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Irradiation as an alternative route for protein crosslinking: Cosolvent free BSA nanoparticles
Abstract
Recent studies reported the development of protein-based nanoparticles by the use of ɣ-irradiation for the production of advanced drug carriers and biomaterials at nanolevel. Basically, the technique combines protein aggregation by means of protein desolvation using a cosolvent, followed by crosslinking using irradiation. We hereby report the effect irradiation dose over the development of protein-based nanoparticles combined or not with cosolvents. BSA was used as a model protein and the samples were irradiated in phosphate buffer (pH=7.2) using a gammacell in absence or presence of ethanol or methanol at 30% and 40% (v/v) respectively. The irradiation dose effect was evaluated following the exposition of BSA to 2.5, 5, 7.5 and 10 kGy over particle size and protein crosslinking, as determined by photon correlation microscopy and fluorescence measurements. Optimized effects were achieved at 10 kGy, under the assayed dose range, with regard to higher particle size and protein crosslinking levels. The use of irradiation was suitable for the synthesis of BSA nanoparticles and tuning of particle size was achieved by controlling the absorbed dose. While the use of ethanol provided an additional control over BSA particle size if compared to the use of methanol at the concentrations assayed, the possibility to perform BSA crosslinking in absence of cosolvents unraveled a novel one-step procedure for the synthesis of protein nanoparticles with no toxicity generated by the use of cosolvents or monomers.
... However, crosslinkers cannot be removed properly and can induce toxicity to biological systems. In addition, the washing step, which requires dialysis process, is time consuming (Varca et al., 2016). For this reason, Varca et al. (2016) investigated another method of crosslinking based on irradiation. ...
... In addition, the washing step, which requires dialysis process, is time consuming (Varca et al., 2016). For this reason, Varca et al. (2016) investigated another method of crosslinking based on irradiation. BSA nanoparticles were exposed to g-irradiation in phosphate buffer (pH 7.2) in the absence and/or presence of ethanol and methanol at 30% and 40% (v/v), respectively. ...
... It was found a correlation between particle size and crosslinking density. This gives the possibility to produce nanoparticles in a one-step procedure and may allow simultaneous crosslinking and sterilization (Varca et al., 2016). ...
Nowadays, nanotechnology has become very integrated in the domain of pharmaceutical sciences since nanoparticle dispersions show various advantages as drug carriers. Among nanoparticles, the protein-based ones are of paramount importance. In fact, protein nanoparticles show many advantages over other types of nanoparticles, they are often non-toxic and biodegradable. In this review, the most common preparation methods of protein nanoparticles were targeted. In addition, the factors affecting their dispersions and the concepts of drug loading and drug release are also highlighted. It was obvious that each method can be optimized for a given protein. This issue was discussed in depth in the light of the current state of art, and supported by evidences for each method from the literature. In addition, it was concluded that the processing parameters strongly affect the properties of nanoparticles dispersion. Graphical Abstract: Preparation methods of nanoparticles and proteins used with them. Red cases: animal protein, Green cases: plant protein
... Initially irradiation attacks the solvent molecules leading to the production of reactive species, mainly • OH and eaq as a result of water radiolysis. In subsequent steps, such cosolvent species react with protein amino acids, forming reactive centres along protein molecules, which upon certain conditions, further react to yield crosslinking [23][24]. ...
... In order to demonstrate this approach, we produced papain crosslinked enzyme aggregates (CLEA) using electron beam irradiation and characterized the nanoparticles by means of particle size and bityrosine formation as a function of ethanol concentration, electron beam irradiation dose and irradiation atmosphere. In addition to the absence of toxicity generated by the use of crosslinkers in conventional methods and alternatively to the use of gamma irradiation for crosslinking [23][24], electron beam irradiation does not require a radioactive source, radiation may be switched off and allows the delivery of high doses in a very short period of time inside the final package, while featuring the same properties from a mechanistic point of view. 2) from Merck® (Germany) was solubilized in phosphate buffer (PBS) 50 mM at pH 7. Appropriate aliquots of papain solution, PBS and ethanol reagent grade from Sigma-Aldrich® (USA) were gently added to glass vials on ice bath to reach final papain concentration of 12.5 mg mL -1 and ethanol content ranging from 0-40% (v/v) to be hermetically sealed and the allowed to stabilize overnight in the refrigerator. ...
... Ethanol has been particularly useful and effective to promote protein desolvation, as it is capable of altering the solvation layer around protein molecules, which leads to changes in particle size as a function of concentration, and crosslinking is applied to provide chemical linkages along the nanoparticle, in order to preserved the acquired rearrangements, rather than physical effects exclusively. The combination of solvation followed by high energy crosslinking has been successfully applied for the synthesis of BSA nanoparticles in recent works [23][24]32], and papain nanoparticles [21][22] exclusively by the use of gamma irradiation Regarding the influence of the irradiation dose over papain particle size, the results revealed that native papain did not undergo size changes as function of the applied irradiation doses. This experimental evidence allowed the conclusion that the desolvation effects promoted by ethanol were essential for the nanoparticle formation. ...
Crosslinked enzyme aggregates comprise more stable and highly concentrated enzymatic preparations of current biotechnological and biomedical relevance. This work reports the development of crosslinked nanosized papain aggregates using electron beam irradiation as an alternative route for controlled enzyme crosslinking. The nanoparticles were synthesized in phosphate buffer using various ethanol concentrations and electron beam irradiation doses. Particle size increase was monitored using dynamic light scattering. The crosslinking formation by means of bityrosine linkages were measured by fluorescence spectra and the enzymatic activity was monitored using Na-Benzoyl-DL-arginine p-nitroanilide hydrochloride as a substrate. The process led to crosslinked papain nanoparticles with controlled sizes ranging from 6 to 11nm depending upon the dose and ethanol concentration. The irradiation atmosphere played an important role in the final bioactivity of the nanoparticles, whereas argon and nitrous oxide saturated systems were more effective than at atmospheric conditions in terms of preserving papain enzymatic activity. Highlighted advantages of the technique include the lack of monomers and crosslinking agents, quick processing with reduced bioactivity changes, and the possibility to be performed inside the final package simultaneously with sterilization.
... On the other hand, angular light scattering shows a sharp decrease in BSA molecular weight between 0.5 and 1 kGy (Gaber, 2005): 73 kDa at 0 and 0.5 kGy, 45 kDa at 1 and 5 kGy. The size of BSA particles (as measured by dynamic light scattering) is 6.6 nm at 0 kGy Varca et al., 2016), does not change considerably at 2.5 kGy , 8.8 nm at 5 kGy , 12.4 nm at 7.5 kGy and 16.6 nm at 10 kGy Varca et al., 2016). ...
... On the other hand, angular light scattering shows a sharp decrease in BSA molecular weight between 0.5 and 1 kGy (Gaber, 2005): 73 kDa at 0 and 0.5 kGy, 45 kDa at 1 and 5 kGy. The size of BSA particles (as measured by dynamic light scattering) is 6.6 nm at 0 kGy Varca et al., 2016), does not change considerably at 2.5 kGy , 8.8 nm at 5 kGy , 12.4 nm at 7.5 kGy and 16.6 nm at 10 kGy Varca et al., 2016). ...
Formation of patterns on the surface of dried films of saline biopolymer solutions is influenced by many factors, including particle size and structure. Proteins may be modified under the influence of ionizing radiation. By irradiating protein solutions with gamma rays, it is possible to affect the formation of zigzag (Z) structures on the film surface. In our study, the films were obtained by desiccation of bovine serum albumin (BSA) solutions, which were irradiated by a ⁶⁰Co gamma-source at doses ranging from 1Gy to 12kGy. The analysis of the resulting textures on the surface of the films was carried out by calculating the specific length of Z-structures. The results are compared against the absorption and fluorescence spectroscopy and dynamic light scattering (DLS) data. Gamma-irradiation of BSA solutions in the 1-200Gy range practically does not influence the amount of Z-structures on the film surface. The decrease in fluorescence intensity and increase in absorbance intensity point to the destruction of BSA structure at 2 and 12kGy, and DLS shows a more than 160% increase in particle size as a result of BSA aggregation at 2kGy. This prevents the formation of Z-structures, which is reflected in the decrease of their specific length.
... 3 Most of the reported studies focused on the BSA nanoparticles since they have great acceptance in the industry and are used as carrier systems or just as protein models in numerous fundamental studies. 4,5 Meanwhile, BSA with a long half-life can assist drugs in improving the blood drug concentration for a relatively long time. 6 Cancer is considered a major cause of death in the world. ...
In recent decades, biodegradable polymeric nano-particles have been used as a nanocarrier for the delivery of anticancer drugs. In the present study, we synthesize bovine serum albumin (BSA) nanospheres and evaluate their ability to incorporate a plant extract with anticancer activity. The plant extract used was the methanol fruit extract of Cucumis prophetarum, which is a medicinal herb. The fruit-extract-encapsulated BSA nanospheres (Cp-BSA nanospheres) were prepared using a desolvation method at various pH values of 5, 7, and 9. The nanosphere formulations were characterized using various techniques such as dynamic light scattering (DLS), ζ-potential, Fourier transform infrared spectroscopy (FTIR), and field-effect scanning electron microscopy (FESEM). The results show that the Cp-BSA nanospheres prepared at pH 7 were spherical with a uniform particle size, low polydispersity index (PDI), ζ-potential, and high entrapment efficiency (82.3%) and showed sustained release of fruit extract from Cp-BSA nanospheres in phosphate-buffered saline (PBS), pH 5. The anticancer activity was evaluated on A549, HepG2, MCF-7 cancer cell lines and HEK 293 normal cell lines. In vitro, antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, intracellular reactive oxygen species (ROS) production, and mitochondrial membrane potential were estimated. An in vitro cellular uptake study was performed using fluorescein isothiocyanate (FITC) dye at a different time of incubation, and DNA fragmentation was observed in a dose-dependent manner. The gene expression level of Bax and the suppression level of Bcl-2 were observed upon the treatment of Cp-BSA nanospheres. Thus, the Cp-BSA nanospheres triggered ROS-dependent mitochondrial apoptosis in different human cancer cell lines when compared to the noncancerous cell lines and could be used as a potential candidate for anticancer agents.
... The proteins and drugs in an aqueous solution are passed through a jet at very high pressure, resulting in nanoparticles sized 100-200 nm (Desai, 2016). The cross-linking technique with chemical, enzymatic, ionic, and thermal cross-linkers is used to improve the sustainability of protein-based nanoparticles for drug delivery (Varca, Queiroz, and Lugão 2016). ...
Consumer awareness of the relationship between health and nutrition has caused a substantial increase in the demand for nutraceuticals and functional foods containing bioactive compounds (BACs) with potential health benefits. However, the direct incorporation of many BACs into commercial food and beverage products is challenging because of their poor matrix compatibility, chemical instability, low bioavailability, or adverse impact on food quality. Advanced encapsulation technologies are therefore being employed to overcome these problems. In this article, we focus on the utilization of plant and animal derived proteins to fabricate micro and nano-particles that can be used for the oral delivery of BACs such as omega-3 oils, vitamins and nutraceuticals. This review comprehensively discusses different methods being implemented for fabrications of protein-based delivery vehicles, types of proteins used, and their compatibility for the purpose. Finally, some of the challenges and limitations of different protein matrices for encapsulation of BACs are deliberated upon. Various approaches have been developed for the fabrication of protein-based microparticles and nanoparticles, including injection-gelation, controlled denaturation, and antisolvent precipitation methods. These methods can be used to construct particle-based delivery systems with different compositions, sizes, surface hydrophobicity, and electrical characteristics, thereby enabling them to be used in a wide range of applications.
... Therefore, the toxicity of gelatin nanoparticles stabilized with glutaraldehyde needs to be thoroughly tested. Moreover, other approaches to gelatin cross-linking should be considered, e.g., reagentless irradiative cross-linking [81] or stabilization by polymer entrapment [3]. ...
Gelatin nanoparticles found numerous applications in drug delivery, bioimaging, immunotherapy, and vaccine development as well as in biotechnology and food science. Synthesis of gelatin nanoparticles is usually made by a two-step desolvation method, which, despite providing stable and homogeneous nanoparticles, has many limitations, namely complex procedure, low yields, and poor reproducibility of the first desolvation step. Herein, we present a modified one-step desolvation method, which enables the quick, simple, and reproducible synthesis of gelatin nanoparticles. Using the proposed method one can prepare gelatin nanoparticles from any type of gelatin with any bloom number, even with the lowest ones, which remains unattainable for the traditional two-step technique. The method relies on quick one-time addition of poor solvent (preferably isopropyl alcohol) to gelatin solution in the absence of stirring. We applied the modified desolvation method to synthesize nanoparticles from porcine, bovine, and fish gelatin with bloom values from 62 to 225 on the hundreds-of-milligram scale. Synthesized nanoparticles had average diameters between 130 and 190 nm and narrow size distribution. Yields of synthesis were 62–82% and can be further increased. Gelatin nanoparticles have good colloidal stability and withstand autoclaving. Moreover, they were non-toxic to human immune cells.
... 3 Most of the reported studies focused on the BSA nanoparticles since they have great acceptance in the industry and are used as carrier systems or just as protein models in numerous fundamental studies. 4,5 Meanwhile, BSA with a long half-life can assist drugs in improving the blood drug concentration for a relatively long time. 6 Cancer is considered a major cause of death in the world. ...
In recent decades, biodegradable polymeric nanoparticles have been used as a nanocarrier for the delivery of anticancer drugs. In the present study, we synthesize bovine serum albumin (BSA) nanospheres and evaluate their ability to incorporate a plant extract with anticancer activity. The plant extract used was the methanol fruit extract of Cucumis prophetarum, which is a medicinal herb. The fruit-extract-encapsulated BSA nanospheres (Cp-BSA nanospheres) were prepared using a desolvation method at various pH values of 5, 7, and 9. The nanosphere formulations were characterized using various techniques such as dynamic light scattering (DLS), ζ-potential, Fourier transform infrared spectroscopy (FTIR), and field-effect scanning electron microscopy (FESEM). The results show that the Cp-BSA nanospheres prepared at pH 7 were spherical with a uniform particle size, low polydispersity index (PDI), ζ-potential, and high entrapment efficiency (82.3%) and showed sustained release of fruit extract from Cp-BSA nanospheres in phosphate-buffered saline (PBS), pH 5. The anticancer activity was evaluated on A549, HepG2, MCF-7 cancer cell lines and HEK 293 normal cell lines. In vitro, antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, intracellular reactive oxygen species (ROS) production, and mitochondrial membrane potential were estimated. An in vitro cellular uptake study was performed using fluorescein isothiocyanate (FITC) dye at a different time of incubation, and DNA fragmentation was observed in a dose-dependent manner. The gene expression level of Bax and the suppression level of Bcl-2 were observed upon the treatment of Cp-BSA nanospheres. Thus, the Cp-BSA nanospheres triggered ROS-dependent mitochondrial apoptosis in different human cancer cell lines when compared to the noncancerous cell lines and could be used as a potential candidate for anticancer agents.
... It has been shown that the ability of casein to resist against changes in their environment can be improved through Millard's reaction [3,4] or applying cross-linkers. [5] However, the use of cross linkers or Millard's reaction may be imply undesirable effects [6] or decrease the nutritious value of proteins. [7] Thus, other alternative methods may be a good way to enhance the protection of nano-carriers (NCs). ...
Casein is an excellent carrier for curcumin, but it is susceptible to hydrolysis in gastrointestinal digestion. This study intended to design a tailored nano-carrier composed of camel casein/bovine whey protein to protect casein and curcumin during digestion. After loading curcumin to the casein, whey protein was added to solution followed by heating at 80°C for 10 min in order to form casein/whey protein aggregations. Binding of curcumin to the solution was assayed by fluorescence. The results showed that in the existence of aggregated whey proteins, stability of curcumin against gastrointestinal condition significantly improved. These results attributed to the protective effect of the aggregated whey proteins during the gastric digestion and denaturation in the accessibility of pepsin to the casein. It was demonstrated that this method might be a good design to combat susceptibility of casein without the use of any synthetic material.
... Moreover, albumin's primary structure has a high content of charged amino acid residues, which could lead to attractive electrostatic interaction with positively-or negatively-charged active molecules on protein-based nanoparticles without the addition of any intermediate molecules [5]. The majority of reported studies have been focused on BSA since it has good acceptance in the pharmaceutical industry and is used as a carrier system or just as a protein model in numerous fundamental studies [6][7][8][9][10][11]. ...
Nanoparticles are nowadays largely investigated in the field of drug delivery. Among nanoparticles, protein-based particles are of paramount importance since they are natural, biodegradable, biocompatible, and nontoxic. There are several methods to prepare proteins containing nanoparticles, but only a few studies have been dedicated to the preparation of protein- based nanoparticles. Then, the aim of this work was to report on the preparation of bovine serum albumin (BSA)-based nanoparticles using a well-defined nanoprecipitation process. Special attention has been dedicated to a systematic study in order to understand separately the effect of each operating parameter of the method (such as protein concentration, solvent/non-solvent volume ratio, non-solvent injection rate, ionic strength of the buffer solution, pH, and cross-linking) on the colloidal properties of the obtained nanoparticles. In addition, the mixing processes (batch or drop-wise) were also investigated. Using a well-defined formulation, submicron protein-based nanoparticles have been obtained. All prepared particles have been characterized in terms of size, size distribution, morphology, and electrokinetic properties. In addition, the stability of nanoparticles was investigated using Ultraviolet (UV) scan and electrophoresis, and the optimal conditions for preparing BSA nanoparticles by the nanoprecipitation method were concluded
Background
Food proteins are of particular interest in the design of delivery systems due to their biodegradability, biocompatibility, high nutritional value, abundance and desirable functional properties. However, controlling the rate of release of the bioactive compounds from food protein carriers in simulated media presents challenges for the added value of food and nutraceutical technology.
Scope and approach
This review focuses on recent advances in the development of food protein-based delivery systems and highlights an approach for the quantitative prediction of the delivery of bioactive compounds from novel matrices. Moreover, the microstructural properties of such materials and their role in hydrogel network design are discussed along with identifying key parameters of the release mechanisms. This review seeks to provide a better understanding of swelling mechanisms by describing the effect of morphological characteristics, including network mesh size, on molecular transport.
Key findings and conclusions
Swellable matrices represent delivery systems in which an appropriate combination of structural relaxation, swelling and erosion mechanisms can be adapted to the delivery programme. The ability to predict and control the swelling behaviour of protein matrices via mathematical modelling along with a better understanding of network morphological characteristics/parameters will facilitate the development of tailored delivery systems for functional food and nutraceutical applications.
Papain-based nanoparticles were recently developed using radiation technologies and proven effective to generate nanosized crosslinked papain particles with preserved enzymatic activity. The applications of such nanostructured systems are expected to be similar to native papain with considerable biopharmaceutical advantages and concern drug loading among other biotechnological applications. The nature of such crosslinks and the possibility to provide simultaneous sterilization have been hypothesized but remain not totally clarified. This manuscript advances the discussion on the radiation-induced synthesis of protein nanoparticles by approaching the nature of the crosslinking and the possible contribution of bityrosine linkages and disulfide bridges to the overall nanoparticle assembly as well as the feasibility of the simultaneous sterilization process under the pre-established conditions of processing. Papain nanoparticles were synthesized and characterized according to size, proteolytic activity, bityrosine, cysteine content and molecular weight by SDS-PAGE upon sonication at 40 kHz. Bacterial identification and the sterility tests were performed in accordance with ISO 11737 prior to and after inoculating 10⁶ CFU of Corynebacterium xerosis. Our experiments evidenced the crosslinking of rather intra- than intermolecular nature and a contribution of cysteine bridges and bityrosine linkages to the stabilization and formation of the papain nanoparticle assembly. The technique was effective to promote simultaneous crosslinking and sterilization at the established conditions of processing and may be validated in accordance with the ISO 11137.
Albumin is an attractive macromolecular carrier and is widely used in preparing nanoparticles due to its biodegradability, nontoxicity, and nonimmmunogenicity. Albumin nanoparticles themselves are biodegradable and can easily be prepared in defined sizes. Several protocols for preparing albumin nanoparticles that have been developed include: emulsification, desolvation, nab technology, thermal gelation, nano spray drying, and self-assembly techniques.
After preparing albumin nanoparticles, physicochemical properties such as particle size, particle size distribution, zeta potential, and morphology need to be carefully checked if they are intended to be used in a specific delivery system.
Albumin itself has several reactive amino acid groups on its surface, and, as a result, albumin nanoparticles also participate in the electrostatic adsorption of positively or negatively charged molecules. In addition, modifying the reactive amino acid groups on the surface can confer a variety of functionalities to albumin nanoparticles such as prolonged circulation half-life, enhanced nanosystem stability, sustained drug release, or targeting release.
Therefore, albumin nanoparticles represent one of the most important drug carriers for the delivery of therapeutic drugs.
Nanosuspension technology is currently undergoing dramatic expansion in pharmaceutical science research and development. However, most of the research efforts generally focus on formulation and potential beneficial description, while the research into potential toxicological effects and implications (i.e., in vivo safety and health effects) is lacking. This review identifies some of the key factors for studying nanosuspension safety and the potential undesired effects related to nanosuspension exposure. The key factors for discussion herein include particle characterization, preparation approach, composition, and excipients of the formulation and sterilization methods. A few comments on the primary and required safety aspects of each administration route are also reviewed.
Nanotechnology has broadened the options for the delivery of agents of biotechnological and clinical relevance. Currently, attention has been driven towards the development of protein-based nanocarriers due to high the biocompatibility and site-specific delivery. In this work we report radiation-synthesized bovine serum albumin nanoparticles as an attempt to overcome limitations of available albumin particles, as a novel route for the development of crosslinked protein nanocarriers for the administration of chemotherapic agents or radiopharmaceuticals. Albumin containing phosphate buffer solutions were irradiated using γ-irradiation at distinct solvent concentrations - ethanol or methanol. Nanoparticle size was followed by DLS and bityrosine crosslinking formation using fluorescence measurements and SDS-PAGE. In addition, computational experiments were performed to elucidate the mechanism and pathways for the nanoparticle formation. The synthesis of BSA nanoparticles using γ-irradiation in the presence of a cosolvent allowed the formation of the nanoparticles from 7 to 70nm without the use of any chemical crosslinker as confirmed by SDS-PAGE and DLS analysis. The combination of solvent and γ-irradiation allowed a fine tuning with regard to protein size.
Protein oxidation is increasingly recognised as an important modulator of biochemical pathways controlling both physiological and pathological processes. While much attention has focused on cysteine modifications in reversible redox signalling, there is increasing evidence that other protein residues are oxidised in vivo with impact on cellular homeostasis and redox signalling pathways. A notable example is tyrosine, which can undergo a number of oxidative post-translational modifications to form 3-hydroxy-tyrosine, tyrosine crosslinks, 3-nitrotyrosine and halogenated tyrosine, with different effects on cellular functions. Tyrosine oxidation has been studied extensively in vitro, and this has generated detailed information about the molecular mechanisms that may occur in vivo. An important aspect of studying tyrosine oxidation both in vitro and in biological systems is the ability to monitor the formation of oxidised derivatives, which depends on a variety of analytical techniques. While antibody-dependent techniques such as ELISAs are commonly used, these have limitations, and more specific assays based on spectroscopic or spectrometric techniques are required to provide information on the exact residues modified and the nature of the modification. These approaches have helped understanding of the consequences of tyrosine oxidation in biological systems, especially its effects on cell signalling and cell dysfunction, linking to roles in disease. There is mounting evidence that tyrosine oxidation processes are important in vivo and can contribute to cellular pathology.
Currently, the discovery of effective chemotherapeutic agents poses a major challenge to the field of cancer biology. The present study focuses on enhancing the therapeutic and anti cancer properties of atorvastatin calcium loaded BSA (ATV-BSA) nanoparticles in vitro.
BSA-ATV nanoparticles were prepared using desolvation technique. The process parameters were optimized based on the amount of desolvating agent, stabilization conditions as well as the concentration of the cross linker. The anti cancer properties of the protein coated ATV nanoparticles were tested on MiaPaCa-2 cell lines. In vitro release behavior of the drug from the carrier suggests that about 85% of the drug gets released after 72 hrs. Our studies show that ATV-BSA nanoparticles showed specific targeting and enhanced cytotoxicity to MiaPaCa-2 cells when compared to the bare ATV.
We hereby propose that the possible mechanism of cellular uptake of albumin bound ATV could be through caveolin mediated endocytosis. Hence our studies open up new facet for an existing cholesterol drug as a potent anti-cancer agent.
To investigate the effect of oxygen radicals on the molecular properties of bovine serum albumin (BSA), the secondary and tertiary structures, molecular weight and optical anisotropy of BSA were examined after the irradiation of the protein at various doses. gamma-Irradiation of the protein solution caused the disruption of the ordered structure of protein molecules as well as degradation, cross-linking and aggregation of polypeptide chains. Fluorescence spectroscopy indicated that irradiation quenched the emission intensity excited at 280 nm. Fourier transform infrared spectroscopy (FTIR) indicated that irradiation caused transformation from beta-turns into beta-sheets. A light scattering study showed that increasing the radiation dose decreased the molecular weight of the protein. Optical anisotropy data showed that radiation changed the ordered structure of the protein. Ultraviolet absorption spectroscopy indicated that fragmentation and aggregation might occur in response to radiation exposure.
The continued advancement in the sciences of functional foods and nutraceuticals has clearly established a strong correlation between consumption of bioactives and improved human health and performance. However, the efficacy and bioavailability of these bioactive ingredients (e.g., omega-3 oils, carotenoid antioxidants, vitamins, and probiotic bacteria) in foods often remains a challenge, due to their instability in food products and gastrointestinal tract, as well as their limited bioavailability. In some cases, these bioactive ingredients may impart an undesirable organoleptic characteristic to the final product, which hinders acceptance by consumers. In addressing these challenges, development of effective delivery systems is critical to meet the consumer needs for effective bioactives. The scientific knowledge behind developing effective delivery of bioactive components into modern and wide-ranging food products will be essential to reap their health-promoting benefits and to support the sustained growth of the functional foods market. Nanotechnology and Functional Foods: Effective Delivery of Bioactive Ingredients explores the current data on all aspects of nanoscale packing, carrying and delivery mechanisms of bioactives ingredients to functional foods. The book presents various delivery systems (including nano-emulsions, solid lipid nanoparticles, and polymeric nano-particles), their properties and interactions with other food components, and fate in the human body. Later chapters emphasize the importance of consumers? attitude towards nano-delivery for the success of the technology and investigate the challenges faced by regulatory agencies to control risks and harmonize approaches worldwide. The wide applicability of bioactive delivery systems with the purpose of improving food quality, food safety and human health will make this book a worthy reference for a diverse range of readers in industry, research and academia.
Synthesis of small polymeric particles can be achieved by ionizing radiation technology via intramolecular crosslinking by gamma rays onto soluble polymer molecules in random coil conformation. Differently soluble globular proteins are naturally densely packed structures. Fragmentation and aggregation processes have been reported for irradiated globular proteins solutions with ionizing radiations.In this work we describe protein-based nanoparticles prepared by gamma irradiation of a soluble and globular protein, such as seroalbumin, as the basic building blocks keeping its original conformational shape. Protein nanoparticles in the range 20–40 nm were detected after gamma irradiation of the aqueous protein solution in the presence of polar organic solvents. Nanoparticles were characterized by DLS, fluorescence, and UV and CD spectroscopy, showing that the protein molecules keep their general three-dimensional structure into the created nanoparticle.
Catalytic effects of BSA and BSA-Cu(II) complexes on dismutation of superoxide radicals and the effect of Cu(II) addition to BSA on its reactivity towards the hydrated electrons were studied by pulse radiolysis. No catalytic activity was found for BSA. The catalytic activity of BSA-Cu(II) (1:1) complex is low, comparable to that of native ceruloplasmin. Addition of Cu(II) to BSA increases its reactivity towards the hydrated electrons.
Albumin has been used as an intravenous fluid for many years. Its uses reflect its perceived physiology. As a fluid it is used for resuscitation but as a colloid it not only stays longer in the intravascular space but also supports and sustains the colloid oncotic pressure. Its myriad of physiological properties such as transport and buffering or its association with clotting pathways, membrane integrity and free oxygen scavenging have all been exalted as advantageous properties. Indeed the accumulation of all of the potential benefits heavily support its use. Critical examination of the paltry amount of clinical information relating to this ubiquitous fluid challenges this contention. A meta-analysis has gone further and suggest that it may even be harmful. The results of a massive 7000 patient study from Australia will, in January 2004, add further weight to either one or other side of the argument. The arguments as they currently stand have been outlined.
The size effect of nanomaterials is of major interest, since it may affect their bioavailability and toxicity. In this study, bovine serum albumin (BSA) nanoparticles were prepared using a modified desolvation method. Bare BSA nanoparticles and calcium (Ca)-loaded BSA nanoparticles were fabricated at the targeted sizes, 100, 400, and 800 nm. The mean diameters of the prepared BSA nanoparticles were 125, 393, and 713 nm; those of the Ca-BSA nanoparticles were 260, 353, and 919 nm. The surface-area-to-volume-ratios of the prepared BSA nanoparticles were 4.82, 1.53, and 1.03 nm−1; those of the Ca-BSA nanoparticles were 2.34, 1.72, and 0.90 nm−1. The size and the surface-area-to-volume-ratio of the BSA nanoparticles were controlled by adjusting BSA concentration, pH, and NaCl content, which affected the coagulation of the BSA molecules. The surface-area-to-volume-ratio is a more useful parameter than the mean diameter of particles for comparing effectiveness of nanoparticles.Research highlights► Nanoparticles of a controlled size are important to ascertain size effect in food. ► Difference in manufacturing processes affects particle properties. ► Different-size similar-surface particles are good tools to study size effect. ► Size-controlled BSA nanoparticles are prepared by similar manufacturing processes. ► Size-controlled BSA nanoparticles prepared are useful as standard materials.
Differential precipitation of human plasma by ethanol is one of the most important processes for purifying therapeutic proteins, including human serum albumin. Better understanding of the effects of ethanol on the structure and stability of proteins is critical for effective and safe application of ethanol-induced protein precipitation. Here, we examined the effects of ethanol on the structure and solubility of bovine serum albumin (BSA) and SH-modified BSA. Ethanol caused BSA denaturation in a bimodal fashion, i.e., reduction of α-helix at low concentration and subsequent induction of the α-helical structure at higher concentration. In contrast, the solubility of BSA decreased monotonically. The secondary structure of SH-modified BSA was different from that of native BSA. Ethanol resulted in enhanced secondary structures of SH-modified BSA and decreased solubility monotonically. These results suggest the favorable interaction of ethanol with hydrophobic residues, leading to protein denaturation, but the unfavorable interaction with charged residues, leading to a reduction of protein solubility.
A original strategy to obtain intramolecular crosslinking is discussed. This strategy consisted of three consecutive steps to direct the reaction to the formation of intramolecular crosslinks: (a) enzyme are partially modified with the bifunctional reagent in a very controlled fashion; (b) the excess of reagent is removed; and (c) the modified enzyme is incubated long-term to allow a crosslinking reaction without the competition of additional single-point modifications. In this way, we were able to obtain interesting stabilizations of immobilized derivatives of penicillin G acylase using glutaraldehyde as a crosslinking reagent under very controlled conditions.
Protein–protein crosslinks play an important role in determining the functional properties of food proteins. Manipulation of the number and nature of such protein crosslinks during food processing offers a means by which the food industry can manipulate the functional properties of food, often without damaging the nutritional quality. This review discusses advances in our understanding of protein crosslinking over the last decade, and examines current and future applications of this chemistry in food processing.
Among serum proteins albumin and transferrin have attracted the most interest as drug carriers in the past two decades. Prior to that, their potential use was overshadowed by the advent of monoclonal antibodies that was initiated by Milstein and Koehler in 1975. Meanwhile intensive pursuit of exploiting transferrin, but above all albumin as an exogenous or endogenous carrier protein for treating various diseases, primarily cancer, rheumatoid arthritis, diabetes and hepatitis has resulted in several marketed products and numerous clinical trials. While the use of transferrin has clinically been primarily restricted to immunotoxins, albumin-based drug delivery systems ranging from albumin drug nanoparticles, albumin fusion protein, prodrugs and peptide derivatives that bind covalently to albumin as well as physically binding antibody fragments and therapeutically active peptides are in advanced clinical trials or approved products. For treating diabetes, Levemir and Victoza that are myristic acid derivatives of human insulin or glucagon-like peptide 1 (GLP-1) act as long-acting peptides by binding to the fatty acid binding sites on circulating albumin to control glucose levels. Levemir from Novo Nordisk has already developed into a blockbuster since its market approval in 2004. Abraxane, an albumin paclitaxel nanoparticle as a water-soluble galenic formulation avoiding the use of cremophor/ethanol, transports paclitaxel through passive targeting as an albumin paclitaxel complex to the tumor site and is superior to conventional Taxol against metastatic breast cancer. INNO-206, an albumin-binding doxorubicin prodrug that also accumulates in solid tumors due to the enhanced permeability and retention (EPR) effect but releases the parent drug through acid cleavage, either intra- or extracellularly, is entering phase II studies against sarcoma. An expanding field is the use of albumin-binding antibody moieties which do not contain the fragment crystallizable (Fc) portion of, conventional immunoglobulin G (IgG) but are comprised of monovalent or bivalent light and/or heavy chains and incorporate an additional albumin-binding peptide or antibody domain. The most advanced antibody of this kind is ATN-103 (Ozoralizumab), a trivalent albumin-binding nanobody that neutralizes the pro-inflammatory tumor necrosis factor alpha (TNF-α) as a causative agent for exacerbating rheumatoid arthritis. ATN-103 is currently in multi-center phase II trials against this debilitating disease. In summary, because albumin as the most abundant circulating protein cannot only be used to improve the pharmacokinetic profile of therapeutically relevant peptides and the targeting moiety of antibodies but also for peptide-based targeting as well as low-molecular weight drugs to inflamed or malignant tissue, it is anticipated that R&D efforts of academia and the pharmaceutical industry in this field of drug delivery will prosper.
Albumin, a versatile protein carrier for drug delivery, has been shown to be nontoxic, non-immunogenic, biocompatible and biodegradable. Therefore, it is ideal material to fabricate nanoparticles for drug delivery. Albumin nanoparticles have gained considerable attention owing to their high binding capacity of various drugs and being well tolerated without any serious side-effects. The current review embodies an in-depth discussion of albumin nanoparticles with respect to types, formulation aspects, major outcomes of in vitro and in vivo investigations as well as site-specific drug targeting using various ligands modifying the surface of albumin nanoparticles with special insights to the field of oncology. Specialized nanotechnological techniques like desolvation, emulsification, thermal gelation and recently nano-spray drying, nab-technology and self-assembly that have been investigated for fabrication of albumin nanoparticles, are also discussed. Nanocomplexes of albumin with other components in the area of drug delivery are also included in this review.
The objective of the present study was to optimize the fabrication of bovine serum albumin (BSA) nanoparticle by applying the Taguchi method with characterization of the nanoparticle bioproducts. BSA nanoparticles have been extensively studied in our previous works as suitable carrier for drug delivery, since they are biodegradable, non-toxic and non antigenic. A statistical experimental design method (Taguchi method with L16 orthogonal array robust design) was implemented to optimize experimental conditions of the purpose. Agitation speed, initial BSA concentration, pH and temperature were considered as process parameters to be optimized. As the result of Taguchi analysis in this study, temperature and agitation speed were the most influencing parameters on the particle size. The minimum size of nanoparticles (~74 nm) were obtained at 4°C, pH 7.5, 15 mg ml-1 BSA concentration and agitation speed of 500 rpm. As for characterization of the products, Atomic Force microscopy (AFM), Scanning Electron microscopy (SEM) and Sodium Dodecyl Sulphate- Poly Acrylamide Gel Electrophoresis (SDS-PAGE) as well as Fourier Transform Infra-Red (FTIR) techniques were employed.
Dityrosine is found in several proteins as a product of UV irradiation, gamma-irradiation, aging, exposure to oxygen free radicals, nitrogen dioxide, peroxynitrite, and lipid hydroperoxides. Interest of dityrosine in proteins is based on its potential as a specific marker for oxidatively damaged proteins and their selective proteolysis, hence it could be used as a marker for oxidative stress. Dityrosine is also the product of normal post-translational processes affecting specific structural proteins. Since post-translational modification of a given amino acid in a protein is equivalent to the substitution of that residue by an analogue, it has been proposed that the covalent modification of amino acids may serve as a "marking" step for protein degradation.
Fluorescence spectroscopy in combination with circular dichroism (CD) and UV-vis absorption spectroscopy were employed to investigate the binding of anti-inflammatory drug cromolyn sodium (Intal) to bovine serum albumin (BSA) under the physiological conditions with Intal concentrations of 0-6.4 x 10(-5)mol L(-1). In the mechanism discussion, it was proved that the fluorescence quenching of BSA by Intal is a result of the formation of Intal-BSA complex. Quenching constants were determined using the Stern-Volmer equation to provide a measure of the binding affinity between Intal and BSA. The thermodynamic parameters Delta G, Delta H, Delta S at different temperatures (298, 304, and 310 K) were calculated and the results indicate the electrostatic interactions play a major role in Intal-BSA association. Binding studies concerning the number of binding sites (n=1) and apparent binding constant K(b) were performed by fluorescence quenching method. Utilizing fluorescence resonant energy transfer (FRET) the distance R between the donor (BSA) and acceptor (Intal) has been obtained. Furthermore, CD and synchronous fluorescence spectrum were used to investigate the structural change of BSA molecules with addition of Intal, the results indicate that the secondary structure of BSA molecules was changed in the presence of Intal.
Dityrosine is a fluorescent molecule formed as a result of normal posttranslational processing. In many structural proteins, dityrosine confers resistance to proteolysis and physicochemical trauma as a stabilizing crosslink. Dityrosine has also been found in oxidative/nitrative stress under a variety of conditions and biological systems. In this regard, it has been used as an important biomarker for oxidatively modified proteins during UV and gamma-irradiation, aging, and exposure to oxygen free radicals, nitrogen dioxide, peroxynitrite, and lipid hydroperoxides. Renewed interest in dityrosine and other tyrosine oxidation products as clinical indicators of oxidative modification has driven the development of important techniques for the specific analysis and quantification of these molecules. The presence of elevated levels of dityrosine in mammalian tissue and urine samples has been measured by chromatographic separation followed by mass spectrometry GC-MS and HPLC-MS/MS. Increases in dityrosine levels have been associated with pathologies such as eye cataracts, atherosclerosis, acute inflammation, and Alzheimer's disease. The continued development of, and increased accessibility to, improved mass spectrometric instrumentation will expand the capability, feasibility, and sensitivity with which specific biomarkers like dityrosine can be measured.
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