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β-Lactoglobulin: An efficient nanocarrier for advanced delivery systems
β-lactoglobulin: an efficient nanocarrier for advanced delivery systems Zahra Shafaei, Behafarid Ghalandari, Akbar Vaseghi, Adeleh Divsalar, Thomas Haertl´e, Ali Akbar Saboury, Lindsay Sawyer PII: DOI: Reference:
S1549-9634(17)30050-3 doi: 10.1016/j.nano.2017.03.007 NANO 1549
To appear in:
Nanomedicine: Nanotechnology, Biology, and Medicine
Received date: Revised date: Accepted date:
11 September 2016 18 February 2017 14 March 2017
Please cite this article as: Shafaei Zahra, Ghalandari Behafarid, Vaseghi Akbar, Divsalar Adeleh, Haertl´e Thomas, Saboury Ali Akbar, Sawyer Lindsay, β-lactoglobulin: an efficient nanocarrier for advanced delivery systems, Nanomedicine: Nanotechnology, Biology, and Medicine (2017), doi: 10.1016/j.nano.2017.03.007
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ACCEPTED MANUSCRIPT β-lactoglobulin: an efficient nanocarrier for advanced delivery systems Zahra Shafaeia, Behafarid Ghalandarib, Akbar Vaseghic, Adeleh Divsalara*, Thomas Haertlé d, Ali Akbar Sabourye, f & Lindsay Sawyerg a
Department of Cell and Molecular Biology‚ Faculty of Biological Sciences‚ Kharazmi University, Tehran, Iran Applied Biophotonics Research Center, Science and Research Branch, Islamic Azad University, Tehran, Iran c Department of Biotechnology, Faculty of Advanced Science and Technologies of Isfahan, Isfahan, Iran d FIP, BIA UR1268, Institut National de la Recherche Agronomique, Nantes, France e Institute of Biochemistry and Biophysics‚ University of Tehran, Tehran, Iran f Center of Excellence in Biothermodynamics, University of Tehran, Tehran, Iran g School of Biological Sciences, The University of Edinburgh, Edinburgh, UK
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b
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Corresponding Author: Email: [email protected] Tel:+982634579600
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Fax:+982634579600
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Word count for Abstract: 138
Word count for Manuscript: 7524
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Number of Figures: 3
Number of References: 122 Number of Tables: 0
Conflict of interest statement The authors declare that there are no conflict of interest associated with this work.
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Contents
1. Introduction ………………………………………………..….…….…….……
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2. β-LG as a carrier protein…………………………………….…...….….……..
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2.1. β-LG structure………………………………………………….……….……. 2.2. β-LG transporting role……………………………………….…………….…
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3. β-LG nanoparticles as delivery systems …………………….…...………….. 3.1. β-LG nanoparticles formation, characterization and application…….….
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3.2. β-LG nanoparticles and release profile……………………………………. 4. Summery ..........................................................................................…....
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Acknowledgment………………………………………………………….….......
Abstract
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References……………………………………………………………….….…..…
Thanks to the progress of nanotechnology there are several agent-delivery systems that can be selected to achieve rapid and specific delivery of a wide variety of biologically active agents. Consequently, the manipulation and engineering of biopolymers has become one of the most exciting subjects for those who study delivery systems on the nano-scale. In this regard, both nanoparticle formation and a carrier role have been observed in the case of the globular milk whey protein, β-lactoglobulin (β-LG), setting it apart from many other proteins. To date, many efforts adopting different approaches have created β-LG nanoparticles useful in forming delivery systems for various agents with specific targets. In this review, the potential of β-LG to play the role of an efficient and diverse carrier protein, as well as its ability to form a well-targeted nano-scale delivery system is discussed. Keywords: β-lactoglobulin, nanocarrier, delivery system, β-LG nanoparticles 2
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1. Introduction
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The Nobel Prize physicist Richard Feynman in his lecture entitled “There’s plenty of room at the bottom” at the California Institute of Technology (Caltech) in 1959 at the annual
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meeting of the American Physical Society [1, 2] proposed the existence of a new world for humans based on the dream of constructing new structures on the nanometer scale. Many
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materials and systems exhibit unique physicochemical properties in nanoscale [3]. Moreover, the quest for nanoscale structures with specific practical application has increased such that
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fabrication of nanostructures has now become a reality [4].
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The term “nanotechnology” was introduced by Norio Taniguchi on 1974 in field of materials applied to structures on the nanometer scale [5]. The Oxford English Dictionary
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defines nanotechnology as that “branch of technology that deals with dimensions and tolerances of 1 to 100 nanometers, or, generally, with the manipulation of individual atoms and molecules”. In other words, based on the National Nanotechnology Initiative nanotechnology is defined as technology for engineering and controlling of matter and understanding its behavior at dimensions between approximately 1 and 100 nanometers so that materials have unique properties and novel applications [6]. In recent years, nanotechnology has exerted significant effects in the medical sciences as well as other domains of science [7-9]. The considerable progress achieved by the use of nanotechnology in medicine has elevated it to one of the important medical research fields in recent years. Advanced delivery system (ADS) for diagnostic and therapeutic agents, tissue engineering, gene therapy, in vitro and in vivo diagnostic and imaging techniques are just a few examples 3
ACCEPTED MANUSCRIPT of the effective presence of nanotechnology in medical science [2, 8, 9]. Currently, the focus of much research is more towards ADS than on other nanotechnological topics and
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consequently many studies of nano-carriers have been published [7, 10, 11].
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Previous studies have shown there to be many kinds of nanocarriers, like liposomes and polyethyleneglycol (PEG) particles to name but two, which are used for delivery of
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diagnostic and therapeutic agents [2, 12]. The various nano-carriers used for delivery systems have molecular properties which are tailored to their individual uses. Thus, to achieve this
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result, either top-down or bottom-up methods based upon the physicochemical nature of the
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carrier are selected for their production. By top-down, we mean, using bigger machines for materials separation and manipulation in the solid or liquid phase, and by bottom-up we mean
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changing the environmental conditions whereby the atoms and molecules will produce the nano-carrier. At the same time, biomedical studies on delivery systems constantly try to
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improve the properties such as size, complementary features with the target site, drug loading capacity, biodegradability, biocompatibility, and continued controlled release of the drug and
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the noninvasive behavior of the nano-carrier. Hence, numerous efforts have led to the successful creation of stable colloidal nanoparticles. In addition, many studies have been devoted to the optimization of previous methods to enhance the applicability of nano-carriers to delivery systems. Moreover, many reviews have been published describing the unique delivery properties and focusing both on their specific production methods and a better characterization of the nano-carrier [13-15]. Different polymeric nano-carriers have been used as delivery systems. Nano-carriers based on polymers can be divided into two types of nanoparticle: nano-capsules and nanospheres [16]. The difference between them arises from their formation. The carried agent is located in the core environment of a nano-capsule where it is surrounded by a polymeric shell. In contrast, in a nano-sphere there is no extra polymer layer and the agent is not 4
ACCEPTED MANUSCRIPT concentrated at a specific site within the polymer [17]. Furthermore, because of their biocompatibility and biodegradability as well as their being ‘generally recognized as safe’
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(GRAS) properties [16-19], biopolymers rather than other polymers have been selected as
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nano-carriers. Among the biopolymers used are proteins which have been adapted for delivery systems [20] and the better known polysaccharides such as chitosan and pectin [21-
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24].
Proteins, because of their particular physicochemical and physiological properties, and
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their unique structures as well as their biocompatibility and biodegradability, combined with
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their low toxicity, are considered as the most suitable candidates for oral delivery systems [18, 25, 26]. Different proteins are used in delivery systems and they can be broadly
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classified on the basis of their origin, into animal proteins and plant proteins. Plant proteins include: gliadin, soy proteins, zein and lectin, all of which have been used in nanoparticle
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formation. Zein and gliadin are used as protein vehicles because of their high hydrophobicity and lower production cost compared to animal proteins [27].
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In addition to these plant proteins, the most common animal proteins to have been used are gelatin, collagen, albumin, casein, the whey proteins and elastin. Gelatin has a long and safe history in pharmacy and today is used as a nanoparticle stabilizer, because of its low antigenicity and various functional groups, in the delivery of different drugs such as antimalarial, antimicrobial, and anticancer [28] medications. Moreover, for clinical applications such as vaccines, gelatin sponge and some protein formulations gelatin is used as stabilizer. Gelatin has specific motifs such as Arg-Gly-Asp sequences in its chains so that cell recognition and then cell adhesion modulation have make by them; whereas, other polymers are lack of this property. Hence, it is well known as another class of colloidal plasma expander as well as hydroxyethyl starch (HES). On the other hand, it is confirmed by United States Food and Drug Administration (FDA) as GRAS material. Also, gelatin explored that 5
ACCEPTED MANUSCRIPT biodegradable and biocompatible polymeric nanoparticles. These properties lead to its applications for clinically application individual for presence in drug delivery [29-37].
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Collagen/gelatin-based systems are used also because of their biodegradability and their
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superior biocompatibility. The reticuloendothelial system (RES, also known as the mononuclear phagocyte system) can take them up relatively easily and so they can deliver
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exogenous compounds such as anti-HIV drugs, which is an additional advantage [38]. Albumins are intrinsic carriers and the use of albumin-based nanoparticles as drug
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delivery systems has been reviewed previously [39]. Albumin has several drug binding sites
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and may serve for targeted delivery of anticancer drugs to tumor cells, which make it a suitable drug carrier [40]. Casein, the major milk protein, is readily available and non-toxic.
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The gastric digestibility of β-casein was suggested as a possible delivery mechanism for stomach cancer [18]. Recent studies have shown the nanotechnological usefulness of milk
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proteins (both casein and the whey proteins) in various industries including the pharmaceutical and food sectors. [18, 19, 25, 26, 41-44].
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Recent reports have signaled that among the milk proteins, β-lactoglobulin (β-LG) is an excellent candidate for nanoparticle formation [43, 45, 46]. Therefore, this review will present and discuss the results of studies that have investigated ability of the milk whey globular protein, β-LG, to play a nano-carrier role in delivery systems illustrating its potential as an ADS by focusing on its potency (the effect per mg, cf. LD50) and carrier capacity. Many publications with different objectives have been selected which have revealed the various potential applications of β-LG nanoparticles. The main aim of this review, therefore, is to describe and summarize the potential use of β-LG as a well-targeted delivery system. 2. β-LG as a carrier protein 2.1. β-LG structure
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ACCEPTED MANUSCRIPT β-LG is the major whey protein in ruminant milk and is a member of the lipocalin superfamily. This globular protein exists in the milk of most mammalian species but is absent
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from that of human. It is made up of 162 amino acid residues with a molecular weight of 18.3
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kDa [47, 48]. The secondary structure of β-LG consists of nine β-strands and one α-helix (Fig. 1). Eight of the nine β-strands (strands A-H) fold up into a flattened β-barrel that is also
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called a calyx. This calyx is conical and includes two surfaces. One surface is formed by strands A-D and the other one is formed by strands E-H. The ninth strand (strand I) forms a
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significant part of dimer interface and has a key role in dimer interactions. β-LG is resistant
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to acidic conditions. At ambient temperature and at pH values below 3.5 and above 7.5, this protein is a monomer but at physiological pH, the dimeric form is dominant. The interior of the β-barrel is hydrophobic and acts as the main site for binding of hydrophobic ligands. The
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connection between the β-strands A-H of β-LG is formed by seven loops that are known as
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AB, BC, CD, DE, EF, FG and GH, respectively. The BC, DE, and FG loops are short and located at the closed end of the β-barrel. The AB, CD, EF and GH loops are longer and also
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more flexible being located at the open end of the β-barrel. The EF loop acts as a gate over the binding site. Five cysteines are found in the β-LG sequence at positions 66, 106, 119, 121 and 160, resulting in two disulfide bonds (66-160 and 106-119) with Cys 121 forming the free thiol group with pH dependent activity and roles in aggregation and denaturation of protein. Moreover, β-LG contains two tryptophans (19 and 61) of which it appears that Trp 19 is responsible for most of intrinsic fluorescence intensity of β-LG [49-59]. Additional to any putative transport role, an important nutritional role is played by β-LG since it has significant amounts of all nine amino acid residues (Leu, Ile, Val, Phe, Thr, Met, Trp, Lys and His) that are essential for human health and tissue preservation [60]. Also, cysteines, which are present in β-LG, are essential amino acids for stimulation of glutathione
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ACCEPTED MANUSCRIPT synthase in the liver. β-LG has been reported as exerting an inhibitory effect on spleen phosphoprotein phosphatase [61].
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2.2. β-LG transporting role
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Previous studies show that β-LG can bind and transport various amphiphilic and hydrophobic ligands [56, 62-65]. β-LG interactions with tea polyphenols [66], phospholipids
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[67], curcumin and diacetyl curcumin [68], retinal [69], oxali-palladium [55], fatty acids [70], vitamin D and cholesterol [71], retinoid [72], spermine and spermidine [73], hemin [74],
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ellipticine [75], carcinogenic hydrocarbon [76], aromatic hydrocarbons [77], folic acid [78],
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Cr+3 [79], dodecyl sulfate and laurate [80], have all been investigated. The ability of β-LG to bind various ligands results from its structure. Many studies have confirmed that the main site for specific hydrophobic ligand binding to β-LG is located in the
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internal cavity, calyx, of the β-barrel. However, other potential binding sites involving
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residues Trp19, Tyr20, Tyr42, Gln44, Gln59, Gln68, Leu156, Glu157, Glu158 and His161 as a second site and Tyr102, Leu104, and Asp129 as a third site (Fig. 2) have been reported
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though so far lack convincing crystallographic data. [78, 81, 82]. Many authors have explored the carrier role of β-LG for different ligands and the following publications are examples of this. Liang et al investigated the interaction of resveratrol, a natural polyphenolic compound found in grapes and red wine that shows many health benefits, with β-LG and observed a 1:1 complex between resveratrol and β-LG and proposed a potential carrier role for resveratrol. The β-LG-resveratrol complex provides some advantages over the unbound molecule like a slight increase in the photostability and a significant increase in its solubility in water media [83]. Recently, an investigation by Ghalandari et al showed that oxali-platin used as a critical anticancer drug can be bound by β-LG in a molar ratio of 1:1. They revealed that oxali-platin interacts with internal cavity of β-LG and the driving force of this interaction is hydrophobic. 8
ACCEPTED MANUSCRIPT Based on these findings these authors proposed that β-LG could act as a carrier of oxali-platin [84]. Zhang et al studied the possibility of simultaneously binding of multiple bioactive
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compounds to β-LG. They illustrated this by showing that two or three ligands can bind to β-
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LG simultaneously. They suggested that β-LG-based carriers could be used as carriers for varieties of active compounds [85].
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Mohammadi et al studied β-LG interactions with curcumin, a phenolic compound which has many beneficial health properties such as anticancer, anti-inflammatory and antioxidant
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activities. Curcumin, because of its low bioavailability which results from its insolubility in
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water, is poorly assimilated. These authors revealed that β-LG may be a potential transporter of curcumin [68]. β-LG interactions with folic acid were studied by Liang and Subirade in order to verify not only its carrier role but also its protection against photo-degradation. Their
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results show that β-LG is a good candidate for the transport and delivery of folic acid. Also,
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the photo-degradation of folic acid decreased considerably after its interaction with β-LG [78]. Divsalar et al have investigated the interactions of series of Pd(II) and Pt(II) complexes
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with β-LG to devise a vector with low side effects for transporting metal anticancer complexes. Their results demonstrated that β-LG is a suitable protein for this purpose. According to their results, β-LG could be used as an efficient carrier of metal anticancer drugs without significant changes in its structure upon interaction with the metal complexes [80-91]. Transport of new anti-diabetic compounds to the small intestine was studied by Mehraban et al. Their results showed that β-LG may be able to play a carrier role for these new antidiabetic compounds [92-94]. Thus, according to what has been discussed above β-LG can employed as a transport vector for various compounds because of its unique secondary structural properties that allow it to bind different amphiphilic and hydrophobic ligands [66, 73]. Hence, all of these characteristics point to β-LG being a good candidate for use as an 9
ACCEPTED MANUSCRIPT ADS. Further, based on its unique properties and carrier functionality it is also a good candidate for nanoparticle formation thus producing a nanocarrier useful in various practical
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applications in particular delivery systems.
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3. β-LG nanoparticles as delivery systems
Over the two past decades many attempts have been made to construct β-LG-based
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nanoparticles for a variety of practical nano-carrier applications, many of them associated with pharmaceutical and nutraceutical delivery systems. Thanks to the stability of β-LG in
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acid conditions and relative instability in alkaline conditions, in most cases studies were
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devoted to oral delivery [46, 95]. β-LG is able to release its contents in a controlled way and is thus known as a biodegradable vehicle [16]. Hence, this biodegradability is used for nano vehicles in the alkaline conditions of the gastrointestinal tract (GIT) [95]. However, in order
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to maximize the efficiency of ligand protection together with controlled release by β-LG
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nanoparticles, an external layer or coating is necessary [96, 97]. 3.1. β-LG nanoparticles formation, characterization and application
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The external layer for coating β-LG nanoparticles must be resistant to degradation in the GIT conditions that justify selection of β-LG as an oral delivery system. Many studies have examined different molecules for outer layer formation in β-LG nanoparticles. According to these previous studies, among the many candidates that exist, low methoxyl pectin (LMP) has found particular favour for oral delivery systems [98-105]. LMP is bound to β-LG by physical interactions that are influenced by the pH of solution [42, 44]. β-LG nanoparticles can be formed by self-assembly or self-organization brought about by changes in the medium conditions of solution pH, temperature, ionic strength, presence or absence of salt ions and of course the relative concentrations of the β-LG and coating molecules. Hence, there are several protocols for the formation of β-LG nanoparticles such as time-dependent heat treatment of β-LG in solution or the effect of pH associated with ionic 10
ACCEPTED MANUSCRIPT strength changes accomplished by stirring at specific time intervals, or a combination of these and other factors. However, it is unclear which one of the different bottom-up protocols for β-
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LG nanoparticle synthesis is better or most suitable for a particular practical application [20,
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41-46, 95].
Jones et al investigated β-LG nanoparticle construction by time-dependent heat treatment.
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Their findings illustrated that in the presence of LMP as the external coating layer for β-LG, nanoparticles with diameters of 100-300 nm were formed at pH values between 4 and 7. In
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addition, their results revealed that the best sizes (100-250 nm) in terms of colloidal stability
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based on zeta-potential data in the presence of negatively charged LMP, were formed at the pI of β-LG (Fig. 3). Hence they concluded that the presence of the optimal concentration of LMP and the pH of the solution are important factors for β-LG nanoparticle formation. Their
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results also showed that the heat treatment protocol is another important factor in β-LG
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nanoparticle formation. They suggested that β-LG nanoparticles are useful as a delivery system as well as for practical applications in the food industry [41-43]. Giroux et al.
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investigated whey nanoparticle formation at specific time intervals by pH treatment at low temperature which leads to cross-linking denaturation. The whey nanoparticles obtained by this method had a size range of 100-300 nm depending upon the pH of the solution. Also, their results show that the presence of calcium is necessary for significantly enhances the stability of the whey protein nanoparticles [45]. A pH treatment performed by Ghalandari et al observed β-LG nanoparticles complexed with LMP formed in sizes less than 200 nm with good colloidal stability and net charge profile at the pI of β-LG. The results of their investigation agree with previous studies which revealed that both ionic strength and pH have an effect upon the size of the β-LG nanoparticles. All of these studies show that physical forces play a critical role in β-LG nanoparticle formation. Also, these data have confirmed that the β-LG nanoparticles content 11
ACCEPTED MANUSCRIPT in the presence of LMP as an extra layer and compression factor was preserved by nanoparticles compression. In other words, their findings emphasize the formation of smaller
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β-LG nanoparticles in the presence of LMP at the pI of the protein [46].
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Hence, the pH of solution, the presence or absence of stabilizers such as LMP as an external layer, or of ions such as calcium, combined with changes in the overall ionic strength
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of the solution, the time of synthesis, the time of thermal treatment, the degree of shaking during synthesis and the pH treatment of the β-LG before synthesis initiation are the most
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critical factors for the formation of β-LG nanoparticles. They are found to affect the size, the
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colloidal stability and the charge profile as determined by zeta potential measurements. Thus, β-LG nanoparticles are formed by an external stimulation that creates a balance of repulsive
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(e.g., electrostatic interactions between similarly charged groups) and attractive (e.g. van der Waals and hydrophobic forces, and electrostatic interactions between oppositely charged
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groups) physical forces during synthesis [106-109]. The size of a nano-carrier that can act as a nano-capsule must be smaller than 400 nm [95]. The results of the above studies show that
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β-LG has potential for nano-capsule formation which should allow it to function as a nanocarrier ADS in the GIT.
Shape is another important factor influencing the function and practical application of βLG nanoparticles as an ADS for the GIT, since it affects the tissue uptake, and hence the biodistribution/bioavailability, of the nanoparticles [110-112]. According to the results of morphological studies which have been done on β-LG nanoparticles, they appear to be spherical [95, 113-115]. Thus, the spherical shape of the β-LG nanoparticle allows it to move well in the GIT fluid and to reach the target position efficiently thereby increasing uptake at the target site and subsequent bio-distribution. Recently cytotoxicity and cellular uptake of β-LG nanoparticles was studied by Lee et al. They investigated the effects of two key factors namely concentration and incubation time on 12
ACCEPTED MANUSCRIPT the cellular uptake and cytotoxicity of β-LG nanoparticles on Caco-2 cells. Their results showed that by increasing the β-LG nanoparticles concentration from 100 to 250 μg/mL, its
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cellular uptake was significantly increased. While by increasing its concentration from 250 to
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500 μg/mL, the cellular uptake of nanoparticles weren’t significantly affected. Their results indicated that the concentration of 250 μg/mL of β-LG nanoparticle is a saturation limit of β-
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LG nanoparticle. Also their result showed that increasing the incubation time from 0.5 to 2 h had a significant increase in the cellular uptake of β-LG nanoparticles, whilst after 2 h, it
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showed no significant differences. However, their investigation on β-LG cytotoxicity in
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Caco-2 cells showed that increasing β-LG nanoparticles concentration from 100 to 500 μg/mL and also incubation time from 0.5 to 4 h did not significantly affect the cell survival ratio. It means that β-LG nanoparticles had no cytotoxicity to Caco-2 cells [116].
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3.2. β-LG nanoparticles and release profile
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The release profile of a ligand from a β-LG nanoparticle is perhaps the critical feature of any ADS for practical application in the GIT. Yi et al have investigated the time-dependent
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release of β-carotene in vitro from β-LG and β-LG-dextran nanoparticles in simulated GIT conditions at pH values of 2 and 7 in the presence of pepsin and trypsin, respectively. Their results illustrated that β-LG and β-LG-dextran nanoparticles in acidic conditions are stable so that only a small amount of β-carotene is released. At pH 7, in the presence of trypsin 60.9±2.9 and 51.8±4.3% of the total content of encapsulated β-carotene is released from β-LG and β-LG-dextran nanoparticles, respectively. Their findings reveal, therefore, that this β-LG nanoparticle is suitable as a delivery system for the alkaline conditions of the GIT [113]. Vitamin D3 delivery has also been investigated using β-LG-coagulum nanoparticles by Diarrassouba et al. The release profile of vitamin D3 from β-LG-coagulum nanoparticles was obtained after a specific time and at the pH values of simulated intestinal fluid. The β-LG13
ACCEPTED MANUSCRIPT coagulum nanoparticles were stable in the stomach but at basic pH, degradation released their contents into the intestine. Hence, the β-LG-coagulum nanoparticles also appear suitable as a
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delivery system that protects vitamin D3 from rapid digestion allowing transfer to the target
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site for intestinal absorption [117]. 4. Summary
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In this text, the capacity of β-LG, a member of a lipocalin superfamily, for ligand delivery in nanoscale delivery systems in order to be used in ADS has been presented. As has been
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discussed, β-LG has unique properties to bind various ligands with no significant effect on its
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structure, it can transport them through hostile environments and release them subsequently. These findings position β-LG as a good candidate for the delivery of bioactive compounds
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and other ligands. Indeed, the fact that it can be engineered to modify the ligand binding, as has been shown for another lipocalin [120-122], increases this potential considerably. In
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addition, because β-LG due to its structure is resistant to acids, but degrades in alkaline condition and contains a good balance of essential amino acids, is well suited to form a
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biodegradable and biocompatible advanced delivery system especially for the GIT. The many studies on β-LG nanoparticle formation cited here have shown that β-LG has enormous potential as a nanocarrier with good colloidal stability, net charge profile and spherical shape. Overall, significant new insights into various nano-scale ligand delivery systems have been afforded by these studies on β-LG nanoparticles. Of course, safe use of such nanoparticles as the basis of any ADS will require rigorous toxicity testing before general release. Acknowledgement The financial support of the Research Council of the Kharazmi University is highly appreciated.
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ACCEPTED MANUSCRIPT Legends Fig. 1. Molecular structure of β-LG and representation of its nine β-strands (A-I). Image was
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Fig. 2. The diagram of three possible binding site (A: internal cavity of β-barrel, B: second site is involving of residues Trp19, Tyr20, Tyr42, Gln44, Gln59, Gln68, Leu156, Glu157,
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Fig. 3. AFM image of β-LG nanoparticles complex with LMP by time-dependent heat
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ACCEPTED MANUSCRIPT β-lactoglobulin: an efficient nanocarrier for advanced delivery systems
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Zahra Shafaei1, Akbar Vaseghi2, Behafarid Ghalandari3, Adeleh Divsalar1*, Thomas Haertlé 4 , Ali Akbar Saboury5, 6& Lindsay Sawyer7
Graphical abstract
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S1549-9634(17)30050-3 doi: 10.1016/j.nano.2017.03.007 NANO 1549
To appear in:
Nanomedicine: Nanotechnology, Biology, and Medicine
Received date: Revised date: Accepted date:
11 September 2016 18 February 2017 14 March 2017
Please cite this article as: Shafaei Zahra, Ghalandari Behafarid, Vaseghi Akbar, Divsalar Adeleh, Haertl´e Thomas, Saboury Ali Akbar, Sawyer Lindsay, β-lactoglobulin: an efficient nanocarrier for advanced delivery systems, Nanomedicine: Nanotechnology, Biology, and Medicine (2017), doi: 10.1016/j.nano.2017.03.007
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ACCEPTED MANUSCRIPT β-lactoglobulin: an efficient nanocarrier for advanced delivery systems Zahra Shafaeia, Behafarid Ghalandarib, Akbar Vaseghic, Adeleh Divsalara*, Thomas Haertlé d, Ali Akbar Sabourye, f & Lindsay Sawyerg a
Department of Cell and Molecular Biology‚ Faculty of Biological Sciences‚ Kharazmi University, Tehran, Iran Applied Biophotonics Research Center, Science and Research Branch, Islamic Azad University, Tehran, Iran c Department of Biotechnology, Faculty of Advanced Science and Technologies of Isfahan, Isfahan, Iran d FIP, BIA UR1268, Institut National de la Recherche Agronomique, Nantes, France e Institute of Biochemistry and Biophysics‚ University of Tehran, Tehran, Iran f Center of Excellence in Biothermodynamics, University of Tehran, Tehran, Iran g School of Biological Sciences, The University of Edinburgh, Edinburgh, UK
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Corresponding Author: Email: [email protected] Tel:+982634579600
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Fax:+982634579600
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Word count for Abstract: 138
Word count for Manuscript: 7524
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Number of Figures: 3
Number of References: 122 Number of Tables: 0
Conflict of interest statement The authors declare that there are no conflict of interest associated with this work.
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Contents
1. Introduction ………………………………………………..….…….…….……
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2. β-LG as a carrier protein…………………………………….…...….….……..
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2.1. β-LG structure………………………………………………….……….……. 2.2. β-LG transporting role……………………………………….…………….…
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3. β-LG nanoparticles as delivery systems …………………….…...………….. 3.1. β-LG nanoparticles formation, characterization and application…….….
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3.2. β-LG nanoparticles and release profile……………………………………. 4. Summery ..........................................................................................…....
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Acknowledgment………………………………………………………….….......
Abstract
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References……………………………………………………………….….…..…
Thanks to the progress of nanotechnology there are several agent-delivery systems that can be selected to achieve rapid and specific delivery of a wide variety of biologically active agents. Consequently, the manipulation and engineering of biopolymers has become one of the most exciting subjects for those who study delivery systems on the nano-scale. In this regard, both nanoparticle formation and a carrier role have been observed in the case of the globular milk whey protein, β-lactoglobulin (β-LG), setting it apart from many other proteins. To date, many efforts adopting different approaches have created β-LG nanoparticles useful in forming delivery systems for various agents with specific targets. In this review, the potential of β-LG to play the role of an efficient and diverse carrier protein, as well as its ability to form a well-targeted nano-scale delivery system is discussed. Keywords: β-lactoglobulin, nanocarrier, delivery system, β-LG nanoparticles 2
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1. Introduction
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The Nobel Prize physicist Richard Feynman in his lecture entitled “There’s plenty of room at the bottom” at the California Institute of Technology (Caltech) in 1959 at the annual
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meeting of the American Physical Society [1, 2] proposed the existence of a new world for humans based on the dream of constructing new structures on the nanometer scale. Many
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materials and systems exhibit unique physicochemical properties in nanoscale [3]. Moreover, the quest for nanoscale structures with specific practical application has increased such that
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fabrication of nanostructures has now become a reality [4].
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The term “nanotechnology” was introduced by Norio Taniguchi on 1974 in field of materials applied to structures on the nanometer scale [5]. The Oxford English Dictionary
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defines nanotechnology as that “branch of technology that deals with dimensions and tolerances of 1 to 100 nanometers, or, generally, with the manipulation of individual atoms and molecules”. In other words, based on the National Nanotechnology Initiative nanotechnology is defined as technology for engineering and controlling of matter and understanding its behavior at dimensions between approximately 1 and 100 nanometers so that materials have unique properties and novel applications [6]. In recent years, nanotechnology has exerted significant effects in the medical sciences as well as other domains of science [7-9]. The considerable progress achieved by the use of nanotechnology in medicine has elevated it to one of the important medical research fields in recent years. Advanced delivery system (ADS) for diagnostic and therapeutic agents, tissue engineering, gene therapy, in vitro and in vivo diagnostic and imaging techniques are just a few examples 3
ACCEPTED MANUSCRIPT of the effective presence of nanotechnology in medical science [2, 8, 9]. Currently, the focus of much research is more towards ADS than on other nanotechnological topics and
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consequently many studies of nano-carriers have been published [7, 10, 11].
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Previous studies have shown there to be many kinds of nanocarriers, like liposomes and polyethyleneglycol (PEG) particles to name but two, which are used for delivery of
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diagnostic and therapeutic agents [2, 12]. The various nano-carriers used for delivery systems have molecular properties which are tailored to their individual uses. Thus, to achieve this
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result, either top-down or bottom-up methods based upon the physicochemical nature of the
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carrier are selected for their production. By top-down, we mean, using bigger machines for materials separation and manipulation in the solid or liquid phase, and by bottom-up we mean
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changing the environmental conditions whereby the atoms and molecules will produce the nano-carrier. At the same time, biomedical studies on delivery systems constantly try to
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improve the properties such as size, complementary features with the target site, drug loading capacity, biodegradability, biocompatibility, and continued controlled release of the drug and
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the noninvasive behavior of the nano-carrier. Hence, numerous efforts have led to the successful creation of stable colloidal nanoparticles. In addition, many studies have been devoted to the optimization of previous methods to enhance the applicability of nano-carriers to delivery systems. Moreover, many reviews have been published describing the unique delivery properties and focusing both on their specific production methods and a better characterization of the nano-carrier [13-15]. Different polymeric nano-carriers have been used as delivery systems. Nano-carriers based on polymers can be divided into two types of nanoparticle: nano-capsules and nanospheres [16]. The difference between them arises from their formation. The carried agent is located in the core environment of a nano-capsule where it is surrounded by a polymeric shell. In contrast, in a nano-sphere there is no extra polymer layer and the agent is not 4
ACCEPTED MANUSCRIPT concentrated at a specific site within the polymer [17]. Furthermore, because of their biocompatibility and biodegradability as well as their being ‘generally recognized as safe’
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(GRAS) properties [16-19], biopolymers rather than other polymers have been selected as
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nano-carriers. Among the biopolymers used are proteins which have been adapted for delivery systems [20] and the better known polysaccharides such as chitosan and pectin [21-
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24].
Proteins, because of their particular physicochemical and physiological properties, and
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their unique structures as well as their biocompatibility and biodegradability, combined with
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their low toxicity, are considered as the most suitable candidates for oral delivery systems [18, 25, 26]. Different proteins are used in delivery systems and they can be broadly
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classified on the basis of their origin, into animal proteins and plant proteins. Plant proteins include: gliadin, soy proteins, zein and lectin, all of which have been used in nanoparticle
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formation. Zein and gliadin are used as protein vehicles because of their high hydrophobicity and lower production cost compared to animal proteins [27].
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In addition to these plant proteins, the most common animal proteins to have been used are gelatin, collagen, albumin, casein, the whey proteins and elastin. Gelatin has a long and safe history in pharmacy and today is used as a nanoparticle stabilizer, because of its low antigenicity and various functional groups, in the delivery of different drugs such as antimalarial, antimicrobial, and anticancer [28] medications. Moreover, for clinical applications such as vaccines, gelatin sponge and some protein formulations gelatin is used as stabilizer. Gelatin has specific motifs such as Arg-Gly-Asp sequences in its chains so that cell recognition and then cell adhesion modulation have make by them; whereas, other polymers are lack of this property. Hence, it is well known as another class of colloidal plasma expander as well as hydroxyethyl starch (HES). On the other hand, it is confirmed by United States Food and Drug Administration (FDA) as GRAS material. Also, gelatin explored that 5
ACCEPTED MANUSCRIPT biodegradable and biocompatible polymeric nanoparticles. These properties lead to its applications for clinically application individual for presence in drug delivery [29-37].
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Collagen/gelatin-based systems are used also because of their biodegradability and their
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superior biocompatibility. The reticuloendothelial system (RES, also known as the mononuclear phagocyte system) can take them up relatively easily and so they can deliver
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exogenous compounds such as anti-HIV drugs, which is an additional advantage [38]. Albumins are intrinsic carriers and the use of albumin-based nanoparticles as drug
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delivery systems has been reviewed previously [39]. Albumin has several drug binding sites
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and may serve for targeted delivery of anticancer drugs to tumor cells, which make it a suitable drug carrier [40]. Casein, the major milk protein, is readily available and non-toxic.
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The gastric digestibility of β-casein was suggested as a possible delivery mechanism for stomach cancer [18]. Recent studies have shown the nanotechnological usefulness of milk
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proteins (both casein and the whey proteins) in various industries including the pharmaceutical and food sectors. [18, 19, 25, 26, 41-44].
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Recent reports have signaled that among the milk proteins, β-lactoglobulin (β-LG) is an excellent candidate for nanoparticle formation [43, 45, 46]. Therefore, this review will present and discuss the results of studies that have investigated ability of the milk whey globular protein, β-LG, to play a nano-carrier role in delivery systems illustrating its potential as an ADS by focusing on its potency (the effect per mg, cf. LD50) and carrier capacity. Many publications with different objectives have been selected which have revealed the various potential applications of β-LG nanoparticles. The main aim of this review, therefore, is to describe and summarize the potential use of β-LG as a well-targeted delivery system. 2. β-LG as a carrier protein 2.1. β-LG structure
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ACCEPTED MANUSCRIPT β-LG is the major whey protein in ruminant milk and is a member of the lipocalin superfamily. This globular protein exists in the milk of most mammalian species but is absent
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from that of human. It is made up of 162 amino acid residues with a molecular weight of 18.3
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kDa [47, 48]. The secondary structure of β-LG consists of nine β-strands and one α-helix (Fig. 1). Eight of the nine β-strands (strands A-H) fold up into a flattened β-barrel that is also
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called a calyx. This calyx is conical and includes two surfaces. One surface is formed by strands A-D and the other one is formed by strands E-H. The ninth strand (strand I) forms a
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significant part of dimer interface and has a key role in dimer interactions. β-LG is resistant
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to acidic conditions. At ambient temperature and at pH values below 3.5 and above 7.5, this protein is a monomer but at physiological pH, the dimeric form is dominant. The interior of the β-barrel is hydrophobic and acts as the main site for binding of hydrophobic ligands. The
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connection between the β-strands A-H of β-LG is formed by seven loops that are known as
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AB, BC, CD, DE, EF, FG and GH, respectively. The BC, DE, and FG loops are short and located at the closed end of the β-barrel. The AB, CD, EF and GH loops are longer and also
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more flexible being located at the open end of the β-barrel. The EF loop acts as a gate over the binding site. Five cysteines are found in the β-LG sequence at positions 66, 106, 119, 121 and 160, resulting in two disulfide bonds (66-160 and 106-119) with Cys 121 forming the free thiol group with pH dependent activity and roles in aggregation and denaturation of protein. Moreover, β-LG contains two tryptophans (19 and 61) of which it appears that Trp 19 is responsible for most of intrinsic fluorescence intensity of β-LG [49-59]. Additional to any putative transport role, an important nutritional role is played by β-LG since it has significant amounts of all nine amino acid residues (Leu, Ile, Val, Phe, Thr, Met, Trp, Lys and His) that are essential for human health and tissue preservation [60]. Also, cysteines, which are present in β-LG, are essential amino acids for stimulation of glutathione
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2.2. β-LG transporting role
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Previous studies show that β-LG can bind and transport various amphiphilic and hydrophobic ligands [56, 62-65]. β-LG interactions with tea polyphenols [66], phospholipids
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[67], curcumin and diacetyl curcumin [68], retinal [69], oxali-palladium [55], fatty acids [70], vitamin D and cholesterol [71], retinoid [72], spermine and spermidine [73], hemin [74],
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ellipticine [75], carcinogenic hydrocarbon [76], aromatic hydrocarbons [77], folic acid [78],
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Cr+3 [79], dodecyl sulfate and laurate [80], have all been investigated. The ability of β-LG to bind various ligands results from its structure. Many studies have confirmed that the main site for specific hydrophobic ligand binding to β-LG is located in the
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internal cavity, calyx, of the β-barrel. However, other potential binding sites involving
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residues Trp19, Tyr20, Tyr42, Gln44, Gln59, Gln68, Leu156, Glu157, Glu158 and His161 as a second site and Tyr102, Leu104, and Asp129 as a third site (Fig. 2) have been reported
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though so far lack convincing crystallographic data. [78, 81, 82]. Many authors have explored the carrier role of β-LG for different ligands and the following publications are examples of this. Liang et al investigated the interaction of resveratrol, a natural polyphenolic compound found in grapes and red wine that shows many health benefits, with β-LG and observed a 1:1 complex between resveratrol and β-LG and proposed a potential carrier role for resveratrol. The β-LG-resveratrol complex provides some advantages over the unbound molecule like a slight increase in the photostability and a significant increase in its solubility in water media [83]. Recently, an investigation by Ghalandari et al showed that oxali-platin used as a critical anticancer drug can be bound by β-LG in a molar ratio of 1:1. They revealed that oxali-platin interacts with internal cavity of β-LG and the driving force of this interaction is hydrophobic. 8
ACCEPTED MANUSCRIPT Based on these findings these authors proposed that β-LG could act as a carrier of oxali-platin [84]. Zhang et al studied the possibility of simultaneously binding of multiple bioactive
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compounds to β-LG. They illustrated this by showing that two or three ligands can bind to β-
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LG simultaneously. They suggested that β-LG-based carriers could be used as carriers for varieties of active compounds [85].
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Mohammadi et al studied β-LG interactions with curcumin, a phenolic compound which has many beneficial health properties such as anticancer, anti-inflammatory and antioxidant
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activities. Curcumin, because of its low bioavailability which results from its insolubility in
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water, is poorly assimilated. These authors revealed that β-LG may be a potential transporter of curcumin [68]. β-LG interactions with folic acid were studied by Liang and Subirade in order to verify not only its carrier role but also its protection against photo-degradation. Their
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results show that β-LG is a good candidate for the transport and delivery of folic acid. Also,
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the photo-degradation of folic acid decreased considerably after its interaction with β-LG [78]. Divsalar et al have investigated the interactions of series of Pd(II) and Pt(II) complexes
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with β-LG to devise a vector with low side effects for transporting metal anticancer complexes. Their results demonstrated that β-LG is a suitable protein for this purpose. According to their results, β-LG could be used as an efficient carrier of metal anticancer drugs without significant changes in its structure upon interaction with the metal complexes [80-91]. Transport of new anti-diabetic compounds to the small intestine was studied by Mehraban et al. Their results showed that β-LG may be able to play a carrier role for these new antidiabetic compounds [92-94]. Thus, according to what has been discussed above β-LG can employed as a transport vector for various compounds because of its unique secondary structural properties that allow it to bind different amphiphilic and hydrophobic ligands [66, 73]. Hence, all of these characteristics point to β-LG being a good candidate for use as an 9
ACCEPTED MANUSCRIPT ADS. Further, based on its unique properties and carrier functionality it is also a good candidate for nanoparticle formation thus producing a nanocarrier useful in various practical
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applications in particular delivery systems.
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3. β-LG nanoparticles as delivery systems
Over the two past decades many attempts have been made to construct β-LG-based
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nanoparticles for a variety of practical nano-carrier applications, many of them associated with pharmaceutical and nutraceutical delivery systems. Thanks to the stability of β-LG in
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acid conditions and relative instability in alkaline conditions, in most cases studies were
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devoted to oral delivery [46, 95]. β-LG is able to release its contents in a controlled way and is thus known as a biodegradable vehicle [16]. Hence, this biodegradability is used for nano vehicles in the alkaline conditions of the gastrointestinal tract (GIT) [95]. However, in order
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to maximize the efficiency of ligand protection together with controlled release by β-LG
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nanoparticles, an external layer or coating is necessary [96, 97]. 3.1. β-LG nanoparticles formation, characterization and application
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The external layer for coating β-LG nanoparticles must be resistant to degradation in the GIT conditions that justify selection of β-LG as an oral delivery system. Many studies have examined different molecules for outer layer formation in β-LG nanoparticles. According to these previous studies, among the many candidates that exist, low methoxyl pectin (LMP) has found particular favour for oral delivery systems [98-105]. LMP is bound to β-LG by physical interactions that are influenced by the pH of solution [42, 44]. β-LG nanoparticles can be formed by self-assembly or self-organization brought about by changes in the medium conditions of solution pH, temperature, ionic strength, presence or absence of salt ions and of course the relative concentrations of the β-LG and coating molecules. Hence, there are several protocols for the formation of β-LG nanoparticles such as time-dependent heat treatment of β-LG in solution or the effect of pH associated with ionic 10
ACCEPTED MANUSCRIPT strength changes accomplished by stirring at specific time intervals, or a combination of these and other factors. However, it is unclear which one of the different bottom-up protocols for β-
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LG nanoparticle synthesis is better or most suitable for a particular practical application [20,
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41-46, 95].
Jones et al investigated β-LG nanoparticle construction by time-dependent heat treatment.
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Their findings illustrated that in the presence of LMP as the external coating layer for β-LG, nanoparticles with diameters of 100-300 nm were formed at pH values between 4 and 7. In
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addition, their results revealed that the best sizes (100-250 nm) in terms of colloidal stability
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based on zeta-potential data in the presence of negatively charged LMP, were formed at the pI of β-LG (Fig. 3). Hence they concluded that the presence of the optimal concentration of LMP and the pH of the solution are important factors for β-LG nanoparticle formation. Their
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results also showed that the heat treatment protocol is another important factor in β-LG
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nanoparticle formation. They suggested that β-LG nanoparticles are useful as a delivery system as well as for practical applications in the food industry [41-43]. Giroux et al.
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investigated whey nanoparticle formation at specific time intervals by pH treatment at low temperature which leads to cross-linking denaturation. The whey nanoparticles obtained by this method had a size range of 100-300 nm depending upon the pH of the solution. Also, their results show that the presence of calcium is necessary for significantly enhances the stability of the whey protein nanoparticles [45]. A pH treatment performed by Ghalandari et al observed β-LG nanoparticles complexed with LMP formed in sizes less than 200 nm with good colloidal stability and net charge profile at the pI of β-LG. The results of their investigation agree with previous studies which revealed that both ionic strength and pH have an effect upon the size of the β-LG nanoparticles. All of these studies show that physical forces play a critical role in β-LG nanoparticle formation. Also, these data have confirmed that the β-LG nanoparticles content 11
ACCEPTED MANUSCRIPT in the presence of LMP as an extra layer and compression factor was preserved by nanoparticles compression. In other words, their findings emphasize the formation of smaller
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β-LG nanoparticles in the presence of LMP at the pI of the protein [46].
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Hence, the pH of solution, the presence or absence of stabilizers such as LMP as an external layer, or of ions such as calcium, combined with changes in the overall ionic strength
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of the solution, the time of synthesis, the time of thermal treatment, the degree of shaking during synthesis and the pH treatment of the β-LG before synthesis initiation are the most
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critical factors for the formation of β-LG nanoparticles. They are found to affect the size, the
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colloidal stability and the charge profile as determined by zeta potential measurements. Thus, β-LG nanoparticles are formed by an external stimulation that creates a balance of repulsive
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(e.g., electrostatic interactions between similarly charged groups) and attractive (e.g. van der Waals and hydrophobic forces, and electrostatic interactions between oppositely charged
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groups) physical forces during synthesis [106-109]. The size of a nano-carrier that can act as a nano-capsule must be smaller than 400 nm [95]. The results of the above studies show that
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β-LG has potential for nano-capsule formation which should allow it to function as a nanocarrier ADS in the GIT.
Shape is another important factor influencing the function and practical application of βLG nanoparticles as an ADS for the GIT, since it affects the tissue uptake, and hence the biodistribution/bioavailability, of the nanoparticles [110-112]. According to the results of morphological studies which have been done on β-LG nanoparticles, they appear to be spherical [95, 113-115]. Thus, the spherical shape of the β-LG nanoparticle allows it to move well in the GIT fluid and to reach the target position efficiently thereby increasing uptake at the target site and subsequent bio-distribution. Recently cytotoxicity and cellular uptake of β-LG nanoparticles was studied by Lee et al. They investigated the effects of two key factors namely concentration and incubation time on 12
ACCEPTED MANUSCRIPT the cellular uptake and cytotoxicity of β-LG nanoparticles on Caco-2 cells. Their results showed that by increasing the β-LG nanoparticles concentration from 100 to 250 μg/mL, its
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cellular uptake was significantly increased. While by increasing its concentration from 250 to
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500 μg/mL, the cellular uptake of nanoparticles weren’t significantly affected. Their results indicated that the concentration of 250 μg/mL of β-LG nanoparticle is a saturation limit of β-
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LG nanoparticle. Also their result showed that increasing the incubation time from 0.5 to 2 h had a significant increase in the cellular uptake of β-LG nanoparticles, whilst after 2 h, it
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showed no significant differences. However, their investigation on β-LG cytotoxicity in
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Caco-2 cells showed that increasing β-LG nanoparticles concentration from 100 to 500 μg/mL and also incubation time from 0.5 to 4 h did not significantly affect the cell survival ratio. It means that β-LG nanoparticles had no cytotoxicity to Caco-2 cells [116].
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3.2. β-LG nanoparticles and release profile
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The release profile of a ligand from a β-LG nanoparticle is perhaps the critical feature of any ADS for practical application in the GIT. Yi et al have investigated the time-dependent
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release of β-carotene in vitro from β-LG and β-LG-dextran nanoparticles in simulated GIT conditions at pH values of 2 and 7 in the presence of pepsin and trypsin, respectively. Their results illustrated that β-LG and β-LG-dextran nanoparticles in acidic conditions are stable so that only a small amount of β-carotene is released. At pH 7, in the presence of trypsin 60.9±2.9 and 51.8±4.3% of the total content of encapsulated β-carotene is released from β-LG and β-LG-dextran nanoparticles, respectively. Their findings reveal, therefore, that this β-LG nanoparticle is suitable as a delivery system for the alkaline conditions of the GIT [113]. Vitamin D3 delivery has also been investigated using β-LG-coagulum nanoparticles by Diarrassouba et al. The release profile of vitamin D3 from β-LG-coagulum nanoparticles was obtained after a specific time and at the pH values of simulated intestinal fluid. The β-LG13
ACCEPTED MANUSCRIPT coagulum nanoparticles were stable in the stomach but at basic pH, degradation released their contents into the intestine. Hence, the β-LG-coagulum nanoparticles also appear suitable as a
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delivery system that protects vitamin D3 from rapid digestion allowing transfer to the target
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site for intestinal absorption [117]. 4. Summary
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In this text, the capacity of β-LG, a member of a lipocalin superfamily, for ligand delivery in nanoscale delivery systems in order to be used in ADS has been presented. As has been
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discussed, β-LG has unique properties to bind various ligands with no significant effect on its
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structure, it can transport them through hostile environments and release them subsequently. These findings position β-LG as a good candidate for the delivery of bioactive compounds
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and other ligands. Indeed, the fact that it can be engineered to modify the ligand binding, as has been shown for another lipocalin [120-122], increases this potential considerably. In
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addition, because β-LG due to its structure is resistant to acids, but degrades in alkaline condition and contains a good balance of essential amino acids, is well suited to form a
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biodegradable and biocompatible advanced delivery system especially for the GIT. The many studies on β-LG nanoparticle formation cited here have shown that β-LG has enormous potential as a nanocarrier with good colloidal stability, net charge profile and spherical shape. Overall, significant new insights into various nano-scale ligand delivery systems have been afforded by these studies on β-LG nanoparticles. Of course, safe use of such nanoparticles as the basis of any ADS will require rigorous toxicity testing before general release. Acknowledgement The financial support of the Research Council of the Kharazmi University is highly appreciated.
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ACCEPTED MANUSCRIPT Legends Fig. 1. Molecular structure of β-LG and representation of its nine β-strands (A-I). Image was
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Fig. 2. The diagram of three possible binding site (A: internal cavity of β-barrel, B: second site is involving of residues Trp19, Tyr20, Tyr42, Gln44, Gln59, Gln68, Leu156, Glu157,
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Fig. 3. AFM image of β-LG nanoparticles complex with LMP by time-dependent heat
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ACCEPTED MANUSCRIPT β-lactoglobulin: an efficient nanocarrier for advanced delivery systems
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Zahra Shafaei1, Akbar Vaseghi2, Behafarid Ghalandari3, Adeleh Divsalar1*, Thomas Haertlé 4 , Ali Akbar Saboury5, 6& Lindsay Sawyer7
Graphical abstract
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