here, we present results of studies on the growth of biofilms of two different strains each, of the Gram negative bacteria Escherichia coli and Klebsiella pneumoniae, on four polymers used commonly in indwelling medical devices - Polyethene, Polypropylene, Polycarbonate, and Polytetrafluoroethylene immersed in Bovine Serum Albumin (BSA) for 24 hrs. The polymer substrates are studied before and after immersing in BSA for 9 hrs and 24 hrs, using contact angle measurement (CAM) and Field Emission Scanning Electron Microscopy (FE-SEM) to extract, respectively, the philicity (defined as{phi} {equiv} sin ({theta}-90{degrees}), where{theta} is contact angle of the liquid on the solid at a particular temperature and ambient pressure) and spatial Hirsch parameter H (defined from the relation, F(r) ~ r2H, where F(r) is the mean squared density fluctuation at the sample surface). H =, <0.5 or >0.5 signifies no correlation, anti-correlation, and correlation, respectively. The substrates are seen to transform from large hydrophobicity to near amphiphilicity with the formation of BSA conditioning surface layer, and the H-values distinguish the length scales of ~ 100 nm, 500 nm, and 2000 nm, with the anti-correlation increasing with length scale. Biofilms grown on the BSA-covered surfaces are studied with CAM, FE-SEM, Fourier Transform Infrared (FTIR) and Surface Enhanced Raman Spectroscopy (SERS). Most notably, the{phi} -values are independent of the bacterial species and strain but dependent on the polymer, as is also shown strikingly by both types of spectra, while H-values show some bacterial variation. Thus, the EPS composition and consequently the wetting properties of the corresponding bacterial biofilms seems to be decided by the interaction of the conditioning BSA layer with a specific polymer used as the growth substrate.

Nanomaterials have been proposed as drug delivery vehicles to enhance targeting and efficiency of traditional and novel therapeutics and have subsequently been studied for potential ecotoxicity. Previous studies have identified size, surface charge, and volume exclusion as factors that influence nanomaterial diffusion and retention. However, there is little accepted or successful quantification of how these parameters influence nanomaterial penetration relative to biological adaptation and biological response. Part of the challenge is the response of living biological interfaces to many of these nanomaterial delivery vehicles and nanosized drugs. This study aimed to emulate key physicochemical barriers to diffusion found in living biomaterials by developing a tunable, synthetic hydrogel. Through the controlled exposure of 150 kDa and 2 MDa nanodextrans with neutral and negative surface charge, we evaluated the systems ability to emulate three core physicochemical features often implicated in biofilm-associated transport resistance: size exclusion, charge interactions, and volume exclusion. We demonstrated a 30% statistically significant decrease in partition coefficients for 2 MDa nanodextran from 150 kDa nanodextran, confirming the ability of the nanocellulose-based microcaps to mimic the permeability of hydrated biomaterial matrices. These findings reflect patterns observed in, for example, living biofilm studies, where size-based diffusion hinderance is commonly reported, but charge-based interaction and volume exclusion are more context-dependent. This controllable system can be coupled with in silico modeling to understand interfacial transport phenomena for nanomaterial-biomaterial interactions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/703274v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@13c1a34org.highwire.dtl.DTLVardef@dc6c5borg.highwire.dtl.DTLVardef@14dcbd4org.highwire.dtl.DTLVardef@80f70c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Recently, therapeutic uses of bacteriophage (phage) are gaining increased attention, yet common liquid phage formulations require cold chain storage that limits their potential use. Phage therapy is considered as an alternative to antibiotics for bacterial infections and more significantly a promising solution for the ever-increasing prevalence of multi-drug resistance (MDR) pathogens. One of the most promising applications of this therapy is to treat pulmonary bacterial infections. To efficiently deliver therapeutic phage to the lungs, phage formulations that allow for nebulization or dry powder inhalation are under active development. Several conventional particle engineering technologies have been applied in the development of dry powder inhalers (DPI), including spray drying, spray freeze drying, and atmospheric spray freeze drying, but these processes have their own disadvantages that limit their use with bacteriophage formulations and delivery. In our work, we hypothesize that thin film freeze-drying (TFFD) can be used to produce brittle matrix powders containing phage that may be suitable for delivery by several routes of administration, including by nebulization after reconstitution and by intranasal or inhalation delivery of the resulting dry powder. Here we selected T7 bacteriophage as our model phage in a preliminary screening study and found that a binary excipient matrix of sucrose and leucine at ratios of 80:20 or 75:25 by weight, protected bacteriophage from the stresses encountered during the TFFD process. In addition, we confirm that incorporating a buffer system during the TFFD process significantly improved the survival of phage during the ultra-rapid freezing step of the TFFD process and subsequent sublimation step in the lyophilization process. This preservation of phage bioactivity was significantly better than that observed for formulations without a buffer system. The titer loss of phage in standard SM buffer (Tris/NaCl/MgSO4/gelatin) containing formulation was as low as 0.2 log plaque forming units (pfu), which indicates that phage functionality was preserved after the TFFD process. Moreover, the presence of buffers markedly reduced the geometric particle sizes as determined by a dry dispersion method using laser diffraction, which indicates that the TFFD phage powder formulations were easily sheared into smaller powder aggregates, an ideal property for facilitating pulmonary delivery through DPIs. From these findings, we show that TFFD is a particle engineering method that can successfully produce phage containing powders that possess the desired properties for bioactivity and inhalation therapy.

The lining of the alveoli is covered by pulmonary surfactant, a complex mixture of surface-active lipids and proteins that enables efficient gas exchange between inhaled air and the circulation. Despite decades of advancements in the study of the pulmonary surfactant, the molecular scale behavior of the surfactant and the inherent role of the number of different lipids and proteins in surfactant behavior are not fully understood. The most important proteins in this complex system are the surfactant proteins SP-B and SP-C. Given this, in this work we performed non-equilibrium all-atom molecular dynamics simulations to study the interplay of SP-B and SP-C with multi-component lipid monolayers mimicking the pulmonary surfactant in composition. The simulations were complemented by z-scan fluorescence correlation spectroscopy and atomic force microscopy measurements. Our state-of-the-art simulation model reproduces experimental pressure-area isotherms and lateral diffusion coefficients. In agreement with previous research, the inclusion of either SP-B and SP-C increases surface pressure, and our simulations provide a molecular scale explanation for this effect: The proteins display preferential lipid interactions with phosphatidylglycerol, they reside predominantly in the lipid acyl chain region, and they partition into the liquid expanded phase or even induce it in an otherwise packed monolayer. The latter effect is also visible in our atomic force microscopy images. The research done contributes to a better understanding of the roles of specific lipids and proteins in surfactant function, thus helping to develop better synthetic products for surfactant replacement therapy used in the treatment of many fatal lung-related injuries and diseases.

Reentrant condensation (RC) is a phase behavior of protein solution comprising at least two components. In RC, a protein state varies from one phase to two phases and then back to one phase as the concentration of one component monotonically increases. To understand the phase behavior of multicomponent complex solutions of biomolecules, it is worth constructing an experimental multicomponent system that exhibits RC behavior. Here, we used a cola/milk mixture to investigate RC of a multicomponent complex system and explained the RC mechanism by reducing the system to two pure components, polyphosphate (polyP) and casein. In the multicomponent complex system, RC was observed with 20-60% cola and 1% milk. In the pure system, RC occurred with 0.01-2 mM tetraphosphate and 0.5 mg/ml casein. Moreover, the phase diagram showed that the condensation of casein depended on the chain length of the polyP. The present study succeeded in experimentally inducing RC in a multicomponent system and reproducing RC even when the system was reduced to its pure components. The fact that RC can be experimentally induced using common materials will provide important insights into the understanding of phase-separation behavior of biomolecules.

The top layer of skin, the stratum corneum, provides a formidable barrier to the skin. Nanoparticles are utilized and further explored for personal and health care applications related to the skin. In past years several researchers have studied the translocation and permeation of nanoparticles of various shapes, sizes, and surface chemistry through the cell membranes. Most of these studies focused on a single nanoparticle and a simple bilayer system, whereas skin has a highly complex lipid membrane architecture. Moreover, it is highly unlikely that a nanoparticle formulation applied on the skin will not have multiple nanoparticle-nanoparticle and skin-nanoparticle interactions. In this study, we have utilized coarse-grained MARTINI molecular dynamics simulations to assess the interactions of two types (bare and dodecane-thiol coated) of nanoparticles with two models (single bilayer and double bilayer) of skin lipid membranes. The nanoparticles were found to be partitioned from the water layer to the lipid membrane as an individual entity as well as in the cluster form. It was discovered that each nanoparticle reached the interior of both single bilayer and double bilayer membrane irrespective of nanoparticle type and concentration, though coated particles were observed to efficiently traverse across bilayer when compared with bare particles. The coated nanoparticles also created a single large cluster inside the membrane, whereas bare nanoparticles were found in small clusters. Both the nanoparticles exhibited preferential interactions with cholesterol molecules present in the lipid membrane as compared to other lipid components of the membrane. We have also observed that the single membrane model exhibited unrealistic instability at moderate to the higher concentration of nanoparticles, and hence for translocation study, at minimum double bilayer model should be employed.

Liquid proteinaceous materials have been frequently found in cells or tissues and are crucial for various biological processes. Unlike their solid-state counterparts, liquid-state protein compartments are challenging to engineer and control at the microscale. Conventionally, gelation (sol-gel transition) of biological molecules has been thought to be the intermediate step between liquid-liquid phase separation (LLPS) states and insoluble aggregates that are related to protein functions, malfunctions and even diseases. However, the opposite process, i.e., the gel-sol transition of materials, has not been broadly explored. Here we describe a thermoresponsive gel-sol transition of a protein in a crowded environment that results in a demixed LLPS state, contradicting the common consequence of a one-phase protein solution by the end of such transition at elevated temperature without crowding agents. We also demonstrate a simple method to monitor the gel-sol transition by showing that elongated gelatin microgels can evolve towards a spherical morphology in the crowding agents because of interfacial tension. The LLPS system was explored for the diffusion of small particles for drug-release application scenarios. Our results demonstrate a route for the rapid construction of LLPS models, where the gel-sol transition of the protein-rich phase is monitorable. The models are featured with tunable size and dimensional monodispersity of dispersed condensates. The present study can be employed in biophysics and bioengineering with practices such as 3D printing and temperature sensing.

Lipid nanoparticles (LNPs) are the most successful drug delivery carrier to date, but optimizing lipid formulations to improve membrane fusion capabilities for effective drug release has been challenging due to lack of a quantitative measure for fusogenicity. Here we introduce a new framework based on small angle X-ray scattering to experimentally measure [Formula] for lipids used in LNP formulations such as glycerol monooleate (GMO) and ionizable lipids (SM-102 and ALC-0315). Q intrinsically captures spontaneous curvature (J0), which is traditionally used to assess fusogenicity. The change of cubic lattice parameters with temperature was measured for GMO-containing lipid mixtures, and the Q extracted quantitatively correlated with LNP fusogenicity power validated by fluorescence-based fusion assays and cryogenic electron microscopy. Fusogenicity of SM-102 and ALC-0315 was quantified by adding them to host membranes and assessing change in Q. This framework provides researchers with the ability to optimize the fusogenicity of LNP formulations for potent drug release and enhances understanding of parameters governing fusion in all biomembranes.