Adsorption characteristics of bovine serum albumin onto alumina with a specific crystalline structure

Bone cement containing alumina particles with a specific crystalline structure exhibits the ability to bond with bone. These particles (AL-P) are mainly composed of delta-type alumina (δ-Al₂O₃). It is likely that some of the proteins present in the body environment are adsorbed onto the cement and influence the expression of its bioactivity. However, the effect that this adsorption of proteins has on the bone-bonding mechanism of bone cement has not yet been elucidated. In this study, we investigated the characteristics of the adsorption of bovine serum albumin (BSA) onto AL-P and compared them with those of its adsorption onto hydroxyapatite (HA), which also exhibits bone-bonding ability, as well as with those of adsorption onto alpha-type alumina (α-Al₂O₃), which does not bond with bone. The adsorption characteristics of BSA onto AL-P were very different from those onto α-Al₂O₃ but quite similar to those onto HA. It is speculated that BSA is adsorbed onto AL-P and HA by interionic interactions, while it is adsorbed onto α-Al₂O₃ by electrostatic attraction. The results suggest that the specific adsorption of albumin onto implant materials might play a role in the expression of the bone-bonding abilities of the materials.     

Competitive adsorption of bovine serum albumin and lysozyme on characterized calcium phosphates by polyacrylamide gel electrophoresis method

Characterizations of hydroxyapatite (HA), biphasic calcium phosphate (BCP) and beta tricalcium phosphate (β-TCP) ceramic particles were carried out using X-ray diffusion (XRD), Scanning electron micrograph (SEM), Particle Sizer and Zeta potential analyzer. Competitive adsorption of bovine serum albumin (BSA) and lysozyme (LSZ) on the three calcium phosphates were investigated by polyacrylamide gel electrophoresis (PAGE) method. The results showed that HA, BCP and β-TCP ceramic particles with irregular shapes and similar size distributions all had negative surface net charges in pH7.4 phosphate buffered saline (PBS) solution and exhibited alike behaviors of BSA and LSZ adsorption. LSZ had higher affinity for calcium phosphate ceramics than BSA and its adsorption on them didn’t be almost influenced by the increasing of BSA concentration in the solution. Electrostatic interaction played an important role on the competitive adsorption of BSA and LSZ on the surface of calcium phosphate ceramic particles.     

Probing Adsorption Behaviors of BSA onto Chiral Surfaces of Nanoparticles

Chiral properties of nanoscale materials are of importance as they dominate interactions with proteins in physiological environments; however, they have rarely been investigated. In this study, a systematic investigation is conducted for the adsorption behaviors of bovine serum albumin (BSA) onto the chiral surfaces of gold nanoparticles (AuNPs), involving multiple techniques and molecular dynamic (MD) simulation. The adsorption of BSA onto both L‐ and D‐chiral surfaces of AuNPs shows discernible differences involving thermodynamics, adsorption orientation, exposed charges, and affinity. As a powerful supplement, MD simulation provides a molecular‐level understanding of protein adsorption onto nanochiral surfaces. Salt bridge interaction is proposed as a major driving force at protein–nanochiral interface interaction. The spatial distribution features of functional groups (? COO^?, ? NH₃⁺, and ? CH₃) of chiral molecules on the nanosurface play a key role in the formation and location of salt bridges, which determine the BSA adsorption orientation and binding strength to chiral surfaces. Sequentially, BSA corona coated on nanochiral surfaces affects their uptake by cells. The results enhance the understanding of protein corona, which are important for biological effects of nanochirality in living organisms.     

Protein adsorption on single-crystal hydroxyapatite particles with preferred orientation to a(b)- and c-axes

Adsorption of bovine serum albumin (BSA) and lysozyme (LSZ) proteins with preferred orientation to a(b)- and c-axes of single-crystal hydroxyapatite (HAp) particles, was investigated. Fiber-like HAp single crystal particles (aHAp) and plate-like HAp single crystal particles (cHAp) were used as models for a(b)-plane and c-plane oriented HAps, respectively, together with randomly shaped HAp particles (iHAp) as a control. The selective adsorption behaviors of negatively charged BSA and positively charged LSZ on these HAp particles were examined in a phosphate buffered saline at pH 7.3 and 25 °C for 48 h. The amount of BSA adsorption, normalized for specific surface area, was in the order of aHAp > iHAp > cHAp; however, the order for LSZ was reversed as cHAp > iHAp ≒ aHAp. These results indicate that the a(b)- and c-planes of HAp crystal have high specificity for the adsorption of acidic or basic proteins.     

羟基磷灰石微纳结构对蛋白吸附的影响

本研究采用水热反应法, 在不同浓度环己烷六羧酸(H6E)模板调控作用下, 合成了具有不同表面微纳结构的羟基磷灰石(HAP)微粒, 并采用XRD、BET、FTIR和SEM对其进行表征。对HAP微粒进行了牛血清白蛋白(BSA)、纤维蛋白原(FN)和溶菌酶(LYS)的吸附及释放实验。结果表明: H6E能够在HAP微粒表面构建微纳结构, 不同微纳结构对不同蛋白质具有选择性吸附作用; 在H6E浓度为50 mmol/L的合成条件下制备的中空结构HAP微粒(HAP50)其载蛋白后体外释放具有明显的蛋白缓释性能。     

Investigation of the Relative Affinity of Doxorubicin for Neutral and Negatively Charged Particulate Carriers

Microparticles produced from polyisobutylcyano-acrylate (IBCA) and polyglutaraldehyde (PGA) were investigated for their relative affinity and surface characteristics using doxorubicin (DOX) as a model drug. IBCA microparticles have been reported to exhibit neutral hydrophobic surface features, whereas PGA microparticles have been shown to possess negatively charged carboxyl groups on their surface. The adsorption of DOX on the surface of these particles was studied by adding the drug to preformed microparticles. The amount of drug adsorbed was determined by centrifugation and analysis of the supernatant for the free drug by HPLC. The adsorption data was examined by Langmuir, Scatchard, and Hill equations. The results indicate that IBCA micropatricles have a higher adsorption capacity for DOX, however PGA microparticles demonstrated a higher relative affinity for the drug molecule. Additionally, both microparticles presented curvilinear Scatchard plots indicating the possibility of more than one type of binding sites for the drug on the surface of these particles. It appears that strong electrostatic attraction may exist between the positively charged amino group on DOX and the negatively charged carboxyl groups of PGA microparticles.     

Secondary structure analysis of proteins embedded in spherical polyelectrolyte brushes by FT-IR spectroscopy

The adsorption of bovine serum albumin (BSA), bovine beta-lactoglobulin, and bovine pancreatic ribonuclease A onto spherical polyelectrolyte brushes (SPB) is reported. The SPB consist of narrowly distributed poly(styrene) core particles (diameter approximately 100 nm) onto which linear chains of anionic polyelectrolytes are grafted. The polyelectrolyte shell consists of either the weak polyelectrolyte poly(acrylic acid) or the strong polyacid poly(styrenesulfonate). The SPB particles are dispersed in H(2)O at room temperature. The secondary structure of the proteins was investigated by Fourier transform infrared spectroscopy in transmission mode before and during adsorption to these colloidal brushes. The alpha-helix and beta-sheet content of the proteins was nearly fully retained in the adsorbed state for all systems. Only in the case of BSA interacting with poly(styrenesulfonic) brushes could a slight loss of alpha-helix structure be observed. As the interaction of SPB and proteins can be controlled by the ionic strength in the buffer, additional experiments were performed to release the adsorbed protein. The amount of released protein was quantified and was found to be strongly dependent on the kind of protein and brush used. The secondary structure of the released proteins could be analyzed as well. An almost full preservation of secondary structure was found. This demonstrates that SPB are well-suited to immobilize proteins. The SPB can be charged and decharged under retention of the secondary structure of the biomolecules.     

Synthesis and characterization of amino acid containing Cu(II) chelated nanoparticles for lysozyme adsorption

Immobilized metal ion affinity chromatography (IMAC) is a useful method for adsorption of proteins that have an affinity for transition metal ions. In this study, poly(hydroxyethyl methacrylate-methacryloyl-l-tryptophan) (PHEMATrp) nanoparticles were prepared by surfactant free emulsion polymerization. Then, Cu(II) ions were chelated on the PHEMATrp nanoparticles to be used in lysozyme adsorption studies in batch system. The maximum lysozyme adsorption capacity of the PHEMATrp nanoparticles was found to be 326.9 mg/g polymer at pH 7.0. The nonspecific lysozyme adsorption onto the PHEMA nanoparticles was negligible. In terms of protein desorption, it was observed that adsorbed lysozyme was readily desorbed in medium containing 1.0 M NaCl. The results showed that the metal-chelated PHEMATrp nanoparticles can be considered as a good adsorbent for lysozyme purification.     

中空层状羟基磷灰石微球对溶菌酶的吸附与缓释研究

利用锂钙硼玻璃在磷酸盐溶液中的原位转化反应制备表面多孔且具有中空层状结构的羟基磷灰石(HA)微球,以溶菌酶为蛋白的药物模型,研究了中空层状结构的羟基磷灰石微球对溶菌酶的吸附及缓释特性,结果显示,中空微球对不同浓度的溶菌酶溶液,具有不同的吸附机理,当溶菌酶溶液的浓度低于0.8mg/mL时,溶菌酶的吸附主要发生在微球的外表面,符合Langmuir模型,释放速率较快,48h内基本释放完全;当溶菌酶溶液的浓度高于0.8mg/mL时,溶菌酶扩散进入微球内部及球壁的微孔中,使得吸附量显著增加,满足Henry吸附模型,溶菌酶的释放周期明显增加,可持续释放800h,微球对蛋白具有很好的缓释效果。     

Evaluation of hydroxyapatite microspheres made from a borate glass to separate protein mixtures

A hydroxyapatite (HA, Ca₁₀(PO₄)₆(OH)₂), transformed from a calcium-containing borate glass, has been investigated for its protein adsorption and chromatographic characteristics. Microspheres of the borate glass were transformed into HA by reacting them with a 0.25 M phosphate (K₂HPO₄) solution for 24 h at 37 °C (pH 9.0). The HA microspheres with a diameter of 45–90 μm were hand packed into a steel column (4.6 mm × 80 mm) and used to separate a binary protein mixture of bovine serum albumin (BSA) and lysozyme. HA microspheres, with a diameter <45 μm, were used for separating a protein mixture of BSA, myoglobin, and lysozyme. These microspheres had a diameter that was 20–30 times larger than commercial HA column packing spherical particles, 2–3 μm, but these microspheres had a six times larger surface area and a more uniform spherical shape. These advantages compensated for their larger size and the separation results were comparable to those commercially available HA columns in the separation of the proteins studied. These unique HA microspheres, made from microspheres of a borate glass, are considered to be useful as packing materials for protein separation in chromatography.     

Multilayer-assembled microchip for enzyme immobilization as reactor toward low-level protein identification

A microchip reactor has been developed on the basis of a layer-by-layer approach for fast and sensitive digestion of proteins. The resulting peptide analysis has been carried out by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Natural polysaccharides, positively charged chitosan (CS), and negatively charged hyaluronic acid (HA) were multilayer-assembled onto the surface of a poly(ethylene terephthalate) (PET) microfluidic chip to form a microstructured and biocompatible network for enzyme immobilization. The construction of CS/HA assembled multilayers on the PET substrate was characterized by AFM imaging, ATR-IR, and contact angle measurements. The controlled adsorption of trypsin in the multilayer membrane was monitored using a quartz crystal microbalance and an enzymatic activity assay. The maximum proteolytic velocity of the adsorbed trypsin was approximately 600 mM/min mug, thousands of times faster than that in solution. BSA, myoglobin, and cytochrome c were used as model substrates for the tryptic digestion. The standard proteins were identified at a low femtomole per analysis at a concentration of 0.5 ng/muL with the digestion time <5s. This simple technique may offer a potential solution for low-level protein analysis.     

Sonochemical synthesis of (3-aminopropyl)triethoxysilane-modified monodispersed silica nanoparticles for protein immobilization

3-Aminopropyltriethoxysilane modified monodispersed silica nanoparticles were synthesized by a rapid sonochemical co-condensation synthesis procedure. The chemical nature of surface organic modifier on the obtained modified silica nanoparticle was characterized by ¹³C and ²⁹Si MAS Nuclear Magnetic Resonance (NMR) spectroscopies, Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA)- differential scanning calorimetry (DSC). Due to the strengthened positive surface charge of the silica nanoparticles by the modification with aminopropyl groups, the capability for bovine serum albumin (BSA) adsorption was significantly increased as compared with bare silica nanoparticles. 80 mg/g BSA was adsorbed on modified silica nanoparticles, whereas only 20 mg/g BSA could be loaded on pure silica nanoparticles. The enhanced positive surface charge repelled proteins with net positive charge and the modified silica nanoparticles exhibited negligible adsorption of lysozyme, thus a selective adsorption of proteins could be achieved.     

Poly(ethylene glycol) decorated poly(methylmethacrylate) nanoparticles for protein adsorption

Poly(ethylene glycol) decorated poly(methyl methacrylate) particles were synthesized by means of emulsion polymerization using poly(ethylene glycol) sorbitan monolaurate (Tween-20) as surfactant. PMMA/PEG particles presented mean diameter (195 ± 15) nm, indicating narrow size distribution. The adsorption behavior of bovine serum albumin (BSA) and concanavalin A (ConA) onto PMMA/PEG particles was investigated by means of spectrophotometry. Adsorption isotherms obtained for BSA onto PMMA/PEG particles fitted well sigmoidal function, which is typical for multilayer adsorption. Con A adsorbed irreversibly onto PMMA/PEG particles. The efficiency of ConA covered particles to induce dengue virus quick agglutination was evaluated.     

Preparation of guanidine group functionalized magnetic nanoparticles and the application in preconcentration and separation of acidic protein

Guanidine group (Gnd) functionalized magnetic nanoparticles (Fe3O4@SiO2@NH2-Gnd) were synthesized and characterized in this work for the first time. The characterization of Fe3O4@SiO2@ NH2-Gnd nanoparticles was demonstrated by transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectra, vibration sample magnetometer, and zeta potential analyzer. The novel multifunctional nanoparticles were served as a solid-phase extraction sorbent for easy isolation and preconcentration of acidic protein from aqueous solution only using a magnet. Bovine serum albumin (BSA) was selected as a model protein and the main experimental parameters influencing the adsorption and desorption efficiency were investigated and optimized. Under the optimum conditions, the particles reached saturated adsorption within 20 min and exhibited significant specific recognition for the acidic proteins. Fifteen fold enrichment efficiency was achieved and the detection limits was 45 ng x mL(-1) for BSA by capillary electrophoresis (CE). The practical application of the novel nanoparticles as a sorbent for the isolation and preconcentration of acidic proteins from basic proteins was demonstrated by effective separation and enrichment of bovine serum albumin from lysozyme and cytochrome C mixture, which was assayed by CE.     

Mesoporous hydroxyapatite by hard templating of silica and carbon foams for protein release

Calcium phosphates, particularly hydroxyapatite Ca₁₀(PO₄)₆(OH)₂ (HA), are widely used for bone regeneration due to their biocompatibility and good resorption properties. However, their performance upon implantation is improved when they are associated with bioactive molecules such as growth factors. Using mesoporous HA leads to improved protein adsorption and release kinetics because the diameter of the mesopores (2–50 nm) is in the same range as their size. We prepared this type of material by the nanocasting method using three different templates: a silica foam and two carbon templates derived from it using propylene or sucrose as carbon source. We investigated the influence of the template, the calcination temperature and of the conditions during template removal. We obtained HA materials with a surface area of up to 90 m² g^?1 and with an intergranular mesopore volume of up to 0.4 cm³ g^?1. In this paper, we show for the first time that the synthesis of mesoporous HA from a mesoporous silica foam template allows eliminating the template at lower temperatures (in an alkaline medium), thus preventing the sintering of the HA. These materials have interesting properties for drug delivery applications. The protein adsorption and release capacities of these HAs were tested with two model proteins, bovine serum albumin (BSA), and Cytochrome C. These materials are an important milestone for future bone regeneration systems based on HA associated with human growth factor proteins.     

Layer by layer deposition of hydroxyapatite onto the collagen matrix

Layer by layer (LbL) deposition is a useful method for deposition of many inorganic (including metals, oxides and phosphates) and organic (including polymers and proteins) components on a large range of substrates. The LbL deposition of hydroxyapatite (HA) onto a collagen matrix involves HA synthesis on the collagen matrix starting from electrically charged precursors such as Ca²⁺ and PO₄³⁻ at a proper pH to precipitate the desired calcium phosphate.The LbL deposition process was continuously monitored in order to study the amount of HA deposited in each layer. The deposition of the first layers of HA was concluded to be highly influenced by the collagen matrix. When the collagen matrix is crosslinked with glutaraldehyde, the matrix structure is not modified during the deposition, and the porosity will decrease with the number of layers until saturation is reached. Following pore saturation, HA will be only deposited onto the mineralized collagen matrix surface. The obtained composite materials were characterized by XRD, SEM, DTA-TG and FTIR.     

Temperature-swing adsorption of proteins in water using N-isopropylacrylamide copolymer gel particles

The adsorption and desorption behaviors of bovine serum albumin (BSA) in water for temperature-responsive polymer gel particles have been investigated by the temperature-swing operation between 298 and 313 K, where the cationic N-isopropylacrylamide (NIPA) gels copolymerized with vinylbenzyl trimethylammonium chloride (VBTA) or 2-(dimethylamino)ethyl methacrylate (DMAEMA) were used. The NIPA-VBTA and the NIPA-DMAEMA copolymer gels adsorbed BSA while the NIPA homopolymer gel hardly adsorbed BSA, indicating that the copolymer gels adsorb BSA through the electrostatic attraction between the positively charged groups in the gels and the negatively charged BSA. The adsorption amounts for the NIPA-DMAEMA gels were smaller than those for the NIPA-VBTA gels. This may be because almost every VBTA group, which is a quaternary ammonium salt, can be positively charged in water, while only some of the tertiary amine DMAEMA groups are protonated in water. Moreover, it was found that both the copolymer gels with a large mesh size of the polymer network repeatedly adsorbed BSA at 298 K and desorbed some of pre-adsorbed BSA at 313 K by the temperature-swing operation. This BSA desorption may result from the decrease of the number of the positively charged groups accessible to BSA due to the shrinking of the constituent polymer chains.     

Effects of Nanoparticle Electrostatics and Protein–Protein Interactions on Corona Formation: Conformation and Hydrodynamics

All‐atom molecular dynamics simulations of plasma proteins (human serum albumin, fibrinogen, immunoglobulin gamma‐1 chain‐C, complement C3, and apolipoprotein A‐I) adsorbed onto 10 nm sized cationic, anionic, and neutral polystyrene (PS) particles in water are performed. In simulations of a single protein with a PS particle, proteins eventually bind to all PS particles, regardless of particle charge, in agreement with experiments showing the binding between anionic proteins and particles, which is further confirmed by calculating the binding free energies from umbrella sampling simulations. Simulations of mixtures of multiple proteins and a PS particle show the formation of the protein layer on the surface via the adsorption competition between proteins, which influences the binding affinity and structure of adsorbed proteins. In particular, diffusivities are much higher for proteins bound to the particle surface or to the boundary of the protein layer than for those bound to both the particle surface and other proteins, indicating the dependence of protein mobility on their positions in the layer. These findings help to explain in detail experimental observations regarding the replacement of plasma proteins at the early stage of corona formation and the difference in the binding strength of proteins in inner and outer protein‐layers.     

The importance of amino acid interactions in the crystallization of hydroxyapatite

Non-collagenous proteins (NCPs) inhibit hydroxyapatite (HA; Ca₅(PO₄)₃OH) formation in living organisms by binding to nascent nuclei of HA and preventing their further growth. Polar and charged amino acids (AAs) are highly expressed in NCPs, and the negatively charged ones, such as glutamic acid (Glu) and phosphoserine (P-Ser) seem to be mainly responsible for the inhibitory effect of NCPs. Despite the recognized importance of these AAs on the behaviour of NCPs, their specific effect on HA crystallization is still unclear, and controversial results have been reported concerning the efficacy of HA inhibition of positively versus negatively charged AAs. We focused on a positively charged (arginine, Arg) and a negatively charged (Glu) AA, and their combination in the same solution. We studied their inhibitory effect on HA nucleation and growth at physiological temperature and pH and we determined the mechanism by which they can affect HA crystallization. Our results showed a strong inhibitory effect of Arg on HA nucleation; however, Glu was more effective in inhibiting HA crystal growth during the growth stage. The combination of Glu and Arg was less effective in controlling HA nucleation, but it inhibited HA crystal growth. We attributed these differences to the stability of complexes formed between AAs and calcium and phosphate ions at the nucleation stage, and in bonding strength of AAs to HA crystal faces during the growth stage. The AAs also influenced the morphology of synthesized HA. Presence of either Arg or Glu resulted in the formation of spherulites consisting of preferentially oriented nanoplatelets orientation. This was attributed to kinetic factors favoring growth front nucleation (GFN) mechanism.     

Molecular imprinted particles for lysozyme purification

Molecular recognition-based separation techniques have received much attention in chemistry and biology because of their high selectivity for target molecules. The aim of this study is to prepare lysozyme-imprinted polymers which can be used for the purification of lysozyme from aqueous solutions and egg white. N-methacryloyl-(l)-histidinemethylester (MAH) was chosen as the metal-complexing monomer. In the first step, Cu²⁺ was complexed with MAH and the lysozyme-imprinted poly(HEMA–MAH) [Lys-MIP] particles were synthesized by UV-initiated bulk polymerization. After that, the template (i.e., lysozyme) were removed using 0.1 M NaCl solution. The specific surface area of the Lys-MIP particles was found to be 22.9 m²/g with a size range of 20–63 μm in diameter and the swelling ratio was 57%. According to the elemental analysis results, the particles contained 421 μmol MAH/g polymer. The maximum lysozyme adsorption capacity was 12.1 mg/g polymer. The relative selectivity coefficients of imprinted particles for lysozyme/human serum albumin and lysozyme/cytochrome c were 3.6 and 4.1 times greater than NIP particles, respectively. Purification of lysozyme from egg-white was also monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purity of the desorbed lysozyme was about 89% with recovery about 84%. The Lys-MIP particles could be used many times without decreasing their adsorption capacities significantly.