Polyelectrolyte-protein synergism: pH-responsive polyelectrolyte/insulin complexes as versatile carriers for targeted protein and drug delivery.

The co-assembly of polyelectrolytes (PE) with proteins offers a promising approach for designing complex structures with customizable morphologies, charge distribution, and stability for targeted cargo delivery. However, the complexity of protein structure limits our ability to predict the properties of the formed nanoparticles, and our goal is to identify the key triggers of the morphological transition in protein/PE complexes and evaluate their ability to encapsulate multivalent ionic drugs. A positively charged PE can assemble with a protein at pH above isoelectric point due to the electrostatic attraction and disassemble at pH below isoelectric point due to the repulsion. The additional hydrophilic block of the polymer should stabilize the particles in solution and enable them to encapsulate a negatively charged drug in the presence of PE excess. We demonstrated that diblock copolymers, poly(ethylene oxide)-block-poly(N,N-dimethylaminoethyl methacrylate) and poly(ethylene oxide)-block-poly(N,N,N-trimethylammonioethyl methacrylate), consisting of a polycation block and a neutral hydrophilic block, reversibly co-assemble with insulin in pH range between 5 and 8. Using small-angle neutron and X-ray scattering (SANS, SAXS), we showed that insulin arrangement within formed particles is controlled by intermolecular electrostatic forces between protein molecules, and can be tuned by varying ionic strength. For the first time, we observed by fluorescence that formed protein/PE complexes with excess of positive charges exhibited potential for encapsulating and controlled release of negatively charged bivalent drugs, protoporphyrin-IX and zinc(II) protoporphyrin-IX, enabling the development of nanocarriers for combination therapies with adjustable charge, stability, internal structure, and size.

Design of Nickel-Containing Nanocomposites Based on Ordered Mesoporous Silica: Synthesis, Structure, and Methylene Blue Adsorption.

Mesoporous materials containing heteroelements have a huge potential for use as catalysts, exchangers, and adsorbents due to their tunable nanometer-sized pores and exceptionally large internal surfaces accessible to bulky organic molecules. In the present work, ordered mesoporous silica containing Ni atoms as active sites was synthesized by a new low-temperature method of condensation of silica precursors on a micellar template from aqueous solutions in the presence of nickel salt. The homogeneity of the resulting product was achieved by introducing ammonia and ammonium salt as a buffer to maintain a constant pH value. The obtained materials were characterized by nitrogen sorption, X-ray and neutron diffraction, scanning electron microscopy, infrared spectroscopy, and thermal analysis. Their morphology consists of polydisperse spherical particles 50-300 nm in size, with a hexagonally ordered channel structure, high specific surface area (ABET = 900-1200 m2/g), large pore volume (Vp = 0.70-0.90 cm3/g), average mesopore diameter of about 3 nm, and narrow pore size distribution. Adsorption tests for methylene blue show sorption capacities reaching 39-42 mg/g at alkaline pH. The advantages of producing nickel silicates by this method, in contrast to precipitation from silicon alkoxides, are the low cost of reagents, fire safety, room-temperature processing, and the absence of specific problems associated with the use of ethanol as a solvent, as well as the absence of the inevitable capture of organic matter in the precipitation process.

Study of Precipitates in Oxide Dispersion-Strengthened Steels by SANS, TEM, and APT.

In this work, the nanostructure of oxide dispersion-strengthened steels was studied by small-angle neutron scattering (SANS), transmission electron microscopy (TEM), and atom probe tomography (APT). The steels under study have different alloying systems differing in their contents of Cr, V, Ti, Al, and Zr. The methods of local analysis of TEM and APT revealed a significant number of nanosized oxide particles and clusters. Their sizes, number densities, and compositions were determined. A calculation of hardness from SANS data collected without an external magnetic field, or under a 1.1 T field, showed good agreement with the microhardness of the materials. The importance of taking into account two types of inclusions (oxides and clusters) and both nuclear and magnetic scattering was shown by the analysis of the scattering data.

Synthesis, Characterization, and In Vitro Cytotoxicity Evaluation of Doxorubicin-Loaded Magnetite Nanoparticles on Triple-Negative Breast Cancer Cell Lines.

In this study, we investigated the cytotoxicity of doxorubicin (DOX)-loaded magnetic nanofluids on 4T1 mouse tumor epithelial cells and MDA-MB-468 human triple-negative breast cancer (TNBC) cells. Superparamagnetic iron oxide nanoparticles were synthesized using sonochemical coprecipitation by applying electrohydraulic discharge treatment (EHD) in an automated chemical reactor, modified with citric acid and loaded with DOX. The resulting magnetic nanofluids exhibited strong magnetic properties and maintained sedimentation stability in physiological pH conditions. The obtained samples were characterized using X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy, UV-spectrophotometry, dynamic light scattering (DLS), electrophoretic light scattering (ELS), vibrating sample magnetometry (VSM), and transmission electron microscopy (TEM). In vitro studies using the MTT method revealed a synergistic effect of the DOX-loaded citric-acid-modified magnetic nanoparticles on the inhibition of cancer cell growth and proliferation compared to treatment with pure DOX. The combination of the drug and magnetic nanosystem showed promising potential for targeted drug delivery, with the possibility of optimizing the dosage to reduce side-effects and enhance the cytotoxic effect on cancer cells. The nanoparticles' cytotoxic effects were attributed to the generation of reactive oxygen species and the enhancement of DOX-induced apoptosis. The findings suggest a novel approach for enhancing the therapeutic efficacy of anticancer drugs and reducing their associated side-effects. Overall, the results demonstrate the potential of DOX-loaded citric-acid-modified magnetic nanoparticles as a promising strategy in tumor therapy, and provide insights into their synergistic effects.

Densification of Two Forms of Nanostructured TATB under Uniaxial Die Pressures: A USAXS-SAXS Study.

Sequential ultra-small-angle and small-angle and X-ray scattering (USAXS and SAXS) measurements of hierarchical microstructure of a common energetic material, the high explosive 2,4,6-Triamino-1,3,5-trinitrobenzene (TATB), were performed to follow the microstructure evolution upon applied pressure. The pellets were prepared by two different routes-die pressed from a nanoparticle form and a nano-network form of TATB powder. The derived structural parameters, such as void size, porosity, and the interface area, reflected the response of TATB under compaction. Three populations of voids were observed in the probed q range from 0.007 to 7 nm-1. The inter-granular voids with size larger than 50 nm were sensitive to low pressures and had a smooth interface with the TATB matrix. The inter-granular voids with size of ~10 nm exhibited a less volume-filling ratio at high pressures (>15 kN) as indicated by a decrease of the volume fractal exponent. The response of these structural parameters to external pressures implied that the main densification mechanisms under die compaction were the flow, fracture, and plastic deformation of the TATB granules. Compared to the nanoparticle TATB, the applied pressure strongly influenced the nano-network TATB due to its more uniform structure. The findings and research methods of this work provide insights into the structural evolution of TATB during densification.

Micelle Formation in Aqueous Solutions of the Cholesterol-Based Detergent Chobimalt Studied by Small-Angle Scattering.

The structure and interaction parameters of the water-soluble cholesterol-based surfactant, Chobimalt, are investigated by small-angle neutron and X-ray scattering techniques. The obtained data are analyzed by a model-independent approach applying the inverse Fourier transformation procedure as well as considering a model fitting procedure, using a core-shell form factor and hard-sphere structure factor. The analysis reveals the formation of the polydisperse spherical or moderately elongated ellipsoidal shapes of the Chobimalt micelles with the hard sphere interaction in the studied concentration range 0.17-6.88 mM. The aggregation numbers are estimated from the micelle geometry observed by small-angle scattering and are found to be in the range of 200-300. The low pH of the solution does not have a noticeable effect on the structure of the Chobimalt micelles. The critical micelle concentrations of the synthetic surfactant Chobimalt in water and in H2O-HCl solutions were obtained according to fluorescence measurements as ~3 µM and ~2.5 µM, respectively. In-depth knowledge of the basic structural properties of the detergent micelles is necessary for further applications in bioscience and biotechnology.

Synthesis and Characterization of Citric Acid-Modified Iron Oxide Nanoparticles Prepared with Electrohydraulic Discharge Treatment.

Chemical co-precipitation from ferrous and ferric salts at a 1:1.9 stoichiometric ratio in NH4OH base with ultrasonication (sonolysis) in a low vacuum environment has been used for obtaining colloidal suspensions of Fe3O4 nanoparticles coated with citric acid. Before coating, the nanoparticles were processed by electrohydraulic discharges with a high discharge current (several tens of amperes) in a water medium using a pulsed direct current. Magnetite nanoparticles were obtained with an average crystallite diameter D = 25-28 nm as obtained by XRD and particle sizes of 25 nm as measured by small-angle X-ray scattering. Magnetometry showed that all samples were superparamagnetic. The saturation magnetization for the citric acid covered samples after electrohydraulic processing showed higher value (58 emu/g) than for the directly coated samples (50 emu/g). Ultraviolet-visible spectroscopy and Fourier transform infrared spectroscopy showed the presence and binding of citric acid to the magnetite surface by chemisorption of carboxylate ions. Hydrodynamic sizes obtained from DLS and zeta potentials were 93 and 115 nm, -26 and -32 mV for the citric acid covered nanoparticles and 226 nm and 21 mV for the bare nanoparticles, respectively. The hydraulic discharge treatment resulted in a higher citric acid coverage and better particle dispersion. The developed method can be used in nanoparticle synthesis for biomedical applications.

The intriguing dose-dependent effect of selected amphiphilic compounds on insulin amyloid aggregation: Focus on a cholesterol-based detergent, Chobimalt.

The amyloidogenic self-assembly of many peptides and proteins largely depends on external conditions. Among amyloid-prone proteins, insulin attracts attention because of its physiological and therapeutic importance. In the present work, the amyloid aggregation of insulin is studied in the presence of cholesterol-based detergent, Chobimalt. The strategy to elucidate the Chobimalt-induced effect on insulin fibrillogenesis is based on performing the concentration- and time-dependent analysis using a combination of different experimental techniques, such as ThT fluorescence assay, CD, AFM, SANS, and SAXS. While at the lowest Chobimalt concentration (0.1 µM; insulin to Chobimalt molar ratio of 1:0.004) the formation of insulin fibrils was not affected, the gradual increase of Chobimalt concentration (up to 100 µM; molar ratio of 1:4) led to a significant increase in ThT fluorescence, and the maximal ThT fluorescence was 3-4-fold higher than the control insulin fibril's ThT fluorescence intensity. Kinetic studies confirm the dose-dependent experimental results. Depending on the concentration of Chobimalt, either (i) no effect is observed, or (ii) significantly, ∼10-times prolonged lag-phases accompanied by the substantial, ∼ 3-fold higher relative ThT fluorescence intensities at the steady-state phase are recorded. In addition, at certain concentrations of Chobimalt, changes in the elongation-phase are noticed. An increase in the Chobimalt concentrations also triggers the formation of insulin fibrils with sharply altered morphological appearance. The fibrils appear to be more flexible and wavy-like with a tendency to form circles. SANS and SAXS data also revealed the morphology changes of amyloid fibrils in the presence of Chobimalt. Amyloid aggregation requires the formation of unfolded intermediates, which subsequently generate amyloidogenic nuclei. We hypothesize that the different morphology of the formed insulin fibrils is the result of the gradual binding of Chobimalt to different binding sites on unfolded insulin. A similar explanation and the existence of such binding sites with different binding energies was shown previously for the nonionic detergent. Thus, the data also emphasize the importance of a protein partially-unfolded state which undergoes the process of fibrils formation; i.e., certain experimental conditions or the presence of additives may dramatically change not only kinetics but also the morphology of fibrillar aggregates.

Ordered Mesoporous Silica Prepared in Different Solvent Conditions: Application for Cu(II) and Pb(II) Adsorption.

In this work, the synthesis of ordered mesoporous silica of MCM-41 type was investigated aimed at improving its morphology by varying the synthesis conditions in a one-pot process, employing different temperatures and solvent conditions. 2-methoxyethanol was used as co-solvent to ethanol. The co-solvent ratio and the synthesis temperature were varied. The pore morphology of the materials was characterized by nitrogen porosimetry and small angle neutron scattering (SANS), and the particle morphology by transmission electron microscopy (TEM) and ultra-small angle neutron scattering (USANS). The thermal behavior was investigated by simultaneous thermogravimetry-differential scanning calorimetry (TG-DSC) measurements. The SANS and N2 sorption results demonstrated that a well-ordered mesoporous structure was obtained at all conditions in the synthesis at room temperature. Addition of methoxyethanol led to an increase of the pore wall thickness. Simultaneously, an increase of methoxyethanol content led to lowering of the mean particle size from 300 to 230 nm, according to the ultra-small angle scattering data. The ordered porosity and high specific surfaces make these materials suitable for applications such as adsorbents in environmental remediation. Batch adsorption measurements of metal ion removal from aqueous solutions of Cu(II) and Pb(II) showed that the materials exhibit dominantly monolayer surface adsorption characteristics. The adsorption capacities were 9.7 mg/g for Cu(II) and 18.8 mg/g for Pb(II) at pH 5, making these materials competitive in performance to various composite materials.

Imidazolium Ionic Liquids as Designer Solvents Confined in Silica Nanopores.

Composite silica xerogels were prepared via acid catalysed sol-gel route using tetraethoxysilan (TEOS) as silica precursor, and 1-butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF4] or 1-butyl-3-methylimidazolium chloride [BMIM][Cl] ionic liquids, used simultaneously as co-solvents, catalysts and pore templates, at various IL-to-silica ratios. Morphology of the xerogels prepared using the different IL templating agents were investigated using scanning electron microscopy (SEM), nitrogen sorption and small angle neutron scattering (SANS). The thermal behavior of the composites was analyzed by thermal gravimetry, whereas the compositions were checked by infrared spectroscopy and EDX. The differences in the morphology and thermal behavior of the composites due to the different IL additives were revealed.

Interfacial Water and Microheterogeneity in Aqueous Solutions of Ionic Liquids.

In this work, aqueous solutions of two prototypical ionic liquids (ILs), [BMIM][BF4] and [BMIM][TfO], were investigated by UV Raman spectroscopy and small-angle neutron scattering (SANS) in the water-rich domain, where strong heterogeneities at mesoscopic length scales (microheterogeneity) were expected. Analyzing Raman data by a differential method, the solute-correlated (SC) spectrum was extracted from the OH stretching profiles, emphasizing specific hydration features of the anions. SC-UV Raman spectra pointed out the molecular structuring of the interfacial water in these microheterogeneous IL/water mixtures, in which IL aggregates coexist with bulk water domains. The organization of the interfacial water differs for the [BMIM][BF4] and [BMIM][TfO] solutions, being affected by specific anion-water interactions. In particular, in the case of [BMIM][BF4], which forms weaker H-bonds with water, the aggregation properties clearly depend on concentration, as reflected by local changes in the interfacial water. On the other hand, stronger water-anion hydrogen bonds and more persistent hydration layers were observed for [BMIM][TfO], which likely prevent changes in IL aggregates. The modeling of SANS profiles, extended to [BPy][BF4] and [BPy][TfO], evidences the occurrence of significant concentration fluctuations for all of the systems: this appears as a rather general phenomenon that can be ascribed to the presence of IL aggregation, mainly induced by (cation-driven) hydrophobic interactions. Nevertheless, larger concentration fluctuations were observed for [BMIM][BF4], suggesting that anion-water interactions are relevant in modulating the microheterogeneity of the mixture.

Structure and Intermolecular Interactions in Aqueous Solutions of Polyethylene Glycol.

Aqueous solutions of polyethylene glycol are studied by small-angle neutron scattering over a broad range of polymer molecular masses and concentrations. The scattering data were modeled by a Gaussian chain form factor combined with random phase approximation, which provided good fits over the whole studied concentration range. The results showed that polyethylene glycol in the molecular mass range 0.4-20 kDa in water at physiological temperature T = 37 °C behaves like a random coil in nearly theta solvent conditions. The obtained results serve as a reference for the description of complex mixtures with PEG used in various applications.

Advancement of Fluorescent and Structural Properties of Bovine Serum Albumin-Gold Bioconjugates in Normal and Heavy Water with pH Conditioning and Ageing.

The red-emitting fluorescent properties of bovine serum albumin (BSA)-gold conjugates are commonly attributed to gold nanoclusters formed by metallic and ionized gold atoms, stabilized by the protein. Others argue that red fluorescence originates from gold cation-protein complexes instead, not gold nanoclusters. Our fluorescence and infrared spectroscopy, neutron, and X-ray small-angle scattering measurements show that the fluorescence and structural behavior of BSA-Au conjugates are different in normal and heavy water, strengthening the argument for the existence of loose ionic gold-protein complexes. The quantum yield for red-emitting luminescence is higher in heavy water (3.5%) than normal water (2.4%), emphasizing the impact of hydration effects. Changes in red luminescence are associated with the perturbations of BSA conformations and alterations to interatomic gold-sulfur and gold-oxygen interactions. The relative alignment of domains I and II, II and III, III and IV of BSA, determined from small-angle scattering measurements, indicate a loose ("expanded-like") structure at pH 12 (pD ~12); by contrast, at pH 7 (pD ~7), a more regular formation appears with an increased distance between the I and II domains, suggesting the localization of gold atoms in these regions.

The Impact of Redox, Hydrolysis and Dehydration Chemistry on the Structural and Magnetic Properties of Magnetoferritin Prepared in Variable Thermal Conditions.

Ferritin, a spherically shaped protein complex, is responsible for iron storage in bacteria, plants, animals, and humans. Various ferritin iron core compositions in organisms are associated with specific living requirements, health state, and different biochemical roles of ferritin isomers. Magnetoferritin, a synthetic ferritin derivative, serves as an artificial model system of unusual iron phase structures found in humans. We present the results of a complex structural study of magnetoferritins prepared by controlled in vitro synthesis. Using various complementary methods, it was observed that manipulation of the synthesis technology can improve the physicochemical parameters of the system, which is useful in applications. Thus, a higher synthesis temperature leads to an increase in magnetization due to the formation of the magnetite phase. An increase in the iron loading factor has a more pronounced impact on the protein shell structure in comparison with the pH of the aqueous medium. On the other hand, a higher loading factor at physiological temperature enhances the formation of an amorphous phase instead of magnetite crystallization. It was confirmed that the iron-overloading effect alone (observed during pathological events) cannot contribute to the formation of magnetite.

Butyl-Methyl-Pyridinium Tetrafluoroborate Confined in Mesoporous Silica Xerogels: Thermal Behaviour and Matrix-Template Interaction.

Organic-inorganic silica composites have been prepared via acid catalyzed sol-gel route using tetramethoxysilan (TMOS) and methyl-trimethoxysilane (MTMS) as silica precursors and n-butyl-3-methylpyridinium tetrafluoroborate ([bmPy][BF4]) as co-solvent and pore template, by varying the content of the ionic liquid (IL). Morphology of the xerogels prepared using the ionic liquid templating agent were investigated using scanning electron microscopy and small angle neutron scattering (SANS). Thermal analysis has been used in order to evaluate the thermal and structural stability of the materials, in both nitrogen and synthetic air atmosphere. In nitrogen atmosphere, the IL decomposition took place in one step starting above 150 °C and completed in the 150-460 °C temperature interval. In synthetic air atmosphere, the IL decomposition produced two-step mass loss, mainly in the 170-430 °C temperature interval. The decomposition mechanism of the IL inside the silica matrix was studied by mass spectrometric evolved gas analysis (MSEGA). The measurements showed that the degradation of the IL's longer side chain (butyl) starts at low temperature (above 150 °C) through a C-N bond cleavage, initiated by the nucleophilic attack of a fluorine ion.

Physicochemical Characterization and Drug Release Properties of Methyl-Substituted Silica Xerogels Made Using Sol-Gel Process.

In this work, a multi-analytical approach involving nitrogen porosimetry, small angle neutron and X-ray scattering, Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopies, X-ray diffraction, thermal analysis and electron microscopy was applied to organically modified silica-based xerogels obtained through the sol-gel process. Starting from a tetraethoxysilane (TEOS) precursor, methyltriethoxysilane (MTES) was added to the reaction mixture at two different pH values (2.0 and 4.5) producing hybrid xerogels with different TEOS/MTES molar ratios. Significant differences in the structure were revealed in terms of the chemical composition of the silica network, hydrophilic/hydrophobic profile, particle dimension, pore shape/size and surface characteristics. The combined use of structural characterization methods allowed us to reveal a relation between the cavity dimensions, the synthesis pH value and the grade of methyl substitution. The effect of the structural properties on the controlled Captopril release efficiency has also been tested. This knowledge facilitates tailoring the pore network for specific usage in biological/medical applications. Knowledge on structural aspects, as reported in this work, represents a key starting point for the production of high-performance silica-based hybrid materials showing enhanced efficacy compared to bare silica prepared using only TEOS.

Correction: Structural insights into fusion mechanisms of small extracellular vesicles with model plasma membranes.

Correction for 'Structural insights into fusion mechanisms of small extracellular vesicles with model plasma membranes' by Fabio Perissinotto et al., Nanoscale, 2021, 13, 5224-5233, DOI: .

Structural insights into fusion mechanisms of small extracellular vesicles with model plasma membranes.

Extracellular vesicles (EVs) are a potent intercellular communication system. Such small vesicles transport biomolecules between cells and throughout the body, strongly influencing the fate of recipient cells. Due to their specific biological functions they have been proposed as biomarkers for various diseases and as optimal candidates for therapeutic applications. Despite their extreme biological relevance, their mechanisms of interaction with the membranes of recipient cells are still hotly debated. Here, we propose a multiscale investigation based on atomic force microscopy, small angle X-ray scattering, small angle neutron scattering and neutron reflectometry to reveal structure-function correlations of purified EVs in interaction with model membrane systems of variable complex compositions and to spot the role of different membrane phases on the vesicle internalization routes. Our analysis reveals strong interactions of EVs with the model membranes and preferentially with the borders of protruding phase domains. Moreover, we found that upon vesicle breaking on the model membrane surface, the biomolecules carried by/on EVs diffuse with different kinetics rates, in a process distinct from simple fusion. The biophysical platform proposed here has clear implications on the modulation of EV internalization routes by targeting specific domains at the plasma cell membrane and, as a consequence, on EV-based therapies.

Comparison of Structure and Adsorption Properties of Mesoporous Silica Functionalized with Aminopropyl Groups by the Co-Condensation and the Post Grafting Methods.

The adsorptive potential has been evaluated for the aminopropyl functionalized mesoporous silica materials obtained by co-condensation and post grafting methods. Nitrogen sorption, small angle neutron and X-ray scattering (SANS and SAXS) demonstrated high surface area and well-ordered hexagonal pore structure suitable for applications as adsorbents of metals from waste waters. A comparison of Cr(VI) adsorption properties of the materials prepared by different functionalization methods has been performed. The obtained results demonstrated the adsorption capacity due to the affinity of the chromium ions to the amino groups, and showed that co-condensation of tetraethoxysilane (TEOS) and 3-aminopropyl triethoxysilane (APTES) resulted in higher metal sorption capacity of the materials compared to post-synthesis grafting of aminopropyl groups onto the mesoporous silica particles.

Role of Protein-Water Interface in the Stacking Interactions of Granum Thylakoid Membranes-As Revealed by the Effects of Hofmeister Salts.

The thylakoid membranes of vascular plants are differentiated into stacked granum and unstacked stroma regions. The formation of grana is triggered by the macrodomain formation of photosystem II and light-harvesting complex II (PSII-LHCII) and thus their lateral segregation from the photosystem I-light-harvesting complex I (PSI-LHCI) super-complexes and the ATP-synthase; which is then stabilized by stacking interactions of the adjacent PSII-LHCII enriched regions of the thylakoid membranes. The self-assembly and dynamics of this highly organized membrane system and the nature of forces acting between the PSII-LHCII macrodomains are not well understood. By using circular dichroism (CD) spectroscopy, small-angle neutron scattering (SANS) and transmission electron microscopy (TEM), we investigated the effects of Hofmeister salts on the organization of pigment-protein complexes and on the ultrastructure of thylakoid membranes. We found that the kosmotropic agent (NH4)2SO4 and the Hofmeister-neutral NaCl, up to 2 M concentrations, hardly affected the macro-organization of the protein complexes and the membrane ultrastructure. In contrast, chaotropic salts, NaClO4, and NaSCN destroyed the mesoscopic structures, the multilamellar organization of the thylakoid membranes and the chiral macrodomains of the protein complexes but without noticeably affecting the short-range, pigment-pigment excitonic interactions. Comparison of the concentration- and time-dependences of SANS, TEM and CD parameters revealed the main steps of the disassembly of grana in the presence of chaotropes. It begins with a rapid diminishment of the long-range periodic order of the grana membranes, apparently due to an increased stacking disorder of the thylakoid membranes, as reflected by SANS experiments. SANS measurements also allowed discrimination between the cationic and anionic effects-in stacking and disorder, respectively. This step is followed by a somewhat slower disorganization of the TEM ultrastructure, due to the gradual loss of stacked membrane pairs. Occurring last is the stepwise decrease and disappearance of the long-range chiral order of the protein complexes, the rate of which was faster in LHCII-deficient membranes. These data are interpreted in terms of a theory, from our laboratory, according to which Hofmeister salts primarily affect the hydrophylic-hydrophobic interactions of proteins, and the stroma-exposed regions of the intrinsic membrane proteins, in particular-pointing to the role of protein-water interface in the stacking interactions of granum thylakoid membranes.