Electrospun core-shell nanofibers from homogeneous solution of poly(vinyl alcohol)/bovine serum albumin

We report on the preparation and characterization of core-shell structure of bovine serum albumin (BSA) blended poly(vinyl alcohol) (PVA) composite nanofibers by using electrospinning process. The core-shell structure nanofibers have been electrospun from the homogeneous solution of BSA (as shell) and PVA (as core). The morphology, chemical compositions, structure and thermal properties of the resultant products were characterized by scanning electron microscopy (SEM), energy dispersive X-ray spectrometer (EDX), high-resolution transmission electron microscopy (HR-TEM), Fourier transform infrared (FT-IR) spectroscopy, differential scanning calorimetry, thermogravimetric analysis (TGA) and X-ray photoelectron spectroscopy (XPS) techniques. The blending ratio of PVA and BSA, molecular weight of BSA and the applied voltage of electrospinning process were observed to be the key influence factors on the formation of core-shell nanofibers structure. Based on the experimental findings, we proposed a possible physical mechanism for the formation of core-shell nanofibers structure of PVA blended BSA composite.     

Interaction of fractionated poly(acrylic acid)s with bovine serum albumin

The interaction of fractionated poly(acrylic acid)s (PAA) with bovine serum albumin (BSA) has been studied by measuring the hydrolysis rate of p-nitrophenyl acetate catalysed by BSA in the presence of PAA. The binding of PAA with BSA, which prohibits the catalytic action of BSA, increases with increasing molecular weight of PAA. The change in the electronic spectra of BSA-PAA solutions supports this molecular weight dependence. Circular dichroism of BSA shows that the binding of PAA does not induce any conformational change in BSA.     

Interaction,cytotoxicity and sustained release assessment of a novel anti-tumor agent using bovine serum albumin nanocarrier

Abstract

The interaction ability of bovine serum albumin (BSA) with 2,6-divanillylidenecyclohexanone (DVH) as a stable curcumin derivative was investigated using fluorescence and circular dichroism (CD) spectroscopy techniques under simulative physiological conditions (pH = 7.2). Following the obtained results of binding studies, bovine serum albumin nanoparticles (BSANPs) were synthesized and characterized using Fourier transform infrared spectroscopy (FT-IR), filed emission scanning electron microscopy (FE-SEM), atomic force microscope (AFM) and dynamic light scattering (DLS). The stable BSANPs showed a spherical shape with a diameter of 149.14?±?46.69?nm and the formulation of BSA had no change during the fabrication process. DVH was loaded on BSANPs (DVH@BSANPs) and the release studies showed sustained release of DVH from BSANPs. The validation of DVH@BSANPs system confirmed that the Fickian release mechanism of DVH followed on Korsmeyer–Pepas model. The in vitro studies on HFFF2 and MDA-MB-231 were investigated using MTT assay, DAPI and annexinV/PI staining that showed biocompatible BSANPs reduced the cytotoxicity of DVH in normal cell lines significantly, and antitumor activity of DVH was increased when it was loaded onto BSANPs without necrosis. These results suggest that DVH@BSANPs are a novel biocompatible sustained release system for effective therapeutic approach.

Communicated by Ramaswamy H. Sarma     

Complex formation of bovine serum albumin with a poly(ethylene glycol) lipid conjugate

In this work, we report the formation of complexes by self-assembly of bovine serum albumin (BSA) with a poly(ethylene glycol) lipid conjugate (PEG2000-PE) in phosphate saline buffer solution (pH 7.4). Three different sets of samples have been studied. The BSA concentration remained fixed (1, 0.01, or 0.001 wt % BSA) within each set of samples, while the PEG2000-PE concentration was varied. Dynamic light scattering (DLS), rheology, and small-angle X-ray scattering (SAXS) were used to study samples with 1 wt % BSA. DLS showed that BSA/PEG2000-PE aggregates have a size intermediate between a BSA monomer and a PEG2000-PE micelle. Rheology suggested that BSA/PEG2000-PE complexes might be surrounded by a relatively compact PEG-lipid shell, while SAXS results showed that depletion forces do not take an important role in the stabilization of the complexes. Samples containing 0.01 wt % BSA were studied by circular dichroism (CD) and ultraviolet fluorescence spectroscopy (UV). UV results showed that at low concentrations of PEG-lipid, PEG2000-PE binds to tryptophan (Trp) groups in BSA, while at high concentrations of PEG-lipid the Trp groups are exposed to water. CD results showed that changes in Trp environment take place with a minimal variation of the BSA secondary structure elements. Finally, samples containing 0.001 wt % BSA were studied by zeta-potential experiments. Results showed that steric interactions might play an important role in the stabilization of the BSA/PEG2000-PE complexes.     

Effect of the labelling ratio on the photophysics of fluorescein isothiocyanate (FITC) conjugated to bovine serum albumin.

The non-linearity of the fluorescence emission on increasing the probe to protein ratio has long been regarded as problematic and has lead to the development of dyes to overcome this effect. One of the causes of this non-linear response can be ascribed to the overlap of the label's own absorption and emission spectra. At higher labelling ratios, this affords the possibility of a reasonably efficient energy migration pathway, thus reducing the observed quantum yield of the dye. In this work we study the photophysics of fluorescein isothiocyanate (FITC) when conjugated to bovine serum albumin (BSA) at different labelling ratios (in the range FITC : BSA 1 : 17-15 : 1) using both steady state and time-resolved fluorescence techniques where on going from under labelled to over labelled samples a decrease in the initial (and steady state) anisotropy is observed, accompanied by an increase in the complexity of the decay kinetics and a decrease in the average lifetime. The band structure, elucidated by synchronous scan fluorescence spectroscopy, is also found to change on increased labelling. These results can be applied to the study of protein conformation and were confirmed by the analysis of denaturing BSA using urea.     

Enzymatic degradation of and bovine serum albumin release from starch-acetate films

The effect of acetylation of potato starch on swelling, enzymatic degradation, and bovine serum albumin (BSA, molecular mass 68 kDa) release rate from polymer films was studied. Potato starch and potato starch acetates (SA), having a degree of substitution of 1.9 or 2.6, were investigated. Polymer films were incubated in phosphate buffer solution pH 7.4 in the absence and presence of enzymes (alpha-amylase, amyloglucosidase, esterase) or in human serum. The acetylation of potato starch decreased its swelling considerably. Increased acetylation of starch also considerably retarded its enzymatic degradation. Due to the decreased swelling and degradation of SA films, BSA was released much slower from SA films than from potato starch films, both in the presence and absence of enzymes.     

Preparation of poly(ε-caprolactone)/poly(trimethylene carbonate) blend nanofibers by electrospinning

Poly(ε-caprolactone) (PCL)/poly(trimethylene carbonate) (PTMC) blend nanofibers have been prepared for the first time using an electrospinning process. The mixed dichloromethane (DCM) and N,N-dimethylformamide (DMF) (75/25, v/v) was found to be the most suitable solvent for electrospinning. Various blends of PCL/PTMC solutions were investigated for the formation of nano-scale fibers and it was found that the average diameter of the fibers was reduced and the morphology became finer when PTMC content was increased. FT-IR and DSC analysis indicated that the molecular interactions between PCL and PTMC were weak and they were phase-separated in the fibers. Due to the biocompatible properties of PCL and PTMC, the spun nanofibers developed here could have applications in the biomedical field.     

Bacterial cellulose nanofibers for albumin depletion from human serum

Cibacron Blue F3GA (CB) was covalently attached onto the bacterial cellulose (BC) nanofibers for human serum albumin (HSA) depletion from human serum. The BC nanofibers were produced by Acetobacter xylinum in the Hestrin–Schramm medium in a static condition for 14 days. The CB content of the BC nanofibers was 178 μmol/g. The specific surface area of the BC nanofibers was determined to be 914 m²/g. HSA adsorption experiments were performed by stirred-batch adsorption. The non-specific adsorption of HSA on the BC nanofibers was very low (1.4 mg/g polymer). CB attachment onto the BC nanofibers significantly increased the HSA adsorption (1800 mg/g). The maximum HSA adsorption was observed at pH 5.0. The HSA adsorption capacity decreased drastically with an increase of the aqueous phase concentration of sodium chloride. The elution studies were performed by adding 1 M NaCl to the HSA solutions in which adsorption equilibria had been reached. The elution results demonstrated that the binding of HSA to the adsorbent was reversible. The depletion efficiencies for HSA were above 96.5% for all studied concentrations. Proteins in the serum and eluted portion were analyzed by SDS-PAGE for testing the efficiency of HSA depletion from human serum. Eluted proteins include mainly HSA.     

Compatibilization effect of poly(epsilon-caprolactone)-b-poly(ethylene glycol) block copolymers and phase morphology analysis in immiscible poly(lactide)/poly(epsilon-caprolactone) blends

The miscibility and phase behavior of two stereoisomer forms of poly(lactide) (PLA: poly (L-lactide) (PLLA) and poly(DL-lactide) (PDLLA)) blends with poly(epsilon-caprolactone)-b-poly(ethylene glycol) (PCL-b-PEG) and PCL-b-monomethoxy-PEG (PCL-b-MPEG) block copolymers have been investigated by differential scanning calorimetry (DSC). The DSC thermal behavior of both the blend systems revealed that PLA is miscible with the PEG segment phase of PCL-b-(M)PEG but is still immiscible with its PCL segment phase although PCL was block-copolymerized with PEG. On the basis of these results, PCL-b-PEG was added as a compatibilizer to PLA/PCL binary blends. The improvement in mechanical properties of PLA/PCL blends was achieved as anticipated upon the addition of PCL-b-PEG. In addition, atomic force microscopy (AFM) measurements have been performed in order to study the compositional synergism to be observed in mechanical tests. AFM observations of the morphological dependency on blend composition indicate that PLA/PCL blends are immiscible but compatible to some extent and that synergism of compatibilizing may be maximized in the compositional blend ratio before apparent phase separation and coarsening.     

Degradation behavior of poly(epsilon-caprolactone)-b-poly(ethylene glycol)-b-poly(epsilon-caprolactone) micelles in aqueous solution

Poly(epsilon-caprolactone)-b-poly(ethylene glycol)-b-poly(epsilon-caprolactone) triblock copolymers were synthesized by the ring-opening polymerization of epsilon-caprolactone in the presence of hydroxyl-terminated poly(ethylene glycol) with different molecular weights, using stannous octoate catalyst. Micelles prepared by the precipitation method with these triblock copolymers exhibit a core-shell structure. The degradation behaviors of these core-shell micelles in aqueous solution were investigated by FT-IR, 1H NMR, GPC, DLS, TEM, and AFM. It was found that the degradation behavior of micelles in aqueous solution was quite different from that of bulk materials. The size of the micelles increased in the initial degradation stages and decreased gradually when the degradation period was extended. The caprolactone/ethylene oxide (CL/EO) ratio in micelles measured by NMR also shows an increase at the initial degradation stage and a decrease at later stages. The morphology of these micelles became more and more irregular during the degradation period. We explain the observed behavior by a two-stage degradation mechanism with interfacial erosion between the cores and the shells followed by core erosion.     

The immobilizaton of enzymes, bovine serum albumin, and phenylpropylamine to poly(acrylic acid)-polyethylene-based copolymers

A poly(acrylic acid)-polyethylene graft copolymer was prepared and used initially to couple to acid phosphatase, using soluble carbodiimides. Yields which were quite good were obtained with CMC but not with EDAC. The copolymers was used to couple trypsin using EEDQ. Several organic solvents were investigated for the preparation of the "activated" poly(acrylic acid) intermediate. Using the activated system, high concentrations of trypsin were bound but the relative activities were not very high. The yield was good with bovine serum albumin (BSA). When the method was used for invertase, acid phosphatase, and alkaline phosphatase, the yields were poor and the copolymer was shown to absorb protein by an ion-exchange mechanism. However, the activated system gave a good yield of coupling to phenylpropylamine. A polyethylene-coacrylic-acid polymer containing 13% of acrylic acid (by weight) was then converted to the acid chloride by refluxing with thionyl chloride. The chlorinated copolymer which contained 0.7% chlorine and a thionyl-chloride-treated polyethylene control which contained no chlorine were investigated in immobilization studies. Such coupling involved bovine serum albumin (BSA), alkaline phosphatase, trypsin, beta-galactosidase, and invertase. Bovine serum albumin coupled well to the support, but none of the enzymes gave high levels of enzymes activity. Phenylpropylamine coupled well and all of the acid chloride groups were involved. Tyrosine reacted with 63% of the available acid chloride groups.     

A convenient method for the synthesis of amine-terminated poly(ethylene oxide) and poly(epsilon-caprolactone)

A convenient synthetic route to prepare amine-terminated poly(ethylene oxide) (PEO) and poly(epsilon-caprolactone) (PCL) was described. The strategy involved two-step reactions, the condensation of hydroxyl-terminated PEO and PCL with N-benzyloxycarbonyl amino acid followed by the catalytic hydrogenation under mild conditions. NMR and GPC measurements indicated that the reactions proceeded nearly quantitatively. Amine-terminated PEO thus prepared was used to initiate the polymerization of alpha-(N(epsilon)-benzyloxycarbonyl-L-lysine) N-carboxy anhydride [lys(Z)-NCA], and the results confirmed that the reactivity of the amino group was high.     

Stabilization of chromium(V) species in bovine serum albumin adduct

A detailed investigation of molecular interactions of the protein bovine serum albumin (BSA) with potassium dichromate has been made. Compelling evidence for the formation of a relatively stable Cr(V) species which decays only slowly to afford Cr(III) products has been obtained. The resulting final chromium(III) product mediates in cross-linking of BSA. The implications of the formation of a relatively stable chromium(V) as well as chromium(III)-mediated cross-links in the marker protein are discussed in the context of the metal ion-induced biotoxicity.     

Intramolecular complex formation of poly(N-isopropylacrylamide) with human serum albumin

Complexation of human serum albumin (HSA) with poly(N-isopropylacrylamide) (PNIPA) ranging in molecular weight (M(PNIPA)) from 2.1 x 10(4) to 1.72 x 10(6) was studied in an aqueous system (pH 3) containing NaCl as a supporting salt. Dynamic light scattering, static light scattering, electrophoretic light scattering, and dialyzing techniques were used as the experimental tool in a suitable combination. The measurements were performed mainly at 25 degrees C and at 0.01 M NaCl as a function of mixing ratio (r(m), molar ratio of PNIPA to HSA). The results of DLS and ELS evidently demonstrated the formation of a water-soluble complex through mixing of HSA and PNIPA. A detailed analysis of SLS data with the aid of dialysis data revealed that the resulting complex is an "intramolecular" complex consisting of a PNIPA chain with several of bound HSA molecules. Both hydrodynamic radius (R(h)) and radius gyration (R(g)) of intramolecular complexes decreased as r(m) was increased. This result correlated well to the fact that the number (n) of bound proteins per polymer decreases with increasing r(m). The size and the molar mass of the complex became large depending on M(PNIPA), but the increase of M(PNIPA) led to a decrease in n at r(m) < 1. The increase in NaCl concentration from 0.01 to 0.3 M brought about the increase in the size and the molar mass of an intramolecular HSA-PNIPA complex prepared at r(m) = 1.1. This was found to be due to an increase of n. A similar trend was observed when temperature rose from 25 to 32 degrees C (close to lower critical solution temperature of PNIPA). However, the effect of temperature on the increase of was strong in comparison with that of ionic strength. On the basis of these results obtained, the complexation mechanism was discussed in detail.     

Addition of biological functionality to poly(epsilon-caprolactone) films

Biodegradable polyesters such as poly(epsilon-caprolactone) (PCL) have a number of biomedical applications; however, their usage is often limited by a lack of biological functionality. In this paper, a PCL-based polymer containing pendent groups activated by 4-nitrophenyl chloroformate (NPC) and reactive toward primary amines has been cast into thin films. The reactivity of the films toward poly(l-lysine) and the cell adhesion peptide, GRGDS, was assessed, and their cell adhesive capabilities were characterized. ATR-FTIR analysis found that NPC functional groups were present on the surface of the cast film, and the synthesis, conjugation, and visualization of a fluorescent molecule on these films further demonstrated the success of this functionalization methodology. The immersion of these films into a solution of either poly(l-lysine) (PLL) or GRGDS in PBS (pH 7.4) and subsequent 3T3 fibroblast adhesion studies demonstrated significant improvement in cell adhesion and spreading over films cast from unmodified PCL. This investigation has shown that this novel NPC-containing polymer can be utilized in many applications where increased cellular adhesion is required, or the coupling of specific molecules to polymer surfaces is of interest.     

Tb(III) as a fluorescent probe for the structure of bovine serum albumin

Tb(III) was used as a fluorescent probe in the study of the calcium-binding sites on Bovine Serum Albumin (BSA). The fluorescence of Tb(III) is enhanced markedly when bound to BSA and nonradiative energy transfer between two fluorescent tryptophan(Trp) residues and Tb(III) bound to calcium-binding sites on BSA occurred. Experimental results show that the major groups in BSA bound to metal ion are the carboxyl side groups of glutamic acid (Glu) and aspartic acid (Asp). The average distance between the bound Tb(III) and the two tryptophan residues in BSA calculated by a F?ster dipole-dipole nonradiative energy transfer mechanism is 1.48 nm.     

残余牛血清白蛋白含量检测试剂盒抗干扰性研究

为了对目前使用的残余牛血清蛋白(BSA)含量检测试剂盒的抗干扰性进行评价,选用19个企业的12个品种,共计28份样品进行检测,包括冻干疫苗和液体疫苗两种剂型。分别检测15ng/ml BSA对照样品、二倍稀释的疫苗样品和添加15ng/ml BSA的疫苗样品。将添加BSA的疫苗样品的检测结果减去未添加BSA的疫苗样品的结果,其数值应当位于BSA对照样品均值的95%可信区间内。多数品种的疫苗添加BSA后回收率在85%和115%之间。个别制品的回收率在82%~83%之间。实验研究结果证明目前使用的BSA检测试剂盒具有较好的抗干扰作用。