Combination of Chemical and Physical Cancer Therapeutics Developed with Microcapsules Made of κ-Casein and Gold Nanoparticles in the Presence of Drug-Loaded Phase Change Materials.

Tumor heterogeneity requires development of an anticancer system equipped with both chemical and physical therapeutics to eradicate cancer exhibiting drug resistance and clonal evolution into diverse tumor cells. Assortment of various toxic components into one platform without compromising their individual toxic activity remains a formidable task. Herein, a novel drug delivery system (DDS) exerting potent cytotoxicity toward cancer cells was fabricated with gold nanoparticles (AuNPs) coated with an innocuous self-assembly protein of κ-casein (κC). Pickering emulsions of the κC-AuNP conjugates in the presence of chloroform inside led to the κC-AuNP microcapsules being stabilized via robust ß-sheet formation between κC molecules located on the single-layered shell made of κC-AuNPs. Phase change material (PCM) comprising a eutectic mixture of lauric acid and myristic acid with the melting point of 43 °C was encapsulated in the presence of a hydrophilic anticancer drug of doxorubicin (Dox), in which the PCM has played multiple functions such as drug-holding matrix and thermoresponsive gating material for drug release. Once liberated with the heat generated by the AuNPs upon a near-infrared (NIR) irradiation at 808 nm, the PCM by itself exhibited not only chemical cytotoxicity but also physical toxic effects such as membrane destabilization of the cells and a possible cellular fixative effect toward cancer cells by the solidified PCM at body temperature. Moreover, the PCM was shown to facilitate the intranuclear localization of Dox. As a result, the DDS comprising κC-AuNP microcapsules containing Dox-loaded PCM was demonstrated to show a powerful anticancer effect upon the NIR irradiation, which unleashed several toxic agents such as Dox, PCM, heat-generating AuNPs, and tissue-immobilizing solidified PCM. Therefore, the κC-AuNP microcapsules would serve as an anticancer system into which diverse chemical and physical therapeutic agents could be combined to effectively remove the heterogeneous and drug resistant cancer cells.

Multifactorial drug carrier system bringing both chemical and physical therapeutics to the treatment of tumor heterogeneity.

Tumor heterogeneity and drug resistance have been invincible features of cancer for its complete cure. Despite the advent of immunotherapy, the expansion and diversification of cancer cells evolved even in the absence or presence of drug treatment discourage additional therapeutic interventions. For the eradication of cancer cells, therefore, an 'all-at-once' strategy is required, which exploits both target-selective chemotherapy and non-selective physicotherapy. Multifactorial microcapsules comprising gold nanoparticles (AuNPs) and a self-assembly protein of α-synuclein (αS) were fabricated, in which hydrophobic and hydrophilic drugs could be separately encapsulated by employing lipid-based inverted micelles (IMs). Their combined physico-chemical therapeutic effects were examined since they also contained both membrane-disrupting IMs and heat-generating AuNPs upon irradiation as physicotherapeutic agents. For the optimal enclosure of IMs containing hydrophilic drugs, a porous inner skeleton made of poly(lactic-co-glycolic acid) was introduced, which would play the roles of not only compartmentalizing the internal space but also enhancing proteolytic disintegration of the microcapsules to discharge and stabilize IMs to the outside. In fact, hydrophobic paclitaxel and hydrophilic doxorubicin showed markedly enhanced drug efficacy when delivered in the IM-containing microcapsules exhibiting the 'quantal' release of both drugs into the cells whose integrity could be also affected by the IMs. In addition, the remnants of αS-AuNP microcapsules produced via proteolysis also caused cell death through photothermal effect. The multifactorial microcapsules are therefore considered as a promising anti-cancer drug carrier capable of performing combinatorial selective and non-selective chemical and physical therapies to overcome tumor heterogeneity and drug resistance.

Disulfide-Mediated Elongation of Amyloid Fibrils of α-Synuclein For Use in Producing Self-Healing Hydrogel and Dye-Absorbing Aerogel.

Due to their mechanical robustness, biocompatibility, and nanoscale size, amyloid fibrils (AFs) have been considered as a potential nanomaterial for biological applications. Unfortunately, however, AFs are usually not fully extended because of their pre-mature breakage, which hampers their use to generate biocompatible suprastructures, although the amounts of AFs could be amplified via their self-propagation property. Here, we have demonstrated the full extension of AFs of α-synuclein (αS) by introducing a cysteine residue to its C-terminus which prevents the shear-induced fragmentation of AFs via site-directed disulfide bond formation on the exposed surface of AFs. These heat- and cold-resistant elongated AFs were entangled into self-healable hydrogels through a mild disulfide-exchange process in the presence of tris(2-carboxyethyl) phosphine, which subsequently developed into dye-absorbing aerogels upon freeze-drying without collapsing the three-dimensional internal fibrillar network. The resulting αS aerogel with high porosity and increased surface area was shown to be capable of absorbing both hydrophilic and hydrophobic substances. In addition, the aerogel was further engineered with 8-arm polyethylene glycol containing a sulfhydryl group to increase its drug loading capacity and protease susceptibility for drug unloading. The elongated AFs, therefore, have been suggested to play a pivotal component for the development of bio-nano-matrix for diverse biological applications including drug delivery, tissue engineering, and environmental remediation. STATEMENT OF SIGNIFICANCE: Due to accurate protein self-assembly process, α-synuclein forms an amyloid fibril which are the major component of Lewy bodies. In general, α-synuclein amyloid fibrils break under thermal fluctuations as these nanofibrils elongate to reach certain length. In this study, we have demonstrated the full extension of α-synuclein amyloid fibrils by introducing a cysteine residue to its C-terminus by forming site-directed disulfide bonds on the exposed surface of amyloid fibrils for the first time. The resulting elongated amyloid fibrils were mechanically robust and stable. By using elongated amyloid fibrils, we have made self-healable amyloid fibril hydrogel and dye-absorbing aerogel.

Fabrication of a Dual Stimuli-Responsive Assorted Film Comprising Magnetic- and Gold-Nanoparticles with a Self-Assembly Protein of α-Synuclein.

Development of sensing elements for controllable soft materials is crucial to improve their responsiveness toward remotely provided external stimuli. Magnetic nanoparticles (MNPs) and gold nanoparticles (AuNPs) have been coassembled into a flexible free-floating 2D film to produce a shape deformable mobile structure in the presence of magnetic field and light irradiation by employing a self-assembly protein of α-synuclein (αS). αS was demonstrated to be essential for the preparation of a multisensory system because the intrinsically disordered protein led to a complete dispersion of MNPs to an average size of 10 nm in aqueous solution, pH-dependent closely packed single layer adsorption of αS-MNPs, and α-helix-mediated free-floating MNP monolayer film formation upon dissolving the underlying polycarbonate substrate with chloroform. As AuNPs were incorporated into the assorted hybrid film in the presence of MNPs, however, the ß-sheet component became prominent. By placing the assorted film between a spin-coated thin layer of thermoresponsive P(AAc-co-NIPAAm) hydrogel comprising acrylic acid and N-isopropylacrylamide and a passive layer of silicone elastomer, the resulting triply structure exhibited not only magnet-induced locomotion but also shape deformation due to asymmetric contraction of the sandwiching two layers caused by the heat generated by AuNPs upon near IR irradiation. In fact, two adjoining planar layers of another triply structure were shown to form a three-dimensional lotus flower with the light. This multisensory system is suggested to be further functionalized by modifying the αS molecules and incorporating additional nanoparticles to react to diverse stimuli, which would make the system be utilized in the areas of not only soft robotics but also foldable electronics, high-performance sensors/actuators, and medical/wearable applications.

Alternative Assembly of α-Synuclein Leading to Protein Film Formation and Its Application for Developing Polydiacetylene-Based Sensing Materials.

Understanding the self-assembly process of amyloidogenic protein is valuable not only to find its pathological implication but also to prepare protein-based biomaterials. α-Synuclein (αS), a pathological component of Parkinson's disease, producing one-dimensional (1D) amyloid fibrils, has been employed to generate two-dimensional (2D) protein films by encouraging an alternative self-assembly process. At a high temperature of 50 °C, αS molecules self-assembled into 2D films instead of 1D amyloid fibrils, whereas the fibrils were the major product at 37 °C. Based on circular dichroism and Fourier transform infrared spectroscopy analyses, the film was produced via a structural transition from the initial random to still undefined but mostly the turn or loop structure, which was distinctive from the ß-sheet formation observed with the amyloid fibrils. The αS 2D film was also routinely prepared at the oil-water interface and used as a matrix to produce polydiacetylene-based sensing materials. 10,12-Pentacosadiynoic acids (PCDA) were aligned on the film and photopolymerized to form a π-conjugated molecular assembly yielding a blue color. Its colorimetric transition to red was induced by increasing the temperature. This functionalized protein film increased its height from 40 to 55 nm upon PCDA immobilization and exhibited enhanced physical and chemical stability. In addition, the modified film showed remarkably high electrical conductivity only in the red state. This film, therefore, can be considered as a robust protein-based hybrid biomaterial capable of simultaneously recognizing various external stimuli (heat, pH, and solvents) with changes in color and conductivity, and it is expected to be utilized as a basic material for the development of biocompatible sensors.

Morphological Evaluation of Meta-stable Oligomers of α-Synuclein with Small-Angle Neutron Scattering.

Amyloidogenesis of α-synuclein (αS) is considered to be a pathological phenomenon related to Parkinson's disease (PD). As a key component to reveal the fibrillation mechanism and toxicity, we have investigated an oligomeric species of αS capable of exhibiting the unit-assembly process leading to accelerated amyloid fibril formation. These oligomers previously shown to exist in a meta-stable state with mostly disordered structure and unable to seed the fibrillation were converted to either temperature-sensitive self-associative oligomers or NaCl-induced non-fibrillating oligomeric species. Despite their transient and disordered nature, the structural information of meta-stable αS oligomers (Meta-αS-Os) was successfully evaluated with small-angle neutron scattering (SANS) technique. By fitting the neutron scattering data with polydisperse Gaussian Coil (pGC) model, Meta-αS-O was analyzed as a sphere with approximate diameter of 100 Å. Its overall shape altered drastically with subtle changes in temperature between 37 °C and 43 °C, which would be responsible for fibrillar polymorphism. Based on their bifurcating property of Meta-αS-Os leading to either on-pathway or off-pathway species, the oligomers could be suggested as a crucial intermediate responsible for the oligomeric diversification and multiple fibrillation processes. Therefore, Meta-αS-Os could be considered as a principal target to control the amyloidogenesis and its pathogenesis.

Fabrication of Protease-Sensitive and Light-Responsive Microcapsules Encompassed with Single Layer of Gold Nanoparticles by Using Self-Assembly Protein of α-Synuclein.

A bioapplicable cargo delivery system requires the following characteristics of biocompatibility, in vivo stability, and selective cargo release at target sites. We introduce herein the microcapsules enclosed with a single-layered shell of gold nanoparticles (AuNPs) mutually connected by an amyloidogenic protein of α-synuclein (αS). The microcapsules were fabricated by producing oil(chloroform)-in-water Pickering emulsions of the αS-encapsulated AuNPs and subsequent molecular engagement of the outlying αS molecules, leading to formidable ß-sheet formation in the presence of chloroform. The wrinkled skin of microcapsules obtained after evaporation of the internal chloroform also reflects robustness of the protein-protein interaction, which was experimentally confirmed by their rheological stability. For the emulsions loaded with rhodamine 6G, their dye release was demonstrated to be controlled by proteases. Along with their photothermal activity, the AuNP-containing microcapsules and their proteolyzed fragments were therefore suggested to be capable of eliminating aberrant cells in the protease-activated pathologically affected areas. Orthogonal cargo loading was also achieved by encapsulating both hydrophobic and hydrophilic substances either directly dissolved in chloroform or prepackaged in inverted micelles, respectively. Microcapsule's functionality was further expanded by localizing quantum dots, magnetic nanoparticles, and antibodies inside or on the surface of the microcapsules. Taken together, these multimodal AuNP microcapsules are suggested to be an ideal cargo carrier system, which could be employed in not only biomedical theranostic applications as they exhibit structural robustness, specific targeting, triggered release, and photothermal activity but also sensor development in general.

Graphene quantum dots prevent α-synucleinopathy in Parkinson's disease.

Though emerging evidence indicates that the pathogenesis of Parkinson's disease is strongly correlated to the accumulation1,2 and transmission3,4 of α-synuclein (α-syn) aggregates in the midbrain, no anti-aggregation agents have been successful at treating the disease in the clinic. Here, we show that graphene quantum dots (GQDs) inhibit fibrillization of α-syn and interact directly with mature fibrils, triggering their disaggregation. Moreover, GQDs can rescue neuronal death and synaptic loss, reduce Lewy body and Lewy neurite formation, ameliorate mitochondrial dysfunctions, and prevent neuron-to-neuron transmission of α-syn pathology provoked by α-syn preformed fibrils5,6. We observe, in vivo, that GQDs penetrate the blood-brain barrier and protect against dopamine neuron loss induced by α-syn preformed fibrils, Lewy body/Lewy neurite pathology and behavioural deficits.

EGCG-mediated Protection of the Membrane Disruption and Cytotoxicity Caused by the 'Active Oligomer' of α-Synuclein.

(-)-Epigallocatechin gallate (EGCG), the major component of green tea, has been re-evaluated with α-synuclein (αS), a pathological constituent of Parkinson's disease, to elaborate its therapeutic value. EGCG has been demonstrated to not only induce the off-pathway 'compact' oligomers of αS as suggested previously, but also drastically enhance the amyloid fibril formation of αS. Considering that the EGCG-induced amyloid fibrils could be a product of on-pathway SDS-sensitive 'transient' oligomers, the polyphenol effect on the transient 'active' oligomers (AOs) was investigated. By facilitating the fibril formation and thus eliminating the toxic AOs, EGCG was shown to suppress the membrane disrupting radiating amyloid fibril formation on the surface of liposomal membranes and thus protect the cells which could be readily affected by AOs. Taken together, EGCG has been suggested to exhibit its protective effect against the αS-mediated cytotoxicity by not only producing the off-pathway 'compact' oligomers, but also facilitating the conversion of 'active' oligomers into amyloid fibrils.

High-Density Single-Layer Coating of Gold Nanoparticles onto Multiple Substrates by Using an Intrinsically Disordered Protein of α-Synuclein for Nanoapplications.

Functional graffiti of nanoparticles onto target surface is an important issue in the development of nanodevices. A general strategy has been introduced here to decorate chemically diverse substrates with gold nanoparticles (AuNPs) in the form of a close-packed single layer by using an omni-adhesive protein of α-synuclein (αS) as conjugated with the particles. Since the adsorption was highly sensitive to pH, the amino acid sequence of αS exposed from the conjugates and its conformationally disordered state capable of exhibiting structural plasticity are considered to be responsible for the single-layer coating over diverse surfaces. Merited by the simple solution-based adsorption procedure, the particles have been imprinted to various geometric shapes in 2-D and physically inaccessible surfaces of 3-D objects. The αS-encapsulated AuNPs to form a high-density single-layer coat has been employed in the development of nonvolatile memory, fule-cell, solar-cell, and cell-culture platform, where the outlying αS has played versatile roles such as a dielectric layer for charge retention, a sacrificial layer to expose AuNPs for chemical catalysis, a reaction center for silicification, and biointerface for cell attachment, respectively. Multiple utilizations of the αS-based hybrid NPs, therefore, could offer great versatility to fabricate a variety of NP-integrated advanced materials which would serve as an indispensable component for widespread applications of high-performance nanodevices.

Polydiacetylenyl ß-cyclodextrin based smart vesicles for colorimetric assay of arginine and lysine.

Selective visualization of arginine and lysine has been explored among 20 amino acids using the hybrid conjugate of ß-cyclodextrin (ß-CD) and polydiacetylene (PDA). The mono pentacosa-10,12-diynyl aminomethyl group was successfully coupled to either the primary or the secondary face of ß-CD, where mono-6-amino-6-deoxy-ß-CD or mono-3-amino-3-deoxy-ß-CD reacted with the N-hydroxysuccinimide ester of 10,12-pentacosadiynoic acid. In this combinatorial system, the cylindrical ß-cyclodextrin functions as a channel for the introduction of the cationic amino acids to the artificial membrane. The membrane perturbation and aggregation by the target amino acids could be exclusively visualized as a blue to red color change based on the responsive polydiacetylene domain. These interesting findings demonstrated that the developed ß-CD conjugated PDA system may offer a new method of cell-penetrating mechanism, a promising vector system, as well as impact the production industry of arginine or lysine.

Exposure to bacterial endotoxin generates a distinct strain of α-synuclein fibril.

A single amyloidogenic protein is implicated in multiple neurological diseases and capable of generating a number of aggregate "strains" with distinct structures. Among the amyloidogenic proteins, α-synuclein generates multiple patterns of proteinopathies in a group of diseases, such as Parkinson disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). However, the link between specific conformations and distinct pathologies, the key concept of the strain hypothesis, remains elusive. Here we show that in the presence of bacterial endotoxin, lipopolysaccharide (LPS), α-synuclein generated a self-renewable, structurally distinct fibril strain that consistently induced specific patterns of synucleinopathies in mice. These results suggest that amyloid fibrils with self-renewable structures cause distinct types of proteinopathies despite the identical primary structure and that exposure to exogenous pathogens may contribute to the diversity of synucleinopathies.

Controlled Charge Trapping and Retention in Large-Area Monodisperse Protein Metal-Nanoparticle Conjugates.

Here, we report on charge-retention transistors based on novel protein-mediated Au nanoparticle (NP) arrays, with precise control over dimension and distribution. Individual NPs are coated with alpha-synuclein, an amyloidogenic protein responsible for Lewy body formation in Parkinson's disease. Subsequently, a monolayer of protein-NP conjugates is successfully created via a simple and scalable solution deposition to function as distributed nanoscale capacitors. Controllability over the film structure translates into the tunability of the electrical performance; pentacene-based organic transistors feature widely varying programmability and relaxation dynamics, providing versatility for various unconventional memory applications.

Free-standing gold-nanoparticle monolayer film fabricated by protein self-assembly of α-synuclein.

Free-standing nanoparticle films are of great importance for developing future nano-electronic devices. We introduce a protein-based fabrication strategy of free-standing nanoparticle monolayer films. α-Synuclein, an amyloidogenic protein, was utilized to yield a tightly packed gold-nanoparticle monolayer film interconnected by protein ß-sheet interactions. Owing to the stable protein-protein interaction, the film was successfully expanded to a 4-inch diameter sheet, which has not been achieved with any other free-standing nanoparticle monolayers. The film was flexible in solution, so it formed a conformal contact, surrounding even microspheres. Additionally, the monolayer film was readily patterned at micrometer-scale and thus unprecedented double-component nanoparticle films were fabricated. Therefore, the free-floating gold-nanoparticle monolayer sheets with these properties could make the film useful for the development of bio-integrated nano-devices and high-performance sensors.

Robust polydiacetylene-based colorimetric sensing material developed with amyloid fibrils of α-synuclein.

Robust polydiacetylene-based colorimetric sensing material has been developed with amyloid fibrils of α-synuclein in the presence of 10,12-pentacosadiynoic acid (PCDA) by taking advantage of the specific fatty acid interaction of α-synuclein and structural regularity of the self-assembled product of amyloid fibrils. PCDA facilitated not only self-oligomerization of α-synuclein but also its fibrillation into the fibrils with increased thickness. Upon UV irradiation, the PCDA-containing amyloid fibrils (AF-PCDAs) turned blue, which then became red following heat treatment. The blue-to-red color transition was also observed with other stimuli of pH and ethanol. AF-PCDAs were demonstrated to be mechanically stable since not only the individual colors of blue and red but also their colorimetric transition were not affected by a number of sonications which readily disrupted the polydiaceylene (PDA) vesicles with the instant loss of color. Therefore, AF-PCDA can be considered to be a novel PDA-based colorimetric sensing material with high mechanical strength, which has the potential to be employed in various areas involving advanced sensing technologies.

Molecular inscription of environmental information into protein suprastructures: temperature effects on unit assembly of α-synuclein oligomers into polymorphic amyloid fibrils.

Molecular-level storage of environmental information in biological structures in tangible forms, and their subsequent transfer to the next generation, has been studied using the phenomenon of amyloidogenesis, which defines a biochemical condition generating highly ordered protein aggregates known as amyloid fibrils. α-Synuclein oligomers shown to experience unit assembly as the formation of amyloid fibrils were used in the present study as an environment-sensing agent. With temperature varying in 2 °C intervals between 37 °C and 43 °C, the oligomeric unit assembly led to fibrillar polymorphism from a straight to a curly appearance, as assessed using TEM and small-angle neutron scattering; the different effects on the secondary structures were evaluated using attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy. The resulting diversified amyloid fibrils, which have distinctive molecular characteristics, were shown to be inherited by the next generation through the self-propagating property of amyloidogenesis. Storage of intangible temperature information in the diversified protein suprastructures and perpetuation of the stored information in the form of polymorphic amyloid fibrils could represent molecular inscription of environmental information into biological systems; this could further extend our understanding of any physiological/pathological significance of amyloidogenic polymorphism and be utilized in the area of nanobiotechnology to process various external signals.

Ca2+-dependent intracellular drug delivery system developed with "raspberry-type" particles-on-a-particle comprising mesoporous silica core and α-synuclein-coated gold nanoparticles.

For the development of an intracellular cargo release system with mesoporous silica nanoparticles (MSN), gold nanoparticles coated with an amyloidogenic protein of α-synuclein were employed to prepare a protein-mediated nanocomposite into the "raspberry-type" particles-on-a-particle (PoP). The PoPs were successfully fabricated only at pH 4.4 by yielding the MSN coverage to 75.3% with 5 nm gold nanoparticles covalently coated with a mutant form of α-synuclein containing a cysteine residue at the C-terminus. The entrapped cargo of rhodamine 6G was shown to be selectively released from PoPs upon exposure to divalent cations including the α-synuclein-specific pathophysiological ligand of Ca(2+). Intracellular uptake of the PoPs preloaded with doxorubicin as an anticancer drug and its subsequent Ca(2+)-dependent release were demonstrated with HeLa cells in the presence of intracellular Ca(2+)-regulating agents. Therefore, the fabrication of PoPs with the self-interactive protein of α-synuclein is expected to serve as a platform technology for preparation of diversified nanocomposites with various nanoparticles and/or bioactive molecules for eventual applications in the areas of theranostics.

Supramolecular self-assembled aggregates formed by pentacosa-10,12-diynyl amidomethyl-ß-cyclodextrin.

Mono[6-deoxy-6-(pentacosa-10,12-diynyl amidomethyl)]-ß-cyclodextrin was successfully synthesized by reacting mono-6-amino-6-deoxy-ß-cyclodextrin with N-hydroxysuccinimide ester of 10,12-pentacosadiynoic acid in DMF. The modified ß-cyclodextrin self-assembled and aggregated to form a worm-like supramolecular structure, and the novel supramolecular aggregates were studied using 2D nuclear magnetic resonance spectroscopy, X-ray powder diffraction, thermogravimetry, and electron microscopy. Interestingly, the synthesized pentacosa-10,12-diynyl amidomethyl-ß-cyclodextrin formed columnar type self-aggregates and it was clearly differentiated from cage-like structure of native ß-cyclodextrin.

Tyrosine-mediated two-dimensional peptide assembly and its role as a bio-inspired catalytic scaffold.

In two-dimensional interfacial assemblies, there is an interplay between molecular ordering and interface geometry, which determines the final morphology and order of entire systems. Here we present the interfacial phenomenon of spontaneous facet formation in a water droplet driven by designed peptide assembly. The identified peptides can flatten the rounded top of a hemispherical droplet into a plane by forming a macroscopic two-dimensional crystal structure. Such ordering is driven by the folding geometry of the peptide, interactions of tyrosine and crosslinked stabilization by cysteine. We discover the key sequence motifs and folding structures and study their sequence-specific assembly. The well-ordered, densely packed, redox-active tyrosine units in the YYACAYY (H-Tyr-Tyr-Ala-Cys-Ala-Tyr-Tyr-OH) film can trigger or enhance chemical/electrochemical reactions, and can potentially serve as a platform to fabricate a molecularly tunable, self-repairable, flat peptide or hybrid film.

Cholesterol reduction from milk using ß-cyclodextrin immobilized on glass.

ß-Cyclodextrin (ß-CD) was converted into ß-CD-undecenyl ether by chemical modification and subsequently covalently attached to a glass surface. The functionalized glass surface was characterized by static water contact angle and x-ray photoelectron spectroscopy. Both techniques confirmed that an excellent monolayer of ß-CD was formed on the glass surface. The ß-CD solid surface was used to reduce cholesterol levels in milk. In 4h, 73.6% of the cholesterol was extracted at 25°C with shaking at 170rpm. This is the highest value ever reported for milk using ß-CD immobilized on a solid surface. The same surface was repeatedly used for 10 cycles and maintained its efficiency with 72±2% cholesterol reduction observed in all the cycles. X-ray photoelectron spectroscopy analysis completed after 5 and 10 cycles of cholesterol reduction showed that the ß-CD on the glass surface was not degraded. The high efficiency and long-term stability of the functional monolayer was attributed to the specific structure of ß-CD, which is composed of a relatively low number of functional groups and long spacer chain lengths that provide great flexibility.