INTERPHOTORECEPTOR RETINOID-BINDING PROTEIN AND α-TOCOPHEROL PRESERVE THE ISOMERIC AND OXIDATION STATE OF RETINOL

Retinol decomposes rapidly into a number of products, including its aldehyde form, retinal, when introduced into buffer in phospholipid vesicles or ethanol. Interphotoreceptor retinoid-binding protein at low concentrations is found to protect retinol from isomerization and oxidation. The addition of alpha-tocopherol to either liposomes or an ethanolic-buffer solution also prevents decomposition. Neither of these agents interferes with the successful regeneration of pigment with 9-cis retinal in rod outer segment preparations or the restoration of sensitivity by retinoids in isolated rod photoreceptors.     

Electrically polarized biphasic calcium phosphates: adsorption and release of bovine serum albumin

In this study, we applied electrical polarization technique to increase adsorption and control protein release from biphasic calcium phosphate (BCP). Three different biphasic calcium phosphate (BCP) composites, with hydroxyapatite (HAp) and β-tricalcium phosphate (β-TCP), were processed and electrically polarized. Our study showed that stored charge was increased in the composites with the increase in HAp percentage. Adsorption of bovine serum albumin (BSA), as a model protein, on the poled as well as unpoled surfaces of the composites was studied. The highest amount of BSA adsorption was obtained on positively poled surfaces of each composite. Adsorption isotherm study suggested a multilayer adsorption of BSA on the BCP composites. The effect of electrical polarization on BSA release kinetics from positively charged BCP surfaces was studied. A gradual increase in percent BSA release from positively charged BCP surfaces with decreasing stored charge was observed. Our study showed that the BCP based composites have the potential to be used as a drug or growth factor delivery vehicle.     

Topical delivery of retinoids counteracts the UVB-induced epidermal vitamin A depletion in hairless mouse

UVB irradiation depletes all-trans-retinol (ROL) and all-trans-retinyl esters (RE) from the hairless mouse epidermis. Prevention of this may be of relevance in counter-acting the long-term side effects of UVB exposure. We studied the effects of a topical treatment with natural retinoids before and after UVB exposure on three parameters involved in vitamin A metabolism: the amount of epidermal ROL and RE, the level of functional cellular retinol-binding protein I (CRBP-I), which is likely to protect ROL from UVB, as well as the cytosolic and microsomal enzyme activities which generate ROL and RE, i.e. all-trans-retinaldehyde (RAL) reductase, acylCoA:retinol acyltransferase (ARAT) and retinyl-ester hydrolase (REH). Topical pretreatment with retinoids promoted a dramatic increase of epidermal ROL, RE and CRBP-I levels, a transient increase of RAL reductase and ARAT activities as well as a decreased activity of REH, indicating a direction of epidermal vitamin A metabolism toward storage. In untreated mice UVB irradiation induced a depletion of epidermal ROL and RE in 10 min and a 50% decrease of CRBP-I after 24 h. In mice treated with topical retinoids, and then exposed to UVB, epidermal RE levels were higher than in vehicle-treated, nonirradiated mice. In contrast, ROL was as much depleted after UVB in pretreated as in untreated animals in spite of an induction of CRBP-I, indicating that CRBP-I does not actually protect ROL from UVB-induced depletion in this model. However, the reconstitution of both epidermal ROL and RE, after their depletion induced by UVB, was accelerated by previous topical treatment with RAL. Our results indicate that topical delivery of retinoids partly counteracts UVB-induced vitamin A depletion and promotes recovery.     

THE REGENERATION OF VISUAL PIGMENTS AND THE CHANGE OF ROD HYPERSENSITIVITY AFTER IRRADIATION BY BLEACHING LIGHT IN FROG RETINA

Abstract— The regeneration processes of visual pigments and the dark adaptation processes of rod photoreceptor after irradiation by bleaching light were studied by spectrophotometric, electroretinographic(ERG) methods and the measurement of early receptor potentials (ERPs) in bullfrog retina. After irradiation by bleaching light, rhodopsin in the isolated retina regenerated to an extent depending on the wavelength and intensity of the bleaching light as well as pH. Intense blue light and a weak alkaline environment (pH 7.5–9.5) favoured the regeneration. The regeneration of pigment in the green rods could not be detected in these experiments on the isolated retina. The regeneration of cone pigment was studied by measuring ERPs from both isolated retinas and retinas with pigment epithelium-choroid complex separated from scleras, which are called PEC-retinas. In the PEC-retinas, cone pigment regenerated more rapidly and with better efficiency than in the isolated retinas.
Rod photoreceptors desensitized permanently by bleaching light did not demonstrate hypersensitivity at 0.1 m M [Ca²⁺]_out, which induced hypersensitivity in non-desensitized photoreceptor, but showed the hypersensitivity when the [Ca²⁺]_out, was lowered further by the addition of EGTA.     

Calcium Phosphate Nanocarriers Dual‐Loaded with Bovine Serum Albumin and Ibuprofen: Facile Synthesis,Sequential Drug Loading and Sustained Drug Release

A simple and green strategy is reported for the preparation, drug loading, and release properties of a drug delivery system consisting of calcium phosphate (CP) nanocarriers dual‐loaded with bovine serum albumin (BSA) and hydrophobic drug ibuprofen (IBU). The sequential loading of BSA and IBU in calcium phosphate nanocarriers and in vitro simultaneous release of BSA and IBU are realized and investigated. In this method, BSA, which is used as a model protein drug, is encapsulated in situ in calcium phosphate nanocarriers. Subsequently, the typical hydrophobic drug IBU is loaded in the BSA/CP drug delivery system, forming the IBU/BSA/CP dual drug delivery system. The experiments reveal that the preloaded BSA not only reduces the cytotoxicity of calcium phosphate nanocarriers but also significantly improves the IBU drug loading capacity in calcium phosphate nanocarriers and greatly extends the duration of drug release. Thus, the as‐prepared IBU/BSA/CP dual drug delivery system is promising for drug delivery applications.     

Antibody modified Bovine Serum Albumin microspheres for targeted delivery of anticancer agent Gemcitabine

Inhibition of the EGFR signaling pathway is one of the attractive therapeutic targets for pancreatic cancer as recent studies demonstrated that EGFR is over‐expressed in pancreatic cancer. In this article we have demonstrated the design of targeted drug delivery system containing Bovine Serum Albumin (BSA) microspheres as delivery vehicle, gemcitabine as anticancer drug and anti‐EGFR (epidermal growth factor receptor) monoclonal antibody as targeting agent. The conjugated BSA microspheres were characterized by several physico‐chemical techniques such as scanning electron microscope, optical microscopy, fluorescent microscopy etc. Administration of these BSA microspheres containing gemcitabine and anti‐EGFR (BSA‐Gem‐EGFR) shows significant inhibition of pancreatic cancer cells (AsPC1) compared to the cells treated with only BSA microspheres, BSA with gemcitabine (BSA‐Gem), and free gemcitabine. This strategy could be used as a generalized approach for the treatment of pancreatic cancer along with other cancers which overexpress EGFR on cell surface. Copyright © 2012 John Wiley & Sons, Ltd.     

Preparation and characterization of liposomes-in-alginate (LIA) for protein delivery system

This paper describes the preparation and characterization of a novel drug delivery system for protein, liposomes-in-alginate (LIA) of biodegradable polymers, which is conceived from a combination of the polymer and the lipid-based delivery systems. LIA were prepared by first entrapping bovine serum albumin (BSA), a model protein within multivesicular liposomes (MVLs) by double emulsification process, which are then encapsulated within alginate hydrogel microcapsule, with untrapped BSA which are added during preparation of MVLs. Factors impacting encapsulation efficiency of MVLs are investigated and release of protein from the microcapsules in vitro is studied. At the same time, characterization of MVLs, microcapsules encapsulated protein formulation and integrality analyse of BSA in microcapsules are also studied, with the aim of improving the entrapment efficiency and prolonging release time. It is found that encapsulation efficiency and size of MVLs are affected by the composition and fabrication parameters of LIA. The data also show LIA have high encapsulation efficiency (up to 95%), little chemical change in drug caused by the formulation process, narrow particle size distribution and spherical particle morphology. Drug release assays conducted in vitro indicates that these formulations provide sustained release of encapsulated drug over a period, about 2 weeks.     

Biomimetic Mineralization of Protein Nanogels for Enzyme Protection

Protein nanogels have found a wide variety of applications, ranging from biocatalysis to drug/protein delivery. However, in practical applications, proteins in nanogels may suffer from enzymic hydrolysis and denaturation. Inspired by the structure and functionalities of the fowl eggshells, biomimetic mineralization of protein nanogels was studied in this research. Protein nanogels with embedded porcine pancreas lipase (PPL) in the cross-linked nanostructures were synthesized through the thiol–disulfide reaction between thiol-functionalized PPL and poly(N-isopropylacrylamide) with pendant pyridyl disulfide groups. The nanogels were further reacted with reduced bovine serum albumin (BSA) and BSA molecules were coated on the nanogels. Mineralization of BSA leads to the synthesis of biomineralized shells on the nanogels. With the growth of CaCO₃ on the shells, the nanogels aggregate into suprastructures. Thermogravimetric analysis, XRD, dynamic light scattering, and TEM were employed to study the mechanism of the biomineralization process and analyze the structures of the mineralized nanogels. The biomineralized shells can effectively protect the PPL molecules from hydrolysis by trypsin; meanwhile, the nanosized channels on the mineralized shells allow the transport of small-molecule substrates across the shells. Bioactivity measurements indicate that PPL in the nanogels maintains more than 80 % bioactivity after biomineralization.     

Preparation of chitosan microspheres by ionotropic gelation under a high voltage electrostatic field for protein delivery

Biodegradable microspheres have been widely used in drug/protein delivery system. In this paper, a modified ionotropic gelation method combined with a high voltage electrostatic field was developed to prepare protein-loaded chitosan microspheres. Bovine serum albumin (BSA) was chosen as a model protein. The preparation process and major parameters were discussed and optimized. The morphology, particle size, encapsulation efficiency and in vitro release behavior of the prepared microspheres were investigated. The results revealed that the microspheres exhibited good sphericity and dispersity when the mixture of sodium tripolyphosphate (TPP) and ethanol was applied as coagulation solution. Higher encapsulation efficiency (>90%) was achieved for the weight ratio of BSA to chitosan below 5%. 35% of BSA was released from the microspheres cured in 3% coagulation solution, and more than 50% of BSA was released from the microspheres cured in 1% coagulation solution at pH 8.8. However, only 15% of BSA was released from the microspheres cured in 1% coagulation solution at pH 4. The results suggested that ionotropic gelation method combined with a high voltage electrostatic field will be an effective method for fabricating chitosan microspheres for sustained delivery of protein.     

LIGHT-SENSITIVITY MODULATING PROTEIN IN FROG RODS

Cyclic GMP is the second messenger in the phototransduction mechanism in rod photoreceptors. Light-induced activation of cGMP phosphodiesterase (PDE), the hydrolyzing enzyme of cGMP, reduces cytoplasmic cGMP concentration to close the cGMP-activated channel and thereby causes a hyperpolarizing light response. Ca2+ concentration decreases during light-adaptation and this decrease is thought to be at least one of the underlying mechanisms of light-adaptation. Our previous electrophysiological work suggested that PDE in frog rod photoreceptors is regulated by this Ca2+ concentration decrease. In the present work, we isolated a protein that binds to disk membranes at high Ca2+ concentrations. In the presence of this protein (a 26 kDa protein), PDE light sensitivity becomes high at high Ca2+ concentrations. The effect was observed at physiological ranges of Ca2+ concentrations. Thus we could explain high light-sensitivity of photoreceptors under the dark-adapted condition. According to its function, we termed the 26 kDa protein 'sensitivity-modulating protein' or 'S-modulin'. During the purification we noticed that there are additional mechanisms present that may contribute to light-adaptation in frog rod photoreceptors.     

Ellipsometric approach for the real-time detection of label-free protein adsorption by second harmonic generation

Second harmonic generation (SHG) was performed using a novel ellipsometric detection approach to selectively probe the real-time surface binding kinetics of an unlabeled protein. The coherence of nonlinear optical processes introduces new possibilities for exploiting polarization that are unavailable with incoherent methods, such as absorbance and fluorescence. Adsorption of bovine serum albumin (BSA) at silica/aqueous solution interfaces resulted in changes in the polarization state of the frequency-doubled light through weak, dynamic interactions with a coadsorbed nonlinear optical probe molecule (rhodamine 6G). Using a remarkably simple instrumental approach, signals arising exclusively from surface interactions with BSA were spatially isolated and selectively detected with high signal-to-noise. The relative intensities acquired during the kinetics experiments using both circularly and linearly polarized incident beams were in excellent agreement with the responses predicted from SHG ellipsometry polarization measurements. Analysis of the polarization-dependent SHG generated during BSA adsorption at glass/aqueous solution interfaces provided direct evidence for slow conformational changes within the protein layer after adsorption, consistent with protein denaturation. This polarization selection approach is sufficiently general to be easily extended to virtually all coherent nonlinear optical processes and a variety of different surface interactions and architectures.     

Synthesis and characterization of a microsphere‐based coating for textiles with potential as an in situ bioactive delivery system

Proteinaceous microspheres have a wide range of biomedical applications, including their use as drug delivery systems. On the other hand, bioactive and antimicrobial textiles are promising substrates for medical care, in particular, as wound‐dressings. This work relates the development of a new process for the functionalization of textiles through the simultaneous formation and linkage of protein‐based microspheres onto textile fibers by sonochemical techniques. The microspheres developed by this process possess antimicrobial properties by themselves, but other may be incorporated by the encapsulation of various pharmaceutical formulations. This new type of microspheres and particularly their fixation onto textile materials encourage the development of textiles that can be used as delivery systems in a simple, fast, and non‐toxic process. Here it is reported the production of microspheres with a combination of bovine serum albumin (BSA), L ‐Cysteine (L ‐Cys), and n‐dodecane, using the ultrasound technology. The size distribution and morphology of the microspheres was determined as a function of several parameters such as irradiation time and BSA and L ‐Cys concentrations. The produced microspheres were analyzed using a laser light scattering size analyzer, an optical microscope and a scanning electron microscope. The new coating of BSA + L ‐Cys microspheres revealed a high stability and excellent antibacterial properties being a promising alternative to design textile‐based bioactive delivery systems with potential application in the development of textile‐based wound‐dressings. Copyright © 2011 John Wiley & Sons, Ltd.     

Formation and characterization of polyglutamate core-shell microspheres

The need for organ-targeted delivery of drugs and imaging agents creates an interest in biocompatible, biodegradable vesicles. We make protein microspheres using high-intensity ultrasound; these microspheres have a protein shell and a hydrophobic interior, making them ideal for delivering hydrophobic materials. We have previously shown that various proteins, e.g., bovine serum albumin (BSA), form a microsphere shell stabilized by interprotein cross-linking of cysteine residues. In this study, polyglutamate was used to form core-shell microspheres at slightly basic pH using sonication. These particles are smaller than our previous protein microspheres and are stable under conditions encountered in vivo. The stability of polyglutamate microspheres appears to be due to hydrogen bonding networks and not covalent cross-linking.     

基于壳聚糖衍生物的蛋白质药物载体材料的制备及性能

在离子液体均相体系中合成了一种新型两亲性窄分子量分布的低聚壳聚糖衍生物月桂基-琥珀酰化壳聚糖(LSCOS). 以LSCOS为载体材料, 以牛血清蛋白(BSA)为模板蛋白, 以戊二醛为交联剂, 用油包水(W/O)乳化交联法制备了包载BSA的BSA/LSCOS缓释载药微球. 通过扫描电子显微镜(SEM)、 透射电子显微镜(TEM)及紫外-可见光谱(UV-Vis)研究了BSA/LSCOS比率和戊二醛/LSCOS比率对微球的形貌结构、 包埋率、 载药率和体外药物释放特性的影响. 结果表明, 在离子液体中合成的LSCOS包覆了BSA, 形成的微球粒径约为1 μm, 微球表面随BSA用量的增加变得光滑, 随戊二醛用量的增加变得粗糙. BSA的累积释放率与BSA包载量成正比, 与交联剂添加量成反比, 因此, 可通过控制蛋白质药物的添加比率和交联剂用量来控制蛋白质药物体外释放率.     

Design and preparation of pH-responsive curdlan hydrogels as a novel protein delivery vector

New pH-responsive saccharide hydrogels were designed and prepared using curdlan derivatives(curdlan-Bochistidine, CUR-HIS). The CUR-HIS hydrogels possessed highly porous structures. The swelling ratios of CUR-HIS hydrogels increased with the degree of substitution of Boc-histidine groups. And the addition of 0.5 mol/L Na Cl provoked a sharp reduction of swelling ratio of CUR-HIS hydrogels. Bovine serum albumin(BSA) can be efficiently encapsulated into CUR-HIS hydrogels. Moreover, the release profiles of BSA at different p H values from CUR-HIS hydrogels were significantly different. These hydrogels showed good biocompatibility in the cytotoxicity assays. The CUR-HIS hydrogels are of great potential in biomedical applications such as protein delivery systems.     

Encapsulation and release of biomolecules from Ca-P/PHBV nanocomposite microspheres and three-dimensional scaffolds fabricated by selective laser sintering

This study focused on the fabrication of calcium phosphate (Ca-P)/poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) nanocomposite scaffolds loaded with biomolecules using the selective laser sintering (SLS) technique and their evaluation. Ca-P/PHBV nanocomposite microspheres loaded with bovine serum albumin (BSA) as the model protein were fabricated using the double emulsion solvent evaporation method. The encapsulation efficiency of BSA in PHBV polymer microspheres and Ca-P/PHBV nanocomposite microspheres were 18.06 ± 0.86% and 24.51 ± 0.60%, respectively. The BSA loaded Ca-P/PHBV nanocomposite microspheres were successfully produced into three-dimensional porous scaffolds with good dimensional accuracy using the SLS technique. The nanocomposite microspheres served as protective carriers and maintained the bioactivity of BSA during SLS. The effects of SLS parameters such as laser power and scan spacing on the encapsulation efficiency of BSA in the scaffolds and in vitro BSA release were studied. An initial burst release was observed, which was followed by a slow release of BSA. After 28-day release, The PHBV matrix was slightly degraded after 28-day in vitro release study. It was shown that nanocomposite scaffolds with controlled architecture obtained via SLS could be incorporated with biomolecules, enhancing them with more functions for bone tissue engineering application or making them suitable for localized delivery of therapeutics.     

Protein interactions with negatively charged inorganic surfaces

Protein adsorption on charged inorganic solid materials has recently attracted enormous interest owing to its various possible applications, including drug delivery and biomaterial design. The need to combine experimental and computational approaches to get a detailed picture of the adsorbed protein properties is increasingly recognised and emphasised in this review. We discuss the methods frequently used to study protein adsorption and the information they can provide. We focus on model systems containing a silica surface, which is negatively charged and hydrophilic at physiological pH, and two contrasting proteins: bovine serum albumin (BSA) and lysozyme (LSZ) that are both water soluble. At pH 7, BSA has a net negative charge, whereas LSZ is positive. In addition, BSA is moderately sized and flexible, whereas LSZ is small and relatively rigid. These differences in charge and structural nature capture the role of electrostatics and hydrophobic interactions on the adsorption of these proteins, along with the impact of adsorption on protein orientation and function. Understanding these model systems will undoubtedly enhance the potential to extrapolate our knowledge to other systems of interest.     

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.     

Composite microparticle drug delivery systems based on chitosan, alginate and pectin with improved pH-sensitive drug release property

Composite microparticle drug delivery systems based on chitosan, alginate and pectin with improved pH sensitivity were developed for oral delivery of protein drugs, using bovine serum albumin (BSA) as a model drug. The composite drug-loaded microparticles with a mean particle size less than 200 μm were prepared by a convenient shredding method. Since the microparticles were formed by tripolyphosphate cross-linking, electrostatic complexation by alginate and/or pectin, as well as ionotropic gelation with calcium ions, the microparticles exhibited an improved pH-sensitive drug release property. The in vitro drug release behaviors of the microparticles were studied in simulated gastric (pH 1.2 and pH 5.0), intestinal (pH 7.4) and colonic (pH 6.0 and pH 6.8 with enzyme) media. For the composite microparticles with suitable compositions, the releases of BSA at pH 1.2 and pH 5.0 could be effectively sustained, while the releases at pH 7.4, pH 6.8 and pH 6.0 increased significantly, especially in the presence of pectinase. These results clearly suggested that the microparticles had potential for site-specific protein drug delivery through oral administration.     

Preparation of protein-silicate hybrids from polyamine intercalation of layered montmorillonite

Hybrids of the model BSA protein and layered silicate clay with d spacing of approximately 62 A were prepared from either direct or stepwise intercalation. The pristine montmorilloinite (Na+-MMT) was first modified by poly(oxyalkylene)-amine salts (POP- and POE-amine) of 2000 g/mol Mw to a gallery-expanded silicate (d spacing=53 and 18 A, respectively), which became accessible for BSA protein embedding. Subsequent BSA substitution allowed the embedding of the protein into the layered clay galleries in an uncompressed conformation. The stepwise process of embedding large molecules into the silicate gallery provides a new method for synthesizing biomaterial/clay hybrids potentially useful in drug delivery or biomedical design.