Cell-specific delivery of polymeric nanoparticles to carbohydrate-tagging cells

Carbohydrates on cell surfaces contribute a variety of communications between the cell and its environment, and they have been assumed to act as markers for cellular recognition. In this research, 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer nanoparticles, which can react with specific carbohydrates of target cells, were newly prepared to serve as novel drug carriers. A water-soluble MPC polymer bearing hydrazide groups (PMBH) was synthesized by conventional radical polymerization. The MPC polymer showed amphiphilic nature and worked as an emulsifier to form nanoparticles. The nanoparticles covered with PMBH were prepared by the solvent evaporation method and exhibited monodispersity. They were approximately 200 nm in diameter and -2.0 mV in surface potential. According to a surface analysis of the nanoparticles, phosphorylcholine and hydrazide groups were observed, and the surface was fully covered with PMBH. Unnatural carbohydrates having ketone groups on human cervical carcinoma cell (HeLa) surfaces were expressed by treatment with levulinoyl mannosamine (ManLev). When the PMBH nanoparticles were in contact with the ManLev-treated HeLa cells, they accumulated in the cells. In contrast, the nanoparticles were not observed in native HeLa cells (without unnatural carbohydrates). These results indicate that the hydrazide groups of the nanoparticles selectively reacted to the ketone groups of the carbohydrates on the cell surface. The PMBH nanoparticles immobilized with anticancer drugs such as doxorubicin or paclitaxel were in contact with either ManLev-treated or untreated HeLa cells. The viability of the ManLev-treated HeLa cells was effectively reduced, but that of the untreated cells was preserved. This indicated that the anticancer drugs were selectively delivered to the ManLev-treated cells. Nonspecific cellular uptake of the nanoparticles was effectively reduced by MPC polymer coating. Furthermore, the immobilization processes of the drugs differed because of the solubility of the drugs. In conclusion, cellular-specific drug delivery by means of the novel nanoparticles was demonstrated with the selective reaction between unnatural carbohydrates on the cell surface and the hydrazide groups bearing the phosphorylcholine polymer nanoparticles.     

Folate receptor-targeted aggregation-enhanced near-IR emitting silica nanoprobe for one-photon in vivo and two-photon ex vivo fluorescence bioimaging

A two-photon absorbing (2PA) and aggregation-enhanced near-infrared (NIR) emitting pyran derivative, encapsulated in and stabilized by silica nanoparticles (SiNPs), is reported as a nanoprobe for two-photon fluorescence microscopy (2PFM) bioimaging that overcomes the fluorescence quenching associated with high chromophore loading. The new SiNP probe exhibited aggregate-enhanced emission producing nearly twice as strong a signal as the unaggregated dye, a 3-fold increase in two-photon absorption relative to the DFP in solution, and approximately 4-fold increase in photostability. The surface of the nanoparticles was functionalized with a folic acid (FA) derivative for folate-mediated delivery of the nanoprobe for 2PFM bioimaging. Surface modification of SiNPs with the FA derivative was supported by zeta potential variation and (1)H NMR spectral characterization of the SiNPs as a function of surface modification. In vitro studies using HeLa cells expressing a folate receptor (FR) indicated specific cellular uptake of the functionalized nanoparticles. The nanoprobe was demonstrated for FR-targeted one-photon in vivo imaging of HeLa tumor xenograft in mice upon intravenous injection of the probe. The FR-targeting nanoprobe not only exhibited highly selective tumor targeting but also readily extravasated from tumor vessels, penetrated into the tumor parenchyma, and was internalized by the tumor cells. Two-photon fluorescence microscopy bioimaging provided three-dimensional (3D) cellular-level resolution imaging up to 350 μm deep in the HeLa tumor.     

Conjugation of enzymes on polymer nanoparticles covered with phosphorylcholine groups

We investigated the bioconjugation of enzymes on polymer nanoparticles covered with bioinert phosphorylcholine groups. A water-soluble amphiphilic phospholipid polymer (PMBN) was specially designed for preparation of nanoparticles and conjugation with enzymes on them. The PMBN was prepared by random copolymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC), n-butyl methacrylate, and p-nitrophenylester bearing methacrylate. The PMBN was used as an emulsifier and a surface modifier to prepare the poly(l-lactic acid) nanoparticles by a solvent evaporation technique in aqueous medium. The nanoparticles covered with phosphorylcholine groups were stably dispersed in an aqueous solution and a phosphate buffered saline. The diameter and surface zeta-potential of the nanoparticles were ca. 200 nm and -6 mV, respectively. The p-nitrophenyl ester groups, which are active ester units for the amino groups of the protein, were located at the surface of the nanoparticles. Both acetylcholine esterase and choline oxidase were co-immobilized (dual-mode conjugation) by the reaction between the p-nitrophenyl ester group and the amino group of these enzymes. The enzymatic reactions on the nanoparticles were followed using a microdialysis biosensor system with a microtype hydrogen peroxide electrode in the probe. The nanoparticles conjugated with these enzymes could detect the acetylcholine chloride as hydrogen peroxide, which is a product of the enzymatic reactions on the surface of the nanoparticles in the probe. Namely, continuous enzyme reactions could be occurring on the surface of the nanoparticles. It is concluded that the nanoparticles are a promising tool for a highly sensitive and microdiagnostic system.     

Polymer nanoparticles covered with phosphorylcholine groups and immobilized with antibody for high-affinity separation of proteins

Novel polymer nanoparticles were prepared for the selective capture of a specific protein from a mixture with high effectiveness. The nanoparticle surface was covered with hydrophilic phosphorylcholine groups and active ester groups for easy immobilization of antibodies. Phospholipid polymers (PMBN) composed of 2-methacryloyloxyethyl phosphorylcholine, n-butyl methacrylate, and p-nitrophenyloxycarbonyl polyethyleneglycol methacrylate, were synthesized for the surface modification of poly( l-lactic acid) nanoparticles. Surface analysis of the nanoparticles using laser-Doppler electrophoresis and X-ray photoelectron spectroscopy revealed that the surface of nanoparticles was covered with PMBN. Protein adsorption was evaluated with regard to the nonspecific adsorption on the nanoparticles that was effectively suppressed by the phosphorylcholine groups. The immobilization of antibodies on nanoparticles was carried out under physiological conditions to ensure specific binding of antigens. The antibody immobilized on the nanoparticles exhibited high activity and strong affinity for the antigen similar to that exhibited by an antibody in a solution. The selective binding of a specific protein as an antigen from a protein mixture was relatively high compared to that observed with conventional antibody-immobilized polymer nanoparticles. In conclusion, nanoparticles having both phosphorylcholine and active ester groups for antibody immobilization have strong potential for use in highly selective separation based on the biological affinities between biomolecules.     

Multiple Protein-immobilized Phospholipid Polymer Nanoparticles: Effect of Spacer Length on Residual Enzymatic Activity and Molecular Diagnosis

We report the development of phosphorylcholine (PC) group-covered nanoparticles for multiple immobilization reactions; the surface of these nanoparticles facilitates bioreactions such as enzymatic reactions and molecular diagnoses. The nanoparticles were covered with a bioconjugate PC group containing a polymer backbone, and their surface properties were as follows: (1) suppression of nonspecific protein adsorption and (2) stabilization of immobilized biomolecules. In this study, biomolecules were immobilized on PC-covered nanoparticles by using different spacer lengths between the polymer backbone and biomolecules. The stability of the immobilized biomolecules was evaluated using horseradish peroxidase-labeled IgG, and the bioconjugate nanoparticles were stored at 4, 25, and 40 °C. The residual enzymatic activity of the peroxidase was monitored at a particular time. On the other hand, to test the role of these nanoparticles in molecular diagnosis, we used IgG-conjugated nanoparticles and the fluorescence resonance energy transfer (FRET) phenomenon. The IgG molecules were labeled with either donor or acceptor molecules, and each labeled IgG was simultaneously immobilized on the PC-covered nanoparticles. These labeled IgG molecules induce the FRET phenomenon upon capture of the target antigen provided they are in close proximity. The resulting fluorescence was readable via the FRET phenomenon. In the present study, C-reactive protein (CRP) was used as the target antigen, and the effect of the spacer length is discussed. The bioconjugated nanoparticles covered with PC groups are promising tools for tuning bioreactions.     

Detecting minimal traces of DNA using DNA covalently attached to superparamagnetic nanoparticles and direct PCR-ELISA

A single bond covalent immobilization of aminated DNA probes on magnetic particles suitable for selective molecular hybridization of traces of DNA samples has been developed. Commercial superparamagnetic nanoparticles containing amino groups were activated by coating with a hetero-functional polymer (aldehyde-aspartic-dextran). This new immobilization procedure provides many practical advantages: (a) DNA probes are immobilized far from the support surface preventing steric hindrances; (b) the surface of the nanoparticles cannot adsorb DNA ionically; (c) DNA probes are bound via a very strong covalent bond (a secondary amine) providing very stable immobilized probes (at 100 degrees C, or in 70% formamide, or 0.1N NaOH). Due to the extreme sensitivity of this purification procedure based on DNA hybridization, the detection of hybridized products could be coupled to a PCR-ELISA direct amplification of the DNA bond to the magnetic nanoparticles. As a model system, an aminated DNA probe specific for detecting Hepatitis C Virus cDNA was immobilized according to the optimised procedure described herein. Superparamagnetic nanoparticles containing the immobilized HCV probe were able to give a positive result after PCR-ELISA detection when hybridized with 1 mL of solution containing 10(-18) g/mL of HCV cDNA (two molecules of HCV cDNA). In addition, the detection of HCV cDNA was not impaired by the addition to the sample solution of 2.5 million-fold excess of non-complementary DNA. The experimental data supports the use of magnetic nanoparticles containing DNA probes immobilized by the procedure here described as a convenient and extremely sensitive procedure for purification/detection DNA/RNA from biological samples. The concentration/purification potential of the magnetic nanoparticles, its stability under a wide range of conditions, coupled to the possibility of using the particles directly in amplification by PCR greatly reinforces this methodology as a molecular diagnostic tool.     

Adhesive cell cultivation on polymer particle having grafted epoxy polymer chain

In this study, we synthesized a new cell immobilization support having poly(glycidyl methacrylate) as a graft polymer chain and used this support for cell cultivation. Base polymer particle was synthesized by suspension polymerization and epoxy polymer chain was extended from particle surface on graft polymerization. Produced polymer particles had broad particle size distribution ranging from 20 to 1000 μm and the degree of polymerization of grafted polymer chain was ranged from 500 to 1000. The effects of various factors, such as grafted polymer chain length and its surface density, composition of base polymer network and graft polymer chain, on the cell growth of murine fibroblast cell line (MS-5 cell) on polymer particle were studied. This polymer particle could cultivate not only fibroblast cell line but also epidermal cell line (HeLa cell), osteoblast cell line (MC3T3E1 cell), and chondrocyte cell line (ch-8 cell) on its surface. Growth rate is almost the same as that of cells using poly(styrene) tissue culture dish. To apply this cell cultivation system for examination of cell co-culture, HeLa cell immobilized on 100 μm of polymer particle was successfully co-cultured with MS-5 cell immobilized on 300 μm of polymer particle for four weeks.     

A lateral flow biosensor for rapid detection of DNA-binding protein c-jun

A lateral flow biosensor based on an immuno-chromatographic assay has been developed for the detection of DNA-binding proteins. The biosensor is composed of four parts: a sample pad, a conjugate pad, a strip of nitrocellulose membrane and an absorbent pad. A DNA probe containing a specific protein binding consensus sequence is coated onto gold nanoparticles, while an antibody against the DNA-binding protein is immobilized onto a test zone of the nitrocellulose membrane. The target protein binds to the protein binding DNA sequence that is coated on the gold nanoparticles to form nanoparticle-DNA-protein complexes, and the complexes are then captured by the antibody immobilized on the test zone to form a red line for visual detection of the target protein. This biosensor was successfully applied to a DNA-binding protein, c-jun, and the developed biosensor allows for the rapid detection of down to 0.2 footprint unit of c-jun protein within 10 min. This biosensor was verified using HeLa cells and it visually detected c-jun activity in 100 μg of crude cell lysate protein. The antibody against c-jun used in the biosensor can distinguish c-jun from other nonspecific proteins, with high specificity.     

Cytotoxicity and apoptotic effects of nickel oxide nanoparticles in cultured HeLa cells

The aim of this study was to observe the cytotoxicity and apoptotic effects of nickel oxide nanoparticles on human cervix epithelioid carcinoma cell line (HeLa). Nickel oxide precursors were synthesized by an nickel sulphate-excess urea reaction in boiling aqueous solution. The synthesized NiO nanoparticles (<200 nm) were investigated by X-ray diffraction analysis and transmission electron microscopy techniques. For cytotoxicity experiments, HeLa cells were incubated in 50-500 μg/mL NiO for 2, 6, 12 and 16 hours. The viable cells were counted with a haemacytometer using light microscopy. The cytotoxicity was observed low in 50-200 μg/mL concentration for 16 h, but high in 400-500 μg/mL concentration for 2-6 h. HeLa cells' cytoplasm membrane was lysed and detached from the well surface in 400 μg/mL concentration NiO nanoparticles. Double staining and M30 immunostaining were performed to quantify the number of apoptotic cells in culture on the basis of apoptotic cell nuclei scores. The apoptotic effect was observed 20% for 16 h incubation.     

Nanogel-quantum dot hybrid nanoparticles for live cell imaging

We report here a novel carrier of quantum dots (QDs) for intracellular labeling. Monodisperse hybrid nanoparticles (38 nm in diameter) of QDs were prepared by simple mixing with nanogels of cholesterol-bearing pullulan (CHP) modified with amino groups (CHPNH2). The CHPNH2-QD nanoparticles were effectively internalized into the various human cells examined. The efficiency of cellular uptake was much higher than that of a conventional carrier, cationic liposome. These hybrid nanoparticles could be a promising fluorescent probe for bioimaging.     

Characteristics of filamentous cells immobilized with ionic-hydrophobic polymers prepared by a radiation polymerization method

Filamentous cells of Trichoderma reesei were immobilized using a fibrous carrier covered with ionic-hydrophobic polymers, prepared by radiation copolymerization. The effect of the ionic properties of the polymers was investigated by changing the monomer composition in a trimethylpropane triacrylate (A-TMPT) — acrylic acid (AA) or methacrylic acid diethylaminoethyl ester (DEAEMA) system. More positive charge or less negative charge in the polymers led to an increase in the growth of the cells immobilized on their surface. Enzyme productivity in the immobilized cells with AA-A-TMPT polymer was higher than with a DEAEMA-A-TMPT polymer.     

Generic delivery of payload of nanoparticles intracellularly via hybrid polymer capsules for bioimaging applications

Towards the goal of development of a generic nanomaterial delivery system and delivery of the 'as prepared' nanoparticles without 'further surface modification' in a generic way, we have fabricated a hybrid polymer capsule as a delivery vehicle in which nanoparticles are loaded within their cavity. To this end, a generic approach to prepare nanomaterials-loaded polyelectrolyte multilayered (PEM) capsules has been reported, where polystyrene sulfonate (PSS)/polyallylamine hydrochloride (PAH) polymer capsules were employed as nano/microreactors to synthesize variety of nanomaterials (metal nanoparticles; lanthanide doped inorganic nanoparticles; gadolinium based nanoparticles, cadmium based nanoparticles; different shapes of nanoparticles; co-loading of two types of nanoparticles) in their hollow cavity. These nanoparticles-loaded capsules were employed to demonstrate generic delivery of payload of nanoparticles intracellularly (HeLa cells), without the need of individual nanoparticle surface modification. Validation of intracellular internalization of nanoparticles-loaded capsules by HeLa cells was ascertained by confocal laser scanning microscopy. The green emission from Tb(3+) was observed after internalization of LaF(3):Tb(3+)(5%) nanoparticles-loaded capsules by HeLa cells, which suggests that nanoparticles in hybrid capsules retain their functionality within the cells. In vitro cytotoxicity studies of these nanoparticles-loaded capsules showed less/no cytotoxicity in comparison to blank capsules or untreated cells, thus offering a way of evading direct contact of nanoparticles with cells because of the presence of biocompatible polymeric shell of capsules. The proposed hybrid delivery system can be potentially developed to avoid a series of biological barriers and deliver multiple cargoes (both simultaneous and individual delivery) without the need of individual cargo design/modification.     

Intracellular signal-responsive artificial gene regulation for novel gene delivery

We describe two types of artificial gene-regulation systems responding to cyclic AMP-dependent protein kinase (PKA) or caspase-3. These molecular systems use newly synthesized cationic polymers, PAK and PAC. The PAK polymer includes substrate oligopeptide for PKA, ARRASLG, as receptor of PKA signal, while the PAC polymer possesses oligopeptide that is comprised of a substrate sequence of caspase-3, DEVD, and a cationic oligolysine, KKKKKK. These polymers formed stable complexes with DNA to totally suppress the gene expression. However, PKA or caspase-3 signal disintegrates the PAK-DNA or the PAC-DNA complex, respectively. This liberates the DNA and activated the gene expression. These systems are the first concept of an intracellular signal-responsive gene-regulation system using artificial polymer. We expect that these systems can be applied to the novel highly cell specific gene delivery strategy that is involved in our previously proposed new drug delivery concept, the drug delivery system based on responses to cellular signals.     

Conjugating folic acid to gold nanoparticles through glutathione for targeting and detecting cancer cells

Gold nanoparticles (GNPs) were modified with glutathione (GSH) to form GSH-capped GNPs, which have carboxyl groups on the surface of these nanoparticles. Then folic acid (FA) was conjugated with GNPs through the reaction between amino group of FA and carboxyl group of GSH. These folic acid-conjugated nanoparticles (FA-GSH-GNPs) were stable in aqueous solution over a broad range of pH and ionic strength values. The targeting of FA-GSH-GNPs in human cervices carcinoma cells (HeLa cells) with high-level folate receptor expression was confirmed by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). No cellular uptake of these nanoparticles was observed in A549 cells lack of folate receptor. HeLa cells and mouse fibroblasts incubated with FA-GSH-GNPs were assayed by measuring the relative absorbance of the supernatant collected at low-speed centrifugation. Based on this simple spectroscopic method, HeLa cells have been detected with a detection limit of 10² cells/mL.     

Enhancing the reusability of endoglucanase-gold nanoparticle bioconjugates by tethering to polyurethane microspheres

The synthesis of polyurethane microsphere-gold nanoparticle "core-shell" structures and their use in the immobilization of the enzyme endoglucanase are described. Assembly of gold nanoparticles on the surface of polymer microspheres occurs through interaction of the nitrogens in the polymer with the nanoparticles, thereby precluding the need for modifying the polymer microspheres to enable such nanoparticle binding. Endoglucanse could thereafter be bound to the gold nanoparticles decorating the polyurethane microspheres, leading to a highly stable biocatalyst with excellent reuse characteristics. The immobilized enzyme retains its biocatalytic activity and exhibits improved thermal stability relative to free enzyme in solution. The high surface area of the host gold nanoparticles renders the immobilized enzyme "quasi free", while at the same time retaining advantages of immobilization such as ease of reuse, enhanced temporal and thermal stability, etc.     

Selective cell attachment to a biomimetic polymer surface through the recognition of cell-surface tags

Reactive phosphorylcholine polymers, which can recognize biosynthetic cell-surface tags, were synthesized to control cell attachment. Human promyelocytic leukemia cells (HL-60) with unnatural carbohydrates as cell-surface tags were harvested by treatment with N-levulinoylmannosamine (ManLev). The attachment of ManLev-treated HL-60 cells to 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers with hydrazide groups was studied. HL-60 cells, which are nonadhesive, did not attach to any polymer surface without ManLev treatment. In contrast, ManLev-treated HL-60 cells attached to a poly[MPC-co-n-butyl methacrylate (BMA)-co-methacryloyl hydrazide (MH)] (PMBH) surface following 15 min of incubation. The cells that attached to the PMBH surface retained their native morphology and viability for 24 h of incubation. On the other hand, approximately half of the HL-60 cells that attached to the poly(BMA-co-MH) (PBH) surface died. These results suggest that MH units in the polymer act as anchors for cell attachment and MPC units help to preserve cell viability on a polymer surface. The coculture of ManLev-treated HL-60 and fluorescence-stained human uterine cervical cancer cells (HeLa) was carried out on polymer surfaces. ManLev-treated HL-60 cells specifically attached to the PMBH surface. In contrast, both HL-60 and HeLa cells were observed on the PBH surface. The control of cellular interactions with synthetic polymers may be useful for the future development of cell-integrated biosensors and biomedical devices.     

Carbon dots as a luminescence sensor for ultrasensitive detection of phosphate and their bioimaging properties

Highly blue fluorescence carbon dots were synthesized by one‐step hydrothermal treatment of potatoes. The as‐obtained C‐dots have been applied to bioimaging of HeLa cells, which shows their excellent biocompatibility and low cytotoxicity. The results reveal that C‐dots are promising for real cell imaging applications. In addition, the carbon dots can be utilized as a probe for sensing phosphate. Copyright © 2014 John Wiley & Sons, Ltd.     

Virus-receptor interaction in the adenovirus system I. Identification of virion attachment proteins of the HeLa cell plasma membrane.

Plasma membranes from HeLa cells were isolated in a two-phase polymer system. To compare the efficiency of attachment protein extraction, a normalized assay for the assessment of adenovirus type 2 (Ad2) receptor-active components interfering with the attachment of Ad2 to HeLa cells was developed. An optimized detergent extraction procedure, 0.5% Triton X-100, was used, and solubilized membrane proteins were radioisotope labeled in vitro. Proteins with affinity for Ad2 virions were quantified and identified in a sucrose gradient sedimentation assay and by affinity chromatography with cross-linked Ad2 virions immobilized to AH-Sepharose 4B. From virions recovered in the sucrose gradient system, one major membrane component of high affinity was identified with a polypeptide molecular weight of around 40,000. Glycosylated proteins isolated by wheat germ lectin chromatography with high affinity for immobilized virus particles were isolated, and two major components with apparent molecular weights of 40,000 and 42,000 were identified. We suggest that a glycosylated protein with high affinity for Ad2 virions and a polypeptide molecular weight of 40,000 to 42,000 is one component of the Ad2 attachment site on HeLa cells.     

Anticancer potential of AgNPs synthesized using Acinetobacter sp. and Curcuma aromatica against HeLa cell lines: A comparative study

BackgroundBiogenic nanoparticles are gaining attention due to their low toxicity and numerous biomedical applications. Present study aimed to compare the potential anticancer activity of two biogenic silver nanoparticles (bAgNPs and pAgNPs) against human cervical cancer cell lines (HeLa).MethodsbAgNPs were synthesized using Acinetobacter sp. whereas pAgNPs were synthesized using aqueous root extract of Curcuma aromatica. Effect of these nanoparticles on HeLa cells viability was studied using MTT assay and colony formation assay. Anticancer potential was determined using fluorescence microscopy and flow cytometry studies. Bio-compatibility studies were performed against peripheral blood mononuclear cells (PBMCs).ResultsBoth the nanoparticles showed 50 % viability of peripheral blood mononuclear cells (PBMCs) when used at high concentration (200 μg/mL). IC₅₀ for bAgNPs and pAgNPs against HeLa cells were 17.4 and 14 μg/mL respectively. Colony formation ability of Hela cells was reduced on treatment with both nanoparticles. Acridine orange and ethidium bromide staining demonstrated that bAgNPs were cytostatic whereas pAgNPs were apoptotic. JC-1 dye staining revealed that the mitochondrial membrane potential was affected on treatment with pAgNPs while it remained unchanged on bAgNPs treatment. Flow cytometry confirmed cell cycle arrest in HeLa cells on treatment with nanoparticles further leading to apoptosis in case of pAgNPs. About 77 and 58 % HeLa cells were found in subG1 phase on treatment with bAgNPs and pAgNPs respectively. bAgNPs showed cytostatic effect on HeLa cells arresting the cell growth in subG1 phase, whereas, pAgNPs triggered death of HeLa cells through mitochondrial membrane potential impairment and apoptosis.ConclusionOverall, bAgNPs and pAgNPs could be safe and showed potential to be used as anticancer nano-antibiotics against human cervical cancer cells.     

Probing ras effector interactions on nanoparticle supported lipid bilayers

Many biological processes take place in close proximity to lipid membranes. For a detailed understanding of the underlying mechanisms, tools are needed for the quantitative characterization of such biomolecular interactions. In this work, we describe the development of methods addressing the dynamics and affinities of protein complexes attached to an artificial membrane system. A semisynthetic approach provides the Ras protein with palmitoyl anchors, which allow stable membrane insertion, as a paradigm for membrane associated proteins that interact with multiple effectors. An artificial membrane system is constituted by nanoparticles covered with a lipid bilayer. Such a stable suspension allows for the characterization of the interaction between membrane-bound Ras and effector proteins using conventional fluorescence-based methods.