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Zein, Casein and Lactoferrin as Drug Delivery System
Plant-derived proteins produce renewable and environmental-friendly biodegradable polymers and films relative to synthetic materials (Parris, & Coffin, 5). Zein proteins are located in the endosperm of corn and are used as the major protein stores (Shukla & Cheryan, 10). The proteins serve as sources of nitrogen for embryo during germination. In 1821, Gorhan isolated storage protein from corn known as zein (Lawton, 5). In mid-20th century Zein was useful in pharmaceutical coating and encapsulation (Katayama, & Kanke, 14) of tablets based on its microbial resistance and film-forming properties. Tablets coated with zein had special characteristics over sugar-coated tablets because they were resistance to heat, humidity or abrasion. In addition, zein coating helped to mask the original tablets taste and odour. According to the studies on film-coating materials, zein film indicated minimum water vapour transmission due to the large numbers of nonpolar side chains thus has the highest potential to offer better protection against moisture when applied to dosage forms (Kanig, & Goodman, 7).
Besides zein films have important characteristics on packaging on pharmaceutical products because it has lower permeability to carbon dioxide and oxygen. Additionally, zein films provide better protection against oxygen permeability than other plant-derived films such as wheat gluten and soy protein (Gennadios A, WELLER C, Testin R, 7). Zein offers better encapsulation efficiency and controlled-release profiles of drug-loaded nanoparticles in coating materials (Luo, Zhang, Cheng, & Wang, 12).
Zein coating possesses extra beneficial features since it is biodegradable. Therefore, tablets composed of microspheres have been produced using zein (Chen, Hébrard, Beyssac, Denis & Subirade, 13). The biocompatibility of film of zein and drug has enabled the delivery of drug to vascular devices (Wang, et al, 7). Studies by Dong et al confirmed that zein has good biocompatibility characteristic for development of tissue engineering (Guo H, et, al, 12). Plasticizing zein with oleic acid and precipitating in cold water produces fatty acid-zein in industrial polymeric material (Wang & Padua, 3). Fatty acids such as oleic acid are incorporated in zein film in order to reduce water vapour permeability and retard water absorption. Cross-linking of zein also increases the mechanical strength, water resistance and toughness (Swallen & Zein, 5).
Researches indicate that zein have some potential in drug delivery properties as controlled-release is attained (Liu X, Sun Q, et al, 17). Various techniques such as use of barrier coating modify the drug release rate so that body fluid access to the drug is controlled. This controls both the drug diffusion rate and drug dissolution from the dosage form (Li, Yin, Yang, Tang, & Wei 10)
Zein protein has unique characteristics potentially making it good medium for delivery processes. First zein has the ability to form glossy, tough and hydrophobic coatings which have antimicrobial capabilities (Li, Yin, Yang, Tang, & Wei, 11). Secondly, is insoluble in water but it is soluble in aqueous alcohol solutions hence it is a potential vehicle for controlled drug release (Georget, Barker, Belton, 18). Besides, it has biocompatibility and biodegradable as well as adhesive properties (Dong, Sun, & Wang, 19).
Characteristics of zein
Zein proteins are prolamins meaning they are rich in glutamine and proline and/or aspargine (34). Additionally it has more than 50 % hydrophobic amino acids especially aliphatic amino acids which corresponds to leucines at 20 %, proline 10 % and alinine 10 %. (Anderson, & Lamsal, 15). The structure of zein reflects high aliphatic indexes with high surface hydrophobicity (Ansel HC, et al, 18). Zein is rich in glutamic acid but deficient with basic and acidic amino acids (Lai, Geil & Padua, 5). Zein amino acids consist of low amounts of polar and high amounts of nonpolar amino acids hence they are highly insoluble in water.
The negative nitrogen balance of zein is attributed to the absence of tryptophan and lysine residues in zein. However, zein structures comprise both hydrophilic and hydrophobic domains hence it behaves as a polymeric amphiphile (Shukla R, et al, 3). zein can be categorized based on of their solubility and molecular weight into α, β, γ and δ zein ADDIN EN.CITE <EndNote><Cite><Author>Esen</Author><Year>1986</Year><RecNum>254</RecNum><DisplayText>(34, 39)</DisplayText><record><rec-number>254</rec-number><foreign-keys><key app=”EN” db-id=”axa0x0per9drs8e9w5h5rrtpd9zdvxt90tv0″ timestamp=”1403479019″>254</key></foreign-keys><ref-type name=”Journal Article”>17</ref-type><contributors><authors><author>Esen, Asim</author></authors></contributors><titles><title>Separation of alcohol-soluble proteins (zeins) from maize into three fractions by differential solubility</title><secondary-title>Plant Physiology</secondary-title></titles><periodical><full-title>Plant physiology</full-title></periodical><pages>623-627</pages><volume>80</volume><number>3</number><dates><year>1986</year></dates><isbn>0032-0889</isbn><urls></urls></record></Cite><Cite><Author>Wilson</Author><Year>1991</Year><RecNum>255</RecNum><record><rec-number>255</rec-number><foreign-keys><key app=”EN” db-id=”axa0x0per9drs8e9w5h5rrtpd9zdvxt90tv0″ timestamp=”1403479083″>255</key></foreign-keys><ref-type name=”Journal Article”>17</ref-type><contributors><authors><author>Wilson, Curtis M</author></authors></contributors><titles><title>Multiple zeins from maize endosperms characterized by reversed-phase high performance liquid chromatography</title><secondary-title>Plant physiology</secondary-title></titles><periodical><full-title>Plant physiology</full-title></periodical><pages>777-786</pages><volume>95</volume><number>3</number><dates><year>1991</year></dates><isbn>0032-0889</isbn><urls></urls></record></Cite></EndNote>(34, 39). α-zein comprise of between 75-85 % of the total zein. On the other hand, β-zein accounts for 10-15% of the total zein while γ-zein accounts for approximately 20 % of the total zein (Ansel HC, et al, 19). However, the commercial zein mainly consists of α-zein as the other types contribute to gelling (Lawton, 7)
Solvents
Solvents of zein should contain both ionic and non-ionic polar groups and nonpolar groups since they are predominantly nonpolar amino acids (Shukla R, et al, 5). Zein is readily soluble in alcohol but is insoluble in water. It is also soluble in high concentrations of alkali (pH 11 and above), high concentrations of urea or in anionic detergents. It is soluble in alkaline because of the phenolic hydroxyl groups of tyrosine and the amino acids (Li X, et al, 5). Zein is soluble in binary solutions consisting lower aliphatic alcohols and water e.g. aqueous isopropanol (Lawton, 7). According to Shukla et al. the solubility of zein in an alcohol water mixture is unlimited once a suitable mixture is determined.
For aqueous solvents zein solubility in water can be enhanced by either alkali or acid treatment with NaOH of pH more than 12 or HCL of pH less than 1. These are useful in the conversion of aspargine and glutamine amino acids to the acid or salt form (Liu X, Sun Q, et al, 15). However, these treatments may cause large degradation of the protein as they consume large quantities of chemicals. In addition, zein solubility at lower pH can be enhanced by hydrolysing the amide group to carboxylic group (Cabra V, et al, 8). For instance, deamidated zein through the process of esterification or reaction with fatty alcohol forms fatty acylated zein or fatty esters (Ansel HC, et al, 22). A concentration of 70-90 % of aqueous alcohol is used to dissolve zein.
As the concentration of ethanol increases in the solution from 50-90 %, the viscosity of zein increases. Additionally, zein plasticizers require a proper polar and nonpolar balance similar to its solvents. Triethyglycerol (TEG) was identified as an effective plasticizer for zein (Anderson, & Lamsal, 13).
Zein in drug delivery
Zein has great potential for a wide range of modifications, biodegradability and excellent biocompatibility hence it is useful in drug delivery systems (Anderson, & Lamsal, 20). Modifications of zein such as crosslinking affect the elongation, tensile strength, solubility in solvents and Youngs modulus of zein fibres and film (Li X, et al, 8). Several compounds such as hexamethylene diisoyanate, glyoxal, polymeric dialdehyde starch, glutaraldehyde, epichlorohydrin, citric acid and butanetetracarboxylic acid can be used for crosslinking of zein (Ansel HC, et al, 21). In forming zein microspheres, zein is modified with other drugs using glutaraldehyde and trimethylsilyl chloride which is likely to result to additional crosslinking of zein. (Li X, et al, 9).
Modification of zein has also been performed by derivitization of surfaces, deamination phosphorylation of its amino acid and forming copolymers. Other options include the esterification of zein with fatty alcohols or fatty anhydrides acylation, which alters the base or acid sensitivity to proteins (Kanig, & Goodman, 16). Zein protein hydrophobicity can be altered by phosphorylating or deamination of its own amino acids. Chen and Subirade suggested that zein protein could be deaminated and modified with hexanoic anhydride, fatty acids, lauric anhydrides, or octanoic anhydride (Chen, Subirade, 67).
Degradation of zein occurs through the use of enzymes such as pepsin, thermolysin, trypsin, milenzyme, collagenase, papain, pronase, and chymotrypsin. However, zein is resistant to degradation in absence of enzyme (Hurtado-López, Murdan, 8). Hurtado-Lopez and Murdan conducted a study on degradation of zein and found out that enzymes degrade the nano- and microspheres of encapsulating drug in zein but the degradation products will not adversely affect the cells (Hurtado-López, Murdan, 10).
Furthermore, zein is a natural polymer that has biocompatibility features. It is capable to support cell proliferations as compared to poly lactic acids and Corning plates. Therefore, zein surfaces are very useful biomaterial for drug delivery applications.
Micro- and nanoparticles based zein
Various investigations have been carried out on the zein nano- and microspheres for drug delivery and nutraceutical properties. Coacervation or phase separation is the most common method for forming zein micro- and nanoparticles. Coacervation is the process of separating colloidal systems into two liquid phases (Georget, Barker, Belton, 20). Separations involves development of two phases, one with higher concentration of polymer (coacervate) while the other with lower concentration of polymer (Cabra V, et al, 8). This process begins with dissolving zein in a solvent such as aqueous ethanol. This leads to a decrease in the concentration of ethanol that result in partial desolvation of zein (Ansel HC, et al, 21). The encapsulation efficiency, loading, size and release profiles of encapsulated drugs are dependent on zein microsphere formation and characteristics of incorporated compounds in the traditional Coacervation technique.
Therefore, increasing the drug ratio of zein to encapsulated drug leads to increase in encapsulation efficiency (Anderson, & Lamsal, 15)). According to the studies, Zein microspheres produced by Coacervation indicates faster payload for the first 24 hour followed by slower release of the drug. Studies have indicated that spheres and films are examples of the utility of nano- and microspheres formed by Coacervation technique (Tatham et al, 8). Liu X, et al formed zein microsphere and microsphere film encapsulating ciprofloxacin for application on implanted devices (Liu X, Sun Q, et al, 17). The finding showed that the loading and the encapsulation efficiencies were relatively low due to the hydrophilic properties of the drug. Using appropriate amounts of catalysts using glutaraldehyde as the cross linker forms the mono-disperse microspheres less than one micrometre in diameter.
Microsphere size could be changed by varying parameters such as concentration of protein solution, temperature and rate of agitation. Jin and Zhong proposed different mechanisms for sphere formation (Xiao, Zhong, 18). First, the solution of zein is broken down into droplets by shear force. Secondly, solidification of zein follows during removal of solvents to the aqueous phase. Thirdly is droplet coalescence that has not yet solidified Xiao (Xiao, Zhong, 19). The method of sphere formation is an alternative to traditional Coacervation technique. Another technique that is used to form zein micro-nanosphere undertakes the removal of ethanol through the process of evaporation (Sessa DJ, et al 12). Lopez and Murdan conducted a study on the zein microsphere applicability in oral and inter-muscular administration (Gong, et al, 9). The study findings indicated that zein microspheres were better suited for intra-muscular administration as compared to oral administration (Hurtado-López, Murdan, 9).
Solvent evaporation/extraction method can be used to form zein microspheres (Liu X, Sun Q, et al, 17). It differs with the common coacervation technique because the micro droplets formation is not dependent on the solvent removal but rather on the emulsification process involving a non-solvent. Abamectin was encapsulated with zein microspheres using acetic acid. The zein microspheres protects the encapsulated chemical from photo-degradation (Sessa DJ, et al 12)
The combination of zein with other natural polymers, offers a useful method of altering the properties of the sphere (Wilson 7). When zein sphere/soy protein isolate (SPI) showed sustained release profiles of the encapsulated riboflavin that was dependent on zein: SPI ratio. (Guo, Heinämäki, & Yliruusi, 8). SPI/zein microsphere achieved near-zero order absorption profile in jejunum and ileum. Administering zein microsphere with yogurt delays the release of the drug hence the riboflavin reaches the intestines without absorption.
Reports indicate that Zein nanoparticles have the ability to accumulate in liver after intravenous injections in rats. Zein can be combined with other materials to yield spheres with altered properties because it changes the release kinetics (Sessa DJ, et al 18). For instance, zein nanoparticles are encapsulated into thymol using a liquid dispersion method. Tablets encapsulating ivermectin composed of zein microsphere were administered in dogs for demodicidosis treatment (Wilson 9). The full dose treatment of dogs with ivermectin in zein microsphere tablets resulted in 100 % curative ratio while half dose produced 93% curative ratio.
However, dogs that were given triple dose manifested no side effects. This indicated the drug had high degree of safety and low toxicity (Guo, Heinämäki, & Yliruusi, 18). The study demonstrates that efficacy of zein nano- and microspheres for oral administration in in vitro studies do not translate to in vivo studies.
Caseins
Caseins are heterogeneous group of phosphoprotein from the raw skimmed milk. It belong the class of intrinsically unstructured proteins (IUPs). It occurs in four major compounds namely, Alpha 1-, alpha 2-, gamma-casein, and beta- (Fox, 3). Caseins function as a carrier and store for bio-available metal ions especially magnesium and calcium ions. Besides, it provides the body with essential amino acids. Casein micelles consist of sub micelles that are bound by hydrophobic interactions and caesium-phosphate linkages. Caseins have unique characteristics that are suitable for the development of polymeric biomaterials.
These favourable features include good compatibility in oral delivery application and biodegradability characteristics (Swaisgood, 14). In addition caseins are nontoxic and have reactive sites for chemical modification. When caseins are in aqueous solutions, single molecules behave like disordered, flexible and polyelectrolyte-like molecules (Swaisgood, 9). Therefore caseins are easily incorporated into polyelectrolyte films (Szyk-Warszyńska, et al, 38). Caseins have the ability to bind calcium hence they can be used in biomedical and biotechnology applications to enhance bio-mineralization (Szyk-Warszyńska, Kilan, & Socha, 5) Caseins are also good candidate for the preparation and novel drug delivery systems because of the special physicochemical properties as natural polymeric surfactants.
Casein films exhibit tensile strength hence they are suitable in tablet coating (Semo, Kesselman, Danino, & Livney 7). Beta casein is the most common type of caseins in human milk while in cows milk it contains large concentrations of alpha caseins. In human beings it have an average concentration of 5g/L of milk (Szyk-Warszyńska, et al, 4). However, the beta caseins have limited proteolysis by the naturally occurring enzyme because its structure is particularly open and flexible in the region between the N-terminal polar and C-terminal hydrophobic domain (Swaisgood, 8). In addition, the hydrophobic core of beta casein micelles is applied for the delivery of hydrophobic compounds and drug molecules.
However, casein has allergic properties that limit its use as pharmaceutical excipients in formulations especially from cows milk. Casein allergy ignites an immune response to casein since the body has casein-specific IgE antibodies (Semo, Kesselman, Danino, & Livney 9). Casein has open structure hence it can be easily cross-linked with different cross linkers. Crosslinking is very crucial because it helps to stabilize and reduce the allergenicity of caseins.
Lactoferrin (Lf)
Lactoferrin is a cationic iron-binding glycoprotein in mammals. Lactoferrin belong to the family of transferrin (Tf). It has high affinity of iron that is found in milk. The chemical is produced in most of the mammalian external fluids including saliva and tears. Researches have indicated that Lf have multiple functions such as anti-viral, anti-fungal, anti-oxidants, regulation of cellular growth and antitumor (Adlerova, Bartoskova, & Faldyna, 9). These properties make the Lf an important chemical in many field including pharmaceuticals application and nutrition. Researches indicate that Lf mediates its multiple functions through specific receptors on various cells.
The Lactoferrin receptor (LfR) mediates the uptake of Lf into cells. In addition, the receptor is expressed on the surface of cells, such as apical surfaces of enterocytes, megakaryotes, platelets, endothelial cells of mesencephatic micro-vessels and dopaminergic neurons (González, et al, 2). Lf also acts as a ligand since it modifies the nano-carriers and crosses the blood-brain barrier through Lactoferrin receptor (LfR-mediated) transcytosis. Furthermore, Lf is relatively resistant to proteolysis in the gastrointestinal tract (Adlerova, Bartoskova, & Faldyna, 10). Lactoferrin is significantly useful in delivery systems of gambogic acid. Researches through in vitro findings show that, in NAB technology can economically produce Lf nanoparticles. In vivo study findings confirmed that the effective GL-NPs is a potential approach for oral delivery of gambogic acid. Transferrin (Tf) has similar characteristics with Lactoferrin (Lf) hence it has been investigated for oral delivery of granulocytes colony stimulating factor, human growth hormone and insulin.
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