Synthesis,Conjugation, and Immunological Evaluation of the Serogroup 6 Pneumococcal Oligosaccharides

The first synthesis of the newly discovered oligosaccharide of pneumococcal serotype 6C and its spacer‐containing analogue is reported. Conjugation of the spacer‐containing oligosaccharides of pneumococcal saccharides 6A, 6B, 6C and derivatives thereof with bovine serum albumin (BSA) protein carrier was carried out by using squaric‐acid approach to obtain the oligosaccharide–protein conjugates in excellent yields. The conjugates have been tested with a rabbit antiserum pool (Pool B) used for pneumococcal serotyping. The results showed that synthetic carbohydrate conjugates express epitopes found in native capsular polysaccharides of serotypes 6A, 6B, and 6C.     

Synthesis and characterization of an amphiphilic hyperbranched poly(amine‐ester)‐co‐D,L‐lactide (HPAE‐co‐PLA) copolymers and their nanoparticles for protein drug delivery

A series of hyperbranched poly(amine‐ester)‐co‐D ,L ‐lactide (HPAE‐co‐PLA) copolymer were synthesized by ring‐opening polymerization of D ,L ‐lactide with Sn(Oct)₂ as catalyst to a fourth generation branched poly(amine‐ester) (HPAE‐OHs4). The chemical structures of copolymers were determined by FTIR, ¹H‐NMR, ¹³C‐NMR, and TGA. Double emulsion (DE) and nanoprecipitation (NP) method were used to fabricate the nanoparticles of these copolymers encapsulating bovine serum albumin (BSA) as a model. DSC thermo‐grams indicated that the nanoparticles with BSA kept stable below 40°C. Different factors which influence on particular size and encapsulation efficiency (EE) were investigated. Their EE to BSA could reach 97.8% at an available condition. In vitro release behavior of NPs showed a continuous release after a burst release. The stability maintenance of BSA in the nanoparticle release in vitro was also measured via circular dichroism and fluorescence spectrometry. The results showed that the copolymer nanoparticles have a promising potential in protein delivery system. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Micelles formed by self‐assembly of hyperbranched poly[(amine‐ester)‐co‐(D,L‐lactide)] (HPAE‐co‐PLA) copolymers for protein drug delivery

BACKGROUND: The aim of the work presented was to synthesize a series of amphiphilic hyperbranched poly[(amine‐ester)‐co‐(D ,L ‐lactide)] (HPAE‐co‐PLA) copolymers and study the formation of copolymeric micelles. These copolymeric micelle systems are expected to be potential candidates for applications in protein drug delivery. RESULTS: The chemical structures of the copolymers were confirmed by Fourier transform infrared spectroscopy, ¹³C NMR and thermogravimetric analysis. Fluorescence spectroscopy and dynamic light scattering confirmed the formation of copolymeric micelles of the HPAE‐co‐PLA copolymers. The maintenance of stability of bovine serum albumin (BSA) during release from micelles in vitro was also measured using circular dichroism and fluorescence spectrometry. CONCLUSION: Novel hyperbranched HPAE‐co‐PLA copolymers have been synthesized. Conjugation of PLA to HPAE was proved to be an available method for the preparation of micelles for protein delivery. The BSA‐loaded micelles showed enhanced encapsulation efficiency and the structural stability of BSA was retained during the release process. The hyperbranched polymeric micelles could be useful as drug carriers for protein drug delivery systems. Copyright © 2008 Society of Chemical Industry     

Preparation,characterization, and protein loading properties of N‐acyl chitosan nanoparticles

Various N‐acyl chitosans with propionyl‐, hexanoyl‐, nonanoyl‐, lauroyl‐, pentadecanoyl‐, and stearoyl‐groups were synthesized and self‐aggregated N‐acyl chitosan nanoparticles (CSNPs) were prepared by sonication. By the modification with N‐acyl groups, CSNPs increased their hydrophobic character and changed its structural features to be more suitable as a delivery carrier. The mean diameters of bovine serum albumin (BSA)‐loaded N‐acyl CSNPs ranged from 138 to 551 nm. Uniform particle size distribution of BSA‐loaded N‐acyl CSNPs was observed. The protein loading efficiency of N‐acyl CSNPs was about 94–95% with lower BSA concentration (0.1 mg/mL) and not significantly different with acyl chain length. With higher BSA concentration (1.0 mg/mL), however, the highest protein loading efficiency was observed with lauroyl and pentadecanoyl CSNPs. The results suggest that lauroyl and pentadecanoyl CSs are interesting candidates for protein delivery system. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Effects of drug and polymer molecular weight on drug release from PLGA‐mPEG microspheres

This study investigated the effects of drug and polymer molecular weight on release kinetics from poly (g ‐co‐glycolic acid)‐methoxypoly(ethyleneglycol) (PLGA‐mPEG) microspheres. Bovine serum albumin (BSA, 66 kDa), lysozyme (LZ, 13.4 kDa), and vancomycin (VM, 1.45 kDa) were employed as the model drugs, and encapsulated in PLGA‐mPEG microspheres of different molecular weight. Release of macromolecular BSA was mainly dependent on diffusion of drug at/ near the surface of the matrix initially and dependent on degradation of matrix at later stages, while, the small drug of vancomycin seemed to depend totally on diffusion for the duration of the release study. The release behavior of lysozyme was similar to bovine serum albumin, except a shorter lag period. PLGA‐mPEG molecular weight also affected the release behavior of bovine serum albumin and lysozyme, but not obviously. PLGA‐mPEG microspheres in smaller molecular weight seemed to degrade more quickly to obtain a mass lose and matrix erosion, and thus, an accelerated release rate of bovine serum albumin and lysozyme. Vancomycin released much faster than bovine serum albumin and lysozyme, and exhibited no lag period, as it is thought to be diffusion‐controlled. Besides, vancomycin showed no difference in release behavior as PLGA‐mPEG molecular weight change. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41431.     

Poly(hydroxybutyrate‐co‐hydroxyvalerate) and bovine serum albumin blend prepared by electrospinning

Electrospinning is a method for the preparation of nanosized polymer fibers. Here, electrospinning is used to prepare a blend of a polyester, poly(hydroxybutyrate‐co‐hydroxyvalerate) (PHBV), and a globular protein, bovine serum albumin (BSA). The electrospun blend film is compared with a solution‐cast blend film and with single‐component electrospun films made of PHBV and BSA. In the electrospun blend films, BSA manifests itself as flat ribbons and a fine network formed from fibers less than 50 nm in diameter. The dissolution rate of BSA from the electrospun blended film is lower than from the solution‐cast one. The films are characterized using scanning electron microscopy, differential scanning calorimetry, and contact‐angle measurements. The obtained PHBV+BSA blend films have several emergent properties: a slow BSA dissolution rate, a fine BSA network, and unusual thermal behavior. Thus, the PHBV+BSA blend films introduce a new class of polymer–protein blends. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134, 45090.     

Effect of polymer compositions on the fabrication of poly(ortho‐ester) microspheres for controlled release of protein

Eight kinds of self‐catalyzed poly(ortho‐esters) (POEs) are used to fabricate bovine serum albumin (BSA)‐containing microspheres using a W/O/W double‐emulsion solvent extraction/evaporation method. All eight kinds of POE polymers used in this study are shown able to form microspheres under proposed fabrication conditions. The surface morphology and inner structure of the microspheres are analyzed using scanning electron microscopy (SEM). The microspheres have a size range from 64.7 to 120.2 μm. POE with a higher viscosity leads to bigger microspheres. It was found that the POE composition has a significant effect on BSA release profiles. POEs, which are more hydrophilic and contain a greater amount of glycolide or lactate (latent acid), yield higher BSA release rates. Specifically, POE containing 1,6‐hexanediol diglycolide (HD‐diGL) microspheres have the highest BSA release rate after a 20‐day test through a combination of surface erosion and diffusion mechanisms. POE containing a high percentage of the trans‐cyclohexanedimethanol (CDM) segment tends to yield microspheres with a lower release rate because of its hydrophobic nature. It was also found that the BSA release rate is more rapid at 37°C than at 22°C because of faster polymer degradation and water penetration at 37°C. Experimental results suggest that various protein release rates can be achieved by using different compositions of POEs. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 80: 1630–1642, 2001     

Ethylenediamino bridged bis(β‐cyclodextrin)/ poly(DL‐lactic‐co‐glycolic acid) nanoparticles prepared by modified double emulsion method: Effect of polyvinyl alcohol on nanoparticle properties

This study continues long‐standing efforts to develop protein delivery systems based on cyclodextrin‐conjugated polyester in our laboratory. The crude products of ethylenediamino bridged bis(β‐cyclodextrin)‐conjugated poly(DL ‐lactic‐co‐glycolic acid) were used in this study to make full use of unreacted reactant. With bovine serum albumin (BSA) as a model protein, the encapsulation effects (the encapsulation efficiency and particle size) of nanoparticles were similar to those of using pure conjugated products. Besides, a water‐in‐oil‐in‐water emulsification technique was conveniently modified. By adding polyvinyl alcohol (PVA) in the internal aqueous phase, a more stabilized emulsion was formed. Consequently, less PVA (～ 0.05%) was needed in the outer aqueous phase and less PVA (0.14 g/g nanoparticles) remained in the nanoparticles. This modification resulted in improved encapsulation efficiency (～ 89–94%) of BSA and an enlarged particle size (340–390 nm). Furthermore, the burst release of BSA at the 1st day was less pronounced (7–12% of the encapsulated amount) than that of nanoparticles with no PVA added in the internal aqueous phase. Degradation studies using transmission electron microscope and gel permeation chromatography suggested that the mechanism for protein release was mainly through nanoparticles erosion. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

A carboxylic acid‐functionalized polyfluorene as fluorescent probe for protein sensing

A new water‐soluble fluorescent conjugated polyelectrolyte, poly[9,9‐bis(3′‐butyrate)fluoren‐2,7‐yl] sodium (BBS‐PF), was studied as a fluorescent probe for protein sensing. The conjugated polyelectrolyte BBS‐PF shows high fluorescence sensitivity to cytochrome c, lysozyme, and bovine serum albumin (BSA). The Stern–Volmer constant (K_sv) in cytochrome c was measured to be as high as 6.1 × 10⁷ L/mol, approximately twice as that of the other two. Interestingly, it is found that BSA slightly enhances the fluorescence of BBS‐PF rather than quenches the fluorescence at its micromolar concentrations. The excitation spectra confirm that the interaction of BBS‐PF with the proteins could be different. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Dye‐ligand column chromatography: Albumin adsorption from aqueous media and human plasma with dye‐affinity microbeads

Cibacron Blue F3GA was covalently coupled with poly(ethylene glycol‐dimethacrylate‐2‐hydroxyethylmethacrylate) [poly(EGDMA‐HEMA)] microbeads via the nucleophilic substitution reaction between the chloride of its triazine ring and the hydroxyl groups of the HEMA molecules under alkaline conditions. The affinity sorbent carrying 16.5 μmol Cibacron Blue F3GA/g polymer was then used for bovine serum albumin (BSA) adsorption from aqueous protein solutions and from human plasma in a packed‐bed column. The BSA adsorption capacity of the microbeads decreased with an increase in the recirculation rate. High adsorption rates were observed at the beginning, then equilibrium was gradually achieved in about 60 min. The BSA concentration in the mobile phase was also effective on adsorption. BSA adsorption was first increased with BSA concentration, then reached a plateau that was about 57.3 mg BSA/g. Higher BSA adsorption was observed at lower ionic strength. The maximum adsorption was observed at pH 5.0, which is the isoelectric pH of BSA. Higher human serum albumin adsorption was achieved from human plasma (109.6 mg HSA/g). High desorption ratios (over 94% of the adsorbed albumin) were achieved by using 1.0M NaSCN (pH 8.0) in 30 min. It was observed that albumin could be repeatedly adsorbed and desorbed without a significant loss in adsorption capacity. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 74: 2803–2810, 1999     

Structural interactions of globular proteins—Bovine serum albumin,egg albumin,and lysozyme,in aqueous medium,elucidated with molar volumes,viscosities, energy functions,and IR spectra from 293.15 to 303.15 K

Densities (ρ), apparent molar volumes (V_?), viscosities (η), and IR spectra on 0.0010–0.0018% aqueous solutions of bovine serum albumin (BSA), egg albumin (E‐Alb), and lysozyme at an interval of 0.0004% and at temperatures from 293.15, 298.15, and 303.15 K have obtained. The free energy (ΔG), entropy (ΔS), and enthalpy (ΔH) data with compositions and temperatures are calculated from the values of the flow velocity (v_f) of viscous flow, which decrease with temperature. The densities decrease with concentrations and temperatures except BSA, and the V_? values slightly increase with concentrations for BSA and lysozyme, which depict structural reorientations and transition states of protein molecules with increase in viscosities and decrease in reduce viscosities. The reduce viscosities at 293.15 K for BSA, E‐Alb, and lysozyme are noted positive, and for BSA and lysozyme remain positive at 298.15 and 303.15 K, whereas for E‐Alb it is negative. Activation energies (E*) for lysozyme remain almost constant, and are higher than those of the BSA and E‐Alb, respectively, also slightly higher E* values for the BSA than those of the E‐Alb at 293.15 and 298.15, and lower than of the E‐Alb at 303.15 K, are observed elucidating greater structural interactions for BSA at lower while weaker at temperatures. Stretching frequencies of amide (? NHCO? ), ? NH? , ? CO, and ? CH? groups of proteins are noted from IR spectra with broader stretching frequencies for ? NH? . © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 1420–1429, 2007     

Thermosensitive soy protein/poly(n‐isopropylacrylamide) interpenetrating polymer network hydrogels for drug controlled release

A series of the thermosensitive interpenetrating polymer network hydrogels composed of soy protein and poly(N‐isopropylacrylamide) were successfully prepared. The structure and properties were systematically characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, and differential scanning calorimetry. It was found that the hydrogels had good miscibility and high porosity, and the volume phase transition temperatures of the hydrogels were around 32°C. The release behavior and the release mechanism of a model protein, bovine serum albumin (BSA), were also investigated in detail. The results indicated that the release behavior of BSA had strong temperature dependence and the release percentage of BSA could be controlled by modulating the amount of soy protein or crosslinking agent. The analysis of the release mechanism revealed that the Fickian diffusion controlled release was dominant under the experimental conditions. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011.     

Similarly sized protein separation of charge‐selective ethylene‐vinyl alcohol copolymer membrane by grafting dimethylaminoethyl methacrylate

Similarly sized protein separation was investigated using a charge‐selective membrane, which prepared by grafting dimethylaminoethyl methacrylate (DMAEMA) onto ethylene vinyl alcohol copolymer (EVAL) membrane. Bovine serum albumin (BSA) and bovine hemoglobin (BHb) was used as model proteins. P(DMAEMA), the weak cationic polyelectrolyte with ionizable tertiary amine groups, contributed to the charge‐selective separation for BSA and BHb. At pH 6.0, the grafted EVAL membrane surface was positively charged and BSA was negatively charged, while BHb was positively charged. The BSA was adsorbed onto the membrane surface due to electrostatic interaction and the BHb passed through the membrane into the permeate. The charge‐selective behavior resulted in the separation of the similarly sized protein. The maximum separation factors of static adsorption separation for model protein and binary mixture were 32.4 and 37.2, respectively. In the dynamic separation process, the maximum separation factor value was 6.2. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 46374.     

Thermodynamics of protein aqueous solutions: From the structure factor to the osmotic pressure

An analytical expression for the structure factor for globular proteins in aqueous solution is presented. This expression was obtained considering a potential given by the sum of a hard core, a van der Waals attractive, and a screened Coulomb contribution. Experimental data of small angle x‐ray scattering for bovine serum albumin (BSA) in aqueous solutions containing sodium salts at different protein concentrations and pH values are also presented. The developed expression for the structure factor describes accurately these experimental data provided a dependence of the attractive parameter on protein concentration is established. An expression for the osmotic pressure was derived from the structure factor. With attractive parameters adjusted from x‐ray scattering data, the osmotic pressure of BSA aqueous solutions could be predicted with very good agreement with experimental data. © 2015 American Institute of Chemical Engineers AIChE J, 61: 2871–2880, 2015     

Controlled release of bovine serum albumin from stimuli‐sensitive silk sericin based interpenetrating polymer network hydrogels

In recent years, the use of stimuli‐sensitive interpenetrating polymer network (IPN) hydrogels for the encapsulation and controlled release of protein drugs has received significant attention. The purpose of the present study was to investigate the release of bovine serum albumin (BSA), a model drug, from a series of thermosensitive silk sericin (SS) poly(N‐isopropylacrylamide) and pH‐responsive SS poly(methacrylic acid) IPN hydrogels. The effect of the chemical architecture of the IPN was investigated on the percentage loading of BSA and its subsequent release from the loaded devices. The temperature and pH of the release medium were studied for their impact on the release of BSA. The pulsatile releasing behavior of IPN hydrogels revealed that they can be made into microcapsules or thermo‐valves, which act as an on–off release control. © 2013 Society of Chemical Industry     

Fabrication of ferrogels using different magnetic nanoparticles and their performance on protein adsorption

Magnetic biomaterials were prepared using magnetite and chitosan‐coated magnetite nanoparticles (CSNPs) dispersed in poly(vinyl alcohol) gels. Two different methods were developed to obtain ferrogels: in situ co‐precipitation of magnetite (Ferro‐IS) and by adding previously synthesized CSNPs to the neat matrix (Ferro‐CSNPs). In both cases, the crosslinking was carried out by freezing ? thawing (F‐T). The as‐prepared materials as well as precursor CSNPs were characterized by Fourier transform infrared spectroscopy, electronic microscopy (scanning and transmission), X‐ray diffraction, ζ potential, dynamic light scattering, thermogravimetric analysis, differential scanning calorimetry and magnetic properties. The performance of these gels as protein adsorbents was evaluated. Batch adsorption experiments were carried out using bovine serum albumin (BSA) as a model. Substantially different adsorption behaviour was found using Ferro‐IS and Ferro‐CSNPs. This was assigned to dissimilar bonding mechanisms of BSA to the ferrogel matrix. Hence, biomaterials potentially useful in drug delivery as well as in protein purification fields may be prepared by a relatively simple, non‐toxic and low cost method. © 2013 Society of Chemical Industry     

Development of ultrasmall chitosan/succinyl β‐cyclodextrin nanoparticles as a sustained protein‐delivery system

In this article, we introduce a new method for preparing ultrasmall chitosan (CS)/succinyl β‐cyclodextrin (SCD) nanoparticles (NPs) intended for loading bovine serum albumin (BSA) as a model protein. The proposed method is based on the complex coacervation technique followed by ionotropic gelation with tripolyphosphate. SCD, an anionic derivative of cyclodextrin, was synthesized and used in CS‐based NPs to enhance the entrapment efficiency of BSA. The results show that with this approach, ultrasmall, compact, and neutralized NPs with a mean particle size near 30 nm were obtained. A high degree of protein entrapment in the NPs led to a significant improvement in the BSA release profile with a low initial burst release (ca. 3% w/v of the initially loaded BSA) and a sustained release over time. This enabled a suitable nanocarrier for long‐term protein delivery (30% release over 120 h). © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 39648.     

Cu(II)‐incorporated,histidine‐containing,magnetic‐metal‐complexing beads as specific sorbents for the metal chelate affinity of albumin

N‐Methacryloyl‐(L )‐histidine methyl ester (MAH) was synthesized from metharyloyl chloride and histidine. Spherical beads with an average size of 150–250 μm were obtained by the suspension polymerization of ethylene glycol dimethacrylate and MAH in an aqueous dispersion medium. Magnetic poly(ethylene glycol dimethacrylate‐co‐N‐Methacryloyl‐(L )‐histidine methyl ester) [m‐p(EGDMA‐co‐MAH)] microbeads were characterized with swelling tests, electron spin resonance, elemental analysis, and scanning electron microscopy. The specific surface area of the beads was 80.1 m²/g. m‐p(EGDMA‐co‐MAH) microbeads with a swelling ratio of 40.2% and 43.9 μmol of MAH/g were used for the adsorption of bovine serum albumin (BSA) in a batch system. The Cu(II) concentration was 4.1 μmol/g. The adsorption capacity of BSA on the Cu(II)‐incorporated beads was 19.2 mg of BSA/g. The BSA adsorption first increased with the BSA concentration and then reached a plateau, which was about 19.2 mg of BSA/g. The maximum adsorption was observed at pH 5.0, which was the isoelectric point of BSA. The BSA adsorption increased with decreasing temperature, and the maximum adsorption was achieved at 4°C. High desorption ratios (>90% of the adsorbed BSA) were achieved with 1.0M NaSCN (pH 8.0) in 30 min. The nonspecific adsorption of BSA onto the m‐p(EGDMA‐co‐MAH) beads was negligible. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 93: 2669–2677, 2004     

Synthesis,characterization, and cytotoxicity of star‐shaped polyester‐based elastomers as controlled release systems for proteins

With the growing number of therapeutic proteins on the market, effective delivery systems are receiving particular attention. In this study, biodegradable elastomers, intended for protein drug delivery and based on methacrylic tripoly(ε‐caprolactone‐co‐d ,l ‐lactide) cyclic ester with different ratios of ?‐caprolactone to d ,l ‐lactide and methacrylic bipoly[?‐caprolactone‐b‐poly(ethylene glycol)‐b‐?‐caprolactone], were synthesized and characterized. The degradation behavior, bovine serum albumin (BSA)‐releasing kinetics, and cytotoxicity of the elastomers in vitro were investigated. The elastomers were degraded by the hydrolysis of the ester bond; this resulted in pH changes, which further affected the degradation rate. The BSA‐releasing behavior was strongly dependent on the diffusion mechanism. In the diffusion‐controlled period, nearly sustained and stable BSA release was achieved. Furthermore, the elastomers displayed good biocompatibility, as demonstrated by a 3‐(4,5‐dimethyl thiazol‐2‐yl)?2,5‐diphenyl tetrazolium bromide assay and inflammation–induction experiments, and are considered promising candidates for the controllable delivery of protein drugs. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43393.     

High performance liquid chromatography study of water‐soluble complexes and covalent conjugates of polyacrylic acid with bovine serum albumin

Polycomplex formation of bovine serum albumin (BSA) and polyacrylic acid (PAA) was studied by pH titration, fluorescence, and HPLC methods in water solutions. It was shown that the complex formation and the solubility (phase state) of the polycomplexes depended on pH of the solutions and ratio of the components. The stability of PAA‐BSA complexes was negligibly weak when pH = 6.0–7.0 [pH > pI (isoelectric pH)]. Stable water‐soluble polycomplexes formed at pH = pI (pH 5.0) in a wide range of n_BSA/n_PAA ratios (0.05–50) and coexisted with free protein molecules at the higher ratios of components. Existence of water‐soluble and insoluble PAA‐BSA complexes has been observed at pH ≤ 4.5. The soluble to insoluble state transition of polycomplexes has been confirmed and binding mode of polyelectrolytes to proteins was assumed dependent on the ratio of components as a result of formation of soluble polycomplexes, complex coacervation, or amorphous precipitates. Soluble complexes were formed as fully homogenized mixtures. Fraction composition of the mixtures and insoluble complexes depended on the protein/polymer ratio and over the critical protein/polymer ratio, the soluble polycomplexes coexisted with free BSA molecules. On the other hand, the comparision of covalent conjugate formation and complexation of PAA with BSA has been revealed. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2007