Design,properties, and applications of protein micro- and nanoparticles

The design of protein particles with tailored properties has received an increased attention recently. Several approaches, from simple heat treatment in dilute systems to the combination of heat and mechanical treatments in concentrated protein solutions, have been used to obtain protein particles with varying functional properties. Control of particle size, morphology, surface- and internal properties is crucial for obtaining protein particles with the necessary properties for emerging applications. The latter include not only the use of protein particles in foods, where they can improve the stability of foods at high protein content, but also as food-grade particles for the delivery of bio-actives. By tuning the morphology and size of protein particles, protection or controlled release of various bio-active components may be obtained. We review the various methods that have been used to prepare protein particles and discuss the behavior of the particles in dispersed systems and their possible applications.     

Shape-specific, monodisperse nano-molding of protein particles

Herein we report nano-molding proteins for the fabrication of protein PRINT particles of monodisperse size and shape. Lyophilized protein particles are generally highly dispersed in particle size, aggregated, and often made through costly and complicated processes. Attempts to engineer monodisperse, discrete protein particles using wet-milling, spray-freeze-drying, microemulsion, or super critical fluid methods have realized little success. The PRINT technology enables a gentle, facile route to monodisperse particles of 100% protein as small as 200 nm cylinders. Protein PRINT particles of any shape and size are effortlessly achievable. Our research efforts include making PRINT particles composed of albumin and albumin 0.5 wt % siRNA, and Abraxane, the gold standard therapeutic used in metastatic breast cancer.     

Growth and aggregation of surfactant islands during the displacement of an adsorbed protein monolayer: a Brownian dynamics simulation study

We present Brownian dynamics simulations of the displacement of a protein monolayer by competitive adsorption. The protein film is modelled as a network of spherical bonded particles adsorbed at a fluid interface. Spherical displacer particles, which have a stronger affinity for the interface than the protein film particles, are introduced into the system through the sub-phase. At early stages, these particles diffuse to the interface and are adsorbed in the gaps in the network. Soon thereafter, however, further adsorption initiates displacement of the film particles, ultimately leading to the complete removal of the protein layer from the surface. We study the evolution of the number and size of the displacer islands formed at the interface. The introduction of a direct long-range repulsion between film and displacer particles is shown to lead to a phase-separation-type behaviour at intermediate time scales. Further comparisons with the displacement of a non-bonded monolayer are also presented.     

Coupling Proteins to Amine-terminated Magnetic Particles Activated with 1,4-Phenylene Diisothiocyanate

The homobifunctional crosslinker 1,4‐phenylene diisothiocyanate (PDC) was coupled to amine‐terminated magnetic particles, and human IgG, streptavidin, protein G and protein A were immobilized on the activated magnetic particles. The coupling of PDC to the amine‐terminated magnetic particles was completed in 10 min, and 1 mg of activated magnetic particles was able to immobilize 95 (g of protein G, 120 µg of protein A, 160 µg of streptavidin and 280 µg of IgG. Ultraviolet‐visible spectroscopy, FTIR spectroscopy and electron micrography were used to characterize the functional particles. The results indicated that PDC was successfully coupled to the surface of the amine‐terminated magnetic particles, and the proteins were effectively immobilized on their surface. The activity of protein G immobilized on the activated magnetic particles was confirmed by its ability to purify IgG from plasma.     

Rendering protein-based particles transiently insoluble for therapeutic applications

Herein, we report the fabrication of protein (bovine serum albumin, BSA) particles which were rendered transiently insoluble using a novel, reductively labile disulfide-based cross-linker. After being cross-linked, the protein particles retain their integrity in aqueous solution and dissolve preferentially under a reducing environment. Our data demonstrates that cleavage of the cross-linker leaves no chemical residue on the reactive amino group. Delivery of a self-replicating RNA was achieved via the transiently insoluble PRINT protein particles. These protein particles can provide new opportunities for drug and gene delivery.     

Synthesis of magnetic chelator for high-capacity immobilized metal affinity adsorption of protein by cerium initiated graft polymerization

A novel magnetic chelator with high adsorption capacity of protein by immobilized metal affinity adsorption was prepared by cerium (IV) initiated graft polymerization of tentacle-type polymer chains with iminodiacetic acid (IDA) chelating group on magnetic particles with hydroxyl groups. The micron-sized magnetic poly(vinyl acetate-divinylbenzene) (PVAc-DVB) particles were prepared by a modified suspension polymerization in the presence of oleic acid-coated magnetite nanoparticles and subsequently modified by ester exchange reaction to introduce functional hydroxyl groups. Bovine hemoglobin (BHb) was selected as a model protein to investigate the adsorption capacity of these magnetic particles. The magnetic particles were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), superconducting quantum interference device (SQUID) magnetometry, diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), and X-ray diffraction (XRD). The results showed that the magnetic particles had an average size of 5 microm and superparamagnetism with saturation magnetization of 20.0 emu/g at room temperature. The protein adsorption indicated that the graft polymerization of tentacle type polymer chains on the magnetic particles could produce magnetic adsorbents with high adsorption capacity (1428.21 mg/g) and low nonspecific adsorption of protein. The magnetic particles with grafted tentacle polymer chains have potential application in large-scale affinity separation of proteins.     

Monomodal polyelectrolyte complex nanoparticles of PDADMAC/Poly(styrenesulfonate): preparation and protein interaction

The binding of the model proteins HSA, LYZ and MYO to PEC nanoparticles is reported. PEC particles were prepared by mixing solutions of PDADMAC either with PSS or PMA-MS, followed by consecutive centrifugation. Monomodal anionic (PEC-1.50) and cationic (PEC-0.66) PEC particles were obtained using non-stoichiometric mixing ratios. PEC/protein conjugates were prepared by adding charged protein solutions to dispersions of respective like charged PEC particles, followed by one centrifugation step. Mixing proteins and PEC particles under attractive conditions led to flocculation of the dispersion. From CD, DLS and AFM the following trend for protein binding at PEC particles under repulsive conditions was obtained: HSA/PEC-1.50 > MYO/PEC-1.50 > LYZ/PEC-0.66. Protein uptakes up to 0.33 g x g(-1) (protein/PEC) (CD) and particle diameter enlargements up to 13 nm (AFM) were obtained at c(PROT) = 0.091 mg . mL(-1). Furthermore, novel spin coated films of PEC particles were interacted with proteins under both repulsive and attractive conditions. In-situ ATR FT-IR spectroscopy revealed that the adsorbed amount of HSA and LYZ under attractive conditions was significantly higher than under repulsive ones, which is analogous to protein adsorption at polyelectrolyte multilayers terminated either by polycation or polyanion. Similarly to the dispersed PEC/protein conjugates, under repulsive conditions the uptake of HSA was higher compared to LYZ. The shown protein uptake under repulsive conditions is related to concepts of mild enzyme or protein binding at nonbiogenic substrates.     

Electrostatic interactions between amphoteric latex particles and proteins

The electrostatic interactions between amphoteric polymethyl methacrylate latex particles and proteins with different pI values were investigated. These latex particles possess a net positive charge at low pH, but they become negatively charged at high pH. The nature and degree of interactions between these polymer particles and proteins are primarily controlled by the electrostatic characteristics of the particles and proteins under the experimental conditions. The self-promoting adsorption process from the charge neutralization of latex particles by the proteins, which have the opposite net charge to that of the particles, leads to a rapid reduction in the zeta potential of the particles (in other words colloidal stability), and so strong flocculation occurs. On the other hand, the electrostatic repulsion forces between similarly charged latex particles and the proteins retard the adsorption of protein molecules onto the surfaces of the particles. Therefore, latex particles exhibit excellent colloidal stability over a wide range of protein concentrations. A transition from net negative charge to net positive charge, and vice versa (charge reversal), was observed when the particle surface charge density was not high enough to be predominant in the protein adsorption process.     

Reversed-phase liquid chromatography of proteins and peptides using multimodal copolymer-encapsulated silica

Multimodal copolymer-encapsulated particles for liquid chromatography were prepared by bonding 1-octadecene and unsaturated carboxylic acids on silica particles (5 microm diameter, 300 A pores) for liquid chromatography of proteins. These multimodal copolymer-encapsulated particles can provide both hydrophobic and hydrogen bonding interactions with polar compounds. The chromatographic performance of these multimodal copolymer-encapsulated particles for peptide and protein separations was evaluated under reversed-phase conditions. Compared with typical C8-bonded silica, polymer-encapsulated particles were more stable in acidic mobile phases and provided better recoveries, especially for large proteins (Mr>0.5 x 10(6)). Totally hydrophobic polymer-encapsulated particles were found to produce broad peaks for proteins, and significant improvements were observed by introducing hydrophilic groups (-COOH) onto the polymer-encapsulated surface to form a multimodal phase. For the reversed-phase liquid chromatography of peptides and proteins, improved selectivity and increased solute retention were found using the multimodal polymer-encapsulated particles. More peaks were resolved for the separation of complex peptide mixtures such as protein digests using the multimodal polymer-encapsulated particles as compared to totally hydrophobic polymer-encapsulated particles.     

Patterns of protein adsorption in chromatographic particles visualized by optical microscopy

A new method is presented to image transient patterns of protein adsorption in individual spherical chromatographic particles under strong binding conditions. The method takes advantage of the difference in refractive index between the protein-free and protein-saturated adsorbent matrix. When the particles are viewed with an ordinary microscope using white light illumination, the adsorption front appears as a bright ring that moves in time from the surface of the particle to its center. Experimental data are obtained for the proteins lysozyme and albumin with a commercial agarose-based cation exchanger. Sharp rings are observed in both cases confirming that protein mass transfer within the particles occurs via a shell-progressive diffusion mechanism. Quantitative analysis based on the shrinking core model provides an accurate and precise way of determining the intraparticle diffusivity for individual particles as a function of protein concentration and mobile phase composition.     

圆二色性测定蛋白质在超细粒子上吸附的构象变化

研究了蛋白质在超细粒子固体表面吸附过程中构象的变化;选择的吸附剂粒径很小,可以直接应用圆二色性光谱测定蛋白质吸附层的二级结构变化;研究了pH、固体吸附表面在蛋白质吸附过程中对蛋白质构象的影响,得到了较好的实验数据,为进一步进行蛋白质吸附机理的理论研究提供了基础。     

Effect of Functionalization of Colloidal Gold on Controlled Flocculation by the Ligand-Receptor Mechanism

Modification of colloidal gold particles with octadecanthiol and alkyl biotin, with the subsequent flocculation of these particles via introduction of a tetramer protein avidin, was studied.     

Improved liquid chromatographic separation of different proteins by designing functional surfaces of cattle bone-originated apatite

Spherical particles of cattle bone-originated hydroxyapatite (r-HAp) were prepared by dissolution-precipitation, spray-drying using a two fluid-nozzle apparatus, and subsequent heat treatment. The product had effective pore structures for liquid chromatographic separation of albumin, myoglobin, ribonuclease, lysozyme and cytochrome c. The activated surfaces of the r-HAp particles were easily prepared with desired proportions of P- and C-sites and appropriate acid-basic strength for selective protein adsorption by optimizing the synthesis conditions. Liquid chromatography columns packed with the particles exhibited high resolution and durability in protein separation, reflecting stable distribution of pore size.     

Comparison of protein adsorption patterns onto differently charged hydrophilic superparamagnetic iron oxide particles obtained in vitro and ex vivo.

Protein adsorption patterns of superparamagnetic iron oxides (SPIO) were evaluated by two-dimensional electrophoresis (2-DE) after in vitro incubation of the particles in plasma or serum. SPIO particles having positive (MKK 1211), negative (MKA 1211), or neutral (MKG 1411) charge were used. Protein adsorption patterns of different charged SPIO particles acquired in vitro and recollected 5 min after intravenous injection into rats (ex vivo) were compared. For the uncharged MKG 1411 particles, the differences of protein adsorption patterns were negligible and only minor differences were found for the negatively charged MKA 1211 and positively charged MKK 1211 particles. A good correlation between in vitro and ex vivo data could be shown. For the evaluation of protein adsorption patterns of SPIO particles determining organ distribution and allowing estimation of site-specific delivery (drug targeting), the currently used protocol for 2-DE analysis could be confirmed.     

Generation and structural analysis of reactive empty particles derived from an icosahedral virus

Chemical and genetic modifications on the surface of viral protein cages confer unique properties to the virus particles with potential nano and biotechnological applications. The enclosed space in the interior of the virus particles further increases its versatility as a nanomaterial. In this paper, we report a simple method to generate a high yield of stable cowpea mosaic virus (CPMV) empty capsids from their native nucleoprotein counterparts by removing the encapsidated viral genome without compromising the integrity of the protein coat. Biochemical and structural comparison of artificially generated empty particles did not reveal any distinguishable differences from CPMV particles containing viral RNA. Preliminary results on the use of artificially produced empty CPMV capsids as a carrier capsule are described.     

Protein manipulation with insulator-based dielectrophoresis and direct current electric fields

The present study demonstrates the manipulation of protein particles employing insulator-based dielectrophoresis (iDEP) and direct current (d.c.) electric fields. Fluorescently labeled bovine serum albumin (BSA) protein particles were concentrated inside a microchannel that contained an array of glass cylindrical insulating structures. d.c. electric fields were applied and the dielectrophoretic response of the particles was observed as a function of the suspending medium conductivity (25, 50 and 100muS/cm) and pH (8 and 9). It was shown that the magnitude of the applied electric field (700-1600V/cm) and suspending medium properties have a strong effect on the dielectrophoretic response of the protein particles. The results presented here are the first report on protein manipulation employing d.c.-iDEP.     

Microspectroscopic analysis of green fluorescent proteins infiltrated into mesoporous silica nanochannels

The infiltration of enhanced green fluorescent protein (EGFP) into nanochannels of different diameters in mesoporous silica particles was studied in detail by fluorescence microspectroscopy at room temperature. Silica particles from the MCM-41, ASNCs and SBA-15 families possessing nanometer-sized (3-8 nm in diameter) channels, comparable to the dimensions of the infiltrated guest protein EGFP (barrel structure with dimensions of 2.4 nm × 4.2 nm), were used as hosts. We found that it is necessary to first functionalize the surfaces of the silica particles with an amino-silane for effective encapsulation of EGFP. We demonstrated successful infiltration of the protein into the nanochannels based on fluorescence microspectroscopy and loading capacity calculations, even for nanochannel diameters approaching the protein dimensions. We studied the spatial distributions of the EGFPs within the silica particles by confocal laser scanning microscopy (CLSM) and multimode microscopy. Upon infiltration, the fluorescence lifetime drops as expected for an emitter embedded in a high refractive index medium. Further, the spectral properties of EGFP are preserved, confirming the structural integrity of the infiltrated protein. This inorganic-protein host-guest system is an example of a nanobiophotonic hybrid system that may lead to composite materials with novel optical properties.     

Change of zeta potential of biocompatible colloidal oxide particles upon adsorption of bovine serum albumin and lysozyme

The amounts of negatively charged bovine serum albumin and positively charged lysozyme adsorbed on alumina, silica, titania, and zirconia particles (diameters 73 to 271 nm) in aqueous suspensions are measured. The adsorbed proteins change the zeta potentials and the isoelectric points (IEP) of the oxide particles. The added to adsorbed protein ratios at pH 7.5 are compared with the protein treated particle zeta potentials. It is found that the amounts of adsorbed proteins on the alumina, silica, and titania (but not on the zirconia) particle surfaces are highly correlated with the zeta potential. For the slightly less hydrophilic zirconia particles high amounts of protein adsorption are observed even under repulsive electrostatic conditions. One reason could be that the hydrophobic effect plays a more important role for zirconia than electrostatic interaction.     

A novel support with artificially created recognition for the selective removal of proteins and for affinity chromatography

Summary Acrylamide and N,N-methylenebisacrylamide were copolymerized in the presence of a protein to form a gel which was pressed through a sieve. The gel particles obtained were packed into a chromatographic tube. The experimental conditions for the polymerization are such that the pores of the gel particles are large enough to permit the protein to diffuse out of the particles, so that the entrapped protein can be removed from the bed by washing with an aqueous solution. However the interaction with the matrix is so strong that the protein can be desorbed only by a buffer containing 0.5 M sodium chloride or by a 10% solution of acetic acid containing 10% SDS. When a sample containing the protein present during the polymerization was applied to the column along with other proteins this protein was the only one adsorbed. The technique worked selectively with hemoglobin, cytochrome C and transferrin.     

Application of colloidal palladium nanoparticles for labeling in electron microscopy

The application of palladium nanoparticles as electron-dense markers for labeling in both transmission and scanning electron microscopy requires their conjugation to a specific protein. The conjugation protocol described here includes the dihydrolipoic acid (DHLA) capping of Pd nanoparticles (8 nm equivalent diameter) and their subsequent covalent attachment to functional protein molecules such as streptavidin, protein A, or avidin. The single-step reaction was mediated using the cross-linking agent ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The final Pd conjugates were fully functional, as demonstrated by labeling of ultrathin resin sections of either bovine serum albumin or secretory granules of the salivary gland isolated from the partially fed female Ixodes ricinus tick. The results of bovine serum labeling were quantified, statistically evaluated, and compared with results obtained using commercially available gold particle conjugates (10 nm diameter). The highest values of labeling density were achieved using both streptavidin-Pd (106 ± 7 particles/μm2) and protein A-Au conjugates (130 ± 18 particles/μm2) compared to a commercial streptavidin-Au (66 ± 16 particles/μm2) and protein A-Pd conjugates (70 ± 11 particles/μm2). The concentrations of both DHLA and EDC, pH during conjugation, and finally thorough washing away of unbound proteins crucially influenced conjugation.