Grafting Onto Biopolymers

Chapter

9

Grafting Onto Biopolymers: Application in Targeted Drug Delivery

Saundray R. Soni and Animesh Ghosh Birla Institute of Technology, Mesra, Ranchi, India

1.

INTRODUCTION

Biopolymers are basically macromolecules, which are biodegradable and noncytotoxic in nature. Broadly biopolymers can be classified into polymers of synthetically driven or from natural sources, either of microbial or plant origin. Table 9.1 presents a list of some of the commonly used biopolymers with their structure. In the current scenario of investigation in the field of drug delivery systems, biopolymers have attracted significant attention in the research community due to their various inherent advantages like biodegradability, noncytotoxicity, and biocompatibility, along with their rigid backbones, which is a requisite feature for grafting (Vashist et al., 2014; Thakur and Thakur, 2014b). Biopolymers, mainly of natural origin, are susceptible to microbial contamination, drop in viscosity, and uncontrolled hydration, while synthetic polymers have poor shear-resistant properties (Kaity and Ghosh, 2013). Grafting can be an important approach to overcome the limitations and can be customized as per the end user requirement. The three strategies usually followed for grafting are grafting through, grafting from, and grafting to strategies (Shi et al., 2013; Araki, 2013). Different monomers, such as acrylamide (Vijan et al., 2012), N-Isopropyl acrylamide (Das et al., 2015), methyl acrylate (Thakur et al., 2013a), butyl acrylate (Thakur et al., 2013b), ethyl acrylate (Thakur et al., 2014), hydroxyethyl methacrylate (Das et al., 2013), acrylic acid (Liu et al., 2016), lactic acid (Maharana et al., 2015), and many different monomers have been grafted and studied extensively for their temperature and pH stimulus responsiveness. Applications in targeting of cells based on variation of pH and temperature at different body sites have been found (Schmaljohann, 2006). Various different drug delivery systems such as interpenetrating polymer network (IPN) microspheres (Kaity and Ghosh, 2016), hydrogels (Thakur and Thakur, 2015), liposomes (Holig et al., 2004), nanoparticles (Danhier et al., 2009), Biopolymer Grafting: Applications. http://dx.doi.org/10.1016/B978-0-12-810462-0.00009-0 Copyright © 2018 Elsevier Inc. All rights reserved.

335

Name of the Polymer Locust bean gum

Guar gum

Molecular Structure of Polymer

336 CHAPTER 9 Grafting Onto Biopolymers: Application in Targeted Drug Delivery

Table 9.1 Name and Structure of Some Commonly Used Polymers for Drug Delivery Applications

Tamarind kernel powder

Alginate

Dextran

1. Introduction 337

Continued

Name of the Polymer Gellan

Xanthan

Molecular Structure of Polymer

338 CHAPTER 9 Grafting Onto Biopolymers: Application in Targeted Drug Delivery

Table 9.1 Name and Structure of Some Commonly Used Polymers for Drug Delivery Applications Continued

Pullulan

O

H OH

CH2OH

CH2OH

CH2 O

O

H

H

OH

OH

OH

OH

H O

O

O H

O

H

H

OH

OH

CH2

Chitosan

Poly (vinyl alcohol)

CH2CH OH

n

1. Introduction 339

Continued

Name of the Polymer Poly-ε-caprolactone

Poly lactic-co-glycolic acid

Molecular Structure of Polymer

340 CHAPTER 9 Grafting Onto Biopolymers: Application in Targeted Drug Delivery

Table 9.1 Name and Structure of Some Commonly Used Polymers for Drug Delivery Applications Continued

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and polymeric micelles (Zhan et al., 2010) have been prepared. The targeting concept enhances the therapeutic window of drugs by increasing drug delivery to the target tissue, and the approach is particularly attractive when the drug of interest is active at low concentration along with a narrow therapeutic window (Sudimack and Lee, 2000). This technique of drug delivery has gained a lot of importance as the concern for safety and dose reduction are an important factor for consideration during the development of dosage form, and it is currently trending in the area of research, when cytotoxic anticancerous medicinal agents have to be delivered to cancerous cells. The present chapter aims to give a detailed study report about different biopolymers and their source, isolation, purification, and possible application in the drug delivery arena, as well as different grafting techniques and the application of biopolymer grafting in a targeted drug delivery system with respect to its pH and temperature stimulus responsiveness and different receptor-based targeting, such as folate overexpressing receptors and integrin receptors for different oncologic products, to cancerous cells with some classical examples along with their mechanism, application, and advancement in the drug delivery area.

2.

BIOPOLYMERS

Biopolymers are macromolecules that consist of repeating units of small molecules, the monomers. The linking fashion of these monomers decides the type of polymeric chain, i.e., linear polymer, branched polymer, or cross-linked polymer (Dey et al., 2011). Biopolymers have gained a significant importance because of their various applications as dietary fibers (Fabek et al., 2014), gelling agents (Danalache et al., 2015), thickening agents (Xu et al., 2015), coating agents (Kumeria et al., 2015), stabilizing agents (Dickinson, 2016), texture modifiers (Mao et al., 2000), films forming agents (Prajapati et al., 2013a), emulsifiers (Pinheiro et al., 2016), tissue engineering (Okamoto and John, 2013), drug delivery (Vijan et al., 2012), and as nonviral gene carrier (Rudzinski et al., 2016).

2.1 Classification of Biopolymers Broadly biopolymers can be classified as natural and synthetic polymers (Vashist et al., 2014). Natural biopolymers are further categorized on the basis of sources such as polysaccharides obtained from plant sources (galactomannan, e.g., locust bean gum, or nongalactomannan, e.g., tamarind kernel gum), such as seaweed extract (alginate) and bacterial fermentation (dextran, gellan, xanthan, pullulan), or animal sources (chitosan). Different synthetically derived biopolymers are poly (vinylalcohol), poly (ε-caprolactone), and poly (lactic-co-glycolic acid).

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2.1.1 Plant-Based Biopolymers 2.1.1.1 Galactomannan Galactomannans are present in the endosperm of various plant seeds, mainly leguminous plants, and serve as an energy reservoir other than carbohydrates. It is extracted from the endosperm of leguminous seed by grinding to fine particles, separated from the seed husk, and then extracted and purified using a suitable solvent like ethanol (Cerqueira et al., 2009). Galactomannans are a linear polysaccharide composed of partially substituted D-galactopyranosyl (a-1 / 6) linked as the side groups on the linear backbone of (b-1 / 4) linked D-mannopyranosyl (da Silva and Gonçalves, 1990). Biopolymers that are hydrophilic in nature easily solubilize in water, resulting in the formation of a stable and viscous solution. The mannose/galactose (M/G) ratio, along with distribution of galactose residues on the mannon backbone and their molecular weight, decides the physicochemical and rheological properties of the biopolymers (Mirhosseini and Amid, 2012). The M/G ratio varies with the variation of galactomannan source (M/G ratio 2:1 to 5:1). The higher M/G ratio yields a more viscous and thicker solution (Hallagan et al., 1997). Some of the important galactomannans are tara gum (origin: Caesalpina spinosa), locust bean gum (origin: Ceratonia siliqua L.), cassia gum (origin: Cassia tora) and guar gum (origin: Cyampsis tetragonolobus), which find various application in different industries like food, agriculture, textile, paper, cosmetics, and pharmaceutical industries (Thombare et al., 2016; Siqueira et al., 2015). From the galactomannan series of biopolymers, the most explored biopolymer by different researchers with their proved potential to be differently developed as a dosage form include guar gum and locust bean gum (Kaity et al., 2013a; Kaity and Ghosh, 2013; Kajjari et al., 2012).

2.1.1.1.1 Locust Bean Gum. It is a heteropolysaccharide obtained from the endosperm of carob tree fruits, which are generally grown in the Mediterranean region. The extraction process starts with the grinding of seeds to fines, followed by separating the endosperm from the seed husk, then mashing and extracting in a solvent like ethanol (Barak and Mudgil, 2014). The polysaccharide is composed of the repeating units of D-galactose as branches attached at six positions to the backbone of (1 / 4)-linked b-D-mannopyranose (Kaity et al., 2013a). The M:G ratio for locust bean gum (LBG) is 4:1 and affects various properties like the rheological properties, viscosity, interaction with different polymers, and solubility. Higher galactose content contributes to higher water solubility, while the ordered mannose chain shows the hydrophobic effect. The concept of increased entropy and steric hindrance supports the theory for reduced water solubility due to ordered mannose chain (Prajapati et al., 2013b). Because of a higher content of

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mannose, LBG shows limited solubility at room temperature, so heating is required for maximum solubilization.

2.1.1.1.2 Guar Gum. Guar gum (GG) is a complex polysaccharide, composed of D-galactose and D-mannose. This particular galactomannan is obtained from the endosperm seed of the (C. tetragonolobus), a leguminous crop. It is extracted from the seeds by grinding to fines then separating the seed husk and germ from the endosperm. This endosperm is called split guar, which is further purified. The endosperm contains about 75%e86% water soluble galactomannan (Thombare et al., 2016). GG is composed of (1 / 4)-b-D mannopyranose and (1 / 6)-a-D-galactopyranose units attached via (1 / 6) glycosidic linkage to the alternating main mannose chain at an M:G ratio of 2:1 (Vidal and Pawlik, 2015). Guar gum is easily soluble in cold water, and its low content of mannose and high molar mass (1000e2000 kg/mol) imparts to thickening properties and the formation of a super structure in aqueous solution (Szopinski et al., 2016). It is unique in terms of its viscofying nature, as the viscosity was found to be highest at the equivalent concentrations compared to various different carbohydrate polymers, which may be 10,000 cps even for 1% solution (Parija et al., 2001).

2.1.1.2 Nongalactomannans Among the class of nongalactomannans, tamarind kernel powder (TKP) has been most investigated for modifications and its applications in drug delivery. It is obtained from the seed kernels of tamarind (Tamarindus indica, L.). The mucilaginous biopolymer belongs to family fabaceaea and is commonly grown in Southeast Asia and often referred as tamarind kernel powder (Kaur et al., 2012). Tamarind kernel powder is a xyloglucan with a high degree of substitution and property resemblance with starch (amyloid), so it is also referred to as a starch-like polysaccharide (Mishra and Malhotra, 2012). It is a neutral polysaccharide, and the structural composition consists of b-D-galactopyranosyl linked and D-xylopyranose linked via a-(1 / 2) as the substituted side chain is highly substituted on the backbone of (1 / 4)b-D-glucan via (1 / 6) glycosidic bond. The ratios of galactose, xylose, and glucose are 1.0:2.25:2.8, respectively (Goyal et al., 2007). TKP swells in water, and upon heating, it forms a mucilaginous solution. The property of forming gel in aqueous solution is usually utilized by the food industry as a thickening and stabilizing agent. TKP has also been studied for its applications in a novel drug delivery system, as it possesses the suitable properties of noncarcinogenicity, biocompatibility, high thermal stability, and high drug holding capacity to be developed as a pharmaceutical dosage form (Mirhosseini and Amid, 2012).

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2.1.2 Seaweed-Derived Biopolymers 2.1.2.1 Alginate Alginates are anionic polysaccharides, which are mainly present in marine brown algae (Phaeophyceae) as structural components and as capsular polysaccharides in some bacteria (Draget and Taylor, 2011). The commercially important algae species from which alginates are produced on a large scale include species like Macrocystis pyrifera, Laminaria hyperborean and Ascophyllum nodosum. The large-scale production utilizes the alkaline extraction process along with various types of chemical treatment for the removal of different kinds of impurities (like heavy metals, proteins, polyphenols, endotoxins, and other carbohydrates). Azotobacter and Pseudomonas species also produce alginate by a fermentation mechanism, but these are not considered as economically viable for commercial purposes (Goh et al., 2012). Alginates are the copolymers of (1 / 4)-b-Dmannuronic acid (M) and a-L-guluronoic acid (G) residues linked together in a linear fashion (Draget and Taylor, 2011). Both the monomers, i.e., Gresidue and M-residue, can be arranged in three different homopolymeric fashions as consecutive G-residue, M-residue, and alternating M- and Gresidues, and their proportion imparts a great role in selective binding with cations. The selective binding of alginates with multivalent cations increases with an increase in proportion of a-L-guluronic acid residues (G-block) in the polymer structure, while poly-mannuronates (M-block) and alternating guluronates and mannuronates (MG-blocks) are highly insensitive toward selectivity. Alginates extracted from different algal species with varying extraction time affect the sequence of monomer block arrangement and thus vary in chemical composition. The mechanical properties of alginate gel depend upon the content of guluronic acid, and its higher content improves the gel strength. The L. hyperborean algal species can provide the alginate of improved mechanical strength, when the extraction and purification is performed from its outer cortex of old stripes. The more homogenous alginates can be produced by bacterial sources (like Pseudomonas aeruginosa), which can contain up to 100% mannuronate compared to the algal sources. The bacterial species Azotobacter vinelandii encodes a family of 7 exocellular isoenzymes, which have the epimerization potential for alginates resulting in alginate formation of very long repeating units of G-blocks to polyalternating (MG blocks) via short Gblock and M-block monomers. Thus alginates with desired properties can be achieved by a postpolymerization enzymatic modification process using C-5 epimerase from A. vinelandii, which sequentially modifies the polymeric chain by converting M to G within the polymeric chain (Draget and Taylor, 2011; Mokhtarzadeh et al., 2016).

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The thickening, gelling, stabilizing, and suspending properties of alginates have been widely investigated and are being used by food industries. Alginates are also used as film formers alone or in combination with carrageenans or pullulan (Tavassoli-Kafrani et al., 2016). The selective binding nature of alginates with multivalent cations (like Ca2þ) has been utilized in drug delivery applications for the formulation of microspheres (Das and Senapati, 2008), hydrogels (Jabeen et al., 2015), etc. Alginates are nontoxic, biodegradable, water soluble, biocompatible, and low immunogenic in nature, which has increased the attention for its applications in the development of nanoparticles with targeted applications, particularly tumor targeting (Wang et al., 2015).

2.1.3 Biopolymer Derived From Microbial Fermentation 2.1.3.1 Dextran It is a natural biopolymer, biosynthesized from sucrose using different lactic acid bacteria like Luconostoc mesenteroides, Lactobacillus brevis, and Streptococcus mutans using enzymes like glucansucrases. It is composed of repeating units of D-glucose residues interconnected with a-(1 / 6) linkage along with side branches linked to the parent chain via a-(1 / 2), a-(1 / 3) or a-(1 / 4). These side chains make dextran a highly branched high molecular weight homopolymer, and the degree of branching depends upon the bacterial strain used for synthesis. Dextran has been approved as a food ingredient by the US FDA, and it is also being used for reducing vascular thrombosis, blood viscosity, inflammatory response, and increased peripheral blood flow (Anirudhan and Binusreejayan, 2016). Dextran is also used as ratecontrolling (drug release rate) excipients in the formulation of matrix tablets (Casettari et al., 2015). Due to its biodegradability, biocompatibility, and nonimmunogenicity, dextran finds application in different types of therapeutic areas like tissue engineering and controlled release formulations (Anirudhan and Binusreejayan, 2016). The possible formulations are due to the availability of a large number of synthetically useful aldehydic groups, which are available for modification with other functional groups to form pH-sensitive hydrogels, microgels, or scaffolds (McCann et al., 2015).

2.1.3.2 Gellan Gum Gellan gum is an anionic, linear, and high molecular weight polymer. It is bacterial exopolysaccharide, produced by nonpathogenic, aerobic, gramnegative bacterial strains like Sphingomonas paucimobilis (ATCC 31461), S. paucimobilis E2 (DSM 6314), S. paucimobilis NK 2000, and Sphingom S. paucimobilis GS1, and the used strain decides the product yield. The strain S. paucimobilis GS1 also formerly known as Pseudomonas eloda was first isolated by the Kelco company, United States, and

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commercially produced gellan gum (Osmałek et al., 2014). The native biopolymer consists of linear chain tetrasaccharide repeating units consisting of molecules of b-(1 / 3) linked D-glucose, b-(1 / 4) linked D-glucuronic acid, b-(1 / 4) linked D-glucose, and a-(1 / 4) linked Lrhamnose. The two acyl substituents (O-acetyl and L-glyceryl) are linked to the glucose residue as a side chain on these repeating units (Dolan et al., 2016). Based upon the amount of acyl group, gellan gum has three types: native, deacetylated, and clarified gellan gum. The native gellan gum contains the acyl group, which can be deacetylated by hot alkaline hydrolysis to get a linear simple deacetylated polymeric chain of gellan gum. The deacetylated gellan gum is heated in the fermentation broth up to around 95 C, which kills the bacterial cells and reduces the broth viscosity, filtered by 0.2 m filters precipitated and ground to fines. This form of gellan gum is the clarified gellan gum (Prajapati et al., 2013). Gellan gum was approved by the US FDA in 1992 as a natural food additive due to its various advantages over other microbial exopolysaccharides like high strength, high transparency, good taste, thermal stability, etc. It is widely used in the food industry as a thickening, stabilizing, texturing, emulsifying, binding, and gelling agent (Zhang et al., 2015). In the medical and pharmaceutical fields, gellan gum and its modified derivatives are used in the formulation of matrix tablets, cross-linked hydrogels, floating beads, pellets, microspheres, transdermal films, etc. The noncytotoxic, biodegradable, and cytocompatible nature makes it a suitable carrier for the applications in tissue engineering areas (Osmałek et al., 2014). The negative charge of gellan gum forms decationized polymeric polyplexes, which reduce toxicity of cationic polymers like polyethylenimine and make it suitable for application as a gene carrier (Goyal et al., 2011).

2.1.3.3 Xanthan Gum This heteropolysaccharide biopolymer is complex and anionic in nature and is produced by microbial fermentation of different types of carbohydrates like glucose, sucrose, maltose, etc., by a gram-negative bacterium Xanthomonas campestris pv. campestris. The yield depends upon the carbon source, i.e., sugar moiety undergoing fermentation. The molecular weight is approximately 2 million but can be as high as up to 13e50 million, so it is considered to be a high molecular weight microbial exopolysaccharide biopolymer (Rosalam and England, 2006). Xanthan gum is an acidic polymer that consists of five repeating units of monosaccharide, collectively called pentasaccharide repeating units, comprising two units of D-glucosyl and D-mannosyl and one unit of D-glucuronyl acid residue with a variable proportion of O-acetyl and pyruvyl residues, imparting the anionic character.

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From the five sugar units, two units of b-(1 / 4)-D-glucose make the main chain and b-D-mannose, b-D-glucuronic acid, and b-D-mannose are attached to it as a terminal side chain with b-D-(1 / 2) and D-(1 / 4) linkages (Rosalam and England, 2006; Palaniraj and Jayaraman, 2011). The safety and toxicity profile of xanthan gum for food and pharmaceutical application has been extensively studied. It is a biodegradable, biocompatible, and nontoxic biopolymer approved by the USFDA in 1969 and European commission in 1986 under the number E415 as a safe food additive for oral consumption without any limitations (Palaniraj and Jayaraman, 2011; Hublik, 2012). Xanthan gum has vast applications in the textile, paint, petroleum, food, cosmetic and pharmaceutical industries. The application is due to its superior properties like high viscosity even at very low concentrations (as low as 600e2000 ppm, high stability at increased temperatures (stable up to 90 C), resistance to degradation, low sensitivity toward viscosity changes due to salinity change, and finally the non-Newtonian flow properties and environmental friendly product (Rosalam and England, 2006). In the pharmaceutical field, it is used for both solid and liquid formulations. In solid dosage forms, it is used to retard the release rate by forming a polymeric matrix in controlled delivery systems, prolongs the contact time when used in lozenges, and acts as film former in coating process. In liquid formulations, due to its high viscosity properties, it is used as a suspending, thickening, and stabilizing agent in suspension (Hublik, 2012). Xanthan gum is being suitably modified or grafted on its backbone with some other polymers to alter its characteristics and to be used in tissue engineering and targeting applications.

2.1.3.4 Pullulan Pullulan is a nonionic, water soluble, and microbial exopolysaccharide produced by fermenting liquefied starch by a nonpathogenic and nontoxigenic strain of yeast, like Aureobasidium pullulans. In 1958, Bernier reported pullulan for the first time, and in 1959, Bender et al. named it as pullulan and elaborated the structure as a linear maltotriose unit, which is connected by a-(1 / 4) glycosidic linkage and the consecutive maltotriose are interconnected by a-(1 / 6) glycosidic linkage. It has a property of random coiling with a high flexibility, and the reported molecular weight is 5000 to 90,000,000 g/mol. The Japanese Company Hayashibara started its commercial production in 1976 and commercialized its films in 1982. Pullulan has been used in Japan as a food ingredient and excipient as pharmaceutical bulking agent in tablet formulations. It is listed in the Japanese standards for ingredients for drugs. Pullulan has gained the status of generally recognized as safe by US regulations for a wide range of applications. The safety

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and toxicity profile for pullulan has been studied on human volunteers, and only a minor abdominal fullness and some mild gastrointestinal fullness have been reported at a dose of 10 g/day (Prajapati et al., 2013a). Pullulan is being used by food and pharmaceutical companies as an emulsifier, stabilizer, film former, lubricant, coagulant, and gelling, thickening, and suspending agent. The nontoxic, biodegradable, nonimmunogenic, and biocompatible nature of pullulan makes it a suitable candidate for use in the biomedical field such as tissue engineering, organ targeting drug delivery, gene carrier, medical imaging, plasma expander, etc. (Aydogdu et al., 2016; Prajapati et al., 2013a; Mishra et al., 2011). The utility of pullulan can be increased by grafting different chemical groups on its hydroxyl group, as these groups are abundantly present and can be easily substituted with other chemicals to explore their biomedical applications (Prajapati et al., 2013a).

2.1.4 Biopolymer Derived From Animal Sources 2.1.4.1 Chitosan Chitosan is a natural polysaccharide, the most widely utilized biomaterial after cellulose. It is produced by alkaline N-deacetylation of chitin, which is the structural element in the exoskeleton of crustaceans such as crabs, lobsters, and shrimps. The chitosan production process involves two steps: the first step involves chitin [N-acetyl-D glucosamine (2-acetamido-2deoxy-b-D-gluconopyranose) joined together by b (1 / 4) linkage] extraction followed by removal of calcium carbonate (CaCO3) and deproteination from chitin using dilute HCl and dilute aqueous NaOH, respectively, which is finally ground into fine powders. The second step involves deacetylation of chitin using 40%e50% aqueous NaOH at 110e115 C for several hours in the absence of oxygen. More than 50% deacetylation of chitin produces chitosan, and chitosan is recognized above 75% deacetylation value of chitin. Thus chitosan is a linear polymer consisting of (1 / 4)-2acetamido-2-deoxy-b-D-gluconopyranose and (1 / 4)-2-amino-2-deoxyb-D-glucosamine monomer units linked together by b-(1 / 4) linkage (Elgadir et al., 2015; LogithKumar et al., 2016). The quality and properties like viscosity, solubility, coagulation, reactivity of proteinaceous material, tensile strength, elasticity, and heavy metal ion chelation of the produced chitosan depends significantly upon the choice of chitin (a, b or g) along with isolation factors and deacetylation value. At a lower pH value around 6.0, chitosan readily solubilizes in diluted acidic solutions because of amine group quaternization, which has a pKa value of 6.3, rendering chitosan as a water soluble cationic polyelectrolyte, while the parent chitin remains insoluble in almost all organic solvents. The

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solubility of chitosan is due to the protonation of amine groups at a low pH, while at higher pH (above 6.0) the chitosan amines remain deprotonated, resulting in loss of charge, and become insoluble. The solubleeinsoluble transition occurs at its pKa value between pH 6 and 6.5. The degree of deacetylation decides the pKa value of chitosan, so solubility is also dependent on degree and method of deacetylation (Dash et al., 2011). The various drawbacks associated with chitosan like low transfection efficiency and low aqueous solubility can be overcome through its chemical modification like graft copolymerization (Thakur and Thakur, 2014a). Chitosan is biodegradable in nature and is a safe excipient for various kinds of drug delivery applications. The mucoadhesive nature of chitosan attracts its utility in the formulation of mucoadhesive microspheres and hydrogels for gastroretentive drug delivery systems. The various other areas of drug delivery where chitosan is widely used are for the delivery of anticancer drugs, proteins/peptides, growth factors, antibiotics, gene therapy, bio imaging applications, etc. (Dash et al., 2011).

2.1.5 Synthetically Derived Biopolymers 2.1.5.1 Polyvinyl Alcohol Polyvinyl alcohol (PVA) is a synthetically driven polymer that is soluble in hot water, slightly soluble in ethanol, and insoluble in organic solvents. For the time, PVA was synthesized in 1924 by German scientists Herrmann and Haehnel. It was synthesized by free radical polymerization technique using vinyl acetate as the monomer and potassium per sulfate as the initiator under reflux conditions at 80 C for 40 min. Polyvinyl acetate was formed as the intermediate, which when subjected to alkaline hydrolysis in methanol replaces acetate group with hydroxyl group. PVA is then precipitated, washed, and dried. The hydrolysis step decides the physical characteristics of PVA, and based upon hydrolysis, it is classified in two groups, partially hydrolyzed and fully hydrolyzed PVA. The partially hydrolyzed PVA (84.2%e89%) contains residual acetate groups, which reduce crystalline properties, lower the melting point and enhance the aqueous solubility, and increase flexibility and adherence to hydrophobic surfaces. The fully hydrolyzed PVA grade (91%e99%) has low aqueous solubility with high degree of crystallinity, stability, tensile strength, and adhesion to hydrophilic surface (Marin et al., 2014). The US FDA has approved PVA as a safe material and allowed it to be used as an indirect food additive, diluent for color additive mixtures under 21 CFR73.1, and as ophthalmic demulcent (0.1%e4%) under 21 CFR 349.12. PVA is absorbed little through the gastrointestinal route, which is supported by a study on male and female Fischer 344 rats at a dose of

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0.01 mg/kg 14C-lebelled PVA. Less than 0.2% of the total dose was detected in urine and only 0.05% of total dose was detected in the major tissues (liver, kidney, blood, and adipose tissues). The acute oral toxicity (LD50) was approximately 20,000 mg/kg in rats and dogs (DeMerlis and Schoneker, 2003). PVA has vast applications in industries due to its versatile physicochemical properties of viscosity enhancing, film forming, emulsifying, and dispersing powder, as well as good flexibility and good tensile strength (Halima, 2016). PVA is a biodegradable and nontoxic biopolymer and due to its excellent biocompatibility and low protein adsorption properties, it is being developed for biomedical and pharmaceutical applications, like contact lenses, synthetic vitreous humor, artificial pancreases, and implantable materials to replace cartilage tissues (Baker et al., 2012). The most important pharmaceutical application includes PVA hydrogels prepared by crosslinking with different biopolymers by chemicals (glutaraldehyde, formaldehyde), physical (freeze-thaw), or radiations (UV light or gamma radiation) (Halima, 2016). The careful cross-linking has been able to develop different types of delivery systems for cancer therapy (Kayal and Ramanujan 2010), encapsulation of enzymes like amylase for digestive problems (Singh and Singh, 2013), scaffolds, tissue engineering (Kanimozhi et al., 2016), and controlled and targeted systems (Kaity et al., 2013a) in the field of drug delivery.

2.1.5.2 Poly-ε-caprolactone Poly (ε-caprolactone) (PCL) is a semicrystalline, aliphatic polyester, which is composed of hexanoate repeating units. ε-caprolactone undergoes a ring opening polymerization reaction in the presence of stannous octoate (catalyst) to yield PCL. The molecular weight of PCL is controlled using low molecular weight alcohols in the polymerization reaction (Labet and Thielemans, 2009). The glass transition and melting temperature of PCL is approximately 60 C and 60 C. PCL is soluble in organic solvents like chloroform, benzene, toluene, cyclohexanone, and dichloromethane at room temperature, while it has low solubility in acetonitrile, acetone, dimethyl formamide, butanone, and ethyl acetate and is insoluble in alcohol, diethyl ether, petroleum ether, and water (Pohlmann et al., 2013). PCL is one of the most widely used synthetic biopolymers in the biomedical and pharmaceutical field. The main reason for its versatile application is the modifications of its physical, chemical, or mechanical properties by copolymerization or blending with different suitable polymers. The copolymerization alters its chemical properties like solubility and degradation patterns with different desirable delivery routes, while blending alters physical

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and mechanical properties, which makes PCL a desirable polymer to be used in the biomedical and pharmaceutical field (Azimi et al., 2014). PCL has been studied with various polymers like polyvinyl alcohol (Wan et al., 2015), polyethylene glycol (Azouz et al., 2016), hydroxyapatite (Kim and Koh, 2013), and polylactic-co-glycolic acid (PLGA) (Li et al., 2005), and its excellent compatibility makes it tailored along with these polymers for the applications in current drug delivery approaches like microspheres, nanoparticles, tissue engineering, and scaffolds, films, and fibers. Although PCL is a biodegradable polymer, the fabricated PCL can resist the degradation for more than months, thus providing sufficient time for bone, cartilage, and vascular regeneration. Some PCL-based formulations are in different phases of preclinical and clinical studies, while a number of fabrications have been commercialized for tissue culture and tissue engineering like Osteoplug, Teoplug (Osteopore), Relison, 3-D Biotek 3-D Insert (Sigma), and Artelon sportmesh. PCL native or copolymer tailored driven property makes a suitable candidate for all kinds of tissue engineering and novel drug delivery systems (Dash and Konkimalla, 2012).

2.1.5.3 Polylactic-co-glycolic Acid It is one of the most successfully and synthetically derived biopolymers for various biomedical and pharmaceutical drug delivery applications. PLGA can be synthesized using lactide and glycolide as monomers and a suitable catalyst such as stannous chloride using the melt copolymerization technique at a temperature of around 160 Ce190 C under vacuum. Finally, PLGA is purified by dissolution in chloroform, followed by precipitating in ethanol and finally dried under vacuum at 40 C until constant weight is achieved. The physical and chemical properties of PLGA vary with the variation of its grade, i.e., the monomer ratio lactic acid:glycolic acid. The resorption period for PLA (100% LA) is approximately around 12 to 24 months, while for PGA (100% GA) the time period is reduced to 6 to 12 months only. Basically the variation is due to slower degradation rate and poor hydrophilicity of PLA over PGA. A 50:50 ratio of LA and GA has a high degradation rate, while increasing the PLA ratio decreases and PGA increases the degradation rate (Kapoor et al., 2015). So, the suitable grade is a quite necessary requisite for its utilization for different formulations. PLGA is soluble in different organic solvents like dichloro methane, chloroform, tetrahydrofuran, acetone, ethyl acetate, and benzyl alcohol. As PLGA is a biodegradable polymer, its degradation is due to the hydrolytic cleavage of ester bond resulting in the formation of monomers lactic acid and glycolic acid as metabolites. These metabolites further enter into

352 CHAPTER 9 Grafting Onto Biopolymers: Application in Targeted Drug Delivery

Krebs cycle and are metabolized endogenously, producing minimal toxicity. Based on this fact, PLGA is considered to be a nontoxic, biodegradable, and biocompatible biopolymer, which has also been approved by the US FDA and European Medicine Agencies for different kinds of drug delivery systems (Danhier et al., 2012a). PLGA has attracted the attention of researchers due to its desirable requisite properties for the application in novel drug delivery systems like biodegradation and biocompatibility, approval of regulatory authorities, and the possibility of formulation as a sustained release or targeted release with better interaction with biological membranes at the targeted organ or site with enhanced recirculation time by protecting from reticuloendothelial system (Danhier et al., 2012a), e.g., various formulations of anticancerous agents have been developed in nanoparticles (Sharma et al., 2016) and microsphere (Li et al., 2005) forms with PEG and PCL respectively. The molecular structures of the aforementioned biopolymers have been presented in Table 9.1.

3.

BIOPOLYMERS GRAFTING

Biopolymers have several advantages, as discussed in the earlier sections, but all those advantages are not sufficient in the current demanding scenario of drug delivery applications. So modification becomes an essential requirement to alter the physical and chemical properties of these biopolymers in order to meet the criteria of current biomedical and pharmaceutical research scenarios. Out of different modifications, grafting of suitable synthetic polymers on the backbone of biopolymers is the highly trending technique and the simplest method to achieve the suitable property as per user requirement (Thakur et al., 2015).

3.1 Grafting Strategy Graft copolymers are the polymers with modified physical and chemical properties, generally comprising two polymeric components. The second component is randomly distributed branches, which are attached to the first component serving as the backbone. Basically, these graft copolymers are prepared by following three main strategies (Fig. 9.1).

3.1.1 Grafting Through The self-assembled monolayer containing polymerizable groups adhered to a polymeric surface is the basis of the grafting through technique. The growth of the polymeric chain is initiated in the solution with the help of a suitable initiator. During the propagation stage, these surface-attached

3. Biopolymers Grafting 353

n FIGURE 9.1 Schematic depiction of different techniques of chemical grafting of polymers to solid

surfaces: (A) “grafting to”, (B) “grafting from”, and (C) “grafting through”, consisting of the attachment step and further chain growth. Reprinted from Henze, M., Madge, D., Prucker, O., Ruhe, J., 2014. “Grafting Through”: mechanistic aspects of radical polymerization reactions with surface-attached monomers. Macromolecules 47 (9), 2929e2937. Copyright 2014, with permission from American Chemical Society.

monomer units integrate into the backbone of a growing chain, resulting in permanent attachment of the monomer to parent chain and thus chain growth proceeds (Henze et al., 2014). Using this grafting technique, macromonomers like polylactic acid (Shinoda and Matyjaszewski, 2001), polycaprolactone (Hawker et al., 1997), and polyethylene (Hong et al., 2002) have been incorporated into polystyrene or polymethacrylate backbone. The method allows control of functionality, polydispersity, copolymer composition, backbone length, branch length, and branch spacing, considering the mole ratio of macromonomers (Shinoda and Matyjaszewski, 2001).

3.1.2 Grafting To Grafting to is the condition when a covalent attachment is achieved between a preformed polymer chain with specific functional groups and surface groups on a polymeric backbone, which may be located at the end or on the side chain of the main polymeric backbone (Henze et al., 2014). The well-defined star-shaped polymeric structure (Gao et al., 2007) or loosely grafted copolymers (Tsarevsky et al., 2007) have been prepared using the grafting to technique.

3.1.3 Grafting From It is also termed as surface initiated polymerization, which is due to the growth of polymer chains from the surface attached or self-assembled moieties (Henze et al., 2014).

3.2 Grafting Techniques Various methods have been developed for preparation of graft copolymers of different monomers on the polymeric backbone. The widely used grafting methods include chemical, radiation, photochemical, plasma-induced grafting, and enzymatic grafting (Bhattacharya and Misra, 2004).

354 CHAPTER 9 Grafting Onto Biopolymers: Application in Targeted Drug Delivery

3.2.1 Grafting Initiated by Chemical Means Graft copolymerization is generally achieved through two main paths: (1) the free radical grafting method and (2) the ionic grafting method. Free radicals or ions are generated through the initiator, which plays an important role in deciding the grafting process. Beside these two general methods, grafting in melt and atom transfer radical polymerization (ATRP) are also important grafting techniques.

3.2.1.1 Free Radical Grafting In this method, graft copolymers are formed by free radical generation. The free radical is generated on the polymer backbone with the help of suitable chemicals, which later react with the monomer to form graft copolymers. Using this technique, different scientists have prepared graft copolymers of locust bean gum with acrylamide (Kaity and Ghosh, 2016), Grewia Optiva, a cellulose biofiber (Thakur et al., 2012a), and pine needles (Thakur et al., 2012b) with methyl acrylate. The direct or indirect method can be applied for free radical generation. The following pathway represents a probable grafting mechanism in the presence of radical initiator (Bhattacharya and Misra, 2004). X2 /2X 



X þ M/2M











X þ P  OH/P  O þ XH XMM þ P  OH/P  O þ XMMH 

P  O þ nM/P  O  Mn





XMM þ nM/XM  Mn  H 



P  O  Mn þ X /P  O  Mn  X 



P  O  Mn þ XMM /P  O  Mnþ2  X

The indirect free radical may be produced by redox reaction using M nþ =H2 O2 or S2 O82 , and the following mechanism has been proposed for the decomposition of H2O2 and potassium persulfates with radical generation in presence of Fe2þ (Misra et al., 1984). Fe2þ þ HOOH/Fe3þ þ OH þ OH



2  3þ Fe2þ þ S2 O2 8 /Fe SO4 þ SO4



3. Biopolymers Grafting 355

Now again the generated radicals OH and SO4 directly react with the polymeric backbone and thus chain elongation takes place. 



SO4 þ P  OH/HSO4 þ P  O 







OH þ P  OH/H2 O þ P  O

The organic hydroperoxides, persulfates, Cu2þ, and Fe3þ can also be used instead of H2O2 with a suitable reducing agent like sodium bisulfate or thiosulfate for the generation of free radicals in the grafting experiments. S2 O8 þ HSO3 /SO4 þ SO4 þ HSO3 



P þ SO4 /P þ HSO4 



SO4 þ H2 O/ HSO4 þ OH 







HSO3 þ H2 O/H2 SO3 þ OH

Grafting on the polymeric backbone can also be performed by inducing free radicals using different transition metals like Ce4þ, Cr6þ, V5þ, Co3þ, which directly oxidize the backbone of polymers. The transition metals with lower oxidation potential have higher grafting efficiency compared to metals with higher oxidation potential. The possible reason for lower grafting efficiency with metals of higher oxidation potential may be due to the formation of homopolymers, instead of attaching on the polymeric backbone. The probable mechanism for such a process includes formation of intermediate metal ion polymer chelate ion complex and has been schematically represented (Bhattacharya and Misra, 2004). Ce4þ þ P  OH /Chelate complex Chelate complex/P  O þ Ce3þ þ H þ 





P  O þ M/P  OM /P  OMM



In the mechanism described for the generation of free radicals through chelate complexes, sometimes secondary free radicals like   CO2  ; C2 O4  are also generated in the system and contribute toward the grafting requirements. Mn4þ þ C2 O4 /Mn3þ þ CO2 þ CO



Mn3þ þ C2 O4 /Mn2þ þ C2 O4

3.2.1.2 Living Polymerization Grafting Living polymerization is another technique for the preparation of graft copolymers, and it has developed a great potential for grafting reactions.

356 CHAPTER 9 Grafting Onto Biopolymers: Application in Targeted Drug Delivery

According to Szwarc (1998), living polymers retain their property to grow and propagate up to the required chain length with negligible degree of termination or chain transfer possibility. The living polymerization technique has been successfully used for the synthesis of block copolymers, uniform polymers (Poisson molecular weight distribution) with controlled size, and star- and comb-shaped functional polymers (Szwarc, 1998). Controlled free radical polymerization is a blend of conventional free radical and ionic polymerization techniques. A continuous generation of free radicals and the termination of growing chain radicals is an essential requirement in the conventional free radical polymerization technique. This technique provides living polymers with low polydispersity index and regulated molecular weights (Bhattacharya and Misra, 2004). ATRP is an effective method for controlled free radical polymerization. In this method, dormant chains are capped by halogen atoms. The free radicals are reversibly transferred when metal complexes are present in its lower oxidation state, while the free radicals are generated when the metal complexes are present in its higher oxidation state. The dynamic equilibrium between the activationedeactivation phenomenon acts as the driving force for ATRP reaction (Bhattacharya and Misra, 2004). 

Pn eX þ CuðIÞ=2L#Pn þ CuðIIÞX=2L

Pn  X is the polymeric halide and copper(I) complex CuX/2L (X ¼ Cl/Br and L ¼ 2,20 bipyridine or a 4,40 disubstituted 2,20 bipyridine). A possible mechanism for the ATRP grafting reaction includes atom transfer equilibrium (the initiation step), the elongation step, and the radical termination step (Matyjaszewski et al., 1997). Initiation: 

R  X þ CuX=2bipy#R þ CuX2 =2bipy ½X ¼ Cl; Br kp0





R þ monomer ! P1

Elongation: 

Pn  X þ CuX=2 bipy#Pn þ CuX2 = 2 bipy Ke





Pn þ monomer ! Pnþ1

Termination: 



Kt



Pn þ Pm ! Pnþm þ Pn þ PHm




3. Biopolymers Grafting 357

Cu(I) abstracts the halogen atoms from the inactive chains in the polymerization reaction and has been reported in the polymerization reaction of styrene. styrene=110 C

1=2 AIBN þ CuBr2 =2dNbipy ƒƒƒƒƒ! controlled=living polymerization.

The exclusive role of Cu(I) complex in ATRP is atom transfer as the reversible halogen agent between the active and dormant polymeric chains. The optimum ratio of ligand: copper(I) halide for these polymers is approximately 2:1, indicating the presence of two bipyridine ligands in the coordination sphere of active copper(I) center (Matyjaszewski et al., 1997). The simultaneous initiation of an individual growing polymer chain with negligible termination along with the simultaneous growth between all polymeric chains makes this technique best suited for the gradient copolymer synthesis. This technique is also quite useful for the preparation of a highly grafted polymer like molecular brushes using the “grafting from” technique (Borner et al., 2002).

3.2.1.3 Ionic Grafting Ionic grafting is another method for preparing graft copolymers and is performed using the ionic approach (cationic or anionic). In this method the different initiators used are organometallic compounds, sodium naphthalenide, and alkali metal suspension in Lewis base liquid. The copolymerization proceeds via formation of carbonium ion. This carbonium ion is formed along the polymer chain due to the interaction of alkyl aluminum (R3Al) and the polymer backbone in its halide form (PCl), finally resulting in copolymerization. The reaction proceeds through cationic mechanism, and the possible mechanism can be as follows: ACl þ R3 Al/Aþ R3 AlCl Aþ þ M/AM þ  M/graft copolymer

The graft copolymerization can also proceed via anionic mechanism as the polymer alkoxide is formed by interaction of the polymer with sodium ammonia or of alkali metal methoxide, which reacts with the monomer to form a graft copolymer. This reaction proceeds via anionic mechanism as described (Bhattacharya and Misra, 2004). P  OH þ NaOR /PO Naþ þ ROH PO þ M/POM  M/Graft copolymer

3.2.2 Grafting by Irradiation Means The irradiation of polymers forms free radicals through homolytic cleavage mechanism. In this method the initiator is not an essential requirement,

358 CHAPTER 9 Grafting Onto Biopolymers: Application in Targeted Drug Delivery

while the medium has a significant impact, e.g., peroxides may be formed on the backbone of polymers if irradiation is carried out in air. Grafting through this method proceeds via three different ways (Bhattacharya and Misra, 2004).

3.2.2.1 Preirradiation In this technique, the polymer is irradiated in the presence of inert gas or in vacuum to generate free radicals, which are then reacted with monomers present in a solution in a suitable solvent or vapor state or liquid state. 

P/P þ M/P  M



As the monomers are not exposed to irradiation the final product obtained is relatively free from homopolymers, a significant advantage compared to other grafting methods. On the other hand, block polymers can be formed in this process due to the direct radiation to the trunk polymer (Chen et al., 2003).

3.2.2.2 Peroxidation In the peroxidation grafting method, hydroperoxides or diperoxides are generated when the polymer trunks are subjected to high energy irradiation in the presence of air or oxygen. The carefully optimized irradiation conditions produce stable peroxy products, which when treated with monomers at high temperature, initiates grafting. Radiation=O2





M

P ƒƒƒƒƒ! P  O  O  H/P  O þ OH /P  O  M

or Radiation= O2



M

P ƒƒƒƒƒ! P  O  O  H / 2 P  O / P  O  M

This technique has the advantage that the intermediate product can be stored for longer periods before performing grafting reactions.

3.2.2.3 Mutual Irradiation Technique In this technique the simultaneous irradiation of polymers and the monomers form free radicals, and the subsequent addition of monomers on the polymeric backbone forms graft copolymers (Kaur et al., 1994).

3.2.3 Photochemical Grafting The grafting process through this method is initiated only when the chromophoric group of macromolecules absorbs light. This phenomenon causes the transition of electron from ground to excited state followed by its dissociation and formation of free radicals (Bhattacharya and Misra, 2004).

4. Applications as Stimuli Responsive Targeted Drug Delivery System 359

Sometimes free radicals may not be generated through the bond dissociation process, then the process can be catalyzed by the addition of photosensitizes like benzoin ethyl ether, dyes like Na-2,7-anthraquinone sulfonate or acrylate azo dye, metal ions (Bellobono et al., 1981), or aromatic ketones such as benzophenones or xanthone (Kubota et al., 2001). The photochemical grafting can proceed by two mechanisms “with sensitizer” or “without sensitizer”. In the grafting process “with sensitizers”, the sensitizers form free radicals, which undergo diffusion and abstract hydrogen from the main polymer and consequently generate radical sites for grafting. The grafting mechanism “without sensitizer” involves generation of free radicals on the polymeric backbone, which reacts with free radical sites of the monomer to form grafted polymer (Tosh and Routray, 2014).

3.2.4 Plasma Radiation-Induced Grafting The grafting reaction through this method proceeds in the similar way and offers about the same possibilities as that of ionizing radiation. The plasma conditions are attained through slow discharge of electrons and possess sufficient energy to induce cleavage of chemical bonds in the polymeric structure to form radicals, initiating the graft copolymerization reaction. The probable mechanism includes electron-induced excitation, ionization, and dissociation (Wenzel et al., 2000; Yamaguchi et al., 1994; Das and Pal, 2015).

3.2.5 Enzymatic Grafting In this method, the enzyme initiates the grafting reaction. The grafting of cellulose by this method is a quiet, new approach. Only a few reports are available for the enzymatic catalyzation ring opening polymerization from cellulose surface (Chen et al., 2000). Lipase is used for the ring opening polymerization of capralocatones in close proximity to cellulose fibers in a filter paper. In this process, filter paper acts as substrate and the enzyme is immobilized on its surface and then polymerization is performed. This method does not produce covalently linked grafted polymers; instead, polycaprolactone formed is coated on the cellulose surface (Gustavsson et al., 2004; Das and Pal, 2015).

4.

APPLICATIONS AS STIMULI RESPONSIVE TARGETED DRUG DELIVERY SYSTEM

The smart polymers are environmentally responsive polymers, which are generally composed of grafted copolymeric or cross-linked polymeric networks. The most important property of these polymers is to undergo stimuli-triggered behavioral changes. The most common external stimuli to which these polymers

360 CHAPTER 9 Grafting Onto Biopolymers: Application in Targeted Drug Delivery

respond are pH and temperature, but various different stimuli, like ultrasonic waves, ionic strength, redox potential, electromagnetic radiation, and chemical or biochemical agents can also induce the polymer behavioral changes in the physiological system (Gil and Hudson, 2004). These stimuli can be classified into two broad divisions: (1) physical stimuli, which includes temperature, ultrasound, light, and magnetic and electrical fields, and (2) chemical stimuli, which includes pH, redox potential, ionic strength, and different chemical agents. The physical stimuli act by directly modulating the energy level of the polymer, which upon achieving certain critical energy level, induces the response in the biological system. In the case of chemical stimuli the response is generated due to the polymeresolvent interaction at the molecular level. The interaction may be due to adjustment of the hydrophilicelipophilic balance or between polymeric chains (influencing polymeric backbone integrity or their cross-linked networks and proclivity for hydrophobic association or electrostatic repulsion) (Gil and Hudson, 2004). The different types of behavioral changes include solubility phase transition, hydrophilicehydrophobic balance, and conformational changes (Schmaljohann, 2006). These changes may progress through various different mechanisms like coileglobule transition of the polymeric chain (Tian et al., 2012), swellingedeswelling of hydrogels (Khare and Peppas, 1995), or self-assembly of amphiphilic polymers (Malmsten and Lindman, 1992). In the current research scenario the stimuli-responsive polymers have immense applications in the development of a targeted delivery system with different formulations like hydrogels (Das et al., 2015), polyplexes (Novo et al., 2014), micelles (Ma et al., 2016), and polymer drug conjugates (Magoshi et al., 2002). The current section will deal with the development in temperature- and pH-responsive polymers and its utility in targeted drug delivery systems along with recent advances and developments in the respective stimulus response achieved by different scientists.

4.1 Temperature Responsive Polymers: Applications in Targeted Drug Delivery System Temperature is the most common environmental stimulus of physiological significance in targeted drug delivery applications. Both the hydrophilic and hydrophobic moieties are present in temperature-responsive polymers and upon achieving a certain temperature range, the balance between both the moieties breaks and causes reversible phase separation (Tian et al., 2012). Based on temperature responsiveness, polymers can be classified as the following: (1) polymers that become insoluble at increased temperature have lower critical solution temperature (LCST), and (2) polymers that become soluble upon heating have upper critical solution temperature (UCST). The various different factors like temperature, molecular weight,

4. Applications as Stimuli Responsive Targeted Drug Delivery System 361

or addition of cosolvent influence the aqueous solubility of polymers. The critical solution temperature for LCST or UCST can be determined by plotting solubility phase diagram of polymer v/s solvent v/s temperature. For biomedical applications, mainly aqueous systems are of prime interest, but LCST and UCST systems are not limited to aqueous system only. Volume phase transition of thermoresponsive polymers occurs due to changes in hydration state, which reflects intra- and intermolecular hydrogen bonding properties of polymer molecules, where hydrogen bonding is favored over solubilization in the aqueous solvent. The transition is then accompanied by coil to globule transition (Schmaljohann, 2006). Typical polymers, which exhibit LCST properties, are based on N-isopropylacrylamide (NIPAM) (Das et al., 2015), N,N-diethylacrylamide (DEAM) (Panayiotou et al., 2007), methylvinylether (MVE) (Verdonck et al., 2005), and N-vinylcaprolactam (VCa) (Mikheeva et al., 1997) as monomers. Similarly, typical UCST systems are based upon combination of acrylamide (AAm) and acrylic acid (AAc) (Le Ngoc and Takaomi, 2011). The most widely utilized application of the thermoresponsive stimulus concept is based on change in room temperature to body temperature. This change in temperature induces a change in physical properties like gelation in topical applications or swelling behavior in hydrogels. Polymers exhibiting LCST and UCST behavior in aqueous systems have been listed in Table 9.2. These polymers have a transition temperature that is suitable for biomedical applications (20e50 C). N-isopropylacrylamide is the most widely studied thermoresponsive monomer, and some of the recent advancements have been discussed in the current section. Das et al. (2015) synthesized thermoresponsive hydrogels using graft copolymers of dextrin and poly (N-isopropylacrylamide) cross-linked with N,N0 -methylene bis(acrylamide) (MBA), for delivery of ciprofloxacin and ornidazole. The in vitro dissolution study showed that the rate of drug release was higher at 25 C, compared with the release profile at 37 C. The result was in good agreement, as percentage equilibrium swelling rate was higher at 25 C compare to 37 C. Below LCST of pNIPAM (25 C), the water molecule was associated with the hydrogel matrix through intermolecular hydrogen, so the tendency of the water molecule to diffuse inside the matrix is higher in contrast to temperature above 37 C, the LCST. So the diffusion of the drug from the hydrogel matrix is greater at 25 C than 37 C, and thus it controlled the drug release more effectively at body temperature. Kajjari et al. (2012) formulated microspheres of sodium alginate with poly (N-isopropylacrylamide)-g-guar gum using the emulsion cross-linking method for the delivery of the antitubercular drug isoniazid. The percentage

362 CHAPTER 9 Grafting Onto Biopolymers: Application in Targeted Drug Delivery

Table 9.2 Polymers With Lower Critical Solution Temperature Behavior in the Temperature Range Suitable for Biomedical Applications Polymer

Phase Transition Temperature in Aqueous Solution ( C)

PNIPAM

30e34

PDEAM

32e34

PMVE

37

PVCa

30e50

Structure

equilibrium swelling and in vitro dissolution study proved the temperature responsiveness and extended the release up to 12 h. The probable mechanism can be explained on the basis that NIPAM swells at temperatures lower than 32 C due to H-bond between the water molecule and (eCONHe) hydrophilic group present in the polymeric chain. Water molecules are oriented in a special fashion around the hydrophobic isopropyl group to form iceberg structure, contribute in negative entropy, and reduce the free energy of the system. Upon increasing the temperature above LCST, the 3-D structure of the hydrogel gets collapsed, hydrophobic groups become free, and the interaction with isopropyl groups loses a large amount of water and changes the volume drastically. This concept has been widely used for temperatureresponsive graft copolymer drug delivery systems.

4. Applications as Stimuli Responsive Targeted Drug Delivery System 363

4.2 pH Responsive Polymers: Applications in Targeted Drug Delivery System pH is another chemical stimulus in the body physiology, to which pH responsive polymers are activated for site-targeted drug delivery. The physiological pH varies systematically in the body; particularly a drastic pH variation is observed throughout the gastrointestinal tract (g.i.t.) with highly acidic pH in stomach ranging from 1.0 to 3.0, while pH shifts toward basic value in duodenum 4.8 to 8.2 and 7.0 to 7.5 in the colon. The pH profile also varies in the cellular compartments; for example, early endosomes have a pH value around 6.0 to 6.5 and late endosomes have a pH value of 5.0 to 6.0. Lysosomes and golgi bodies have a pH of 4.5 to 5.0 and 6.4, respectively (Schmaljohann, 2006). pH at the site of tumors and infections is found to be different from that of normal body sites. After 60 h of inflammatory reaction, pH at the inflammation site falls from 7.4 to 6.5 (Ganta et al., 2008). This behavior of pH variation can be harnessed for the formulation of the stimuli-responsive targeted drug or gene delivery systems. Polymers that ionize at pKa value between 3 and 10 are suitable candidates to be developed as pH responsive systems. Table 9.3 presents a list of different polymers with pH responsiveness along with their structure. Weak acids like carboxylic acids and phosphoric acids and weak bases like amines get ionized and exhibit charge upon pH variation. These ionizable groups are attached to the polymeric backbone, which upon ionization brings conformational changes and swelling behavior of delivery systems like hydrogels. The most widely used monomers to induce pH sensitivity are acrylic acid (AAc), methacrylic acid, maleic anhydride, and phosphoric acid derivatives like methacyloyloxyethyl dihydrogen phosphate (phosmer M) (Schmaljohann, 2006). Das et al. (2015) developed novel biodegradable, noncytotoxic, and pHresponsive chemical hydrogels using AAc (pH responsive monomer) and dextrin (biopolymer) cross-linked using MBA. The hydrogel was finally formulated into tablets, the most common drug delivery route. They used this delivery system for the delivery for ciprofloxacin and ornidazole with an approach to deliver the drug in the colon for treatment of bacterial infections. The formulation demonstrated significant pH sensitivity as it showed maximum swelling of 115  9% at pH 1.2 (stomach), 446  12% at pH 6.8 (duodenum) and 2620  131 at pH 7.4 (colon). The release profile was in accordance to selling behavior and controlled according to pH variation. Das et al. (2013) synthesized hydrogel for colon-targeted delivery of ornidazole by chemical cross-linking method using poly (hydroxyl ethyl methacrylate) as pH-responsive polymer and dextrin as biopolymeric backbone cross-linked with MBA. The equilibrium swelling ratio study of hydrogels

364 CHAPTER 9 Grafting Onto Biopolymers: Application in Targeted Drug Delivery

Table 9.3 Polymers With pH Responsive Behavior With Their Structure Suitable for Biomedical Applications Polymer Poly (acrylic acid)

Poly (methacrylic acid)

Poly (maleic anhydride)

Poly(histidine)

Poly(sulphonamides)

Structure

4. Applications as Stimuli Responsive Targeted Drug Delivery System 365

showed maximum swelling at pH 7.4 of 280  3 in the colon region and the in vitro drug dissolution profile proved the controlled delivery of ornidazole in this region. Loth et al. (2013) reported preparation-free radical polymerization of maleic anhydride with N-isopropylacrylamide and pentaerythriotal diacrylate monostearate. In the graft copolymers, N-isopropylacrylamide acted as temperature responsive and maleic anhydride as pH responsive, while stearate increased hydrogel stability. The synthesized macromers could be suitably cross-linked for hydrogel fabrication from different biopolymers with an advantage to decorate the hydrogels with monoamines that alter the biological or physicochemical properties. Mei et al. (2013) designed novel biocompatible Ca-alginate-based capsular membrane with grafted poly(methacrylic acid) brushes using the coextrusion minifluidic approach, a UV-irradiation induced grafting technique for controlled enzyme reactions. The presence of acrylic acid in the graft copolymer induced the property of pH responsiveness. This was experimentally verified as, when the environmental pH was lower than pKa of PMMA, the grafted copolymer is shrunken, transmembrane resistance for mass transfer is larger, and the enzymatic reaction inside the capsule remains inactivated. But as soon as the pH becomes higher than pKa of PMMA, the polymer swells, the transmembrane resistance for mass transfer reduces, and the enzymatic reaction in the capsule becomes activated due to high permeability of substrates and reactants across the membrane. Anticancer cancer therapy is generally associated with various side effects, including cytotoxicity to normal cells, due to unspecific drug distribution in the body. So, targeting of oncologic drugs to cancerous cells is the ultimate aim in cancer therapy. The pH (Gerweck and Seetharaman, 1996), surface charge (Bolot et al., 1998) and density of lipoprotein (Gal et al., 1981) receptors are some of the factors that show significant pH differences between normal and tumor cells. The mean pH of tumor extracellular fluid is around 7.06 with a range of 5.7e7.8, which is lower than physiological pH 7.4. These properties influence the physicochemical properties and can be harnessed for the targeted delivery system. The pH-sensitive polymeric drug delivery system for cancerous cells can be developed using two approaches (Liu et al., 2013). The first approach is the formation of a pH labile chemical bond between polymer drug conjugate. These bonds may be hydrazone, cis-acotinyl, and acetal bonds. The acid labile bond gets cleaved after activation at suitable pH, either in relatively acidic extracellular fluid or after endocytosis by lysozymes in tumor cells, and releases the drug. These pH responsive bonds are stable at neutral or

366 CHAPTER 9 Grafting Onto Biopolymers: Application in Targeted Drug Delivery

basic pH or blood pH 7.4 but are cleaved at acidic pH and release the drug under acidic conditions (Liu et al., 2013). Hydrazone-linked polymeric conjugates have been extensively studied for the delivery of anticancer drugs by pH triggered the drug release mechanism. Bae et al. (2005) prepared self-assembling polymeric micelles using amphiphilic block copolymers, poly(ethylene glycol)-poly-(aspartate hydrazone adriamycin), and conjugated anticancer drug adriamycin to the hydrophobic segments through acid-sensitive hydrazone linkers. This polymeric design successfully stabilized and preserved the drug under physiological conditions (pH 7.4) and selectively releases them at intracellular pH due to signaling at decreased pH (pH 5.0 to 6.0) in endosomes and lysosomes. The formulation selectively released the drug at endosomal pH, resulting in enhanced tumor with greater therapeutic efficacy and minimal systemic toxicity. Al-Shamkhani and Duncan (1995) prepared covalent conjugate of anticancer drug daunomycin and alginate with the cis-acotinic group at its amine site for stable circulation and drug release in acidic milieu of lysosomal and endosomal compartments of tumor cells. The system was pH responsive, as the cis-acotinic group got hydrolyzed at lower pH to release the drug. The in vitro drug release profile showed that approximately 60% of the drug was released after 48 h under acidic conditions (pH 5e6) while only 4% drug was released at neutral pH (7.4). The second strategy is based on the attached titrable group to copolymers such as carboxylic acids or amines to control the bond cleavage at altered pH level. The basic concept behind the mechanism is based on the fact that at normal physiological pH the pH-sensitive polymers are present as the hydrophobic core in the polymeric micelle, and as it comes into contact with lower pH, the titrable pendent group becomes protonated. This causes destabilization and leads to repulsion between the polymeric chains and dissociation of micelles and ultimately release of drug (Liu et al., 2013). Different pH-sensitive polymers include poly(histidine), poly(acrylic acid), and poly(sulfonamides) that cause polymer protonation and destabilization of complexes leading to drug release. Among them, poly(histidine) is the most commonly used due to its additional advantage of strong endosmolytic property exhibited by its interaction with anionic phospholipids present in the endosomal compartments. Wu et al. (2013) prepared a pH-sensitive polymeric micellar system based on 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinimidyl (polyethylene glycol)-3400] (DSPE-PEG3400-NHS) and poly(ethylene glycol) coupled with poly (L-histidine) for paclitaxel delivery to a tumor site with

5. Applications as Receptor Targeted Drug Delivery System 367

an approach to reduce cytotoxic effect to other tissues and enhanced uptake by selective tumor cells due to poly (L-histidine) responsiveness to more acidic endosomal pH. Lee et al. (2007) synthesized polymic micelles from poly (lactic acid)-b-poly (ethylene glycol)-b-poly (histidine), a triblock copolymer for the delivery of doxorubicin. The poly (histidine) imparted the pH-responsive property to the polymeric system. The formulation showed significantly enhanced release of doxorubicin at pH 6.0 compared to pH 7.4. Cytotoxicity studies at different pH showed a varied result. At pH 7.4 a very minimal cytotoxicity was observed, while with decrease in pH cytotoxicity increased, at pH 6.8 cytotoxicity was 60%, while at pH 6.0 it was 74%. The increased cytotoxicity with increase in pH proved pH triggered release. These works suggests that pH-responsive polymers like poly (L-histidine) could trigger pHdependent drug release with increased uptake at tumor sites. So, the two strategies could be promising for pH-triggered targeted drug delivery system to tumor sites.

5.

APPLICATIONS AS RECEPTOR TARGETED DRUG DELIVERY SYSTEM

The increasing concern for safer drug delivery with increased uptake by affected cells and reduced cytotoxic effect, especially in the case of the delivery of anticancer drugs based on receptor approach, seems to be another promising strategy in the area of targeted drug delivery system. Targeting via folate receptors and arginine-glycine-aspartic acid (RGD) peptides are the most studied approaches and have been discussed in this section.

5.1 Targeting via Folate Receptors Folate targeting was invented in 1986 by Kamen and Capdevila at the University of Texas Southwestern Medical Center. They reported the folate entry through receptor-mediated endocytosis process. Folates belong to the class of pteridine compounds that are an essential requirement for normal growth, cell division, and its maturation. Reduced folic acid coenzymes are involved in one carbon transfer reaction necessary for the biosynthesis of methionine, serine, deoxythymidylic acid, and purines. The normal plasma folate concentration present in the human physiological system is around 10e20 nM (Kamen and Capdevila, 1986). The drug bound with folate binds specifically to externally oriented folate receptors (FRs) located on the plasma cell membrane (Fig. 9.2). The mechanism is highly specific and analogous to lock and key fashion as the key (folate) enters to lock (FRs) and shows response. A number of sequential reactions take place after

368 CHAPTER 9 Grafting Onto Biopolymers: Application in Targeted Drug Delivery

n FIGURE 9.2 Folate receptor (FR)-mediated endocytosis of pteroate drug conjugates. Exogenously

added pteroate drug conjugates bind specifically to the FR protein with high affinity. The plasma membrane invaginates around the conjugate/FR complex to form an intracellular vesicle (endosome). As the lumen of the maturing endosome acidifies to wpH 5, the receptor changes conformation and releases the conjugate. Eventually, the fates of the pteroate ligand, attached drug cargo, and the FR are determined during a sorting process within late endosomal elements. The reduced folate carrier (RFC), which unlike the FR is an anion transporter, can shuttle folate molecules inside the cell. Pteroate drug conjugates, however, are not substrates for the RFC. Reprinted from Leamon, C.P., Reddy, J.A., 2004. Folate-targeted chemotherapy. Advanced Drug Delivery Reviews 56 (8), 1127e1141. Copyright 2004, with permission from Elsevier.

binding folate conjugates with the plasma membrane. The folate complex enters and evades the plasma membrane, forming a distinct internal vesicle, the early endosome. Due to the action of the endosomal proton pump the pH of lumen vesicle falls approximately to 5. The fall in pH causes protonation of various carboxyl moieties on the FR protein and brings about conformational changes, enabling the folate molecule to dissociate and release the drug at the target site (Leamon and Reddy, 2004). The FRs are generally absent in normal tissue with some exceptions, such as the choroid plexus and placenta, and low levels are present in the lung, thyroid, and kidney, but an elevated level of FRs are overexpressed in various types of human cancers like ovarian, endometrial, colorectal, breast, and lung carcinomas. The overexpression of FRs in carcinomas acts as a marker for drug folate conjugate, and the same has been utilized in the concept of tumor-targeted drug delivery systems (Sudimack and Lee, 2000); various works in this arena have been illustrated in Table 9.4.

Table 9.4 Targeted Drug Delivery Using Folic Acid Conjugate for Folate Overexpressing Tumor Cells Anticancer Drug

Targeted Tumor Model

Dox-PLGA-mPEG-FA micelle

Doxorubicin

KB tumor xenograft mice

FA-PEG-EB-Dox micelle

Doxorubicin

4T1.2Breast cancer cells

M-FA-PTX

Paclitaxel

EMT-6 breast cancer model

Fol-AmCD nanoparticles

Paclitaxel

T-47D and ZR-75-1 human breast cancer cells

Therapeutic Improvements Folate receptor mediated receptor endocytosis enhanced Dox transport toward tumor cell compared to free Dox and in vivo experiments in nude mice xenograft model demonstrated significantly reduced tumor volume. Blood circulation time of Dox was significantly improved and Dox preferentially accumulated at the tumor tissue with minimal toxicity compared to free Dox. The inhibition rate of tumor growth for M-FAPTX was 62.3%, while it was only 32.6% and 51.6% for PTX and M-PTX, respectively. The M-FA-PTX have better antitumor activity and are promising in treatment for human breast cancer. These nanoparticles resulted in higher anticancer efficacy with reduced side effects and equivalent efficacy for cellular internalization with folate-dependent mechanism.

References Yoo and Park (2004)

Lu et al. (2014)

Wu et al. (2015)

Erdogar et al. (2016)

Continued

5. Applications as Receptor Targeted Drug Delivery System 369

Drug Delivery Mode

Drug Delivery Mode

Anticancer Drug

Targeted Tumor Model

MPEG-PLA-MTX-NSs

Methotrexate

Nude H22 tumor-bearing mice

MIP-PEG-FA nanopoarticle

Paclitaxel

MDA-MB-231 folate receptor positive cancer cell

Fol-PEG-DPSE liposomes

Doxorubicin

Human nasopharyngeal epidermal carcinoma KB cell

Therapeutic Improvements The shape design mediated an early phase tumor accumulation and late phase cell internalization and Janusfaced function mediated early phase active targeting effect and late phase anticancer effect. This highly convergent and cooperative drug delivery strategy is promising with new shape and function for cancer therapy. MIP-PEG-FA nanoparticles showed enhanced intracellular uptake in folate receptor-positive cancer cells (MDA-MB231cells) in comparison with the nonfolate nanoparticles and free PTX, with IC50value of 4.9  0.9, 7.4  0.5 and 32.8  3.8 nM, respectively. The uptake of Fol-PEGDPSE liposomes was 45 times higher by KB cells compared to nontargeted liposomal dox

References Cui et al. (2015)

Esfandyari-Manesh et al. (2016)

Lee and Low (1995)

AmCD, Amphiphilic cyclodextrin; Dox, Doxorubicin; EB, Embelin; FA, Folic acid; MTX, Methotrexate; NSs, Nanospheres; PEG, Poly ethylene glycol; PLGA, Poly lactic-co-glycolic acid; PTX, Paclitaxel.

370 CHAPTER 9 Grafting Onto Biopolymers: Application in Targeted Drug Delivery

Table 9.4 Targeted Drug Delivery Using Folic Acid Conjugate for Folate Overexpressing Tumor Cells Continued

5. Applications as Receptor Targeted Drug Delivery System 371

5.2 Targeting via RGD Peptide Toward Integrin Receptors Integrins are the cell adhesion receptors for extracellular matrix proteins, growth factor, immunoglobulin, cytokines, and matrix degrading proteases. It is basically divalent cation-dependent heterodynamic membrane glycoproteins composed of noncovalently associated a- and b-subunits. There are 18 a- and 6 b-subunits, which can assemble in 24 different kinds of heterodimers, and their combination determines the ligand-binding specificity and signaling properties (Danhier et al., 2012b). These integrins recognize the extracellular matrix proteins through short peptide sequence such as RGD, Arg-Glu-Asp-Val (REDV), or Glu-Ile-Leu-Asp-Val (EILDV). Integrins act as the mediator for various different cancer stages, such as malignant transformation, tumor growth and progression, invasion, and metastasis during tumor growth, and special b3 integrins are involved with the ability of tumors to metastasize. avb3 integrin is the most widely expressed in the blood vessels of tumor cells, but it is absent in blood vessels of normal tissues. There is various evidence where avb3 integrin is overexpressed and acts as a biomarker, and this concept is utilized in the targeted delivery of anticancer drugs. In the case of breast cancer, avb3 integrin is overexpressed, is associated with bone metastasis, and induces tumor growth in response to osteopontin. In the patients suffering from pancreatic tumors, avb3 integrin is associated with the increased activation of MMP-2 and lymph node metastasis (Danhier et al., 2012b). RGD peptide was discovered for the first time by Ruoslahti and Pierschbacher in the early 1970s as a cell attachment site fibronectin (Ruoslahti and Pierschbacher, 1987). Later this sequence was identified as the integrin sequence, which is present in natural ligands binding avb3 receptor as osteopontin, fibrinogen, vitronectin, fibronectin, plasminogen, thrombospondin, prothrombin, etc. Currently the conformational feature of RGD has been modified, as the linear RGD is prone to degradation, while RGD in its cyclic form are more stable, potent, and specific (Liu, 2006). RGD-based strategy for tumor-targeted drug delivery systems are RGD antagonists, RGD conjugates, and RGD grafted with polymeric nanoparticles (Fig. 9.3). Different nanocarrier systems like nanoparticles, polymeric nanomicelles, and liposomes can be grafted with RGD for recognition of avb3 integrin in an objective way. The different advantages involved with these nanocarriers include the following: (1) the size of nanocarriers are in the range of 50e100 nm, so the passive transportation route follows through the new blood vessels and avoids renal filtration, which prolongs the circulation time and accumulates at the tumor site

372 CHAPTER 9 Grafting Onto Biopolymers: Application in Targeted Drug Delivery

n FIGURE 9.3 Arginine-glycine-aspartic acid (RGD)-based strategies: (A) RGD antagonists and (B) RGD

conjugates. The RGD-based peptide or peptidomimetic is conjugated to drugs or radionuclides with covalent links. (C) RGD peptides or peptidomimetics are grafted at the nanoparticle surface (polymeric nanoparticles, liposomes, polymeric micelles, etc.). These structures contain various agents such as anticancer drugs, peptides or proteins, nucleic acids, radionuclides, contrast agents, or a mixture of contrast agents and anticancer drugs (theranostics). Reprinted from Danhier, F., Breton, A.L., Preat, V., 2012b. RGD-based strategies to target alpha (v) beta (3) integrin in cancer therapy and diagnosis. Molecular Pharmaceutics 9 (11), 2961e2973 Copyright 2012, with permission from American Chemical Society.

through enhanced permeability and retention effect. (2) RGD grafted nanoparticles release the drug at the target site as RGD specifically binds to endothelial cells or cancer cells due to overexpressed avb3 integrins, which promote active targeting (Lv et al., 2012). RGD grafted nanoparticles have been proven to be a better approach for site-targeted delivery of chemotherapeutics, and different classical examples of the work have been illustrated in Table 9.5.

6.

APPLICATION OF GRAFTED BIOPOLYMERS IN CONTROLLED DRUG DELIVERY SYSTEM

The controlled drug delivery approach is mainly applied for preparation of sustained or extended release dosage forms. The main aim is to reduce the dose, dosage regimen, and in vivo drug metabolite load to enhance patient

Table 9.5 RGD Grafted Nanoparticles for Targeted Delivery of Anticancer Drugs Anticancer Drug

Targeted Tumor Model

RGD-g-Chitosan nanoparticle

Paclitaxel

Lewis lung carcinoma

RGD-g-PLGA nanoparticle

Paclitaxel

TLT hepatocarcinoma

cRGD-g-PEG-co-PLA micelle

Paclitaxel

U87 MF glioblastoma

RGD-PTX-LP

Paclitaxel

A549 lung adenocarcinoma

HPMA-co-RGD conjugate

Geldanamycin

DU145 prostate cancer

Therapeutic Improvements Greater tumor growth inhibition effect along with reduced side effects over commercial formulation RGD grafted nanoparticles selectively bounded with tumor vessels and effectively retarded TLT tumor growth and prolonged the survival time for mice treated with PTX-loaded RGD nanoparticles compared to non-RGD grafted nanoparticles The median survival time for U87 MF tumor xenograft significantly increased with cRGD-gPEG-co-PLA-PTX treated mice (48 days) compared to PEG-co-PLA-PTX (41.5 days), TaxolÒ(38.5 days) or saline (34 days). Greater cellular uptake and lower tumor microvessel density was observed in RGD-PTX-LP compared to PTX-LP. RGD conjugated formulation showed increased tumor accumulation compared to the conjugate formulation without RGD peptides.

References Lv et al. (2012)

Danhier et al. (2009)

Zhan et al. (2010)

Meng et al. (2011)

Borgman et al. (2009)

Continued

6. Application of Grafted Biopolymers in Controlled Drug Delivery System 373

Drug Delivery Mode

Drug Delivery Mode

Anticancer Drug

Targeted Tumor Model

RGD-Dox-NPs

Doxorubicin

Pancreatic and renal cell orthotopic mouse tumor model

RGD-g-Liposome

Doxorubicin

C26 murine colon carcinoma

RGD-SSL-Dox

Doxorubicin

B16 melanoma tumor

RGD-Dox-NPs

Doxorubicin

Mammary tumor clone-66

Therapeutic Improvements Targeted delivery of doxorubicin resulted in 15fold increased antimetastatic activity without causing drug associated weight loss, as associated with free drug. Doxorubicin loaded RGDg-liposome resulted in specific and efficient binding with C26 colon carcinoma model compared to free doxorubicin and untargeted liposome. Prolonged circulation time and greater accumulation at melanoma cell was observed for RGD-SSL-Dox, while a significantly lower level of Dox was observed in blood with higher uptake by spleen was found for SSL-Dox. The doxorubicin metabolite doxorubicinol or doxorubicinone was observed in tumor samples, while it was not detected in any other tissue, and thus indicated tumor-specific uptake of doxorubicin.

References Murphy et al. (2008)

Holig et al. (2004)

Xiong et al. (2005)

Bibby et al. (2005)

Dox, Doxorubicin; LP, liposomes; NPs, nanoparticles; PEG, Poly ethylene glycol; PLGA, Poly lactic-co-glycolic acid; PTX, Paclitaxel; RGD, arginine-glycine-aspartic acid.

374 CHAPTER 9 Grafting Onto Biopolymers: Application in Targeted Drug Delivery

Table 9.5 RGD Grafted Nanoparticles for Targeted Delivery of Anticancer Drugs Continued

7. Concluding Remarks 375

compliance with reduced toxicity. For this application, different ratecontrolling polymers like hydroxy propyl cellulose, hydroxyl propyl methyl cellulose, ethyl cellulose, etc. are being used in marketed formulations. In the search for different alternatives to these rate-controlling polymers, various groups of scientists have approached graft copolymers and have successfully formulated controlled drug delivery systems like buflomedil HCl-loaded IPN hydrogels and matrix tablets for buflomedil HCl using acrylamide-g-LBG (Kaity and Ghosh, 2016; Kaity et al., 2013b). The in vitro release profile for buflomedil HCl tablets prepared using acrylamide-g-LBG was compared against standard HPMC tablets. The similarity factor (f2) was found to be 60.37, which indicated different grafted biopolymers can be developed as an alternative to the existing synthetic or semisynthetic polymers. Different examples of such works by various scientists have been presented in Table 9.6.

7.

CONCLUDING REMARKS

Numerous investigations have been done on biopolymers for their applications in drug delivery and biomedical applications. As this chapter is titled “Grafting onto biopolymers: application in targeted drug delivery”, we have discussed different biopolymers and their source, isolation, purification, and possible application in the drug delivery arena. However, the application of each biopolymer has not been discussed in detail. Instead, the discussion is very concise and short, but a detailed discussion has been done on grafting techniques and different strategies for designing targeted drug delivery systems. The discussion on targeted drug delivery systems is based on temperature and pH stimuli responsiveness and different receptor-based targeting such as folate overexpressing receptors and integrin receptors. The grafting technique overcomes various drawbacks of biopolymers and alters its physicochemical properties, making it suitable for biomedical applications. At the same time the biodegradability, biocompatibility, and noncytotoxicity of biopolymers shall be taken care of and should not be compromised at any stage of dosage form development. The targeted drug delivery system is promising in the field of pharmaceuticals, as this concept delivers the drug at the required site with maximum therapeutic efficacy and minimal side effects.

ACKNOWLEDGMENTS The author appreciates and is thankful to all the authors of the publications listed in the references that were used in the preparation of content of the chapter entitled “Grafting onto biopolymers: application in targeted drug delivery”.

Drug Delivery Mode

Grafted Monomer

Grafting Technique

Locust bean gum

Acrylamide

Free radical induced microwave assisted grafting

Acrylamide

Free radical-induced microwave-assisted grafting

Methacrylate

Microwave irradiation grafting

N-isopropylacrylamide

Cerric ammonium nitrate induced free radical polymerization

Guar gum

Application/Therapeutic Improvements Controlled release tablets of Buflomedil HCl tablets were prepared using grafted LBG. The release property was found to be similar with similarity factor (f2) of 60.37when compared with HPMC-K15 M (standard release rate-controlling polymer). This modified polymer could be an alternative to HPMC-K15 M. Buflomedil HCl loaded Am-g-LBG-PVA IPN microspheres were prepared and proved to be promising controlled drug delivery device for highly water soluble drug with short half-life. GG-g-MA macromers were photopolymerized in UV radiation to form hydrogels and encapsulated human endothelial cell line (EA.hy926). The prepared hydrogel revealed excelled endothelial cell proliferation, similar to the control (Matrigel) Blend hydrogel microspheres of sodium alginate PNIPAAm-g-GG was prepared using emulsion cross-linking method. PNIPAAm induced pH and temperature responsiveness to the formulation as confirmed by equilibrium swelling studies and also drug release extended up to 12 h from the matrices.

References Kaity et al. (2013b)

Kaity and Ghosh (2016)

Tiwari et al. (2009)

Kajjari et al. (2012)

376 CHAPTER 9 Grafting Onto Biopolymers: Application in Targeted Drug Delivery

Table 9.6 Biopolymers Grafted With Different Monomers and Their Application as Controlled Drug Delivery System

Acrylamide

Free radical-induced microwave-assisted grafting

Alginate

Acrylic acid

Chemical-induced free radical grafting

Dextran

Acrylamide

Free radical crosslinking copolymerization technique

Gellan gum

Acrylamide

Microwave assisted cerric ammonium nitrate induced free radical grafting

Xanthan

Acrylamide

Cerric ammonium nitrate-induced free radical grafting

Aspirin-loaded hydrogel tablets were prepared using TKP-g-PAM. Drug release from the hydrogel was dependent on % grafting, which decreased with increase in % grafting. Drug release profile was lower in acidic pH compared to basic pH. This hydrogel can be a potential candidate for lower gastrointestinal tract-targeted drug delivery. Microspheres were prepared using Alg-g-PAA. These microspheres improved the survival and release of L. plantarum MA2 in comparison to alginates in vitro and in vivo. The result also demonstrated its pH-sensitive property, which can be used as matrix for the oral delivery of proteins. Lysozyme-loaded IPN hydrogels were prepared using poly acrylamide-dextran sulphate. The slow release of lysozymes from the IPN hydrogel recommend them as potential in the controlled drug delivery system. Polymeric matrix tablet of metformin HCl was prepared using acrylamideg-gellan gum. These matrices sustained the drug release up to 8 h and thus proved that these graft copolymers could be used as rate-controlling polymer for the development of controlled release dosage form. Controlled release matrices for antihypertensive drug atenolol and carvedilol were prepared form AAm-g-XG. The different studies concluded that the prepared matrix tablet is a potential hydrophilic carrier for the design of oral controlled drug delivery system.

Ghosh and Pal (2013)

Liu et al. (2016)

Dinu et al. (2011)

Vijan et al. (2012)

Mundargi et al. (2007)

Continued

7. Concluding Remarks 377

Tamarind kernel powder

Drug Delivery Mode

Grafted Monomer

Grafting Technique

Pullulan

Poly(Nisopropylacrylamide-coacrylamide)

Cerric ammonium nitrate-induced free radical grafting

Chitosan

Poly(Nisopropylacrylamide)

Plasma-induced grafting polymerization

Application/Therapeutic Improvements Upon grafting of pullulan with poly(N-isopropylacrylamide-coacrylamide), the prepared lysozyme-loaded microspheres of pullulan-g-poly(N-isopropylacrylamideco-acrylamide) were temperature and pH responsive. The swelling degree and water regain capacity for grafted polymer increased significantly compared to native pullulan. Wound dressing with temperatureresponsive property was prepared by grafting poly(N-isopropylacrylamide) onto chitosan, which showed better wound healing compared to the dressing containing chitosan only. PP-g-chitosan-g-PNIPAAm wound dressings healed the wound completely in 17 days.

References Fundueanu et al. (2008)

Chen et al. (2012)

378 CHAPTER 9 Grafting Onto Biopolymers: Application in Targeted Drug Delivery

Table 9.6 Biopolymers Grafted With Different Monomers and Their Application as Controlled Drug Delivery System Continued

References 379

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380 CHAPTER 9 Grafting Onto Biopolymers: Application in Targeted Drug Delivery

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