Smart Nanopolysaccharides for the Delivery of Bioactives

3 Smart Nanopolysaccharides for the Delivery of Bioactives S. Maiti, L. Kumari DEPA RTM ENT O F PHARM ACEUTI C S , G U P TA C O L L E G E O F T E C H N O L O G I C A L S C I E N C E S , ASANSOL, WEST BENGAL, INDIA

1 Introduction Over the last few decades, smart biopolymers are gaining popularity to meet the demand for more versatile and dynamic material properties. These materials can respond to external stimuli or their environment (Langer and Tirrell, 2004). Smart polymers are also called stimuli-responsive polymers, environmental-sensitive polymers, or intelligent polymers. They can rapidly undergo changes in their microstructure from a hydrophilic to a hydrophobic state, triggered by small changes in their environment. These changes are reversible in nature, that is, the system is capable of returning to its initial state when the stimuli are removed (Kumar et al., 2007). The stimuli that drive these changes can be categorized into physical (light, temperature, electric fields, pressure, sound, magnetic fields) and chemical (pH, ions) stimuli (Qiu and Park, 2001). A plethora of naturally occurring polymers are available that can change form and/or function in response to changes in environment. Among them, polysaccharides constitute the most common and important class of molecules that exhibit smart behavior (Cascone et al., 2001). Due to the presence of various reactive groups in their structure, polysaccharides can undergo modification, giving them a wide and interesting application spectrum. In nature, polysaccharides can be obtained from various resources of algal origin (eg, alginate), plant origin (eg, pectin, guar gum), microbial origin (eg, dextran, xanthan gum), and animal origin (eg, chitosan, chondroitin) (Sinha and Kumria, 2001). Polysaccharides possess a wide range of molecular weight and varying chemical composition, which contribute to their diversity in structure and in property. Moreover, polysaccharides are highly stable, nontoxic, hydrophilic, and biodegradable natural biomaterials (Liu et al., 2008). Owing to the presence of hydrophilic groups in their structure, such as hydroxyl, carboxyl, and amino groups, polysaccharides can enhance bioadhesion by forming noncovalent bonds with biological tissues, mainly epithelia and mucous membranes (Lee et al., 2000). Further, easy production of polysaccharides makes them cheaper than synthetic polymers (Coviello et al., 2007). Therefore, polysaccharides can be considered promising biomaterials for the design of drug-delivery vehicles. Recently, polysaccharides, either in their native or modified forms, are being investigated as smart biomaterial for the fabrication of nanoparticulate drug-delivery systems. The Nanoarchitectonics for Smart Delivery and Drug Targeting. http://dx.doi.org/10.1016/B978-0-323-47347-7.00003-3 Copyright © 2016 Elsevier Inc. All rights reserved.

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FIGURE 3.1  Bioactives-loaded polysaccharide nanostructures. (a) nanospheres (matrix system); (b) nanocapsules (inner matrix surrounded by polymer membrane); and (c) polysaccharide-based micelles.

size of these colloidal polymer particles ranges between 10 and 1000 nm. The nanoparticles are called either nanospheres or nanocapsules, depending on their structural features. Briefly, the nanospheres are matrix systems containing drug; whereas the nanocapsules are vesicular systems in which the drug-rich cavity is surrounded by a polymeric membrane. These nanostructures possess different credentials for the encapsulation and delivery of therapeutic agents (Barratt, 2000). The nanostructures are depicted in Fig. 3.1. Polymeric materials used for preparing nanoparticles for drug delivery must be biocompatible at least and biodegradable best. Among natural polymeric particles, proteins and polysaccharides tend to internalize and degrade rapidly, thus enabling a moderate intracellular release of bioactive molecules. The nanoparticles have been investigated so far may fall into any of these three categories: (1) a passive stealth nanosystem that avoids their uptake by phagocytic blood cells and localizes into the target site, but they do not have specific recognition of the targets; (2) functionalized or ligand-directed nanosystems that allow molecular recognition of the target tissue or for active or triggered release of the payload at the disease site. The stimuli-responsive systems belong to this category; and (3) nanovectors to overcome natural barriers that the vector needs to bypass to efficiently deliver the drug to the target site (Martínez et al., 2012). The stimuli-responsive nanocarriers offer tremendous promise in target-specific delivery of bioactives in the body. The smart carriers offer benefits when the stimuli are unique to disease pathology, allowing the nanocarrier to respond specifically to the pathological triggers (Ganta et al., 2008). This chapter explores the natural occurrence of various polysaccharides and the structural basis of smartness with respect to the recognized stimulus to which they respond. This is followed by a discussion of the fabrication methodologies of nanodevices using smart polysaccharides. The latest applications of the smart behavior of nanopolysaccharides are discussed herein.

2  Structural Basis for Smartness of Nanopolysaccharides 2.1 Chitosan Chitin is a hydrophilic cationic polyelectrolyte obtained by alkaline N-deacetylation of chitin, isolated from crab and shrimp shells. It has (1–4)-linked 2-amino-2-

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deoxy-β-d-glucopyranose units on its structure (Muzzarelli and Muzzarelli, 2005) (Fig. 3.2a). It possesses free amino groups with pKa value 6.5. The primary amines of chitosan offer beneficial material properties. At low pH (pH < 6.5), amines carry positive charge. The protonated amines make chitosan a cationic polyelectrolyte, which is soluble in water and several dilute organic acids (Rinaudo et al., 1999). When the pH of an aqueous solution of chitosan is raised to near neutrality, all the amino groups are deprotonated and chitosan undergoes progressive transition from a soluble cationic polyelectrolyte to an

FIGURE 3.2  Chemical structures of (a) chitosan, (b) sodium alginate, (c) carrageenan, (d) dextran, and (e) hyaluronic acid.

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insoluble polymer. This transition process renders chitosan a pH-responsive smart polymer. The degree of deacetylation is also an important parameter for its stimuli responsiveness (Sorlier et al., 2001). This biologically derived stimuli-responsive property of chitosan has been widely employed for the formation of nanoparticles. The different functional moieties present in chitosan allow its chemical modification, which confers a wide range of derivatives such as quaternized chitosan (N,N,N-trimethyl chitosan) (Van der Merwe et al., 2004), thiolated chitosan (Bernkop-Schnürch et al., 2004), sugar-bearing chitosan (Park et al., 2003), and carboxyalkyl chitosan (Wang et al., 2008). Such chemical modification imparts amphiphilic character to chitosan which is very crucial for the fabrication of self-assembled nanoparticles. Recently, coating the surface of drug-loaded chitosan nanoparticles with targeting moieties has also been explored (Dufes et al., 2004). Besides the endowment of positive charge on the nanoparticles’ surfaces, chitosan also increases the adhesion time with intestinal epithelium, and thus the paracellular permeation is facilitated (Schipper et al., 1997). Furthermore, chitosan undergoes digestion by chitosanase enzymes secreted by luminal microorganisms after oral administration (Nagpal et al., 2010).

2.2 Alginates Sodium alginate is a natural hydrophilic polysaccharide derived isolated from marine brown algae. It has been widely investigated in the field of drug delivery due to its biocompatible and biodegradable nature. It is an anionic polyelectrolyte with a backbone of (1–4) linked β-d-mannuronic acid (M units) and α-l-guluronic acid (G units). It can form irregular patterns of GG, MG, and MM blocks (Fig. 3.2b). The G-blocks of alginate are known to participate in intermolecular crosslinking with divalent calcium ions. Thus, the mechanical properties of alginate gels are enhanced by increasing the length of its G-block and molecular weight (George and Abraham, 2006). Ca2+ is most frequently employed for alginate gel formation. Therefore, alginates can be considered as calcium-responsive polymers. Such a peculiar nature of alginate has widely been exploited in the field of nanomedicine. The release of drugs from alginate gels crosslinked with Ca2+ depends on pH of the medium and the solubility of drug. Because of their low viscosity, high M-rich alginate nanocapsules have found their application in the design of implantable biohybrid organs and in cell transplantation. Furthermore, a large number of free hydroxyl and carboxyl groups are distributed along the alginate backbone. These are highly reactive and amenable for chemical modifications. Thus, the properties like solubility, hydrophobicity and biological characteristics may be altered to have a lot of potential applications of its derivatives. The modifications are accomplished by various chemical processes including oxidation, sulfation, esterification, amidation, and graft copolymerization (Yang et al., 2011). The alginate nanodevices have been studied extensively in order to control the release of drugs by varying drug–polymer interaction, and chemical immobilization of drug in alginate polymer matrix (Nair and

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Laurencin, 2007). Furthermore, its mucoadhesive property can be utilized for the preparation of controlled drug-delivery systems in order to achieve an enhanced drug bioavailability (Pandey and Ahmad, 2011).

2.3 Carrageenan Carrageenans are marine polysaccharides obtained by extraction from Chondrus crispus and Gigartina stellata resources. It is composed of repeating galactose residues (Fig. 3.2c). Three primary classes of carrageenans are available depending on the number and position of the ester sulfate groups, termed kappa-, iota-, and lambda-carrageenan. In k- and ι-carrageenans, the α-d-galactopyranose residue is present in 3, 6-anhydro form. The ι-carrageenan possess an additional sulfo group in 2-position of the anhydrogalactose residue or it can be substituted for α-d-galactose-6-sulfate or -2, 6-disulfate. γ-carrageenan is basically a nongelling linear polysaccharide and does not contain 3, 6-anhydro-α-dgalactopyranose residues. Among them, γ-carrageenan is the most sulfated form of polysaccharides. Sulfate groups generally exist in the 4 position of β-d-galactopyranose residue and in 2- and 6-positions of α-d-galactopyranose residue. The ι-carrageenan carries two and λ-carrageenan has on an average 2.7 charges per disaccharide unit. ι-carrageenan produces dry and elastic form of gels, whereas, λ-carrageenan forms a pseudoplastic form of gels in water (Shchipunov, 2003). k-carrageenan forms the strongest gels involving a coil to helix conformational transition followed by helix aggregation. The gel melts on heating and resets on cooling. This process is, therefore, thermoreversible and is responsive to monovalent potassium ions (Daniel-da-Silva et al., 2011). Other monovalent cations such as Rb, Cs, and NH+4 also promote the formation of k-carrageenan gels. k-carrageenan possess only one negative charge per disaccaharide unit which is essential for the formation of strong and rigid gel (Park et al., 2001). It has been reported that stronger k-CG gels were formed in presence of KCl compared to other salts such as LiCl, NaCl, MgCl2, CaCl2, and SrCl2 (Kara et al., 2006). The thermoresponsive nature of carrageenans makes them particularly attractive candidates for the formulation of hydrogel nanoparticles (also referred to as nanogels). An aqueous k-CG gel displays an undesirable large extent of syneresis. Moreover, k-CG is highly susceptible to microbial attack (Mishra et al., 2010). Thus chemical modifications have been proposed to modulate its physicochemical properties. Specific targeting, swelling, and bioactive properties of CG can be achieved by grafting hydroxyl groups of CG depending upon the need for drug delivery.

2.4 Dextran Dextran is synthesized by a wide variety of bacterial strains including Leuconostoc mesenteroides, Gluconobacter oxydans, Streptococcus mutans (Heinze et al., 2006). Dextran ­sulfate is another biocompatible, polyanionic, and highly branched polysaccharide possessing 1→6 and 1→4 glycosidic linkage with approximately 2.3 sulfate groups per glucosyl unit (Fig. 3.2d). Dextran, consisting of mainly α (1→6) linked d-glucose units, is the

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most important commercially available polysaccharide produced by bacterial strains (Sun and Mao, 2012). Synthetic glycopolymers bearing diverse pendant saccharide chains could significantly expand their biomedical applications due to their biorecognition properties. Grafted ­polymer chains can undergo transition from hydrophilic to hydrophobic by varying temperature. Till now, a number of dextran-grafted, temperature-responsive segments have been reported with potential applications as drug nanocarriers. These include ­dextran–PMAA–PNIPAM-iron oxide (Feng et al., 2012); cyclic or branched dextran-poly(N-­ isopropylacrylamide) (Otsuka et al., 2012); and dextran-g-poly(N-isopropylacrylamide) copolymers (Patrizi et al., 2009). Different hydrophobic molecules such as aromatic rings, aliphatic or cyclic hydrocarbons can be conjugated to the hydroxyl groups of native dextran. Various amphiphilic dextran derivates with different degrees of substitution are thus obtained (Rotureau et al., 2004). Its application is not limited to the synthesis of nanocarrier systems. It is been employed to coat materials for different nanocarrier systems. Due to its biocompatibility and biodegradability, this biomaterial has drawn the attention of several researchers to evaluate its potential as a nanocarrier for controlled drug delivery.

2.5  Hyaluronic Acid Currently, hyaluronic acid (HA) is commercially produced from animal tissues such as cock’s comb and from microbial fermentations. This naturally occurring mucopolysaccharide is also known as sodium hyaluronic or hyaluronan. It is made up of d-glucuronic acid and N-acetyl d-glucosamine (NAG) disaccharide units interconnected alternately with β (1→4) glycosidic linkage (Ossipov, 2010) (Fig. 3.2e). The major portion of HA in the tissue is taken up and degraded in lymphatic systems. The degraded HA enters the blood, transported to the liver and catabolized. Circulating HA is efficiently sequestered by the receptor-mediated endocytosis to liver endothelial cells. Internalization of HA takes less than a minute. HA degrades to glucuronic acid and Nacetylglucosamine in lysosomes. Finally, they are metabolized by hepatocytes to CO2, H2O, and urea. The physiological turnover of HA is remarkably rapid. The half-life of injected HA in plasma is between 2.5 and 5.5 min. Despite successful commercialization of HA, its short half-life must be overcome for long-term clinical applications. In order to elongate the residence time of HA in the body, carboxyl and hydroxyl groups have been modified via esterification (Campoccia et  al.,  1998), carbodiimide reaction (Kuo et  al.,  1991), and dialdehyde crosslinking (Luo et al., 2000). Because carboxyl groups of HA are the recognition sites for HA receptors and hyaluronidase (Banerji et al., 2007), the chemical modification of HA–COOH would change its biological behaviors in the body. As an example, it was reported that enzymatic degradation of HA derivatives was delayed with increasing degree of HA modification (Zhong et al., 1994). The pendant chains of HA can be decorated with various functional groups. In order to introduce amine groups, adipic acid dihydrazide, hexamethylenediamine, or

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cystamine can be grafted onto HA by the conjugation reaction with its carboxyl groups (Kim et al., 2008). HA is a highly anionic biopolymer present in the extracellular matrix and synovial fluids. It is biodegradable, nontoxic, nonimmunogenic, and noninflammatory, which makes it an ideal carrier polymer for systemic drug-delivery applications (Han et al., 2009). The carboxyl, primary and secondary hydroxyl, and N-acetyl functional groups of HA are amenable to chemical modification (Burdick and Prestwich, 2011). Many tumors overexpress hyaluronan CD44 and RHAMM receptors. Hence, hyaluronan can target the chemotherapeutic agents to cancerous tissues (Yip et al., 2006). HA as drug carriers offers several advantages like negligible interaction with serum components, and efficacious delivery to the liver tissues due to polyanionic characteristics and presence of HA receptors, respectively (Zhou et al., 2003). More recently, HA is being considered as new biomaterials for tissue engineering and regenerative medicine. The negative charge characteristics of HA have been exploited for shielding the positive charge of polycations or cationic nanoparticles. HA-g-diethylaminopropyl nanoparticles have been reported for their pH and enzyme sensitive properties (Kim et al., 2014). By using pentenoate-modified HA nanocarriers, the ability to precisely control the mechanical and swelling properties of HA nanogels have been fruitful by radical coupling of poly(ethylene glycol)-bis(thiol) to hyaluronic acid-pentenoate (Hachet et al., 2014). Therefore, modified hyaluronic acid has been known to possess pH, enzyme and redox responsive potential for the development of nanoscale drug-delivery systems.

3  Fabrication of Polysaccharide Nanostructures Based on structural features of the polysaccharides and literature survey, the following methods have been used for the preparation of nanoparticles.

3.1  Covalent Crosslinking Method Crosslinkers having at least two reactive functional groups allow the formation of bridges between polymeric chains. Dialdehydes such as glyoxal and glutaraldehyde are commonly used crosslinkers for the fabrication of polysaccharide nanoparticles. For example, the aldehyde groups form covalent imine bonds with the amino groups of chitosan, due to the resonance generated with adjacent double ethylenic bonds, mediated via Schiff reaction (Figs. 3.3 and 3.4). However, the cytotoxic nature of glutaraldehyde on cell viability restricts its utilization in drug-delivery systems to a large extent. Recently, some biocompatible crosslinkers, natural di- and tricarboxylic acids including succinic acid, malic acid, tartaric acid, and citric acid have replaced glutaraldehyde for the preparation of intermolecular crosslinked chitosan nanoparticles (Bodnár et al., 2005). This method involves the carbodiimide reaction between pendant amino groups of chitosan and carboxylic groups of natural acids. The nanoparticles with size range of 270–370 nm can be attained by this method. Covalently

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FIGURE 3.3  Covalent crosslinking mechanism of polysaccharide chains in presence of glutaraldehyde crosslinker.

FIGURE 3.4  Schematic representation of imine linkage formation between amine group of chitosan and glutaraldehyde.

crosslinked chitosan nanoparticles developed via reaction between amino groups of the chitosan chain with polyethylene glycol dicarboxylic acid are available in the art (Bodnár et al., 2006). Other condensing agents such as genipin and oxalic acid have also been used to prepare covalently crosslinked networks with chitosan (Hirano et  al.,  1990; Mi et al., 2000). Several other polysaccharides such as alginate, guar gum, and hyaluronic acid are also known to form nanoparticles by the covalent crosslinking method (Bodnár et al., 2009; Martínez et al., 2011).

3.2  Ionic Crosslinking Method Ionic crosslinking is generally accomplished in aqueous media and does not require organic solvents. The charged polysaccharides require the incorporation of low molecular weight polyanions and polycations which could act as ionic crosslinkers for polycationic and polyanionic polysaccharides, respectively. Chitosan is the only naturally existing

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FIGURE 3.5  Scheme for the preparation of divalent metal ion-mediated crosslinked alginate polysaccharide nanoparticles.

cationic polysaccharide in acidic media which reacts with negatively charged components leading to the formation of a network through ionic bridges between polymeric chains, thus forming ionic crosslinked nanoparticles. The most commonly used polyanion crosslinker for chitosan are phosphate-bearing groups, such as β-glycerophosphate and particularly tripolyphosphate (TPP) (Sun and Wan, 2007; Janes et al., 2001). TPP crosslinks with hydroxyl groups of the sugar moiety and forms phosphodiester bonds between two polysaccharide chains. TPP has been used as a crosslinker to obtain mainly chitosan and hyaluronic acid-based nanoparticles. The negatively charged polysaccharides such as sodium alginate, sodium carboxymethyl derivatives of polysaccharides could form nanoparticles in presence of divalent metal ions (Ca+2) by ionic interaction with carboxylic groups on molecular chains of polysaccharide structure (Fig. 3.5). A Ca+2-crosslinked alginate nanoparticulate system by water-in-oil reverse microemulsion/ionic gelation method has also been proposed by Reis et al. (2007).

3.3  Gelation of Emulsion Droplets Polysaccharide nanoparticles are generally obtained by gelation of the polymer dissolved in the water-in-oil emulsion droplets. This method can be applied to water-­soluble polysaccharides exhibiting gelling properties (Vauthier and Couvreur, 2000). Alginate and pectin nanoparticles can be obtained by using a modified emulsification/internal gelation method (Opanasopit et al., 2008). The size of alginate particles depends on the order of counter-ion addition to the polymer solution (Fig. 3.6).

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FIGURE 3.6  Diagrammatic presentation for the production of polysaccharide nanoparticles by emulsion-gelation method.

Several studies revealed that polyelectrolyte complexation, an extra step in this procedure, could be advantageous in achieving better control over the size distribution of nanoparticles. Dextran or chitosan polyelectrolytes can be used for in situ gelation of ­nanoemulsion droplets (Reis et al., 2007). In the case of alginate and pectin, nanogels can be obtained by adding a second component or by modifying the pH of polymer solution. Here, two different nonstable o/w emulsions are prepared by mechanical stirring and then sonicated. The first emulsion contains the gelling polymer in dispersed phase; while the other contains gelling agent or pH controlling agent in the dispersed phase. Both emulsions are mixed together under strong agitation to enhance collisions between droplets which are required to induce gelation of the polymer, thereby forming nanoparticles. In this case, this technique is applied to water-soluble polymers and it allows the formation of hydrogel nanoparticles for the encapsulation of hydrophilic drugs such as insulin (Reis et al., 2008). Tokumitsu et al. (1999) described the coalescence phenomenon of the emulsion droplets for the production of chitosan nanoparticles. An emulsion system is prepared by dispersing aqueous chitosan/drug solution in liquid paraffin containing surfactant. The second emulsion system containing aqueous sodium hydroxide solution is then mixed with the previous emulsion under high-speed stirring. The droplets of each emulsion collide in a random manner, coalesce, and precipitate chitosan droplets in the form of nanoparticles.

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3.4  Self-Aggregation Process In this process, an amphiphilic copolymer is designed by conjugating hydrophobic polymer chains to the backbone of hydrophilic polysaccharides by suitable chemical reactions. The copolymer assembles in water and forms core–shell nanostructures. The inner core consists of hydrophobic portion, which encapsulates the poorly water-soluble drug, whereas the hydrophilic outer shell protects the drug from aqueous environment and stabilizes the polymeric micelles against recognition in vivo by the reticuloendothelial system (RES) (Kwon and Okano, 1996). The hydrophobic moieties that have been used to form micellar systems include poly(llysine), poly(aspartic acid), poly(ε-caprolactone) and poly(lactic acid). Poly(ethylene glycol) due to its highly hydrophilic and nontoxic nature has been widely employed in the pharmaceutical and biomedical fields. It is free from antigenic and immunogenic properties and can easily be modified by chemical modification. The synthesis of poly(ethylene glycol)-g-chitosan nanoparticles has been reported by several workers (Yang et al., 2008; Yoksan et al., 2004; Ouchi et al., 1998). Chitosan, dextran, and heparin can be successfully grafted with these hydrophobic moieties to obtain nanosized micelles. Chitosan nanoparticles obtained through attachment of these polyacrylic groups were less than 35 nm (­Passirani et al., 1998; Bertholon et al., 2006).

3.5  Nanoprecipitation Method Nanoprecipitation is one of the widely employed methods for the preparation of polysaccharide nanoparticles due to its more facile nature, simplicity, reproducibility, and less energy consuming properties. Essential prerequisites for this technique are: polymer, solvent, and nonsolvent for the polymer. This technique relies upon the controlled mixing of polymer solution with a nonsolvent. The rapid diffusion of the polymer solution into the nonsolvent induces instantaneous formation of nanoparticles (Fessi et al., 1989). The polysaccharide derivatives used to form nanoparticles by this method are pullulan acetate, alkyl- and phenyl-dextran ethers, and multiple functionalized dextran derivatives (Jeong et al., 1999; Aumelas et al., 2007; Hornig and Heinze, 2007). PLA-grafted dextran copolymer was synthesized using the nanoprecipitation method. It leads to the formation of small nanoparticles, with high yield and improved colloidal stability even without any stabilizer in the aqueous phase (Gavory et al., 2011). Chitosan nanoparticles can also be produced by nanoprecipitation method. Sodium sulfate is generally used to induce precipitation of chitosan polymer in the form of nanoparticles. With the gradual addition of this inorganic salt into a solution of chitosan and polysorbate 80, the desolvated nanoparticles are produced under continuous stirring and ultrasonication (Berthold et al., 1996). Alginate nanoparticles were prepared using a desolvation method. Alginate powder was added to distilled water. Acetone was added continuously or intermittently into 1% alginate solution at pH 4 under stirring at 700 rpm at room temperature until the solution became just turbid. In continuous addition method acetone was added continuously in the solution

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with rate addition about 1–2 mL/min and for intermittent method 2 mL of acetone was added for every 5 min interval (Azarmi et al., 2006; Sailaja and Amareshwar, 2012).

4  Polysaccharide Nanostructures in the Delivery of Therapeutics 4.1  Chitosan-Based Nanocarriers One of the most widely studied biopolymers in the field of drug delivery and biomedical applications is chitosan. Some of the interesting literature on chitosan nanocarriers is described herein. A chitosan-deoxycholic acid-plasmid DNA self-aggregates liberated DNA from the complex above pH 8.0 since chitosan has a pKb value of 7.7 and the amino groups in chitosan almost remain in unionized form above pH 8.0 (Lee et al., 1998). The modified pH-responsive chitosan particles can act as gene-delivery carrier. Hu et al. (2002) studied in vitro silk peptide (SP) release from chitosan (CS)–poly(acrylic acid) (PAA) complex nanoparticles. The release behavior was influenced by pH of the medium and was continued over 10 days. Under stronger acidic conditions, such as pH < 4.0, most carboxylic groups of PAA are in the form of COOH. The interaction between NH+3 and COO− in the CS–PAA nanoparticles could be disrupted by the acid, which led to chain stretch of CS and PAA. Therefore, the CS–PAA nanoparticles dissolved quickly. The nanoparticles were stable under acidic and neutral conditions (pH 4.0–8.0), and aggregated at pH > 9.0. Due to their pH-sensitive behavior, CS–PAA nanoparticles were appropriate carriers for the delivery of drugs in the gastric cavity. The release of epirubicin from cholesterol-modified chitosan nanoparticles was much faster in acetate buffer (pH 3.5) than in PBS (pH 7.4). The self-aggregated nanoparticles exhibited pH-sensitive behaviors because of the presence of many amino groups in chitosan molecules (Wang et al., 2007). Smart magnetic nanoparticles of chitosan-g-poly(N-isopropylacrylamide-co-N, N-dimethylacrylamide) copolymer responded to changes in external temperature or pH, with characteristics of longer circulation and reduced side effects (Yuan et al., 2008). The system exhibited lower DOX release at temperatures lower than LCST (20°C) and a higher, rapid release at temperature greater than LCST (40°C) during the first 1–4 h, followed by a near-sustained release at longer durations. Chitosan-O-PEG-CONH-Tat peptide nanoparticles (5 nm) were developed by the TPP crosslinking method to deliver siRNA in neuronal cells (Neuro 2a), with no/minimal toxicity. The nanoparticles were tested against Ataxin-1 gene in an in vitro established model of a neurodegenerative spinocerebellar ataxia (SCA1) overexpressing ataxin protein. The results indicated successful suppression of the SCA1 protein following 48 h of transfection and thus showed potential in treating neurodegenerative diseases like SCA, Parkinsons, Alzheimers and others (Malhotra et al., 2013). Recently, redox stimuli-responsive poly(ε-caprolactone) (PCL)-chitosan nanoscale particles have been developed by Guerry et al. (2014) for the delivery of an anticancer

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drug, doxorubicin. The PCL-g-chitosan copolymer assumed micellar structures in water (<20 nm) in such a way that chitosan constituted the shell and PCL formed the core of the micellar structures. This core was further crosslinked with bis-alkynyl cystamine for the design of redox-sensitive nanoparticles. The drug-filled nanoparticles lost their crosslinked structures gradually and effectively in the presence of glutathione (reductive environments as found in cytoplasm and cell nucleus) to liberate doxorubicin. This finding can be exploited for selective delivery of the anticancer agents, in response to intracellular concentration of glutathione. Another group of workers tested pH and oxidation-sensitive chitosan nanoparticles for the delivery of 5-fluorouracil (Xu et al., 2014). Ferrocene-modified chitosan oligosaccharide nanoparticles (∼238 nm) disassembled faster and released the drug molecules due to the synergistic effect of oxidative agent (NaClO, H2O2, and oxygen) and low pH 3.8. The sample under bubbled air had a higher percentage of drug release, up to 59.64%, compared with samples under bubbled N2, at just 49.02%. Chitosan (CS)-g-polylactide-citric acid (PLACA) nanoparticles were prepared by polyelectrolyte complexation method (PEC) applying dextran sulfate as a polyanion (Martino and Sedlarik, 2014). An aqueous solution of dextran sulfate was added dropwise under vigorous stirring to the mixture containing doxorubicin and temozolomide. The copolymer was dissolved in CH3COOH aqueous solution (pH 3.5). The mixture containing dextran sulfate and drugs was added dropwise to PLACA solution under vigorous stirring and slight heating to obtain nanoparticles (150–300 nm). A subtle change in temperature can cause various rearrangements of the polymer chains, influencing the entire system, including the release kinetic of the present bioactive compound. Increasing the temperature causes an upward trend in particle size. Reduction in diameter was observed above critical temperature, probably due to a reduction in hydrogen bonds among CS chains and water molecules, causing a deswelling in the system. Systems containing CS-g-PLACA showed critical temperature in the range 20–30°C. The presence of COO− influences the thermal properties of the carrier, as they can interact with the free amino groups and avoid the formation of a hydrogen bond with the water molecules in the release environment. Due to electrostatic interactions between the COO− groups and the positive-charged molecules of the drug there is slowed diffusion towards the surface of the nanoparticles and, subsequently, in the media. Moreover, the absence of an initial burst indicated that the whole amount of drug was located inside the structure of nanoparticles, meaning that it was protected from the outer environment. The presence of COOH groups on PLA-side chains influenced the dimension and temperature behavior of the particles, as well as the encapsulation efficiency and release rate of the drugs. Chitosan-coated magnetic nanoparticles (CS MNPs) exhibited a pH-dependent targeting of drugs to the tumor site under a magnetic field. The cellular internalization of doxorubicin-loaded CS MNPs were visualized on MCF-7 (MCF-7/S). Consequently, ­ CS MNPs synthesized at various sizes (58–103 nm) can be effectively used for the pH-­ dependent release of doxorubicin. In cancer cells, chitosan-coated magnetic iron oxide nanoparticles (CS MNPs) were synthesized by coprecipitation of Fe(II) and Fe(III) salts in

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the presence of chitosan and tripolyphosphate (TPP) molecules. Doxorubicin release was investigated at different pH values (pH 4.2 and 5.0) which mimic endosomal conditions. A burst release of doxorubicin from CS MNPs was observed at pH 4.2 as 20–30% and at pH 5.0 as 15–20% in 30 min. Then, a slower release was evident after 7 h (Unsoy et al., 2014). Ibuprofen completely released within 12 h from chitosan particles, however the release was not completed even after 48 h from TPP-PEGylated–chitosan nanoparticles in pH 7.4 buffer solution. This was related to the hydrophobic–hydrophobic interactions between drug and PEGylated chitosan that did not occur for chitosan nanoparticles. Therefore, the nanoparticles could be useful for colon-targeted drug delivery (Najafabadi et al., 2014). Doxorubicin (DOX)-loaded nanoparticles of 166 nm size were explored for leukemia therapy (Termsarasa et al., 2014). PEG was conjugated to the backbone of chitosan ­oligosaccharide-arachidic acid (CSOAA) copolymer via amide linkage. CSOAA-PEG nanoparticles sustained the release of DOX at physiological pH. However, the drug-release rate was relatively faster at acidic pH. The cytotoxicity of the conjugate was almost negligible in human leukemia cells (K562) at 100 µg/mL concentration level. The PEGylated CSOAA-based nanoparticles could be beneficial for the treatment of blood malignancies. The poly(l-malic acid-co-d,l-lactic acid) (PML) copolymer nanoparticles were synthesized by complexation with chitosan (CS). The high doxorubicin-loading efficiency (16.5%) and the sustained release patterns in acidic media and faster drug release in alkaline solution encouraged the probable application of nanoparticles (316–590 nm) as a pH-sensitive, controlled drug-release system (Wang et al., 2014). Chitosan nanoparticles immediately released siRNA in plasma while the inclusion of hyaluronic acid and poly(ethylene glycol) in the formulation afforded stability to the particles without altering their biological activity. PEGylated chitosan-based nanoparticles created new perspectives for future gene-­ silencing treatments (Ragelle et al., 2014). Thiolated chitosan-TPP nanoparticles (<300 nm) had pronounced effects on paracellular permeability via mucus-free Caco-2 layers and thiolation enhanced the permeation of FD4 3.0- to 5.3-fold (Dünnhaupt et al., 2015). The chitosan-alginate (CS/ALG) nanoparticles (100–200 nm) retained almost 100% encapsulated insulin in simulated gastric fluid; but slowly liberated their content in simulated intestinal fluid. The relative oral bioavailability of insulin improved in mice models (8.11%) with consequent improvement of hypoglycemic effects. Thus, the efficacy of pH-sensitive CS/ALG core-shell nanoparticles was noteworthy for oral insulin delivery (Mukhopadhyay et al., 2015). Nagpal et al. (2015) explored the central antinociceptive activity of brain targeted tween 80-chitosan nanoparticles (NP) containing minocycline hydrochloride (MH). Their average particle size, and drug entrapment efficiency was found to be 164.50 ± 11.00 nm and 82.74 ± 1.42% respectively. The data retrieved on animal models suggested that the nanoparticles may be utilized for effective brain targeting of MH. Tat peptide derived from the transactivator of transcription (Tat) of human immunodeficiency virus is a cell-penetrating peptide. Tat-tagged and folate-modified N-­ succinylchitosan (Tat-Suc-FA) self-assembled nanoparticles were investigated as a new vector for tumor gene therapy (Yan et al., 2015). Particle sizes of Tat-Suc-FA/DNA complexes

Chapter 3 • Smart Nanopolysaccharides for the Delivery of Bioactives  81

were between 54 and 106 nm. As a gene-transfer vector, it should first interact with DNA to form a stable complex to prevent the gene from DNase degradation, which is critical in DNA transportation and in final gene expression. The formation of polymer/DNA complex is an important prerequisite for gene delivery using cationic polymers. Tat-Suc-FA polymers have higher cytotoxicity to K562 cells than Suc but have lower cytotoxicity than chitosan. It is well known that the cytotoxicity of chitosan arises from its amine groups, which lead it to high positively charged in aqueous solutions. However, the grafting of Tat peptide onto Suc introduced positively charged groups. Therefore, Tat-Suc-FA polymers showed some cell cytotoxicity. But, almost at all tested concentrations (2–500 µg/mL), its cytotoxicity is much lower than chitosan.

4.2  Alginate-Based Nanosystems Drug-delivery carriers have attracted a lot of interest during the past decades, since they can deliver low-molecular-weight drugs, as well as large biomacromolecules such as proteins and genes, either in a localized or in a targeted manner. Alginate has been widely adopted as a carrier to encapsulate drugs, bioactive molecules, proteins, and cells, for its biocompatible and biodegradable nature. To date, a number of researchers have studied alginate-based nanocarriers systems for controlled drug delivery. Calcium alginate nanoparticles have been studied to a small extent. Goswami et al. (2014) pointed out the fact that in harsh acidic environment (pH 1.2) the insulin remains chemically stable and suitable in nanoparticles (40 nm) for oral delivery. In another study, in situ nanoemulsification–polymer crosslinking method was used for the preparation of alginate nanoparticles for the delivery of an anti-HIV drug lopinavir (Angshuman et al., 2010). The pharmacokinetics data in mice showed higher blood concentrations and prolonged residence time of lopinavir nanoparticles (127–336 nm). The particles released only 50% drug in 12 h in pH 7.4 buffer solution. The relatively high brain concentration of lopinavir nanoparticles suggests that this system could be an alternative to the treatment of brain malignancies. The smaller dimension of lopinavir particles in conjunction with its prolonged blood circulating property revealed that they could target lymphatic system through EPR effect. The particles could be a useful targeted drug-delivery system for eradicating the viral sanctuaries in patients infected with HIV-1/AIDS. You and Peng (2005) also studied the alginate nanoparticles (55–100 nm) for nonviral gene delivery into nonphagocytic cells via endocytosis and high transfection efficiency of NIH 3T3 cells was noted. Most of the workers focused their nanosystems based on polyelctrolyte complexation between negatively charged sodium alginate and positively charged chitosan. Their reports are summarized in Table 3.1. In addition to chitosan, other oppositely charged polyelectrolytes have also been utilized for the development of alginate nanoparticles. These include alginate-poly-l-lysine systems for diphtheria toxoid vaccine delivery (Sarei et al., 2013); sustained antifungal activity (Sangeetha et al., 2007), and alginate-PLGA nanosystems (90 nm) by solvent diffusion method for sustained antimicrobial activity of streptomycin (Asadi, 2014).

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Table 3.1  Alginate-Chitosan Polyelectrolyte Complex Nanostructures and Their Potential in the Delivery of Bioactives Therapeutic Agents Rifampicin, isoniazid, pyrazinamide, ethambutol

Size (nm)/DEE (%)/Release of Bioactives 70–90%

Insulin

750 nm, >70%, 50% in 2 h in pH 1.2 acid solution followed by pH 6.8 buffer for 6 h releasing <60% of encapsulated protein Gatifloxacin 205–572 nm, fast release during 1 h followed by gradual drug release over 24 h Nifedipine 20-50 nm, 26.52% at pH 1.5, 69.69% at pH 6.8 and 56.50% at pH 7.4 within 24 h EGFR phosphorothioated 194 nm, 95.6% 21 mer antisense Curcumin ∼13%, 100 ± 20 nm <80% in 100 h in phosphatebuffered saline solution (pH 7.4) Epidermal growth 196 nm, 95.6%, 58.3 in factor receptor (EGFR) 15 min and 100% in 50 h antisense vector in pH 7.4 Amoxicillin 265–638 nm, 88%, ∼76% drug release over a period of 6 h in pH 1.2 buffer solution Tri-peptide glutathione 301–361 nm, 27% (S-nitroso glutathione) Venlafaxine

173.7 nm, 81.3%, 90% in 25 h in phosphate buffer solution (pH 7.4)

Conclusion

References

Single oral dose resulted in therapeutic Ahmad et al. (2006) drug concentrations in the plasma for 7–11 days and in the organs (lungs, liver, and spleen) for 15 days. In TB-infected mice three oral doses of the formulation spaced 15 days apart resulted in complete bacterial clearance from the organs, compared to 45 conventional doses of orally administered free drugs Sarmento et al. pH-responsive mucoadhesive (2007) nanoparticles improved oral absorption and oral bioactivity of insulin Mucoadhesive nanoparticulate carrier systems can prolong ocular delivery of the drug

Motwani et al. (2008)

pH-responsive release of nifedipine was possible from nanoparticles

Li et al. (2008)

Promising for their application in gene Gazori et al. delivery (2009) Cellular internalization of drug-loaded Das et al. (2010) nanoparticles occurred and thus useful for delivery to cancer cells Nanoparticles may be good carriers for the delivery of antisense oligonucleotides Gastro protective nanoparticles can be utilized for transmucosal delivery of antibacterial drugs in eradication of H. pylori Great potential for use in biomedical applications where NO has a therapeutic effect Nanoparticles delivered greater drug to the brain in comparison to drug solution and opens a new window for the treatment of depression

Azizi et al. (2010)

Arora et al. (2011)

Marcato et al. (2011) Haque et al. (2014)

Chapter 3 • Smart Nanopolysaccharides for the Delivery of Bioactives  83

4.3  Carrageenan Nanoparticles Due to different chemical structure and physical properties, this natural source can be used in the different applications, varying from tissue engineering to the preparation of drug vehicles for controlled release. It was reported that CS/k-CG nanoparticles exhibited noncytotoxic behavior under in vitro tests using L929 fibroblasts, and provided a controlled release for up to 3 weeks with ovalbumin as a model protein, and CS/CG ratio had a significant effect on the properties of the nanoparticles (Grenha et al., 2010). Crosslinked k-carrageenan hydrogel nanoparticles (nanogels) with an average size smaller than 100 nm were prepared by water-in-oil (w/o) microemulsions-thermal gelation method (Daniel-da-Silva et al., 2011). The nanogels were thermosensitive in a temperature range acceptable for living cells (37–45°C). The release rate of a model agent methylene blue increased with increasing temperature and achieved a plateau at 60% at both 25° and 37°C, increasing to 95% at 45°C in PBS. The results suggested that k-carrageenan polysaccharide nanogels can be used as thermosensitive drug carriers. Bulmer et al. (2012) prepared recombinant human erythropoietin-loaded CS–kC nanoparticles by ionotropic gelation process. The particles had drug encapsulation efficiency of 47.97  ±  4.10% and provided sustained in vitro release of ∼50% over a 2-week period. The drug-release behavior of kC-NaCMC nanocomposite hydrogels was studied in solutions of different pH (Hezaveh and Muhamad, 2012, 2012a). The drug release from genipin-crosslinked nanoparticles in pH 1.2 medium was lower than in pH 7.4 medium as a result of the formation of hydrogen bond between the carboxymethyl cellulose due to the existence of carboxylic group (─COOH) at low pH. At higher pH, larger swelling force caused by the electrostatic repulsion between the ionized groups results in more release of a model drug, methylene blue. Genipin crosslinking resulted in formation of smaller nanoparticles since the smaller free space for NPs formation was available. By changing the nanoparticle loading and genipin concentration in the composite, the amount of drug released can be monitored. Magnetite-genipin kC-NaCMC nanocomposite, could release its drug loaded in intestine environment slightly more than kC-NaCMC hydrogels; however, it has still the advantage of less release in stomach environment. A novel triple-stimuli poly(acrylic acid)-grafted-k-carrageenan (kC-g-PAA)-super paramagnetic iron oxide nanoparticles were tested for controlled delivery of defrasirox (Bardajee et al., 2013). The hydrogel was sensitive to different temperature, pH, and magnetic field. kC-g-PAA/SPION hydrogel was nontoxic and biocompatible which could also be useful for biomedical applications.

4.4  Dextran NPs Many efforts have been made to gain insight into the preparation and understanding of the behavior of modified dextran polymer for use as nanocarriers for several drugs. Different authors have reported hydrophobic modification and further nanostructure formation of modified dextran over the last decade.

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Biodegradable hydrogel nanoparticles were synthesized from glycidyl methacrylate dextran and dimethacrylate poly(ethylene glycol). Clonazepam (CNZ) released at a higher rate in presence of dextranase enzyme. This result was attributed to degradation of dextran portion of hydrogel nanomatrices by dextranase. The influence of pH on CNZ release from the nanoparticles was examined at three different pH values. Without enzyme, CNZ dissolved more quickly at lower pH than at higher pH. However, the pH effect on drug release reversed with the presence of dextranase. This result can be explained as follows. The optimum pH of dextranase is between pH 5.0 and 7.0. Accordingly, CNZ release rate was increased more by the enzymatic degradation of dextran chain at pH 6.0 than at 2.0 and 4.0. In vitro drug-release rate from the hydrogel nanoparticles depended on the existence of dextranase and the pH of release medium (Kim et al., 2000). The amount of cyclosporine A (CsA) incorporated in dextran-g-polyoxyethylene (10) cetyl ether micelles (14 ± 6 nm) reached 8.5% (w/w). The polymeric micelles were highly stable in gastric and intestinal fluids and no significant cytotoxicity was noted toward Caco-2 cells. The apical to basal permeability of CsA across Caco-2 cells increased significantly in an entrapped state rather than free CsA (Francis et al., 2005). The core-shell carrier combines the advantages of superparamagnetic Fe3O4 core and the temperature-responsive dextran-g-poly(N-isopropylacrylamide-co-N, N-dimethylacrylamide) shell. The polymer was conjugated with doxorubicin, an anticancer agent. Further, the polymer exhibited a lower critical solution temperature (LCST) at ∼38°C (Zhang and Misra, 2007; Zhang et al., 2007). This phase transition behavior allowed for an on–off trigger mechanism. The drug release was controlled by thermal sensitivity of the polymer and the cleavage of acid-labile hydrazone linkage. This carrier can be explored as a targeted drug-delivery system that has longer circulation time, reduced side effects, and controlled drug-release properties. Polylactide-grafted dextran (Dex-g-PLA) nanoparticles were prepared using the emulsion/solvent evaporation process and reported by Nouvel et al. (2009). Enzymatic degradation of Dex-g-PLA in presence of dextranase enzyme was particularly considered. Its rate of enzymatic hydrolysis was lower than native dextran. However, this copolymer remained degradable despite its low water solubility. While dextran was rapidly degraded in a few hours, the total degradation of Dex-g-PLA took about 6 days. The specific properties of these nanoparticles can be widely employed for the encapsulation of hydrophobic drugs and their release. Dextran decanoate esters with varying degree of substitution were used to form nanoparticles via nanoprecipitation technique (Kaewprapan et al., 2012). Two types of enzyme-catalyzed degradation of nanoparticles were investigated: hydrolysis of polysaccharide backbone in the presence of dextranase and hydrolysis of ester links between glucose units and hydrocarbon tails in the presence of porcine pancreatic lipase. Complete degradation of low modified dextrans (DS ∼ 25%) by dextranase occurred within 7 days. They proposed lidocaine-encapsulated dextran esters nanoparticles as potential drug delivery system. Poly(ε-caprolactone)-gdextran copolymer micellar nanoparticles (Dex-g-PCL) were prepared via W/O emulsionsolvent evaporation method. Amoxicillin-loaded copolymer particles showed two stage drug release process in phosphate buffer (pH 7.2) medium. An initial quick release up to

Chapter 3 • Smart Nanopolysaccharides for the Delivery of Bioactives  85

50 h was followed by a remarkably slower release stage thereafter. The drug entrapment efficiency was 78%. Dex-g-PCL micellar nanoaggregates can be used as potential nanocarriers for drug delivery (Saldías et al., 2015).

4.5  Hyaluronic Acid-Based Conjugates The effect of chemical modification of HA on its application has been reported for target specific and long-acting delivery applications of therapeutics. The latest advances in HAbased drug-delivery systems are reviewed. Amphiphilic HA-5β-cholanic acid conjugates (237–424 nm) were efficiently uptaken intracellular in SCC7 cancer cells by receptor-mediated endocytosis (Choi et al., 2010). Irrespective of particle size, significant amounts of HA-NPs circulated for 2 days in the bloodstream and selectively accumulated in the tumor site. The smaller HA-NPs reached the tumor site more effectively than larger HA-NPs. The concentration of HA-NPs in the tumor site reduced dramatically when mice were pretreated with an excess of free-HA. These results implied that HA-NPs accumulated into the tumor site by a combination of passive and active targeting mechanisms. A major drawback of HA-based drug conjugates or nanoparticles for cancer therapy are their preferential accumulation in the liver after systemic administration. The PEGylated HA-NPs self-assembled into negatively charged nanostructures (217–269 nm) in physiological condition (Choi et al., 2011). PEGylation of the NPs reduced their cellular uptake in vitro. However, cancer cells overexpressing CD44, an HA receptor, took up large amounts of NPs than that of normal fibroblast cells. The nanoparticles indicated their high tumortargeting ability after systemic administration. Interestingly, PEGylated HA-NPs were more effectively accumulated into the tumor tissue up to 1.6-fold higher than bare HA-NPs. Hyaluronic acid-ceramide (HA-CE)-based self-assembled nanoparticles were developed for intravenous docetaxel (DCT) delivery (Cho et al., 2011). DCT-loaded nanoparticles composed of HA-CE and Pluronic 85 (P85) with a mean diameter of 110–140 nm was prepared for intracellular DCT uptake in the CD44-overexpressing cell line (­MCF7). DCT-loaded cyanine 5.5 (Cy5.5)-conjugated nanoparticles efficiently targeted CD44-­ overexpressing tumors in mice models and thus proved its promise as anticancer drugdelivery system. HA (<10 kDa)/CS plasmid–DNA nanoparticles were synthesized through the complex coacervation of the cationic polymers with pEGFP (Lu et al., 2011). Maximum transfection efficiency was noted at pH 6.8 for the HA/CS-plasmid nanoparticles. The cell viability of HA/CS-plasmid nanoparticles was >90%. Thus, HA/CS nanoparticles showed their promise as a nonviral vector for gene delivery to chondrocytes. A redox-sensitive ­hyaluronic acid-deoxycholic acid (HA-ss-DOCA) conjugates has been reported for the targeted intracellular delivery of an anticancer drug, paclitaxel (PTX) (Li et al., 2012). The conjugates formed self-assembled nanoparticles in aqueous media and encapsulated 93.2% PTX. HA-ss-DOCA nanoparticles were stable at simulated gastrointestinal fluids but disassembled quickly in presence of 20 mM reducing agent, glutathione. The redox-responsive HA-ss-DOCA micelles can be used as intracellular carriers of lipophilic anticancer drugs.

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Landesman-Milo et al. (2013) developed hyaluronan (HA) coated lipid-based nanoparticles (LNPs). HA-LNPs bound and internalized specifically into cancer cells. SiRNA-loaded HA-LNPs efficiently and specifically reduced mRNA and P-gp protein levels. In addition, no cellular toxicity or cytokine induction was observed. The HA-LNPs offered an alternative approach to cationic lipid based formulations for RNAi delivery into cancer cells in an efficient and safe manner. Ganesh et al. (2013) screened a series of CD44-targeting, HA-based, self-assembling nanosystems for siRNA delivery. The HA polymer was conjugated with lipids and polyamines for assessing siRNA encapsulation. HA-derivatives encapsulated/com­ plexed siRNAs and self-assembled into nanoparticles. Some of these HA derivatives transfected siRNAs into cancer cells overexpressing CD44 receptors. HA-PEI/PEG-siRNA nanoparticles demonstrated dose-dependent and target-specific gene knockdown in A549 lung ­cancer cells overexpressing CD44 receptors. Liu et al. (2013) described oleoyl-­ carboxymethylchitosan-HA nanoparticles using coacervation process for gene delivery. NPs showed higher in vitro DNA release rates and increased cellular uptake by Caco-2 cells due to the HA involved in NPs. The NPs internalized in Caco-2 cells mediated by hyaluronan receptor CD44. The data gave evidence of the potential of NPs for the targeting and further transfer of genes to the epithelial cells. Flt1 peptide-HA conjugates self-assembled into nanoparticles and the particles reduced cytokine levels of lipopolysaccharide (LPS)-stimulated cells more efficiently than free dexamethasone. The conjugates had remarkable therapeutic effects in both eosinophilic and neutrophilic asthma model mice (Kim et al., 2013). Since most of the anticancer drugs act intracellularly, it becomes necessary to deliver them selectively and effectively inside the cancer cells. To serve this purpose, Shin et al. (2014) designed an amphiphilic hyaluronic acid-dodecanethiol (DDT) conjugate (HA-ss-DDT). The disulfide bond was selectively cleaved at the intracellular environment. The amphiphilic HA-ss-DDT conjugate self-assembled into nanoparticles in an aqueous medium. The nanoparticles showed excellent stability under physiological condition (pH 7.4, 37°C) for 5 days. Doxorubicin (DOX) encapsulation efficiency of the nanoparticles was >70%. The particles slowly released their content in PBS solution (pH 7.4). Glutathione is a peptide abundant in the cytoplasm of the cancer cells. In presence of glutathione (10 mM), the drug delivery rate became faster due to cleavage of the disulfide bond and consequently by disintegration of the nanoparticles. Squamous cell carcinoma (SCC7) cells effectively took up the DOX-loaded nanoparticles via receptor-mediated endocytosis pathway, and caused rapid release of DOX in the cell cytoplasm. Overall, the nanoparticles were efficient for intracellular delivery of DOX. Hyaluronan nanoparticles (HA-NPs) bearing a γ-secretase inhibitor (DAPT) were prepared as potential therapeutics for rheumatoid arthritis (Heo et al., 2014). In a collagen-induced arthritis (CIA) mouse model, DAPT-loaded nanoparticles (DNPs) significantly attenuated the severity of RA induction compared to DAPT alone (2 mg/kg). In addition, DNPs dramatically reduced the production of proinflammatory cytokines and collagen-specific autoantibodies (IgG1 and IgG2a) in serum of CIA mice. These results demonstrated that DNPs could be an effective

Chapter 3 • Smart Nanopolysaccharides for the Delivery of Bioactives  87

therapeutics for rheumatoid arthritis. Novel pH-responsive nanoparticles composed of hyaluronic acid-g-diethylaminopropyl (HA-g-DEAP) were developed by Kim et al. (2014). The degradation kinetics of HA by hyaluronidase enzyme modulated the DOX release rate in diseased cells (endosomal pH 5.0). Biodegradable docetaxel-loaded poly(d,l-lactide-coglycolide)/hyaluronic acid block copolymer nanoparticles (<200 nm) showed a biphasic drug-release pattern during 120 h, and enhanced cytotoxicity toward CD44-­overexpressing MDA-MB-231 cells (Huang et al., 2014). PLGA-b-HA nanoparticles were taken up in MDAMB-231 cells by CD44-mediated endocytosis. The nanoparticles demonstrated enhanced tumor targeting and antitumor activity with lower systemic toxicity.

5 Conclusions Owing to natural occurrence, the polysaccharides are highly stable, safe, nontoxic, hydrophilic, and biodegradable. Due to their peculiar nature, polysaccharides and their derivatives have emerged as one of the most explored smart biomaterials in the field of nanomedicine over the last few decades. Ionic polysaccharides have efficiently substituted synthetic polymers in the design of stimuli-responsive, novel drug-delivery systems. These nanopolysaccharides are endowed with smart characteristics, which show immense potential for targeted and self-regulated drug delivery. The pH sensitivity, ionic character, and redox potential of polysaccharides can be strengthened by grafting ionic polymers to their backbone. Crosslinked networks of natural polysaccharides offer sophisticated and widespread applications in the delivery of bioactive molecules.

References Ahmad, Z., Pandey, R., Sharma, S., Khuller, G.K., 2006. Alginate nanoparticles as antituberculosis drug carriers: formulation development, pharmacokinetics, and therapeutic potential. Indian J. Chest Dis. Allied Sci. 48, 171–176. Angshuman, B., Bhattacharjee, S.K., Rita, M., Maity, B., Bandyopadhayay, S.K., 2010. Alginate-based nanoparticulate drug delivery for anti-HIV drug lopinavir. J. Global Pharm. Technol. 2, 126–132. Arora, S., Gupta, S., Narang, R.K., Budhiraja, R.D., 2011. Amoxicillin loaded chitosan–alginate polyelectrolyte complex nanoparticles as mucopenetrating delivery system for H. Pylori. Sci. Pharm. 79, 673–694. Asadi, A., 2014. Streptomycin-loaded PLGA-alginate nanoparticles: preparation, characterization, and assessment. Appl. Nanosci. 4, 455–460. Aumelas, A., Serrero, A., Durand, A., Dellacherie, E., Leonard, M., 2007. Nanoparticles of hydrophobically modified dextrans as potential drug carrier systems. Colloids Surf. B 59, 74–80. Azarmi, S.H., Huang, Y., Chen, H., McQuarrie, S., Abrams, D., Roa, W., Finlay, W.H., Miller, G.G., Löbenberg, R., 2006. Optimization of a two-step desolvation method for preparing gelatin nanoparticles and cell uptake studies in 143B osteosarcoma cancer cells. J. Pharm. Pharm. Sci. 9, 124–132. Azizi, E., Namazi, A., Haririan, I., Fouladdel, S., Khoshayand, M.R., Shotorbani, P.Y., Nomani, A., Gazori, T., 2010. Release profile and stability evaluation of optimized chitosan/alginate nanoparticles as EGFR antisense vector. Release profile and stability evaluation of optimized chitosan/alginate nanoparticles as EGFR antisense vector. Int. J. Nanomed. 5, 455–461.

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Banerji, S., Wright, A.J., Nobel, M., Mahoney, D.J., Campbell, I.D., Day, A.J., Jackson, D.G., 2007. Structures of the Cd44–hyaluronan complex provide insight into a fundamental carbohydrate–protein interaction. Nat. Struct. Mol. Biol. 14, 234–239. Bardajee, G.R., Hooshyar, Z., Rastgo, F., 2013. Kappa carrageenan-g-poly(acrylic acid)/SPION nanocomposite as a novel stimuli-sensitive drug delivery system. Colloid Polym. Sci. 291, 2791–2803. Barratt, G.M., 2000. Therapeutic applications of colloidal drug carriers. Pharm. Sci. Technol. Today 3, 163–171. Bernkop-Schnürch, A., Guggi, D., Pinter, Y., 2004. Thiolated chitosans: development and in vitro evaluation of a mucoadhesive, permeation enhancing oral drug delivery system. J. Control. Release 94, 177–186. Berthold, A., Cremer, K., Kreuter, J.S.T.P., 1996. Preparation and characterization of chitosan microspheres as drug carrier for prednisolone sodium phosphate as model for anti-inflammatory drugs. J. Control. Release 39, 17–25. Bertholon, I., Lesieur, S., Labarre, D., Besnard, M., Vauthier, C., 2006. Characterization of dextranpoly(isobutylcyanoacrylate) copolymers obtained by redox radical and anionic emulsion polymerization. Macromolecules 39, 3559–3567. Bodnár, M., Daróczi, L., Batta, G., Bakó, J., Hartmann, J.F., Borbély, J., 2009. Preparation and characterization of crosslinked hyaluronan nanoparticles. Colloid Polym. Sci. 287, 991–1000. Bodnár, M., Hartmann, J.F., Borbely, J., 2005. Preparation and characterization of chitosan-based nanoparticles. Biomacromolecules 6, 2521–2527. Bodnár, M., Hartmann, J.F., Borbely, J., 2006. Synthesis and study of cross-linked chitosan-N-poly(ethylene glycol) nanoparticles. Biomacromolecules 7, 3030–3036. Bulmer, C., Margaritis, A., Xenocostas, A., 2012. Encapsulation and controlled release of recombinant human erythropoietin from chitosan–carrageenan nanoparticles. Curr. Drug Deliv. 9, 527–537. Burdick, J.A., Prestwich, G.D., 2011. Hyaluronic acid hydrogels for biomedical applications. Adv. Mater. 23 (12), 41–56. Campoccia, D., Doherty, P., Radice, M., Brun, P., Abatangelo, G., Williams, D.F., 1998. Semisynthetic resorbable materials from hyaluronan esterification. Biomaterials 19, 2101–2127. Cascone, M.G., Barbani, N.P., Giusti, C.C., Ciardelli, G., Lazzeri, L., 2001. Bioartificial polymeric materials based on polysaccharides. J. Biomater. Sci. Polym. Ed. 12, 267–281. Cho, H.-J., Yoon, H.Y., Koo, H., Ko, S.-H., Shim, J.-S., Lee, J.-H., Kim, K., Kwon, I.C., Kim, D.-D., 2011. Self-assembled nanoparticles based on hyaluronic acid-ceramide (HA-CE) and pluronic for tumortargeted delivery of docetaxel. Biomaterials 32, 7181–7190. Choi, K.Y., Chung, H., Min, K.H., Yoon, H.Y., Kim, K., Park, J.H., Kwon, I.C., Jeong, S.Y., 2010. Self-assembled hyaluronic acid nanoparticles for active tumor targeting. Biomaterials 31, 106–114. Choi, K.Y., Min, K.H., Yoon, H.Y., Kim, K., Park, J.H., Kwon, I.C., Choi, K., Jeong, S.Y., 2011. PEGylation of hyaluronic acid nanoparticles improves tumor targetability in vivo. Biomaterials 32, 1880–1889. Coviello, T., Matricardi, P., Marianecci, C., Alhaique, F., 2007. Polysaccharide hydrogels for modified release formulations. J. Control. Release 119, 5–24. Daniel-da-Silva, A.L., Ferreira, L., Gil, A.M., Trindade, T., 2011. Synthesis and swelling behavior of temperature responsive k-carrageenan nanogels. J. Colloid Interface Sci. 355, 512–517. Das, R.K., Kasoju, N., Bora, U., 2010. Encapsulation of curcumin in alginate-chitosan-pluronic composite nanoparticles for delivery to cancer cells. Nanomed. Nanotechnol. Biol. Med. 6, 153–160. Dufes, C., Muller, J.M., Couet, W., Olivier, J.C., Uchegbu, I.F., Schätzlein, A.G., 2004. Anticancer drug delivery with transferrin targeted polymeric chitosan vesicles. Pharm. Res. 21, 101–107. Dünnhaupt, S., Barthelmes, J., Köllner, S., Sakloetsakun, D., Shahnaz, G., Düregger, A., Bernkop-Schnürch, A., 2015. Thiolated nanocarriers for oral delivery of hydrophilic macromolecular drugs. Carbohydr. Polym. 117, 577–584.

Chapter 3 • Smart Nanopolysaccharides for the Delivery of Bioactives  89

Feng, W.Q., Lv, W.P., Qi, J.J., Zhang, G.L., Zhang, F.B., Fan, X.B., 2012. Quadruple-responsive nanocomposite based on dextran–PMAA–PNIPAM, iron oxide nanoparticles, and gold nanorods. Macromol. Rapid Commun. 33, 133–139. Fessi, H., Puisieux, F., Devissaguet, J.P., Ammoury, N., Benita, S., 1989. Nanocapsule formation by interfacial polymer deposition following solvent displacement. Int. J. Pharm. 55, R1–R4. Francis, M.F., Cristea, M., Yang, Y., Winnik, F.M., 2005. Engineering polysaccharide-based polymeric micelles to enhance permeability of cyclosporin A across Caco-2 cells. Pharm. Res. 22, 209–219. Ganesh, S., Iyer, A.K., Morrissey, D.V., Amiji, M.M., 2013. Hyaluronic acid based self-assembling nanosystems for CD44 target mediated siRNA delivery to solid tumors. Biomaterials 34, 3489–3502. Ganta, S., Devalapally, H., Shahiwala, A., Amiji, M., 2008. A review of stimuli-responsive nanocarriers for drug and gene delivery. J. Control. Release 126, 187–204. Gavory, C., Durand, A., Six, J.L., Nouvel, C., Marie, E., Leonard, M., 2011. Polysaccharide-covered nanoparticles prepared by nanoprecipitation. Carbohydr. Polym. 84, 133–140. Gazori, T., Khoshayand, M.R., Azizi, E., Yazdizade, P., Nomani, A., Haririan, I., 2009. Evaluation of alginate/chitosan nanoparticles as antisense delivery vector: formulation, optimization and in vitro characterization. Carbohydr. Polym. 77, 599–606. George, M., Abraham, T.E., 2006. Polyionic hydrocolloids for the intestinal delivery of protein drugs: alginate and chitosan—a review. J. Control. Release 114, 1–14. Goswami, S., Bajpai, J., Bajpai, A.K., 2014. Calcium alginate nanocarriers as possible vehicles for oral delivery of insulin. J. Exp. Nanosci. 9, 337–356. Grenha, A., Gomes, M.E., Rodrigues, M., Santo, V.E., Mano, J.F., Neves, N.M., et al., 2010. Development of new chitosan/carrageenan nanoparticles for drug delivery applications. J. Biomed. Mater. Res. Part A 92A, 1265–1272. Guerry, A., Cottaz, S., Fleury, E., Bernard, J., Halila, S., 2014. Redox-stimuli responsive micelles from DOXencapsulating polycaprolactone-g-chitosan oligosaccharide. Carbohydr. Polym. 112, 746–752. Hachet, E., Sereni, N., Pignot-Paintrand, I., Ravaine, V., Szarpak-Jankowska, A., Auzély-Velty, R., 2014. Thiol-ene clickable hyaluronans: from macro-to nanogels. J Colloid Interface Sci. 419, 52–55. Han, S.E., Kang, H., Shim, G.Y., Kim, S.J., Choi, H.G., Kim, J., et al., 2009. Cationic derivatives of biocompatible hyaluronic acids for delivery of siRNA and antisense oligonucleotides. J. Drug Target. 17, 123–132. Haque, S., Md, S., Sahni, J.K., Ali, J., Baboota, S., 2014. Development and evaluation of brain targeted intranasal alginate nanoparticles for treatment of depression. J. Psych. Res. 48, 1–12. Heinze, T., Liebert, T., Heublein, B., Hornig, S., 2006. Functional polymers based on dextran. Adv. Polym. Sci. 205, 199–291. Heo, R., Park, J.-S., Jang, H.J., Kim, S.-H., Shin, J.M., Suh, Y.D., Jeong, J.H., Jae, D.-G.J., Park, H., 2014. Hyaluronan nanoparticles bearing γ-secretase inhibitor: in vivo therapeutic effects on rheumatoid arthritis. J. Control. Release 192, 295–300. Hezaveh, H., Muhamad, I.I., 2012a. Impact of metal oxide nanoparticles on oral release properties of pHsensitive hydrogel nanocomposites. Int. J. Biol. Macromol. 50, 1334–1340. Hezaveh, H., Muhamad, I.I., 2012b. The effect of nanoparticles on gastrointestinal release from modified k-carrageenan nanocomposite hydrogels. Carbohydr. Polym. 89, 138–145. Hirano, S., Yamaguchi, R., Fukui, N., Iwata, M., 1990. A chitosan, oxalate gel: its conversion to an N-acetyl chitosan gel via a chitosan gel. Carbohydr. Res. 201, 145–149. Hornig, S., Heinze, T., 2007. Nanoscale structures of dextran esters. Carbohydr. Polym. 68, 280–286. Hu, Y., Jiang, X., Ding, Y., Ge, H., Yuan, Y., Yang, C., 2002. Synthesis and characterization of chitosan– poly(acrylic acid) nanoparticles. Biomaterials 23, 3193–3201.

90  Nanoarchitectonics for Smart Delivery and Drug Targeting

Huang, J., Zhang, H., Yu, Y., Chen, Y., Wang, D., Zhang, G., Zhou, G., Liu, J., Sun, Z., Sun, D., Lu, Y., Zhong, Y., 2014. Biodegradable self-assembled nanoparticles of poly(d,l-lactide-coglycolide)/hyaluronic acid block copolymers for target delivery of docetaxel to breast cancer. Biomaterials 35, 550–566. Janes, K.A., Calvo, P., Alonso, M.J., 2001. Polysaccharide colloidal particles as delivery systems for macromolecules. Adv. Drug Deliv. Rev. 47, 83–97. Jeong, Y.I., Nah, J.W., Na, H.K., Na, K., Kim, I.S., Cho, C.S., Kim, S.H., 1999. Self-assembling nanospheres of hydrophobized pullulans in water. Drug Dev. Ind. Pharm. 25, 917–927. Kaewprapan, K., Inprakhon, P., Marie, E., Durand, A., 2012. Enzymatically degradable nanoparticles of dextran esters as potential drug delivery systems. Carbohydr. Polym. 88, 875–881. Kara, S., Arda, E., Kavzak, B., Pekcan, O., 2006. Phase transitions of kappa-Carrageenan gels in various types of salts. J. Appl. Polym. Sci. 102, 3008–3016. Kim, I.S., Jeong, Y.I., Kim, S.H., 2000. Self-assembled hydrogel nanoparticles composed of dextran and poly(ethylene glycol) macromer. Int. J. Pharm. 205, 109–116. Kim, J., Kim, K.S., Jiang, G., Kang, H., Kim, S., Kim, B.S., Park, M.H., Hahn, S.K., 2008. In vivo real-time bioimaging of hyaluronic acid derivatives using quantum dots. Biopolymers 89, 1144–1153. Kim, S.W., Oh, K.T., Youn, Y.S., Lee, E.S., 2014. Hyaluronated nanoparticles with pH-and enzymeresponsive drug release properties. Colloids Surf. B 116, 359–364. Kim, H., Park, H.T., Tae, Y.M., Kong, W.H., Sung, D.K., Hwang, B.W., Kim, K.S., Kim, Y.K., Hahn, S.K., 2013. Bioimaging and pulmonary applications of self-assembled Flt1peptide-hyaluronic acid conjugate nanoparticles. Biomaterials 34, 8478–8490. Kumar, A., Srivastava, A., Galaev, I.Y., Mattiasson, B., 2007. Smart polymers: physical forms and bioengineering applications. Prog. Polym. Sci. 32, 205–237. Kuo, J.W., Swann, D.A., Prestwich, G.D., 1991. Chemical modification of hyaluronic acid by carbodiimides. Bioconjugate Chem. 2, 232–241. Kwon, G.S., Okano, T., 1996. Polymeric micelles as new drug carriers. Adv. Drug Deliv. Rev. 21, 107–116. Landesman-Milo, D., Goldsmith, M., Ben-Arye, S.L., Witenberg, B., Brown, E., Leibovitch, S., Azriel, S., Tabak, S., Morad, V., Peer, D., 2013. Hyaluronan grafted lipid-based nanoparticles as RNAi carriers for cancer cells. Cancer Lett. 334, 221–227. Langer, R., Tirrell, D.A., 2004. Designing materials for biology and medicine. Nature 428, 487–492. Lee, K.Y., Kwon, I.C., Kim, Y.H., Jo, W.H., Jeong, S.Y., 1998. Preparation of chitosan self-aggregates as a gene delivery system. J. Control. Release 51, 213–220. Lee, J.W., Park, J.H., Robinson, J.R., 2000. Bioadhesive-based dosage forms: the next generation. J. Pharm. Sci. 89, 850–866. Li, P., Dai, Y.-N., Zhang, J.-P., Wang, A.-Q., Wei, Q., 2008. Chitosan-alginate nanoparticles as a novel drug delivery system for nifedipine. Int. J. Biomed. Sci. 4, 221–228. Li, J., Huo, M., Wang, J., Zhou, J., Mohammad, J.M., Zhang, Y., Zhu, Q., Waddad, A.Y., Zhang, Q., 2012. Redox-sensitive micelles self-assembled from amphiphilic hyaluronic acid-deoxycholic acid conjugates for targeted intracellular delivery of paclitaxel. Biomaterials 33, 2310–2320. Liu, Z., Jiao, Y., Wang, Y., Zhou, C., Zhang, Z., 2008. Polysaccharides-based nanoparticles as drug delivery systems. Adv. Drug Deliv. Rev. 60, 1650–1662. Liu, Y., Kong, M., Cheng, X.J., Wang, Q.Q., Jiang, L.M., Chen, X.G., 2013. Self-assembled nanoparticles based on amphiphilic chitosan derivative and hyaluronic acid for gene delivery. Carbohydr. Polym. 94, 309–316. Lu, H.-D., Zhao, H.-Q., Wang, K., Lv, L.L., 2011. Novel hyaluronic acid–chitosan nanoparticles as non-viral gene delivery vectors targeting osteoarthritis. Int. J. Pharm. 420, 358–365.

Chapter 3 • Smart Nanopolysaccharides for the Delivery of Bioactives  91

Luo, Y., Kirker, K.R., Prestwich, G.D., 2000. Cross-linked hyaluronic acid hydrogel films: new biomaterials for drug delivery. J. Control. Release 69, 169–184. Malhotra, M., Tomaro-Duchesneau, C., Prakash, S., 2013. Synthesis of TAT peptide-tagged PEGylated chitosan nanoparticles for siRNA delivery targeting neurodegenerative diseases. Biomaterials 34, 1270–1280. Marcato, P.D., Adami, L.F., Melo, P.S., de Paula, L.B., Durán, N., Seabra, A.B., 2011. Glutathione and S-nitrosoglutathione in alginate/chitosan nanoparticles: cytotoxicity. J. Phys. Conf. Ser. 304, 1–5. Martínez, A., Fernández, A., Pérez, E., Benito, M., Teijón, J.M., Blanco, M.D., 2012. Polysaccharidebased nanoparticles for controlled release formulations. In: Hashim, A.A. (Ed.), The Delivery of Nanoparticles. InTech, Croatia, pp. 185–222. Martínez, A., Iglesias, I., Lozano, R., Teijón, J.M., Blanco, M.D., 2011. Synthesis and characterization of thiolated alginate-albumin nanoparticles stabilized by disulfide bonds. Evaluation as drug delivery systems. Carbohydr. Polym. 83, 1311–1321. Martino, A.D., Sedlarik, V., 2014. Amphiphilic chitosan-grafted-functionalized polylactic acid based nanoparticles as a delivery system for doxorubicin and temozolomide co-therapy. Int. J. Pharm. 474, 134–145. Mi, F.L., Sung, H.W., Shyu, S.S., 2000. Synthesis and characterization of a novel chitosan-based network prepared using naturally occurring crosslinker. J. Polym. Sci. A 38, 2804–2814. Mishra, M.M., Yadav, M., Sand, A., Tripathy, J., Behari, K., 2010. Water-soluble graft copolymer (kappacarrageenan-g-N-vinyl formamide): preparation, char-acterization, and application. Carbohydr. Polym. 80, 235–241. Motwani, S.K., Chopra, S., Talegaonkar, S., Kohli, K., Ahmad, F.J., Khar, R.K., 2008. Chitosan–sodium alginate nanoparticles as submicroscopic reservoirs for ocular delivery: formulation, optimisation and in vitro characterization. Eur. J. Pharm. Biopharm. 68, 513–525. Mukhopadhyay, P., Chakraborty, S., Bhattacharya, S., Mishra, R., Kundu, P.P., 2015. pH-sensitive chitosan/ alginate core-shell nanoparticles for efficient and safe oral insulin delivery. Int. J. Biol. Macromol. 72, 640–648. Muzzarelli, R.A.A., Muzzarelli, C., 2005. Chitosan chemistry: relevance to the biomedical sciences polysaccharides 1: structure, characterization, and use. Adv. Polym. Sci. 186, 151–209. Nagpal, K., Singh, S.K., Mishra, D.N., 2010. Chitosan nanoparticles: a promising system in novel drug delivery. Chem. Pharm. Bull. 58, 1423–1430. Nagpal, K., Singh, S.K., Mishra, D.N., 2015. Minocycline encapsulated chitosan nanoparticles for central antinociceptive activity. Int. J. Biol. Macromol. 72, 131–135. Nair, L.S., Laurencin, C.T., 2007. Biodegradable polymers as biomaterial. Prog. Polym. Sci. 6, 762–798. Najafabadi, A.H., Abdouss, M., Faghihi, S., 2014. Synthesis and evaluation of PEG-O-chitosan nanoparticles for delivery of poor water-soluble drugs: ibuprofen. Mater. Sci. Eng. C 41, 91–99. Nouvel, C., Raynaud, J., Marie, E., Dellacherie, E., Six, J.L., Durand, A., 2009. Biodegradable nanoparticles made from polylactide-grafted dextran copolymers. J. Colloid Interface Sci. 330, 337–343. Opanasopit, P., Apirakaramwong, A., Ngawhirunpat, T., Rojanarata, T., Ruktanonchai, U., 2008. Development and characterization of pectinate micro/nanoparticles for gene delivery. AAPS PharmSciTech 9, 67–74. Ossipov, D.A., 2010. Nanostructured hyaluronic acid-based materials for active delivery to cancer. Exp. Opin. Drug Deliv. 7, 681–703. Otsuka, I., Travelet, C., Halila, S., Fort, S., Pignot-Paintrand, I., Narumi, A., et al., 2012. Thermoresponsive self-assemblies of cyclic and branched oligosaccharide block-poly(N-isopropylacrylamide) diblock copolymers into nanoparticles. Biomacromolecules 13, 1458–1465.

92  Nanoarchitectonics for Smart Delivery and Drug Targeting

Ouchi, T., Nishizawa, H., Ohya, Y., 1998. Aggregation phenomenon of PEG-grafted chitosan in aqueous solution. Polymer 39, 5171–5175. Pandey, R., Ahmad, Z., 2011. Nanomedicine and experimental tuberculosis: facts, flaws, and future. Nanomedicine 7, 259–272. Park, J.H., Cho, Y.W., Chung, H., Kwon, I.C., Jeong, S.Y., 2003. Synthesis and characterization of sugar-bearing chitosan derivatives: aqueous solubility and biodegradability. Biomacromolecules 4, 1087–1091. Park, S.Y., Lee, B.I., Jung, S.T., Park, H.J., 2001. Biopolymer composite films based on k-carrageenan and chitosan. Mater. Res. Bull. 36, 511–519. Passirani, C., Barratt, G., Devissaguet, J.P., Labarre, D., 1998. Interactions of nanoparticles bearing heparin or dextran covalently bound to poly(methyl methacrylate) with the complement system. Life Sci. 62, 775–785. Patrizi, M.L., Piantanida, G., Coluzza, C., Masci, G., 2009. ATRP synthesis and association properties of temperature responsive dextran copolymers grafted with poly(N-isopropylacrylamide). Eur. Polym. J. 45, 2779–2787. Qiu, Y., Park, K., 2001. Environment-sensitive hydrogels for drug delivery. Adv. Drug Deliv. Rev. 53, 321–339. Ragelle, H., Riva, R., Vandermeulen, G., Naeye, B., Pourcelle, V., Le Duff, C.S., D’Haese, C., Nysten, B., Braeckmans, K., De Smedt, S.C., Jérôme, C., Préat, V., 2014. Chitosan nanoparticles for siRNA delivery: optimizing formulation to increase stability and efficiency. J. Control. Release 176, 54–63. Reis, C.P., Ribeiro, A.J., Houng, S., Veiga, F., Neufeld, R.J., 2007. Nanoparticulate delivery system for insulin: design, characterization and in vitro/in vivo bioactivity. Eur. J. Pharm. Sci. 30, 392–397. Reis, C.P., Ribeiro, A.J., Veiga, F., Neufeld, R.J., Damgé, C., 2008. Polyelectrolyte biomaterial interactions provide nanoparticulate carrier for oral insulin delivery. Drug Deliv. 15, 127–139. Rinaudo, M., Pavlov, G., Desbrieres, J., 1999. Influence of acetic acid concentration on the solubilization of chitosan. Polymer 40, 7029–7032. Rotureau, E., Leonard, M., Dellacherie, E., Durand, A., 2004. Amphiphilic derivatives of dextran: adsorption at air/water and oil/water interfaces. J. Colloid Interface Sci. 279, 68–77. Sailaja, A.K., Amareshwar, P., 2012. Preparation of alginate nanoparticles by desolvation technique using acetone as desolvating agent. Asian J. Pharm. Clin. Res. 5, 132–134. Saldías, C., Velásquez, L., Quezada, C., Leiva, A., 2015. Physicochemical assessment of dextran-g-poly(εcaprolactone) micellar nanoaggregates as drug nanocarriers. Carbohydr. Polym. 117, 458–467. Sangeetha, S., Venkatesh, D.N., Adhiyaman, R., Santhi, K., Suresh, B., 2007. Formulation of sodium alginate nanospheres containing amphotericin B for the treatment of systemic candidiasis. Trop. J. Pharm. Res. 6, 653–659. Sarei, F., Dounighi, N.M., Zolfagharian, H., Khaki, P., Bidhendi, S.M., 2013. Alginate nanoparticles as a promising adjuvant and vaccine delivery system. Indian J. Pharm. Sci. 4, 442–449. Sarmento, B., Ribeiro, A., Veiga, F., Sampaio, P., Neufeld, R., Ferreira, D., 2007. Alginate/chitosan nanoparticles are effective for oral insulin delivery. Pharm. Res. 24, 2198–2206. Schipper, N.G., Olsson, S., Hoogstraate, J.A., deBoer, A.G., Vårum, K.M., Artursson, P., 1997. Chitosans as absorption enhancers for poorly absorbable drugs 2: mechanism of absorption enhancement. Pharm. Res. 14, 923–929. Shchipunov, Y.A., 2003. Sol–gel-derived biomaterials of silica and carrageenans. J. Colloid Interface Sci. 268, 68–76. Shin, J.M., Hwang, S.R., Heo, R., Saravanakumar, G., Park, J.H., 2014. Amphiphilic hyaluronic acid derivative with the bioreducible bond: synthesis and its implication for intracellular drug delivery. Polym. Degrad. Stabil. 109, 398–404. Sinha, V.R., Kumria, R., 2001. Polysaccharides in colon-specific drug delivery. Int. J. Pharm. 224, 19–38.

Chapter 3 • Smart Nanopolysaccharides for the Delivery of Bioactives  93

Sorlier, P., Denuzière, A., Viton, C., Domard, A., 2001. Relation between the degree of acetylation and the electrostatic properties of chitin and chitosan. Biomacromolecules 2, 765–772. Sun, G.M., Mao, J.J., 2012. Engineering dextran-based scaffolds for drug delivery and tissue repair. Nanomedicine 7, 1771–1784. Sun, Y., Wan, A.J., 2007. Preparation of nanoparticles composed of chitosan and its derivatives as delivery systems for macromolecules. J. Appl. Polym. Sci. 105, 552–561. Termsarasa, U., Yoon, I.-S., Park, J.-H., Moon, H.T., Choc, H.-J., Kim, D.-D., 2014. Polyethylene glycolmodified arachidyl chitosan-based nanoparticles for prolonged blood circulation of doxorubicin. Int. J. Pharm. 464, 127–134. Tokumitsu, H., Ichikawa, H., Fukumori, Y., 1999. Chitosan-gadopentetic acid complex nanoparticles for gadolinium neutron-capture therapy of cancer: preparation by novel emulsion-droplet coalescence technique and characterization. Pharm. Res. 16, 1830–1835. Unsoy, G., Khodadust, R., Yalcin, S., Mutlu, P., Gunduz, U., 2014. Synthesis of doxorubicin loaded magnetic chitosan nanoparticles for pH responsive targeted drug delivery. Eur. J. Pharm. Sci. 62, 243–250. Van der Merwe, S.M., Verhoef, J.C., Verheijden, J.H.M., Kotze, A.F., Junginger, H.E., 2004. Trimethylated chitosan as polymeric absorption enhancer for improved peroral delivery of peptide drugs. Eur. J. Pharm. Biopharm. 58, 225–235. Vauthier, C., Couvreur, P., 2000. Development of nanoparticles made of polysaccharides as novel drug carrier systems. In: Wise, D.L. (Ed.), Handbook of Pharmaceutical Controlled Release Technology. Marcel Dekker Inc., New York, pp. 413–429. Wang, Y.S., Jiang, Q., Li, R.S., Liu, L.L., Zhang, Q.Q., Wang, Y.M., Zhao, J., 2008. Self-assembled nanoparticles of cholesterol-modified O-carboxymethyl chitosan as a novel carrier for paclitaxel. Nanotechnology 19, 145101. Wang, Y.S., Liu, L.R., Jiang, Q., Zhang, Q.Q., 2007. Self-aggregated nanoparticles of cholesterol-modified chitosan conjugate as a novel carrier of epirubicin. Eur. Polym. J. 43, 43–51. Wang, J., Ni, C., Zhang, Y., Zhang, M., Li, W., Yao, B., Zhang, L., 2014. Preparation and pH controlled release of polyelectrolyte complex of poly(l-malic acid-co-d,l-lactic acid) and chitosan. Colloids Surf. B 115, 275–279. Xu, Y., Wang, L., Li, Y.K., Wang, C.Q., 2014. Oxidation and pH responsive nanoparticles based on ferrocenemodified chitosan oligosaccharide for 5-fluorouracil delivery. Carbohydr. Polym. 114, 27–35. Yan, C.-Y., Gu, J.-W., Hou, D.-P., Jing, H.-Y., Wang, J., Guo, Y.-Z., Katsumi, H., Sakane, T., Yamamoto, A., 2015. Synthesis of Tat tagged and folate modified N-succinyl-chitosanself-assembly nanoparticles as a novel gene vector. Int. J. Biol. Macromol. 72, 751–756. Yang, J.S., Xie, Y.J., He, W., 2011. Research progress on chemical modification of alginate: a review. Carbohydr. Polym. 84, 33–39. Yang, X., Zhang, Q., Wang, Y., Chen, H., Zhang, H., Gao, F., Liu, L., 2008. Self-aggregated nanoparticles from methoxy poly(ethylene glycol)-modified chitosan: synthesis; characterization; aggregation and methotrexate release in vitro. Colloids Surf. B 61, 125–131. Yip, G.W., Smollich, M., Gotte, M., 2006. Therapeutic value of glycosaminoglycans in cancer. Mol. Cancer Ther. 5, 2139–2148. Yoksan, R., Matsusaki, M., Akashi, M., Chirachanchai, S., 2004. Controlled hydrophobic/hydrophilic chitosan: colloidal phenomena and nanosphere formation. Colloid Polym. Sci. 282, 337–342. You, J.-O., Peng, C.-A., 2005. Calcium alginate nanoparticles for non-viral gene delivery. NSTI-Nanotech 1, 270–273. Yuan, Q., Venkatasubramanian, R., Hein, S., Misra, R.D.K., 2008. A stimulus-responsive magnetic nanoparticle drug carrier: magnetite encapsulated by chitosan-grafted-copolymer. Acta Biomater. 4, 1024–1037.

94  Nanoarchitectonics for Smart Delivery and Drug Targeting

Zhang, J., Misra, R.D.K., 2007. Magnetic drug-targeting carrier encapsulated with thermosensitive smart polymer: core–shell nanoparticle carrier and drug release response. Acta Biomater. 3, 838–850. Zhang, J.L., Srivastava, R.S., Misra, R.D.K., 2007. Core–shell magnetite nanoparticles surface encapsulated with smart stimuli-responsive polymer: synthesis, characterization, and LCST of viable drug-targeting delivery system. Langmuir 23, 6342–6351. Zhong, S.P., Campoccia, D., Doherty, P.J., Williams, R.L., Benedetti, L., Williams, D.F., 1994. Biodegradation of hyaluronic acid derivatives by hyaluronidase. Biomaterials 15, 359–365. Zhou, B., McGary, C.T., Weigel, J.A., Saxena, A., Weigel, P.H., 2003. Purification and molecular identification of the human hyaluronan receptor for endocytosis. Glycobiology 13, 339–349.